Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �1

Module 9

Cell Cycle and Proliferation

Synopsis

Cell proliferation is the process whereby cells reproduce themselves by growing and then dividing intotwo equal copies. Growth factors employ a range of growth factor signalling pathways to activate cellsto enter the cell cycle. In most cases, proliferation is mediated by growth factors operating within ahighly localized environment so that only those cells in the immediate vicinity are instructed to grow. Tounderstand how growth factors control cell proliferation, we have to consider both the nature of thesignalling mechanisms and how they impinge upon the cell cycle machinery that regulates cell growthand cell division. One of the most active areas of cell signalling concerns this growth factorsignalling/cell cycle interface.

An analysis of the information flow during proliferative signalling reveals that there is a complexinterplay between different signalling mechanisms operating throughout the early phase of the cellcycle. In order to activate cell proliferation, growth factors have to do two things. They have to inducecells to enter the cell cycle and they also function in cell growth control by increasing cell mass throughan increase in macromolecular biosynthesis. The integrity of the genome is maintained by a process ofcheckpoint signalling that operates to arrest the cell cycle in response to DNA damage.

Proliferation is most evident during development, when the single-celled zygote begins many roundsof growth and division to make up the cell mass of the developing organism. As the adult organism isformed, many cells stop growing when they differentiate to perform their specialized functions. In thecase of neurons and muscle, this growth arrest is permanent, but, for many other cells, this arrest istemporary in that they retain the option of growing again to replace cells that are lost either throughnormal processes of wear and tear or through damage. There are many examples of such proliferationof specific cell types.

Interest in the signalling pathways that regulate cell proliferation increased when it became apparentthat these were altered in many forms of cancer. These signalling pathways also control tumourangiogenesis. Very often, key components of the signalling pathways are proto-oncogenes thatbecome constitutively active to function as oncogenes. Many of the tumour suppressors function asnegative regulators of growth factor-mediated signalling pathways. Changes in these proliferativecontrol mechanisms are also relevant to other diseased states, such as atherosclerosis, psoriasis,hypertension and polycystic kidney disease.

Cell cycleCells enter the cell cycle through two quite distinct pro-cesses (Module 8: Figure cellular life history). One is theunique process of fertilization, and the other is cell pro-liferation activated by growth factors. Here the primaryfocus will be on the cell cycle events that occur when G0

cells are stimulated to proliferate by growth factors. Thereis a complex cell cycle network of signalling pathways thatinteract with each other to control whether or not cells willgrow and divide. There is an extensive cell cycle toolkit thatcontains both the signalling molecules and the large num-ber of targets that are engaged as the cell passes through the

Green text indicates links to content within this module; blue textindicates links to content in other modules.

Please cite as Berridge, M.J. (2012) Cell Signalling Biology;doi:10.1042/csb0001009

cell cycle. Growth factors act early in G1 through a growthfactor signalling/cell cycle interface to engage the cell cyclesignalling system that then takes over and presides over theorderly sequence of events that culminates in cell divisionto give two daughter cells.

Defects in the cell cycle have been linked to variousdiseases, such as microcephaly.

Cell cycle eventsThe sequence of events that occur when a cell is stimu-lated to grow and divide constitutes the cell cycle. Restingcells, which have zero growth (G0), have to be stimulatedby growth factors in order to enter the cell cycle, whichbegins with the first period of growth (G1) during whichit prepares for a period of DNA synthesis (S) (Module 9:Figure cell cycle). Towards the end of G1, there is a re-striction point (R), which marks the point where the cell

C©2012 Portland Press Limited www.cellsignallingbiology.orgLicensed copy. Copying is not permitted, except with prior permission and as allowed by law.

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �2

becomes irreversibly committed to traverse the rest of thecell cycle. In effect, the control of subsequent events ishanded over from the growth factor-mediated signallingpathways to the internal cell cycle signalling system. Thelatter then controls events during the S phase and the fi-nal events of G2 and mitosis, when the cell divides. Oncedivision is complete, control swings back to the growthfactor-mediated signalling pathways. To understand howproliferation is controlled it is therefore necessary to fol-low the sequence of events during G1 that leads up to R.Once R is breached, cells become independent of growthfactors in that further progression through the cell cycleis now in the hands of the cell cycle control system (bluearrows in Module 9: Figure cell cycle).

Once it has duplicated its chromosomes, the cell entersa second period of growth (G2) when it prepares to divideinto two daughter cells during the period of mitosis (M).This M phase is separated into a series of discrete steps thatbegin with prophase and then pass through prometaphase,metaphase, anaphase and finally the process of cytokinesis,which divides the cell into two equal halves (Module 9:Figure mitotic events).

ProphaseDuring prophase, the paired centrosomes begin to separ-ate. The paired chromosomes that were replicated duringthe S phase begin to condense to form the sister chromat-ids. Multisubunit condensing complexes and histone phos-phorylation are used to condense chromatin to form thetwo sister chromatids that are held together by cohesin mo-lecules located at the centromeres. The nuclear membranebegins to break down. This initiation of mitosis is drivenby the cyclin B/cyclin-dependent kinase 1 (CDK1) com-plex working in conjunction with the Polo-like kinases(Plks) (Module 9: Figure mitotic entry).

PrometaphaseIn prometaphase, the nuclear membrane disappears andthe spindle begins to assemble. There are three types ofspindle microtubules: the astral microtubules, the polarmicrotubules, which overlap each other in the middle topush the poles apart, and the kinetochore spindles thatattach to the kinetochore (yellow dots) on the centromeresof the sister chromatids (Module 9: Figure mitotic events).

MetaphaseIn metaphase, the chromatids align in one plane at themetaphase plate. The sister chromatids are attached via thekinetochore microtubule to opposite poles of the spindle.Considerable tension develops between the two chromat-ids because their microtubules are trying to pull themapart. Separation is prevented by the spindle checkpointthat delays separation until all the chromatids are in place.

AnaphaseWhen the spindle checkpoint control is satisfied that all thechromatids are properly aligned, a specific signal triggersthe separation process that is driven by the anaphase-promoting complex (APC) that cleaves the cohesin mo-lecules that hold the chromatids together. There are in-

dications that anaphase is triggered by a pulse of Ca2 +

(Module 9: Figure chromosome separation).

TelophaseAs the chromosomes separate and move towards thespindle poles, the cell begins the final process of cellcleavage: telophase. Cell division is driven by a processof cytokinesis, which depends upon a contractile ring ofactomyosin that gradually contracts to form a constrictionthat cleaves the cell into two daughter cells (Module 9:Figure cytokinesis).

These events of the cell cycle are controlled by an extens-ive cell cycle toolkit (Module 9: Table cell cycle toolkit).

Cell cycle toolkitThe main components of the cell cycle machinery are afamily of proteins known as cyclins and their associatedcyclin-dependent kinases (CDKs) (Module 9: Table cellcycle toolkit). Various combinations of these two com-ponents regulate the orderly progression through the cellcycle. In addition, there are a number of CDK inhibitors(Cip/Kip and p16 families) and a number of other mo-lecules that either regulate or carry out the downstreamevents, such as the pocket proteins (Rb, p107, p130), theE2F family of transcription factors and the mitotic com-ponents such as the Polo-like kinases (Plks), checkpointkinases, regulatory phosphatases, centrosome and spindlecomponents, chromosome separation proteins and thespindle midzone complex. The challenge is to understandhow all these components come together to control theorderly sequence of events that constitutes the cell cycle.Some of these cell cycle control elements operate early inG1 and represent the targets for the proliferative signallingpathways, which are part of the cell cycle network.

Cell cycle networkThere is a complex cell cycle network that has a numberof signalling systems that control whether or not a cellenters the cell cycle. These signalling systems determine thechoices that are available when cells exit from the cell cycle(Module 9: Figure cell cycle network). They can followfour possible pathways, resulting in markedly differentcell fates:

• Senescence• Apoptosis• Differentiation• Cell proliferation

It is this last option of cell proliferation that is controlledby the proliferative signalling pathways that induce resting(G0) or stem cells to enter the cell cycle. These prolifera-tion signalling pathways are highly dynamic systems, withboth positive and negative elements. Many of the positiveelements are proto-oncogenes, which often are mutatedto become constitutively active to form theoncogenesfound in many tumour cells. Conversely, there are neg-ative elements, and many of these are tumour suppressorsthat are inactivated in many tumour cells. There also are

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �3

Module 9: Figure cell cycle

G1G2

M

G0

Growthfactors

S

Growth factorsignalling

Cell cyclesignalling

Daughtercells

Cyclin

D

Cyclin E

Cyclin

A

Cyclin B

R

Operation of the growth factor and cell cycle signalling system during different phases of the cell cycle.Growth factor signalling (red arrows) operates primarily during G1 to drive the cell towards the restriction (R) point, after which control moves overtowards the internal cell cycle signalling system (blue arrows). The different cyclin proteins, which are a central feature of the cell cycle signallingsystem, operate at different points during the cell cycle (Module 9: Figure cell cycle dynamics). Note how the cell grows in size over the course of thecell cycle.

Module 9: Figure mitotic events

Prometaphase esahpateMesahporPG2 A Telophase

Spindleassembly

Interphase cell

Spindlecheckpoint

Chromosomeseparation Cytokinesis

begins andnucleus reforms

Cytokinesiscomplete and

daughter cellsseparate

Centrosomeseparation,Chromosomecondensation,NEB

Summary of the morphological changes that occur during the mitotic phase.The final phase of the cell cycle begins when cells leave the G2 phase and enter the mitotic (M) phase. The events that occur at mitosis are themorphological manifestation of cell division. As cells pass through the G2/M boundary, they begin to divide into two equal daughter cells as they passthrough a series of steps (see the text for details). NEB, nuclear envelope breakdown.

anti-proliferative signalling pathways that inhibit cellsfrom entering the cell cycle. An important example isthe Smad signalling pathway controlled by the transform-ing growth factor-β (TGF-β) superfamily, which not onlyprevents cells from entering the cell cycle, but also drivescells to differentiate.

Presiding over the operation of the cell cycle is the p53surveillance system, which is constantly checking on theperformance of all the cell cycle processes, and particu-larly those concerned with DNA synthesis. The p53 sys-tem is also responsive to cell stress from external sourcessuch as irradiation, which often results in causing damage

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �4

Module 9: Table cell cycle toolkitMolecular components of the cell cycle signalling system.Cell cycle component CommentsCyclins

Cyclin A1 Associates with CDK2Cyclin A2 Associates with CDK2Cyclin B1 Associates with CDK1Cyclin B2 Associates with CDK1Cyclin D1 Widely expressed, interacts with CDK4

and CDK6Cyclin D2Cyclin D3Cyclin E1 Associates with CDK2Cyclin E2 Associates with CDK2Cyclin H Associates with CDK7 to form the

cyclin-dependent kinase(CDK)-activating kinase (CAK)

Cyclin/CDK-associatedproteinsMat1 Stabilizes the cyclin H/CDK7 complex

that forms the cyclin-dependentkinase (CDK)-activating kinase(CAK) (Module 9: Figure cyclinD/CDK action)

Cyclin-dependent kinases(CDKs)CDK1CDK2CDK4CDK6CDK7 A component of the cyclin-dependent

kinase (CDK)-activating kinase(CAK) (Module 9: Figure cyclinD/CDK action)

CDK inhibitorsCip/Kip family

p21 Expression strongly increased by p53(Module 4: Figure Myc as a generepressor)

p27p57

p16 familyp16INK4a This CDK4 inhibitor shares the same

gene locus as the tumoursuppressor alternative reading frame(ARF)

p15INK4b Expression increased by transforminggrowth factor β (TGF-β)

p18INK4c

p19INK4d

Pocket proteinsRb/p105p107p130/Rbl2

E2F family of transcriptionfactorsE2F1 E2 promoter-binding factor 1E2F2E2F3E2F4E2F5E2F6E2F7

E2F-binding partnersDP1DP2

Regulatory kinasesPolo-like kinases (Plks)

Plk1 Plays a major role in regulating mitosis(Module 9: Figure mitotic entry)

Plk2Plk3Plk4Wee1 Phosphorylates Tyr-15 on CDK1

(Module 9: Figure mitotic entry)Myt1 Phosphorylates Thr-14 and Tyr-15 on

CDK1 (Module 9: Figure mitoticentry)

Module 9: Table continuedCell cycle component Comments

Nek2 A kinase that may plays a priming rolein phosphorylating Plk substrates

Citron kinase Phosphorylates myosin light chain(MLC) during cytokinesis (Module 9:Figure cytokinesis)

Checkpoint kinasesATM Ataxia telangiectasia mutatedATR ATM- and Rad3-relatedChk1 Checkpoint kinase 1Chk2 Checkpoint kinase 2Aurora B Phosphorylates MgRac-GAP and

MYPT1 during cytokinesis (Module9: Figure cytokinesis)

Regulatory phosphatasesCdc25

Cdc25A Dephosphorylates CDK2 in late G1

(Module 9: Figure cell cyclesignalling mechanisms)

Cdc25B Dephosphorylates CDK1 at G2/M(Module 9: Figure mitotic entry)

Cdc25C Dephosphorylates CDK1 at G2/M(Module 9: Figure mitotic entry)

Cdc14a Dephosphorylates components of thespindle midzone complex duringcytokinesis (Module 9: Figurecytokinesis)

Centrosome and spindlecomponentsNlp Ninein-like proteinKatanin A microtubule-severing proteinTCTP A microtubule-stabilizing proteinStathmin A microtubule-destabilizing proteinASPM Abnormal spindle protein-like,

microcephaly-associatedChromosome separation

Cohesin A protein that “glues” chromatidstogether during spindle assembly

Securin A scaffolding protein that inactivatesseparase (Module 9: Figurechromosome separation)

Separase A cysteine protease that cleavescohesin (Module 9: Figurechromosome separation)

Spindle midzone complexMKLP1 Mitotic kinesin-like protein 1MKLP2 Mitotic kinesin-like protein 2Plk1 Translocates MKLP2 to the spindleMgRac-GAP A Rho-GEFECT2 A microtubule-associated protein

(MAP)PRC1 Protein regulator of cytokines 1KIF1 A kinesin-4 family member that

interacts with PRC1INCENP Inner centromere proteinSurvivin See Module 9: Figure cytokinesis

to DNA (Module 4: Figure p53 function). All such celldefects are relayed through p53, which then takes appro-priate action, depending on their severity. If the damage isnot too severe, p53-induced cell cycle arrest occurs whilethe DNA damage is repaired (Module 9: Figure cell cyclenetwork). However, if damage is more severe, the cell isdriven towards senescence or p53-induced apoptosis.

With regard to cell proliferation, the main question tofocus on is how the information flowing into the cell fromthe proliferative signalling pathways activates the endo-genous cell cycle signalling system responsible for guidingthe cell through the rest of the cell cycle. This growthfactor signalling/cell cycle interface is of central import-ance because it deals with how the growth factor signalling

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �5

Module 9: Figure cell cycle network

Apoptosis

Proliferation

Proto-oncogenes

Tumoursuppressors Anti-proliferative

signalling pathways

Proliferativesignalling pathways

Growth factors

G0or

Stem cells

Stress signals(UV irradiation etc)

TGF-

CELLCYCLE

Differentiation

Senescence

Cell cyclearrest

p53 surveillancesystem

+ +

+

+

+

_

_

_

G1

S G2

M

The cell cycle network of signalling systems that control cell proliferation and related cellular processes.The cell cycle is at the centre of a number of major cellular signalling systems that not only control cell proliferation, but also determine the subsequentfate of the daughter cells by controlling the processes of differentiation, apoptosis or senescence. Some of the major signalling components associatedwith this network are shown in more detail in Module 9: Figure proliferation signalling network.

systems instruct the cell cycle signalling system to proceedwith another cycle of cell division.

Such a holistic view of the cell cycle network is criticalfor understanding the causes of cancer, because tumori-genesis develops from multiple alterations in componentsdistributed throughout the signalling network that regu-lates the cell cycle (Module 12: Figure cell cycle networkand cancer).

Growth factor signalling/cell cycleinterfaceThe interface between the growth factor signalling path-ways and the cell cycle signalling system is a critical eventin the control of cell proliferation. It is during this processthat information flowing in from the cell surface sets inmotion the sequence of events that culminates in cell divi-sion. The cell cycle network illustrates how the cell cycleis controlled by a number of signalling systems (Module9: Figure cell cycle network). An overview of how theproliferative signalling pathways engage the cell cycle sig-nalling pathway is shown in Module 9: Figure G1 prolif-erative signalling. A key event is the activation of cyclinD, which then activates the E2F transcriptional system,which is critical for initiating the events that lead to DNAsynthesis. The nature of the molecular interface betweenthese signalling systems and the cell cycle signalling systemis shown in more detail in Module 9: Figure proliferationsignalling network.

The cyclins are the primary targets of the signals com-ing into the cell from growth factor receptors on the cellsurface. Proliferative signalling pathways drive cells intothe cell cycle by activating events early in G1. One of

these pathways depends on Ras and the mitogen-activatedprotein kinase (MAPK) signalling pathway, and anotheris the Wnt signalling pathway that acts through β-cateninto increase the level of the transcription factor Myc. Thelatter then increases the transcription of cyclin D, which isan early component of the cell cycle signalling machinery(Module 9: Figure cell cycle dynamics). The cyclin D thenacts on retinoblastoma protein (Rb) to remove its inhib-itory effect on E2F, which then activates the formation ofcyclin E and cyclin A, which are responsible for drivingthe cell into the cell cycle (Module 9: Figure proliferationsignalling network). Myc also increases the expression ofthe inhibitor of DNA binding (Id) proteins that play a keyrole in the proliferation-differentiation switch (Module 8:Figure proliferation-differentiation switch).

In contrast with the positive effects that certain growthfactors have in initiating the cell cycle, other growthfactors such as transforming growth factor-β (TGF-β)act by inhibiting the cell cycle signalling pathway by in-terfering with cyclin activation. The transforming growthfactor-β inhibition of cell proliferation depends upon theup-regulation of the cyclin-dependent kinase (CDK) in-hibitor p15 that inhibits cyclin E and cyclin A. The Smadsalso act to prevent the formation of the transcription factorMyc, which is a key regulator of cell proliferation (Module4: Figure Myc as a gene activator).

The p53 surveillance system, which has such an import-ant role in checking that the cell cycle proceeds normallywith no errors, also exerts its inhibitory effects on the cellcycle by interfering with the cyclins to induce either G1

or G2/M arrest (Module 9: Figure proliferation signallingnetwork). p53 produces p21, growth-arrest and DNA-damage-inducible protein 45 (GADD45) and the 14-3-3

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �6

Module 9: Figure proliferation signalling network

Apoptosis

Cellproliferation

Growth factors

Growth factorreceptors

Stemcell

Stress signals(UV irradiation etc)

TGF-

TGF- R

p53ARF MDM2 GADD4514-3-3

Bax, Noxa, Puma,APAF1, Bcl-2

p21p16

p15 IdsSmads

Cyclin ECyclin A

Cdc25Cyclin B

Rb

APC

Myc

Ras

PP2A

Raf

MAPKs

Cyclin D

-Catenin

E2F

E2F/Rb DNAdamage

G2/

Mar

rest

CELLCYCLE

Differentiation

Senescence

p53 surveillancesystem

Proliferative signallingpathways

Anti-proliferativesignalling pathway

G1

SG2

MG1 arrest

Control of cell proliferation by a network of signalling mechanisms.The hub of this network is the cell cycle, during which proliferative competent cells, such as stem cells, traverse the cell cycle from which they emergeas two daughter cells that then face a number of possible fates. They can re-enter the cell cycle or they can differentiate. Various stresses actingon the p53 system can direct cells towards senescence or apoptosis. Cell proliferation is controlled by a number of signalling systems that act toregulate different components of the cell cycle. This figure illustrates how the various signalling systems outlined in Module 9: Figure cell cycle networkinterface with the cell cycle by targeting the different cyclin molecules that drive the cell cycle.

protein. The p21 inhibits cyclin E to bring about G1 arrest,whereas 14-3-3 protein and GADD45 are responsible forG2/M arrest. The 14-3-3 protein binds to Cdc25, and thusprevents it from activating cyclin B, whereas GADD45also inactivates cyclin B to prevent it from initiating theonset of mitosis (Module 9: Figure cell cycle signallingmechanisms). In addition, p53 promotes the transcriptionof apoptotic factors that initiate apoptosis.

The PtdIns 3-kinase signalling pathway contributes tothe build-up of cyclin D by increasing the stability andthe expression of the cyclin D mRNA. Another import-ant function of this PtdIns 3-kinase pathway is to controlsome of the key regulators of the cell cycle. For example,the downstream effector of this pathway, protein kinase B(PKB), phosphorylates the forkhead box O (FOXO) tran-scription factor to reduce its ability to activate transcrip-tion of the CDK inhibitor p27. This leads to a reduction inthe level of p27, which remains low throughout the rest ofthe cell cycle (Module 9: Figure cell cycle dynamics), andthus facilitates the operation of the cell cycle signallingevents.

The downstream elements of the proliferative and anti-proliferative signalling pathways thus interface with thecell cycle by controlling the activities of the cyclin/CDKcomplexes, which are central components of the cell cyclesignalling system.

Cell cycle signallingCell cycle signalling is responsible for controlling the or-derly sequence of events that occur when a cell is stimu-

lated to grow and divide. Central features of the signallingsystem are the waves of cyclin expression that occur dur-ing the course of the cell cycle (Module 9: Figure cellcycle dynamics). Since the cyclins lack enzymatic activity,their action is mediated by binding to cyclin-dependentkinases (CDKs) to form cyclin/CDK complexes. Eachcyclin binds to a specific CDK (Module 9: Table cellcycle toolkit). The activity of the CDKs depends uponthis association with the cyclins to form the cyclin/CDKcomplexes that control cell cycle progression (Module 9:Figure cell cycle signalling mechanisms). In addition toactivating the CDKs, some of the cyclins, such as cyclinB, also determine the cellular location of the cyclin/CDKcomplex.

Before the CDKs can begin to function as ser-ine/threonine kinases, they have to be activated bythe cyclin-dependent kinase (CDK)-activating kinase(CAK). The different complexes can also be regulatedby more specific phosphorylation/dephosphorylation re-actions that are particularly important in the cyclinB/cyclin-dependent kinase 1 (CDK1) control of mitosis. Inaddition, the cyclin/CDK complexes can be regulated by avariety of cyclin/CDK-associated proteins. For example,Mat1 stabilizes the cyclin H/CDK7 complex, which is acomponent of CAK. The cyclin-dependent kinase (CDK)inhibitors are particularly important in regulating the cyc-lin/CDK complexes that operate early in the G1 phase. Thesequential activation of different cyclin/CDK regulatorycomplexes is responsible for the orderly progression ofthe cell cycle, as illustrated in Module 9: Figure cell cyclesignalling mechanisms:

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �7

Module 9: Figure cell cycle dynamics

G1G0

p27

S

Growth factors

G2 M

Cyclin D

Cyclin ECyclin A

Cyclin B

Changes in the level of different cyclin isoforms and p27 during the course of the cell cycle.Following the addition of growth factors, cyclin D appears first and remains at a high level during the cell cycle. The rise in cyclin D is followed laterduring G1 by the appearance of cyclin E, which rapidly disappears towards the beginning of DNA synthesis (S phase). Cyclin A begins to appeartowards the end of G1, continues to rise during the S phase and then rapidly declines as the cell progresses through G2. Finally, cyclin B begins toappear during the S phase, and this rise continues into the G2 phase before rapidly falling during the mitotic (M) phase. In contrast with these periodicfluctuations in the cyclins, the level of the cyclin-dependent kinase (CDK) inhibitor p27 drops rapidly early in G1 and remains at a low level throughoutthe cell cycle.

Cyclin D/cyclin-dependent kinase 4 (CDK4)activationThe cell cycle is driven by the waves of cyclin forma-tion that begin when growth factors activate the transcrip-tion of cyclin D. Cyclin D then combines with cyclin-dependent kinase 4 (CDK4) to form an active complex thatphosphorylates the retinoblastoma protein (Rb), whichnormally acts to repress the transcriptional activator E2F.This repression is removed when Rb is phosphorylatedand E2F can begin to activate its various gene targetsthat include E2F itself, cyclin E, cyclin A and Cdc25A.Since E2F increases both E2F and cyclin E, it sets up apositive-feedback loop, which serves to increase the phos-phorylation of Rb to provide further transcription of thecomponents necessary to drive the cycle on. The Cdc25Aphosphatase acts to dephosphorylate CDK2, which thenactivates cyclin E/CDK2, a complex that co-operates withcyclin A/CDK2 to activate DNA synthesis at the G1/Sinterface. The way in which cyclin D controls G1 pro-gression depends upon the transcriptional activation andexpression of cell cycle signalling molecules.

Cyclin B/cyclin-dependent kinase 1 (CDK1)activationOnce S phase is complete, the cell enters the second growthphase (G2) when it prepares to begin mitosis. This entryinto mitosis depends upon the activation of the cyclinB/cyclin-dependent kinase 1 (CDK1) complex, which hasbeen kept inactive by phosphorylation of CDK1 by theWee1 and Myt1 kinases. These inhibitory phosphates areremoved by the Cdc25B/C phosphatase, and the active

cyclin B/CDK1 complex enters the nucleus, where it ac-tivates nuclear envelope breakdown (NEB) and spindleassembly. It also contributes to metaphase arrest, whichensures that anaphase is prevented until all the chromat-ids are assembled on the spindle. When this is achieved,the final phase of chromosome separation and cytokinesisis triggered by the activation of the anaphase-activatingcomplex (APC). An increase in Ca2 + , acting throughCa2 + /calmodulin-dependent protein kinase II (CaMKII),triggers this process. Once activated, the APC degradescyclin B, thus cancelling the inhibitory effect of the cyc-lin B/CDK1 complex on anaphase, and it also hydrolysessecurin to release the enzyme separase that is respons-ible for hydrolysing the cohesin that sticks the chromatidstogether. Once they are separated, the chromatids onceagain become functional chromosomes as they move awayfrom each other towards the opposite poles. Cytokinesis istriggered by the formation of a spindle midzone complexthat activates the RhoA signalling mechanisms responsiblefor activating contraction of actin and myosin to completethe processes of mitosis.

The sequence of events just described in Module 9: Fig-ure cell cycle signalling mechanisms provides a signallingroadmap composed of a number of events that are de-scribed in more detail in subsequent sections. The firstevent is the cyclin D control of G1 progression. The levelof cyclin D, which is low in the resting cell (G0), in-creases rapidly following growth factor stimulation. Asthe level of cyclin D rises, it binds to CDK4 or CDK6and the cyclin D/CDK4/6 complex enters the nucleus,where it drives the transcription of a number of cell cycle

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �8

Module 9: Figure cell cycle signalling mechanisms

Cyclin D

p21GADD45a14-3-3

p53

G1G0 S

DNAsynthesis

NEB

Spindleassembly

Metaphasearrest

G2 M

Chromosomeseparation

Cytokinesis

Cyclin D

Cyclin D

E2F

E2F

p15

p27p130

E2F

E2FCyclin ECyclin ACdc25A

FOXO

FOXO

TGF-

Rb

Rb

Cyclin E Cyclin A

Cyclin B

Cyclin B

CDK4 CDK2 CDK2

p27p57

p15 p16

p18p19

P

P

P

P

PP

P

MITOSIS

GROWTHFACTORS

CDK1

CDK1

Cdc25B/C

Cdc25A CaMKII

Ca2+p21

APC

Separase

CDK Inhibitors

RhoAGTP

ROK

Spindlemidzonecomplex

Actin +myosin II

Wee1Myt1

GADD45

14-3

-3

Summary of the major cell cycle control pathways.This figure is a roadmap that charts the signalling sequence of events that occurs when growth factors activate their receptors (top left) to bring aboutmitosis (bottom right). The orderly sequence of events is orchestrated by the different cyclin/cyclin-dependent kinase (CDK) complexes that act atdifferent times during the course of the cell cycle, as described in the text.

components that control the next phases of the cycle. Forexample, cyclin E controls G1 progression and DNA syn-thesis and is followed by cyclin A control of DNA syn-thesis. Cyclin E/CDK2 acts together with cyclin A/CDK2to initiate DNA synthesis. Once DNA synthesis has beenswitched on, most attention is focused on this process ofreplication at the expense of other cellular processes suchas gene transcription, which are switched off. Little elsehappens until DNA synthesis is complete, whereuponthe cell leaves the S phase and enters the G2/M phases,where cyclin B/cyclin-dependent kinase 1 (CDK1) con-trols mitosis. During this final M phase, there are threemain events:

• Nuclear envelope breakdown and spindle assembly con-trolled by the degradation of cyclin B.

• Chromosome separation at anaphase is controlled by aCa2 + -sensitive process.

• Cytokinesis is controlled by formation of the spindlemidzone complex.

Cyclin D controls G1 progressionCyclin D is a key component of the growth factor sig-nalling/cell cycle interface, because it is the main targetof growth factor signalling pathways (Module 9: Figurecell cycle signalling mechanisms). Of the three cyclin Disoforms, cyclin D1 is the most widely expressed in cells.Their partners cyclin-dependent kinase 4 (CDK4) and 6(CDK6) are constitutively expressed, but remain inactiveuntil the level of cyclin D rises early in G1 and begins to ac-tivate transcription through the sequence of events shownin Module 9: Figure cyclin D/CDK action:

1. Cyclin D in the cytoplasm interacts with the cyclin-dependent kinase 4 or 6 (CDK4/6) to form a complexthat moves into the nucleus.

2. The cyclin D/CDK4/6 complex then interacts withthe cyclin-dependent kinase (CDK)-activating kinase(CAK), which is a complex made up of cyclin H, CDK7and Mat1 that phosphorylates the CDK4/6 to activ-ate its kinase activity. The activated cyclin D/CDK4/6complex then has two actions:

3. It begins to phosphorylate retinoblastoma (Rb), whichthen moves away from E2F1, allowing it to activatetranscription. In addition to controlling the activity ofthe E2F transcription factors, the Rb family also reg-ulates transcription by remodelling chromatin throughits ability to pull in the histone deacetylases (HDACs).The phosphorylation of Rb results in the removal ofthe HDACs, which are replaced by the histone acet-yltransferases (HATs) that open up chromatin so thattranscription of cell cycle components can occur.

4. The activated cyclin D/CDK4/6 also phosphorylatesp107/130, which is then unable to facilitate therepressor activity of E2F4/5. The complex moves awayfrom the promoter site, and E2F1 is now able to activatethe transcription of cyclin E.

In addition to controlling the expression of cyclin E,the Rb/E2F signalling systems also control a number ofother genes, such as cyclin A, and thus set up the nextwaves of cyclin formation to propel the cell cycle forward.One of the processes to occur is that cyclin E controls G1

progression and DNA synthesis.As is apparent in Module 9: Figure cell cycle dynam-

ics, the cyclin D level remains high throughout the cell

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �9

Module 9: Figure cyclin D/CDK action

Ac Ac AcAc Ac AcAc Ac Ac

Deacetylation

Acetylation

Sp1

Sp1Cyclin E

Cyclin Egene

repressed

HAT

X

Cyclin D

CAK

Cyclin D Cyclin D Cyclin D

Cyclin H

Cyclin D

E2F1 E2F4/5

E2F4/5

E2F

Rb

CDK7

CDK4/6

CDK4/6

CDK4/6 CDK4/6 CDK4/6

Mat1

Rb

HDACp107p130

p107p1304

3

_

1

2

+

PP P

P

PPPP P

P P

Cyclin D/cyclin-dependent kinase 4/6 (CDK4/6) activation of cyclin E transcription.The grey box at the bottom represents the repressed state of the cyclin E gene as occurs in G0 when cell proliferation is arrested. There are twopromoter sites where the pocket proteins [retinoblastoma protein (Rb), p107 and p130] and the E2F proteins (Module 9: Figure Rb–E2F interactions)interact to repress gene transcription. At one site, the transcriptional activator E2F is repressed by the Rb, which also contributes to repression bybinding the histone deacetylase (HDAC). At the other site, the pocket proteins p107 and p130 facilitate the binding of the E2F4/5 transcriptionalrepressors. When cells are stimulated to grow, there is an increase in the level of cyclin D that sets off a cascade of events that lead to activation ofthe cyclin E gene, as described in the text.

cycle, which means that the daughter cells are able to im-mediately enter another cell cycle once they complete mi-tosis, assuming that growth factors are present. However,if growth factors are withdrawn during the cell cycle, thelevel of cyclin D will fall, and the daughter cells enter a G0

state.

Cyclin E controls G1 progression and DNAsynthesisCyclin E is a nuclear protein that pairs up with cyclin-dependent kinase 2 (CDK2) to form the cyclin E/CDK2complex that has two main functions (Module 9: Figurecell cycle signalling mechanisms). Firstly, it contributesto G1 progression by phosphorylating the retinoblastoma(Rb) family of proteins. This process sets up a positive-feedback loop, because two of the genes activated by Rbphosphorylation are cyclin E itself and the E2F1–E2F3transcription factors. The cyclin-dependent kinase (CDK)inhibitors also play an important role in cyclin E/CDK2activation. In particular, cyclin E/CDK2 can remove p27,which is one of the major inhibitors that limit its activ-ity. It phosphorylates p27, which is then degraded by theSCFSkp2 ubiquitin ligase.

Its second action is to contribute to the onset of DNAsynthesis. One of its substrates is the transcription factornuclear protein mapped to the ataxia telangiectasia locus(NPAT), which regulates the transcription of histone thatwill be required during the S phase. It also phosphorylatescyclic AMP response element-binding protein (CREB)-binding protein (CBP)/p300 to enhance its histone acetyl-transferase (HAT) activity, which is essential for the tran-

scription of many of the genes that are required for drivingthe cell into DNA synthesis. Finally, it can initiate the pro-cess of centrosome duplication by phosphorylating nucle-ophosmin to cause it to leave the centrosome.

The activity of the cyclin E/CDK2 complex is regulatedby Cdc25A, which is one of the dual-specificity Cdc25phosphatases that act by removing inhibitory phosphateson CDK2.

Cyclin E is somewhat unstable, and its level in the cell isdetermined by the balance between synthesis and proteo-lysis. The latter is carried out by two separate ubiquit-ination pathways. Monomeric cyclin E is degraded bythe pathway that depends on the Cul-3 protein. On theother hand, the cyclin E/CDK2 complex is degraded bythe SCFFbw7 ubiquitin ligases. As the cell cycle proceeds,these proteolytic mechanisms gain the upper hand and thelevel of cyclin E falls, and its role is then taken over by thecyclin A control of DNA synthesis.

Roscovitine, which is a potent inhibitor of cyc-linE/CDK2, is being tested as an anticancer drug. It mayalso prove useful for treating polycystic kidney disease.

Cyclin A control of DNA synthesisCyclin A is a nuclear protein of which there are twoisoforms. The cyclin A1 is normally restricted to germcells, whereas the cyclin A2 functions in most cells tocontrol both the G1/S and G2/M transitions. In effect,it provides a link between the early events in G1 and thefinal events that occur during the G2/M phase, when cyclinB/cyclin-dependent kinase 1 (CDK1) controls mitosis.

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �10

Cyclin B/cyclin-dependent kinase 1 (CDK1)controls mitosisThe cyclin B/cyclin-dependent kinase 1 (CDK1) complexcontrols the final events of the cell cycle, during which thecell divides into two daughter cells (Module 9: Figure cellcycle signalling mechanisms). This mitotic phase can bedivided into three main phases. The first phase is nuclearenvelope breakdown and spindle assembly. The second ischromosome separation at anaphase, and, finally, there isthe process of cytokinesis, during which the cell dividesinto the two daughter cells.

Nuclear envelope breakdown and spindle assemblyThe entry into mitosis at the G2/M interface depends uponthe activation of the cyclin B/cyclin-dependent kinase 1(CDK1) complex. The main events that control passagethrough the G2/M boundary are illustrated in Module 9:Figure mitotic entry:

1. The cyclin B/CDK1 complex is kept quiescent duringinterphase, during which it tends to shuttle in and outof the nucleus. This nuclear shuttling is controlled by anuclear localization signal (NLS) that targets the com-plex in an importin-β-dependent manner. In addition,there is a cytoplasmic retention signal (CRS) that hasa nuclear export signal (NES) responsible for Crm1-dependent export. The nuclear export system predom-inates, and the inactive cyclin B/CDK1 complex is loc-ated mainly in the cytoplasm during G2. In addition,there are two inhibitory phosphorylation sites (Thr-14 and Tyr-15) that are phosphorylated by the kinasesWee1 and Myt1. Wee1 phosphorylates Tyr-15 in thenucleus.

2. Myt1 phosphorylates both Thr-14 and Tyr-15 in thecytoplasm, and these two phosphorylations serve toinactivate cyclin B/CDK1 and to retain it in the cyto-plasm.

3. The critical event for the activation of cyclin B/CDK1is the removal of these inhibitory phosphates by theCdc25 phosphatases, with the Cdc25B acting togetherwith Cdc25C. Cdc25B, which seems to initiate the pro-cess, is activated first following its dephosphorylation.

4. Cdc25B begins to dephosphorylate cyclin B/CDK1.5. Activation of Cdc25C is enhanced by phosphorylation

through both CDK1 and the Polo-like kinases (Plks).This is an example of how these two kinases co-operatewith each other. The CDK1 acts as a priming kinase byadding a phosphate to the Cdc25C, which then acts as abinding site for Plk1 that attaches itself to add anotherphosphate. This phosphorylation of Cdc25C providesa positive-feedback loop that functions to sharpen andenhance the activation of the cyclin B/CDK1 complex.

6. The activated Cdc25C is then able to contribute to thedephosphorylation and activation of cyclin B/CDK1.

7. The dephosphorylated cyclin B/CDK1 enters the nuc-leus, and acts together with Plk1 to begin the mitoticevents.

8. Cyclin B/CDK1 phosphorylates nuclear lamins andtriggers nuclear envelope breakdown. One of the im-portant actions of cyclin B/CDK1 is to inhibit the

anaphase-promoting complex (APC), which is re-sponsible for metaphase arrest. One of the activators ofAPC is Cdh1, which is inactivated following its phos-phorylation by cyclin B/CDK1. One component ofthis metaphase arrest is cyclin B/CDK1, which phos-phorylates and thus inactivates Cdh1, which is an ac-tivator of APC. The APC is also inactivated by thespindle-assembly checkpoint signalling pathway (see10 below).

9. Plk1 controls the maturation of the centrosome by re-cruiting γ-tubulin and by phosphorylating some ofthe centrosomal proteins, such as ninein-like protein(Nlp), katanin (a microtubule-severing protein), trans-lationally controlled tumour protein (TCTP), stathmin(a microtubule-destabilizing protein) and abnormalspindle protein-like, microcephaly-associated (ASPM)protein. Once Nlp is phosphorylated, it leaves thecentrosome and makes way for the other proteins toform the microtubule organizing centre (MTOC) thatorchestrates the polymerization of tubulin to assemblethe spindle. Mutation of ASPM has been linked tomicrocephaly.

10. It is important that the next process of chromosomeseparation is delayed until all of the chromosomesare located correctly on the spindle. The kineto-chores, which are the sites on the centrosome re-gion of the chromosome where the microtubules at-tach, play an important role in the spindle-assemblycheckpoint, which is a surveillance system that en-sures that chromosome separation at anaphase isdelayed until metaphase is complete. Those kineto-chores, which are not attached to microtubules, activatea spindle-assembly checkpoint signalling pathway thatensures that anaphase is delayed until all the chromatidsare lined up on the spindle equator (Module 9: Figurespindle assembly checkpoint).

Microtubule organizing centre (MTOC)As its name implies, the microtubule organizing centre(MTOC) functions to organize the polymerization of mi-crotubules into networks that control various cellular pro-cesses:

• The MTOC forms the basal bodies that organize flagellaand cilia.

• The MTOC is part of the centrosome, which consistsof a pair of centrioles, directs spindle assembly (see step9 in Module 9: Figure mitotic entry).

• During neutrophil chemotaxis, the MTOC functionsto align the microtubules in the direction of movementand helps to stabilize cell polarity. It also directs vesiclestowards the leading edge (Module 11: Figure neutrophilchemotaxis).

• In natural killer (NK) cells and cytotoxic T lymphocytes(CTLs), the MTOC is orientated between the nucleusand the target cell or antigen-presenting cell respectively.This orientation enables the cells to deliver vesicles tothe region of the immunological synapse.

• During FcγR-mediated phagocytosis in macrophages,the MTOC and Golgi are orientated towards the

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �11

Module 9: Figure mitotic entry

APC

Wee1

1

++

+

++

+

ProphaseG2 Prometaphase Metaphase

Cyclin B

Cyclin B

Cyclin B

Cyclin B

Cyclin B

P

P

P

P

P

P

P

P

P

P

METAPHASEARREST

CDK1

CDK1

CDK1

CDK1

CDK1

Cdc25B

Nuclearenvelope breakdown

Microtubule

Cdc20

Em

i2

Cdh1

Nuclearlamins

Chromosomecondensation

Spindle assemblycheckpoint

Spindleassembly

Plk1

P

Cdc25B

Cdc25C

Myt1

2

3

4

5

6

7

9

10

8

Centrosomeduplication

Centrioles

Centrosome

Entry into the final mitotic phase of the cell cycle.Passage through the G2/M boundary depends upon the cyclin B/cyclin-dependent kinase 1 (CDK1) complex that is inactivated during G2, but isactivated by a series of steps as the cells enter prophase (see the text for further details). The operation of the spindle-assembly checkpoint thatprevents premature separation of the chromosomes is described in Module 9: Figure spindle assembly checkpoint.

phagosome and this may facilitate the antigen processingevents.

Spindle-assembly checkpoint signallingErrors in chromosome segregation can lead to aneuploidy(abnormal chromosome numbers), which is a feature ofmany aggressive tumours. Such errors are prevented bya dynamic surveillance mechanism known as the spindle-assembly checkpoint, which ensures that segregation doesnot begin until all the chromatid pairs are correctly alignedon the mitotic spindle (Module 9: Figure spindle as-sembly checkpoint). This checkpoint operates by con-trolling the activity of the anaphase-promoting complex(APC) responsible for initiating chromosome separationat anaphase segregation (Module 9: Figure chromosomeseparation).

The spindle-assembly checkpoint signalling pathway,which is still being worked out, has to detect the exist-ence of free kinetochores and relay this information to theAPC complex to inhibit its activity. The following hypo-thesis has been proposed to explain how this signallingpathway might operate. The proteins Mad2 and BubR1appear to be the sensors that detect the free kinetochoreson the incomplete spindle (Module 9: Figure spindle as-sembly checkpoint). These proteins are also able to bindthe Cdc20 to prevent it from activating APC. Before Cdc20can be activated, it must be released from this inhibitoryaction of Mad2/BubR1 and must also by polyubiquin-

ated by a combination of APC and E2 ubiquitin ligaseUbcH10. The p21Comet facilitates the binding of UbcH10to Cdc20. Even though these two ligases are constitutivelyactive, they are not able to ubiquitinate Cdc20 becauseany added ubiquitin groups are immediately removed byan ubiquitin-specific protease 44 (Usp44). There appearsto be a futile cycle of ubiquitination and deubiquitination,which is biased in favour of the latter, thus ensuring thatthe Cdc20 is kept inactive. As soon as the spindle is com-plete, the complexes located on the kinetochores dissociateto liberate the Cdc20. The latter is then rapidly ubiquit-inated and this enables it to stimulate APC to initiate theprocess of chromosome separation at anaphase (Module 9:Figure chromosome separation).

Chromosome separation at anaphaseThe spindle-assembly checkpoint, which ensures thatanaphase is delayed until all the chromatids are lined up onthe spindle equator (metaphase plate), is switched off onceall the chromatids are assembled on the spindle (Module 9:Figure spindle assembly checkpoint). Chromosome separ-ation can now begin through a process that appears to betriggered by an increase in Ca2 + that initiates a signallingsequence that activates the anaphase-promoting complex(APC) through the sequence of steps shown in Module 9:Figure chromosome separation:

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �12

Module 9: Figure spindle-assembly checkpoint

Spindle-assemblycheckpoint

Spindle-assemblycheckpoint

Chromosomeseparation

APC

Incompletespindle

Completespindle

Usp44Inactive

MicrotubuleChromatid

Kinetochore

Ubiquitination

Deubiquitination

Mad2

Mad2

p21p21

CometComet

BubR1

BubR1

APC

Active

APCUbcH10

Cdc20 Cdc20

X

UbUb

UbUb Ub

Ub

Ub

Spindle-assembly checkpoint signalling pathway.During assembly of the spindle, the free kinetochores, which have not bound a microtubule, assemble a molecular complex (see box) that inhibitsthe Cdc20 protein which is responsible for activating the anaphase-promoting complex (APC) that initiates chromatid separation. This checkpoint isremoved when the spindle is fully assembled. The inhibitory protein complex dissociates and Cdc20 is able to activate APC and anaphase begins asdescribed in (Module 9: Figure chromosome separation). The hypothesis presented in this figure was developed by Stegmeier et al. 2007.

1. Ca2 + activates Ca2 + /calmodulin-dependent proteinkinase II (CaMKII) to phosphorylate Emi2, which isan inhibitor of the APC.

2. The phosphate on Emi2 provides a binding site for thePolo-like kinase 1 (Plk1).

3. Plk1 then phosphorylates an additional site on Emi2,which creates the phosphodegron [a phosphorylatedresidue(s)] that makes this inhibitor susceptible to de-gradation.

4. The ubiquitination complex SCF (Skp/cullin/F-box)then degrades the phosphorylated Emi2 to activate theAPC. The APC is inactivated by components of thecheckpoint control systems. One of these is exertedby Emi2, which inactivates Cdc20, which is one of theAPC activators.

5. One of the functions of the active APC is to de-grade cyclin B to inactivate cyclin-dependent kinase1 (CDK1), which has the effect of allowing the Cdh1to further activate APC.

6. The APC also degrades securin to release the enzymeseparase that is responsible for hydrolysing cohesinmolecules.

7. Sister chromatids (green lines) are held together by co-hesin molecules that are part of the kinetochore com-plex at the centromere. These cohesins are composedof two subunits; Rec8 and stromal antigen 2 (SA2).This SA2 subunit can be phosphorylated by Plk1 andwould lead to dissociation of the complex except thatstability is maintained by a protein called shugosin thathas a binding site for the B56 regulatory subunit of

protein phosphatase 2A (PP2A), which ensures thatSA2 remains unphosphorylated. Once separase is ac-tivated, cohesin is hydrolysed and the two chromatidsnow separate to become the chromosomes of the twodaughter cells. Now the kinetochore microtubules canbegin to pull the chromosomes apart. This separation ofthe chromosomes marks the beginning of cytokinesis.

8. APC plays an important role in the proliferation-differentiation switch by hydrolysing the inhibitorof DNA binding (Id) proteins to liberate transcrip-tion factors such as E2A that promote differentiation(Module 8: Figure proliferation-differentiation switch).

CytokinesisThe final process of mitosis is cytokinesis, which is re-sponsible for cell division when the cell cleaves into twoequal daughter cells. As the chromosomes move towardsthe poles during anaphase, the spindle remains in the formof the overlapping polar microtubules. Proteins that areconcentrated at this midzone of the spindle then determ-ine the division plane during cytokinesis. The exact pro-cess and how it is controlled is still being worked out, butit appears to depend upon four clearly defined processesthat begin with assembly and activation of a spindle mid-zone complex, which results in the activation of RhoA thatthen controls assembly of the actomyosin contractile ringand the activation of contraction. Formation of the cleav-age furrow depends upon the trafficking and insertion ofmembrane vesicles.

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �13

Module 9: Figure chromosome separation

1

+

Metaphase

Cyclin B

Cyclin B

Cyclin BP

P

P

PP

P

P

P

P

P

P

P

P

PCDK1

CDK1

CDK1

CaMKIICaN

Ca2+

Cdh1

Cdh1

Cdh1

Chromosomeseparation

Cohesinhydrolysis

Differentiation

2

3

4 5

6

8

7

Anaphase Telophase

APC

APC

APCSCF

Cdc20

Cdc20

Cdc20

Em

i2E

mi2

Emi2

Plk1

B56

B56

Securin

Ids

Ids

Securin

Separase

Shugosin

Shugosin

Kinetochore

E2Aetc

E2Aetc

X

METAPHASEARREST

_

_

PP2A

Cohesin

PP2A

Separase

Control of chromosome separation at anaphase.The spindle checkpoint ensures that the cell cycle is arrested at metaphase until all the chromatids are lined up on the metaphase plate. The onset ofchromosome separation, which marks the beginning of anaphase, is triggered by a specific stimulus. There are indications that a surge in the levelof Ca2 + may be responsible for activating the anaphase-promoting complex (APC) through the sequence of events described in the text.

Assembly and activation of a spindle midzonecomplexThe polar microtubules that are left behind after thechromosomes move towards the poles function to as-semble the spindle midzone complex that determinesthe division plane (Module 9: Figure cytokinesis). Thismidzone complex is made up of three main groups.There is the Aurora B/inner centromere protein (IN-CENP)/survivin/Csc1 group, the protein-regulating cy-tokinesis 1 (PRC1)/KIF1 group and the mitotic kinesin-like protein 1 (MKLP1)/MgcRac GTPase-activating pro-tein (MgRacGAP) group. Some of these components, suchas PRC1, MKLP1 and INCENP, are phosphorylated bythe cyclin B/cyclin-dependent kinase 1 (CDK1) complexand lie inactive in the cytoplasm. In the case of MKLP1,its phosphorylation inhibits its motor activity, which thusprevents it from binding to the spindle. When the cyc-lin B/CDK1 complex is degraded at anaphase (Module9: Figure chromosome separation), these phosphorylatedproteins are dephosphorylated by the phosphatase Cdc14aand are then incorporated into the spindle midzone com-plex (Module 9: Figure cytokinesis). The site of insertionof the MKLP1/MgcRacGAP complex may depend uponthe Aurora B complex. Aurora B may play a role in stimu-lating MgcRacGAP, which is a component of the signallingsystem that controls the assembly of the actomyosin con-tractile ring.

Assembly of the actomyosin contractile ringThe spindle midzone complex determines the positionwhere the contractile ring is formed, and it also helps

to control the activation of RhoA, which is responsiblefor initiating the assembly of actomyosin fibres (Module9: Figure cytokinesis). Actin is assembled through an in-teraction between the proteins formin and profilin. Theformin normally functions to cap the barbed ends of theactin filaments to prevent them from growing. However,the activated RhoA-GTP binds to the N-terminal regionof formin, its autoinhibition is removed, and the filamentbegins to grow by nucleating the actin monomers carriedin by profilin. As the activation of contraction proceedsand the contractile ring contracts down, there is an over-abundance of actin, and this is then reduced by cofilin,which acts to destabilize and severe the redundant actinfilaments.

Activation of contractionContraction of the contractile ring is controlled by phos-phorylation of the myosin light chains (MLCs) on Ser-19and Thr-18, which is carried out by a number of mechan-isms (Module 9: Figure cytokinesis). This phosphorylationof MLC enables the head of the non-muscle myosin II mo-tor to interact with actin to bring about contraction of thering. One of the phosphorylation pathways is carried outby the RhoA signalling mechanism, which results in stim-ulation of Rho kinase (ROK) that activates contraction bystimulating MLC or by inhibiting myosin phosphatase tar-geting subunit 1 (MYPT1). The latter is a targeting proteinthat positions the protein phosphatase 1c (PP1c) at its siteof action (Module 5: Table PP1 regulatory, targeting andinhibitory subunits and proteins), which, in this case, is on

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �14

Module 9: Figure CaM redistribution at mitosis

Redistribution of calmodulin during the 90 min time period of mitosis in a HeLa cell.At the S/G2 interface, calmodulin is concentrated in the nucleus (A), and then begins to segregate into two sites where the centrosomes are located(B and C). As the spindle begins to form, it becomes highly concentrated in the polar regions (D and E), where it remains during metaphase (F),anaphase (G and H) and during cytokinesis (I--P). Towards the end of anaphase, calmodulin begins to concentrate in the cortex immediately belowthe plasma membrane at the site where the cleavage furrow will form (G). The density in this region builds up progressively at the site where thecleavage processes cut the cell into two. Reproduced from Li, C.J., Heim, R., Lu, P., Tsien, R.Y. and Chang, D.C. (1999) Dynamic redistribution ofcalmodulin in HeLa cells during cell division as revealed by a GFP--calmodulin fusion protein technique. J. Cell Sci. 112:1567–1577, with permissionfrom The Company of Biologists; see Li et al. 1999.

the myosin molecule, where it can remove the activatingphosphates on MLC. The MYPT1 is inhibited followingits phosphorylation by both Rho kinase and by Aurora B.Citron kinase, which is another Rho-effector kinase, hasalso been implicated in the control of myosin activity.

MLC can also be phosphorylated by myosin light chainkinase (MLCK), which is stimulated by Ca2 + actingthrough calmodulin (CaM). There certainly are remark-able changes in the distribution of CaM during mitosis(Module 9: Figure CaM redistribution at mitosis). The highconcentration at the spindle poles suggests some role in theprocess of anaphase. The appearance of CaM at the site ofthe cleavage furrow is consistent with a role for Ca2 + incontrolling cytokinesis (Module 9: Figure cytokinesis).

Imaging experiments have observed localized Ca2 + sig-nals in the vicinity of the cleavage furrow. This localizedCa2 + signal may be released from inositol 1,4,5-trisphos-phate (InsP3) receptors because an extra cleavage furrowwas induced by injecting microsomes rich in these re-ceptors near the surface membrane of a Xenopus embryo(Module 9: Figure InsP3 receptors and cell cleavage).

Trafficking and insertion of membrane vesiclesThe monomeric G protein Rab11, operating through theRab11-family interacting protein 2 (FIP2), plays a role incontrolling the trafficking and insertion of new vesicles toform the cleavage furrow (Module 9: Figure cytokinesis).Annexin A11 also seems to play a role in the trafficking ofvesicles to the plasma membrane, and this would suggestthat it is a Ca2 + -mediated process.

Cyclin-dependent kinase(CDK)-activating kinase (CAK)The cyclin-dependent kinase (CDK)-activating kinase(CAK) is a complex composed of cyclin H, CDK7 andMat1, which is a RING finger protein that stabilizes thecomplex. CAK functions to activate the cyclin/CDK com-plexes by phosphorylating a threonine residue located onthe T-loop region of the CDKs. This T-loop region in-hibits the CDKs by covering up their ATP-binding sites.CAK moves this T-loop out of the way to activate theCDKs. CAK plays a critical role in activating the CDKs.For example, it activates cyclin D/CDK4/6 once it entersthe nucleus (Module 9: Figure cyclin D/CDK action).

Polo-like kinases (Plks)The Polo-like kinases (Plks) (Module 9: Table cell cycletoolkit), which are expressed from G2 onwards and peakat the time of mitosis, are serine/threonine kinases thatplay a critical role during the M phase by controlling manyaspects of mitosis, including nuclear envelope breakdownand spindle assembly, chromosome separation at anaphaseand the process of cytokinesis. They have an N-terminalkinase domain and two C-terminal Polo-box (PB) domains(PB1 and PB2) responsible for binding to their substrates.These PB domains form a phosphopeptide-binding motif.In its resting state, these PB domains bend around to in-teract with the kinase catalytic region to keep it inactive.Activation of the Plks depends upon the phosphorylationof Thr-210 in the T-loop of the kinase domain. They alsohave a D-box motif, which controls their destruction by

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �15

Module 9: Figure InsP3 receptors and cell cleavage

Induction of an extra-cleavage furrow by injecting microsomes rich in inositol 1,4,5-trisphosphate (InsP3) receptors close to the membrane.The microsomes were injected into one cell of a Xenopus embryo after the first cleavage (A). By the time of the next cleavage, an extra furrow appearedat the site where the inositol 1,4,5-trisphosphate (InsP3)-rich microsomes had been deposited. Reproduced from Dev. Biol., Vol. 214, Mitsuyama,F., Sawai, T., Carafoli, E. Furuichi, T. and Mikoshiba, K., Microinjection of Ca2 + store-enriched microsome fractions to dividing newt eggs inducesextra-cleavage furrows via inositol 1,4,5-trisphosphate-induced Ca2 + release, pp. 160--167. Copyright (1999), with permission from Elsevier; seeMitsuyama et al. 1999.

Module 9: Figure cytokinesis

+

+

+

+

+

+

+

+

+

+

MLCK

CYTOKINESIS

MgcRacGAP

RhoA RhoA

AnnexinA11

RhoA

Actin-Profilin

Profilin

Polarmicrotubule

Formin

Myosin

Actin

GDP GTP

GTP

P

P

PP

ROK

Citronkinase

CaM

Ca2+

Ca2+

INCENP

INCENP

PRC1

PRC1KIF1

Rab11FIP3

ECT2

MLC

CSC1

Survivin

Spindle midzonecomplex

AuroraB Aurora

B

MKLP1

MKLP1

Cdh1 APCPlk1

Plk1

Cdc14a

PP1c

PP1c

MYPT1

MYPT1

Actin assembly

Vesiclefusion

P

P

P

P

Control of cytokinesis.Cytokinesis begins at telophase, and is driven by formation of an actomyosin contractile ring and by a process of vesicle fusion. Contraction of thering squeezes the cell such that it finally cleaves in two. Formation of the contractile ring is controlled by the spindle midzone complex that forms inthe region where the polar microtubules intersect. One of the components of this complex is MgcRacGAP, which is a GTPase protein that controls theactivity of RhoA. The latter has a central role in regulating both the formation of the contractile ring and in the activation of the ring during cytokinesis(see the text for further details).

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �16

the anaphase-promoting complex (APC). The Plks, par-ticularly Plk1, play an important role in controlling vari-ous events during the M phase, such as entry into mitosis(Module 9: Figure mitotic entry) and the separation of thechromatids at anaphase (Module 9: Figure chromosomeseparation).

Cyclin-dependent kinase (CDK)inhibitorsThere are two quite distinct families of cyclin-dependentkinase (CDK) inhibitors (Module 9: Table cell cycletoolkit) that play a critical role in regulating the cell cycle:the INK4 family and the Cip/Kip family.

The INK4 family functions to inhibit cyclin D/CDK4/6complexes (Module 9: Figure cell cycle signalling mech-anisms). There is considerable interest in this family be-cause p15 functions in the transforming growth factor β

(TGF-β) inhibition of cell proliferation. There also is in-terest in p16INK4a, which is a specific inhibitor of CDK4,because it is a tumour suppressor that plays a role in thedevelopment of some cancers. p16INK4a has also been im-plicated in the signalling pathways of compensatory hy-pertrophy.

The activity of cyclin E/CDK2 is controlled by theCip/Kip family, which is older than the INK4 family inevolutionary terms, consists of p21, p27 (KIP1) and p57(KIP2). The p53-induced cell cycle arrest depends upon thetranscriptional activation of various cell cycle components,such as p21 (Module 4: Figure p53 function). Similarly,the hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]inhibits parathyroid gland hyperplasia by increasing theexpression of p21 (Module 7: Figure PTH secretion).

Mutations of p57 have been linked to Beckwith--Wiedemann syndrome.

The p27 also plays an important role in the activation ofthe cyclin E/CDK2 complex (Module 9: Figure cell cyclesignalling mechanisms). The onset of cell proliferation de-pends upon a rapid fall in the level of p27 early in G1

(Module 9: Figure cell cycle dynamics).

Pocket proteinsThe pocket proteins, represented by the retinoblastomaprotein (Rb) family (Module 9: Table cell cycle toolkit)contains three proteins: Rb/p105, p107 and p130/Rbl2.Rb play a central role in regulating the cell cycle throughtheir control of the E2F family of transcription factorsthat regulate expression of a number of cell cycle compon-ents, such as cyclin E, cyclin A, Cdc25A and E2F itself(Module 9: Figure cell cycle signalling mechanisms). TheRb family and the E2F family bind to each other (Module9: Figure Rb–E2F interactions) to set up a complex web ofinteractions that function to both activate and repress tran-scription of a large number of genes. The Rb family acts byregulating the activity of the E2F family, with Rb acting asan activator, whereas p107 and p130/Rbl2 are repressors.

The pocket proteins have an important role in main-taining the cell in a G0 state by repressing the E2F genes(Module 9: Figure cyclin D/CDK action). Most attention

has focused on Rb, which is a nuclear protein whose activ-ity is regulated through its phosphorylation state. Duringthe resting G0 phase, hypophosphorylated Rb binds toE2F to repress its transcriptional activity. However, whencyclin D/cyclin-dependent kinase 4/6 (CDK4/6) beginsto phosphorylate Rb, the latter leaves the promoter siteto allow E2F1--E2F3 to transcribe a variety of genes thatfunction in the cell cycle (Module 9: Figure cell cycle sig-nalling mechanisms). In the case where Rb family mem-bers act to control repressors such as E2F4 and E2F5, thephosphorylation of p107 and p130/Rbl2 removes theserepressors. Therefore, there are two main mechanismswhereby the Rb family regulates transcription. Rb al-lows the E2F1--E2F3 isoforms to exert their activator role,whereas p107 and p130 remove the repressor activity ofE2F4 and E2F5 (Module 9: Figure cyclin D/CDK action).

E2F family of transcription factorsThe family of E2F transcription factors (Module 9: Tablecell cycle toolkit) plays a critical role in cell proliferation bycontrolling the transcription of many of the key compon-ents of the cell cycle. The E2Fs function together with theirbinding partners DP1 and DP2. The E2F family can be di-vided into those that are strong transcriptional activators(E2F1, E2F2 and E2F3a) and those that act as repressors(E2F4--E2F7) (Module 9: Figure Rb–E2F interactions).Both the activators and the repressors are controlled bythe family of pocket proteins. Retinoblastoma protein (Rb)acts to regulate E2F1--E2F3, whereas the repressors E2F4and E2F5 are regulated by the pocket proteins p107 andp130 (Module 9: Figure cyclin D/CDK action). E2F6 andE2F7 act independently of the pocket proteins.

The Rb/E2F families set up a network that not only con-trols cell proliferation, but also participates in checkpointcontrols, differentiation and apoptosis.

DNA damage and checkpoint signallingDNA damage occurs frequently during the life of a celland can lead to mutations and the development of can-cer. There are different types of damage that give rise todifferent types of DNA lesions that are then dealt withby a sophisticated set of repair mechanisms. Since theserepair processes can take some time to complete, a seri-ous problem arises in cells that are actively proliferating.It would be disastrous if cells began DNA synthesis ormitosis while repairing their DNA. Consequently thereare DNA damage checkpoint signalling systems that op-erate to co-ordinate the processes of DNA repair with thedifferent phases of the cell cycle.

DNA damageDNA damage is caused by both exogenous and endo-genous agents resulting in different types of lesion. Theselesions are then recognized by components of the differ-ent repair mechanisms. This DNA damage is caused bytwo main groups of agents. Firstly, there are exogenousagents such as chemicals, ultraviolet (UV) and ionizing ra-diation (IR). Secondly, there are endogenous agents such

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �17

Module 9: Figure Rb–E2F interactions

E2F1 and E2F2

E2F3a

E2F3b

E2F4 and E2F5

E2F6

E2F7

DB

NLS

Pocket proteinsE2F Family of transcription factors

NES

DB

DB

DB

DB1 DB2

DB

DIM

DIM

DIM

DIM

DIM

Rb

Rb

Rb

p107

p130

PcG

The domain structure of the E2F family of transcription factors and their Rb family-binding partners.All the E2F transcription factors have a DNA-binding domain (DB), and most of them also have a dimerization domain (DIM). The Rb family-bindingdomain (green) located in the C-terminal region of the transcriptional activators E2F1--E2F3 is responsible for binding retinoblastoma protein (Rb),whereas p107 and p130 bind to a similar domain on the repressors E2F4 and E2F5. E2F6 and E2F7 do not bind pocket proteins, but E2F6 doesbind to the Polycomb group of proteins (PcG). The E2F1–E2F3 isoforms have a nuclear localization signal (NLS), whereas E2F4 and E2F5 have twonuclear export signals (NESs).

as reactive oxygen species (ROS) that induce DNA breaks,oxidation of bases, mismatches caused by replication de-fects and strand breaks caused by collapse of the replicationfork. Double strand breaks (DSBs), which are among thedeadliest of lesions, are caused by multiple factors includ-ing replication fork collapse induced by blocking lesionscaused by ROS, normal genomic rearrangements that oc-cur during meiosis or V(D)J recombination or by the phys-ical stress exerted on dicentric chromosomes as they arepulled apart at mitosis. When such DSBs occur during theS phase, the high levels of CDKs will induce resection ofthe DSBs resulting in the appearance of single-strandedDNA (ssDNA). Stalling of the DNA polymerase can alsoresult in ssDNA tracts when the replicative helicase isuncoupled from the advancing fork. The checkpoint sig-nalling pathways are triggered by either the DSBs or thessDNA. The Fanconi anaemia/BRCA pathway also seemsto play a central role in both DNA repair and cell cyclecontrol.

Checkpoint signallingDNA damage activates a checkpoint signalling system thathas components similar to those found in many othercell signalling pathways. Components of the checkpointsignalling toolkit (Module 9: Table checkpoint signallingtoolkit) are organized into signalling pathways that relayinformation from the sites of DNA damage to variousdownstream effectors that can temporarily arrest the cellcycle. The DNA lesions, such as the double-strand breaks(DSBs) or single-stranded DNA (ssDNA), provide the

stimuli that are recognized by ‘receptors’. The latter arethen coupled through transducers/amplifiers to activatethe checkpoint kinases that function as messengers thatrelay information to the various effectors, which are com-ponents of the cell cycle machinery. The key compon-ents of the checkpoint signalling network are the phos-phoinositide 3-kinase related kinases (PIKKs) represen-ted by Ataxia-telangiectasia mutated (ATM), ATM andRAD3-related, and DNA-dependent protein kinase. It isthese three PIKKs that feature significantly in checkpointsignalling systems that will be illustrated by consideringG1 checkpoint signalling to DNA double-strand breaks(DSBs) and S and G2/M checkpoint signalling to singlestranded DNA.

G1 checkpoint signalling to DNA double-strand breaks(DSBs)DNA double-strand breaks (DSBs) are among the dead-liest of lesions and are responsible for triggering the fol-lowing two G1 checkpoint signalling cascades (Module 9:Figure G1 checkpoint signalling):

DNA-protein kinase (DNA-PK) signalling pathway

(a) The DNA break is recognized by the Ku80 com-ponent of the Ku70--Ku80 complex that provides anucleus to bind other components of the pathway.

(b) The DNA-PKs, artemis and DNA ligase IV asso-ciate with the Ku complex. The DNA-PK is ac-tivated through an autophosphorylation reaction.

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �18

Module 9: Table checkpoint signalling toolkitCheckpoint signalling toolkit.Checkpoint signallingcomponents CommentsReceptors/sensors

Ku 70 and Ku80 One of the sensors that recognizes DSBs(Module 9: Figure G1 checkpointsignalling)

RAD 50 One of the sensors that recognizes DSBs(Module 9: Figure G1 checkpointsignalling)

RPA Replication protein A coats single strandedDNA (ssDNA) (Module 9: Figure S/G2phase checkpoint signalling)

Transducers/amplifiers Phosphoinositide 3-kinase related kinases(PIKKs)

ATM Ataxia-telangiectasia mutated (ATM)ATR ATM and RAD3-relatedDNA-PK DNA-dependent protein kinase

Adaptors/scaffoldingproteinsMRE11 Meiotic recombination protein-11Nbs1 Nijmegen breakage syndrome protein-1

responsible for recruiting ATMMDC1 Mediator of DNA damage checkpoint

protein-1 is an adaptor that functions inG1 checkpoint signalling (Module 9:Figure G1 checkpoint signalling)

ATRIP ATR-interacting protein is an adaptor thatfunctions in S/G2 checkpoint signalling(Module 9: Figure S/G2 phasecheckpoint signalling)

RAD17-RFC RAD17-replication factor C functions as aclamp loader for the 9-1-1 complex

RAD-9 A component of the 9-1-1 complexRAD1 A component of the 9-1-1 complexHUS1 A component of the 9-1-1 complexTOBP1 Topoisomerase-binding protein-1Claspin Adaptor protein that connects ATR to

CHK1 (Module 9: Figure S/G2 phasecheckpoint signalling)

Checkpoint kinases(CHKs)CHK1 Checkpoint kinase 1CHK2 Checkpoint kinase 2

(c) The DNA ligase IV then joins the two ends to-gether to complete the repair process. In addition,the DNA ligase can contribute to the process ofG1 arrest by activating p53 as will be described inthe following section.

The ATM/checkpoint 2 signalling pathway

1. Another way in which the DSB is recognized de-pends on the binding of a MRN complex that ismade up from the meiotic recombination protein-11 (MRE-11), RAD50 and Nijmegen breakagesyndrome protein-1 (Nbs1) proteins. It is theRAD50 that is responsible for recognizing theDSB.

2. The MRN complex recruits ataxia-telangiectasiamutated (ATM) during which it switches froman inactive dimer to an active phosphorylatedmonomer. It is the Nsb1 that binds this activatedATM.

3. One of the first actions of activated ATM is tophosphorylate the C-terminal tails of the histonevariant H2AX, which initiates an amplificationprocess.

4. This amplification process is completed by thephosphorylated H2AX recruiting mediator ofDNA damage checkpoint protein-1 (MDC1),which is an adaptor that recruits additional MRN–ATM complexes to build up a local population ofactivated ATM molecules.

5. The activated ATM then phosphorylates check-point kinase 2 (CHK2), which is responsible forG1 phase arrest through two main pathways.

6. CHK2 hyperphosphorylates Cdc25A, which isone of the dual-specificity Cdc25 phosphatasesthat act by removing inhibitory phosphates onCDK2 that controls CyclinE. The Cyclin E con-trols G1 progression and DNA synthesis and theinhibition of this critical step results in G1 phasearrest (Module 9: Figure cell cycle signalling mech-anisms).

7. The activated CHK2 can also arrest cells byphosphorylating and activating the transcriptionfactor p53 (Module 4: Figure p53 function). Oneof the main mechanisms used by p53 is to ac-tivate G1 arrest by activating p21, which is apotent cyclin-dependent kinase (CDK) inhibitor(Module 9: Figure proliferation signalling net-work). p21 inhibits the CDK2 that activates tran-scription of the E2F-regulated genes that are re-quired for the onset of DNA replication (Module9: Figure cell cycle signalling mechanisms). Anumber of other p53-dependent mechanisms caninhibit the cell cycle as described in the section onp53-induced cell cycle arrest.

S and G2/M checkpoint signalling to single-strandedDNAAn ATR/checkpoint 1 signalling pathway, which occursmainly during the S and G2 phases of the cell cycle, oper-ates during the process of homologous recombination. Theprimary trigger that initiates this checkpoint signalling re-sponse is the existence of single-stranded DNA (ssDNA)as outlined in the following sequence of events (Module 9:Figure S/G2 phase checkpoint signalling):

1. The first step in the signalling pathway is the coating ofthe single-stranded regions with replication protein A(RPA). The latter then functions as a receptor to recruitATR-interacting protein (ATRIP), which functions asan adaptor that brings ATM and RAD3-related (ATR)protein kinase into the site of DNA damage. This RPA–ATRIP--ATR complex is not enough to induce ATRactivation, which requires the recruitment of additionalcomponents.

2. Activation of ATR depends on the 9-1-1 complex,which is a ring-shaped molecule made of RAD9, RAD1and HUS1. The loading of 9-1-1 at the primer-templatejunction is facilitated by the clamp loader RAD17-replication factor C (RAD17-RFC).

3. Once the 9-1-1 complex is in place, the C-terminaltail of the RAD9 component of this complex is phos-phorylated and this provides a binding site that recruitsthe topoisomerase-binding protein-1 (TOPBP1). The

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �19

Module 9: Figure G1 checkpoint signalling

5

CHK2

Cdc25a

G1 phasearrest

p21

3

MRE11

MRE11

MRE11

MRE11

RAD50

RAD50

RAD50

RAD50

Nbs1

Nbs1

Nbs1

Nbs1

P

P

P

P

P

P P

P

PP

P

PPP

P

P P PCyclin E

CDK2

ATM

H2AX

ATMATM

Ku70-Ku80

ATM

DNA-PKArtemis

DNA ligase IV

ATM

MDC1 MDC1

Ionizingradiation

p53

DNA double-strandbreak (DSB)

3

4

2

5

1a

b

c

7

6

G1 checkpoint signalling mechanisms.Ionizing radiation causes double-strand breaks (DSBs) that induce two major DNA damage signalling pathways that arrests cells in the G1 phase ofthe cell cycle. The DNA-protein kinase (DNA-PK) signalling system (a–c). The ATM/checkpoint 2 signalling pathway (1–7).

TOPBP1 is positioned close to ATR and is able to ac-tivate the latter through its ATR activation domain.

4. Once TOPBP1 has activated ATR, the latter can be-gin to phosphorylate and activate checkpoint kinase1 (CHK1), which relays information to the cell cyclecomponents that bring about cell cycle arrest. The in-teraction between CHK1 and ATR is facilitated by theprotein claspin.

5. CHK2 hyperphosphorylates Cdc25A, which is one ofthe dual-specificity Cdc25 phosphatases that act by re-moving inhibitory phosphates on CDK2 that controlsCyclin E. The Cyclin E controls G1 progression andDNA synthesis and the inhibition of this critical stepresults in G1 phase arrest (Module 9: Figure cell cyclesignalling mechanisms).

6. The activated CHK2 can arrest the cell cycle by phos-phorylating and activating the transcription factor p53(Module 4: Figure p53 function). p53-induced cell cyclearrest depends on a number of mechanisms (Module9: Figure proliferation signalling network). One ofthe main mechanisms used by p53 is to activate p21,which is a potent cyclin-dependent kinase (CDK) in-hibitor. p21 inhibits the activity of CDK2 that con-trols transcription of the E2F-regulated genes that arerequired for DNA replication (Module 9: Figure cellcycle signalling mechanisms). Another protein calledGADD45a, which is up-regulated by p53, acts togetherwith the Wilms’ tumour suppressor (WT1) to dissoci-ate the cyclin B/CDK1 complex by binding to CDK1(Module 9: Figure proliferation signalling network).

7. CHK1 also phosphorylates Cdc25C, which removesinhibitory phosphates on CDK1 that controls the cyc-lin B that is responsible for the final events of the cell

cycle and thus contributes to the G2/M phase arrest(Module 9: Figure cell cycle signalling mechanisms).

Fanconi anaemia/BRCA pathwayThe Fanconi anaemia/BRCA pathway operates duringDNA damage that is induced by DNA cross-linking andother forms of genotoxic stress. The components of thispathway came to light during the analysis of Fanconi an-aemia, which is a genetically heterogeneous disease thatresults in chromosome instability. There is a Fanconi an-aemia complementation group (FANC) that has at least12 components (A, B, C, D1, D2, E, F, G, I, J, L and M).These FANC proteins, which seem to function as two sep-arate complexes (the green and yellow groups of proteinsshown in Module 9: Figure Fanconi anaemia pathway),co-operate with each other in the operation of a signallingsystem that responds to various defects in DNA. The path-way is particularly evident during DNA synthesis whenthe replication fork stalls due to defects such as a DNAcross-link. Just how all these proteins function is still be-ing worked out and the following hypothesis illustratessome of the key features of the pathway (Module 9: FigureFanconi anaemia pathway):

1. The A, B, C, E, F, G, L, M and possibly I (colouredgreen) form complex 1 that binds to the stalled laggingstrand. The M protein has been identified as helicase-associated endonuclease for forked structured DNA(Hef).

2. The other group of FANC proteins form complex 2that have three members (coloured yellow): D1 [(alsoknown as breast cancer 2 (BRCA2)], D2 (referred tohere as FANCD2) and the J protein that has been

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �20

Module 9: Figure S/G2 phase checkpoint signalling

5

ClaspinTOPBP1

CHK1

CHK1

5

ATRIPATRIP

ATRIP

ATR

RPA

ssDNARAD9RAD1

9-1-1complex

HUS1

ATR

ATR

3

RAD17-RFC

RAD17-RFC

Cdc25C

Cdc25AS phasearrest

G2/M phasearrest

P

P

P

P

P

P

P

P

PP P P

Cyclin E

Cyclin B

CDK2

CDK1

3

4

2

5

1

7

6p21

GADD45a

Pp53

S and G2 phase checkpoint signalling.An ATR/checkpoint 1 signalling pathway responds to single-stranded DNA (ssDNA) by initiating a signalling cascade that results in either S or G2arrest depending on the phase of the cell cycle. See text for details of the numbered sequence of events

Module 9: Figure Fanconi anaemia pathway

DNArepair

Replication fork stalledat the DNA crosslink

Fanconi anemiacomplementationgroup (FANC)

?Cell cycleregulation

C

C

E

E

F

F

G

G

L

L

FANCD2

FANCD2

FANCD2

J/BACH1

J/BACH1

J/BACH1

Laggingstrand

Leadingstrand

3 4

5

6

2

1

A

A

B

B

M/HEF

M/HEF

D1/BRCA2

D1/BRCA2

D1/BRCA2

BRCA1

BRCA1

Ubiquitin

Complex 2

Complex 1

Ubiquitin

Usp1

ATR

P

P

Ionizingradiation

Fanconi anaemia pathway in DNA repair and cell cycle control.The Fanconi anaemia pathway functions in DNA repair, particularly in response to DNA crosslinks (vertical red line). The Fanconi anaemia comple-mentation group (FANC) proteins operate as part of two macromolecular complexes that are drawn into such regions when DNA replication is stalledat the crosslinks. Complex 1 (green proteins) has the ubiquitin ligase (L) that ubiquitinates FANCD2, which is part of complex 2 (yellow proteins).Monoubiquitination of the FANCD2 is particularly important in the activation of this pathway. The information used to construct this figure was takenfrom Kennedy and D’Andrea (2009).

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �21

identified as BRCA1-interacting protein C-terminalhelicase (BRIP1), which is also known as BACH1.Both phosphorylation and ubiquitination reactionshave been implicated in the activation and attachmentof these proteins to DNA. The Ataxia telangiectasiamutated and Rad3-related (ATR) kinase, which is activ-ated during ionizing radiation (Module 9: Figure S/G2

phase checkpoint signalling), is one of the kinases thatcan phosphorylate FANCD2.

3. The L subunit of complex 1 is an ubiquitin ligase thatubiquitinates and activates FANCD2 to facilitate theattachment of complex 2 to the replication fork.

4. The ubiquitin-specific protease 1 (Usp1) reverses thisactivation process by deubiquitinating FANCD2.

5. The precise function of complex 2 is still not clear, but itappears to contribute to both DNA repair and cell cycleregulation. For example, FANCD2 may participate inDNA repair by interacting with various recombinationenzymes.

6. Breast cancer 1 (BRCA1) is also drawn into the complexand has a role in carrying out the processes responsiblefor DNA repair

Mutations in components of the Fanconi an-aemia/BRCA pathway are responsible for the geneticallyheterogeneous disease Fanconi anaemia.

Cell proliferationResting cells in a zero growth phase (G0) are induced tore-enter the cell cycle by growth factors that function bystimulating a process of cell proliferation (Module 9: Fig-ure cell cycle network).

Spatial and temporal aspects feature significantly in theactivation of cell proliferation by growth factor signallingpathways (Module 9: Figure G1 proliferative signalling).The spatial problem concerns the fact that growth factorsacting at the cell surface have to transfer information tointernal targets, many of which are found in the nucleus.With regard to the temporal problem, the signalling path-ways have to operate over long periods in G1. This ques-tion of the time it takes to stimulate cells to enter thecell cycle is also apparent during the completion of mei-osis at fertilization, where Ca2 + spikes have to persist for2 h before the zygote is committed to begin its growthand development. Another example is Ca2 + signalling inlymphocytes, where a similar long period of stimulationis required to drive cells into the cell cycle. Therefore theinformation flow during proliferative signalling is adaptedto transmit information from growth factor receptors tothe nucleus over long periods of time.

Information flow during proliferativesignallingMost of the signalling pathways used to transmit informa-tion into the cell are made up of sequential elements, someof which are sufficiently stable and diffusible to disperseinformation deep into the cell, thus solving the spatialproblem. These early signalling events are driven by the

so-called competence growth factors in that they set thestage for the action of the later progression growth factors(Module 9: Figure G1 proliferative signalling). Solutionof the temporal problem has two elements. Firstly, thesignalling mechanisms show little desensitization, thus en-abling them to operate over prolonged periods. Secondly,the signalling pathways trigger a cascade of gene transcrip-tional events that serve to maintain the flow of informationduring the critical G1 period. One way of doing this is to in-duce the expression of additional signalling pathways thatoperate later in G1. It is during this transcriptional cascadethat the progression growth factors and their receptors areput in place to provide additional signalling pathways thatcarry information in from the outside to drive some of thelater cell cycle signalling elements responsible for initiatingDNA synthesis.

In summary, growth factors use a number of signallingmechanisms to initiate the process of cell proliferation.Early in G1, these signalling pathways transmit informa-tion into the nucleus to trigger a transcriptional cascadethat culminates in the phosphorylation of retinoblastomaprotein (Rb) at the restriction point. As the role of thegrowth factor-dependent signalling pathways decline dur-ing G1, the onus for driving the cell cycle to completionis gradually handed over on to the endogenous cell cyclesignalling mechanisms based on the cyclin cascade (bluearrows in Module 9: Figure cell cycle). These cell cycleevents are set in motion by a number of growth factorsignalling pathways.

Growth factor signalling pathwaysGrowth factors use a variety of signalling pathways totransmit information into the nucleus. In describing thesepathways, the emphasis will be placed on the way in whichinformation travels from the cell surface to the cytosolicand nuclear targets that co-ordinate the onset of DNAsynthesis, as outlined in Steps 1--10 in Module 9: Figuregrowth factor signalling:

1. The Smad signalling pathway mediates thetransforming growth factor β (TGF-β) inhibitionof cell proliferation. Stimulation of the TGF-βreceptor results in the activation of the Smad transcrip-tion factors (Module 2: Figure TGF-βR activation),which then translocate into the nucleus (Module2: Figure Smad signalling). One of the functionsof the Smads is to increase the expression of thecyclin-dependent kinase (CDK) inhibitor p15 (Module4: Figure Myc as a gene repressor), which acts to inhibitcell proliferation.

2. The Hedgehog (Hh) signalling pathway activates thezinc-finger transcriptional activator GLI1, which trans-locates into the nucleus to activate a number of genes,some of which are components of other signalling sys-tems (Module 2: Figure Hedgehog signalling pathway).

3. The canonical Wnt/β-catenin pathway activates thetranscription factor β-catenin, which translocates intothe nucleus to activate a number of the genes that

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �22

Module 9: Figure G1 proliferative signalling

Growth factors(competence)

Growth factors(progression)

MycEtsJun

GF

GFR

GFRsGFRs

Rb

Rb

P P P

E2F E2F

Autocrineloop

Growth factorsignallingpathways

DNAsynthesis

Cyclin D1

G0 G1 S

Cyclin ECyclin ACdc25ACDK4

Cytoplasm

Nucleus

R

Spatiotemporal aspect of proliferative signalling.Competence growth factors initiate a number of signalling pathways that carry information into the nucleus to activate immediate early genes. Someof these early genes encode information for the expression of progression growth factors (GFs) and their receptors (GFRs) to set up an autocrineloop to drive later events. Other early genes encode transcription factors such as E twenty-six (ETS), Jun and Myc to initiate transcription of latergenes, such as cyclin D, which combines with the cyclin-dependent kinases (CDKs) to phosphorylate the retinoblastoma protein (Rb): one of the keygate-keepers guarding passage through the restriction point (R). A key event at the restriction point (R) is the phosphorylation of Rb, which removesits inhibition of E2F and enables this transcription factor to increase the expression of the cell cycle components (e.g. cyclin E, cyclin A and Cdc25A)responsible for initiating DNA synthesis. Further details of this activation sequence are provided in Module 9: Figure cell cycle signalling mechanisms.

function in cell proliferation (Module 2: Figure Wntcanonical pathway).

4. The mitogen-activated protein kinase (MAPK) sig-nalling pathway plays a central role in cell prolifer-ation. Activation of tyrosine kinase-linked receptorsresults in the phosphorylation of extracellular-signal-regulated kinases 1/2 (ERK1/2), which then translocateinto the nucleus (Module 2: Figure ERK signalling).One of the main targets of ERK1/2 in the nucleus isthe transcription factor cyclic AMP response element-binding protein (CREB) (Module 4: Figure CREB ac-tivation). In addition, ERK1/2 activates a number ofother transcription factors (Module 9: Figure growthfactor signalling).

5. Ca2 + signalling plays a key role in controlling the pro-cess of cell proliferation in many primary cells. Theimportance of Ca2 + as a proliferative signal may alsoexplain the connection between K+ channels and cellproliferation. The opening of K+ channels is criticalfor keeping the membrane hyperpolarized to maintainthe driving force for Ca2 + entry.

Both tyrosine kinase- and G protein-linked growthfactor receptors stimulate the formation of inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG) (Module2: Figure InsP3 and DAG formation). The DAG releasedfollowing hydrolysis of PtdIns4,5P2 by phospholipase C(PLC) has an important role in regulating cell prolifera-tion. One function is to activate protein kinase C (PKC),

which then phosphorylates inhibitor of nuclear factor κB(IκB), causing it to be hydrolysed, and the released nuclearfactor κB (NF-κB) is then free to diffuse into the nucleus(Module 9: Figure growth factor signalling).

The InsP3 releases Ca2 + , which can stimulate cellproliferation in a number of ways. Some of the cyto-plasmic targets of Ca2 + are the transcription factorsnuclear factor of activated T cells (NFAT) (see Step6) and NF-κB (Step 7). In addition to acting inthe cytoplasm, Ca2 + and calmodulin (CaM) can in-vade the nucleus, where they activate various effect-ors such as Ca2 + /calmodulin-dependent protein kinaseIV (CaMKIV), the CREB-binding protein (CBP) or theS100B-dependent nuclear protein kinase (Ndr). One ofthe functions of CaMKIV is to activate CREB (Module 4:Figure CREB activation).

The Ca2 + sensor CaM undergoes a profound redistri-bution during the course of the cell cycle. During G1, muchof the calmodulin is in the cytoplasm, but becomes con-centrated in the nucleus during the S phase and is localizedat the spindles and at the site of cell cleavage during theprocess of mitosis (Module 9: Figure CaM redistributionat mitosis).

One reason the importance of Ca2 + in regulating cellproliferation has not been fully appreciated is that muchof the experimental work has been carried out on immor-talized cell lines, which may have a signalling phenotypevery different from that of primary cells. In particular, it ispossible that many of these cells may have Ca2 + signalling

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �23

Module 9: Figure growth factor signalling

PLCPLC Gq

Raf

MEK

ERK1/2Smads

Smads

Catenin

Catenin

ERK1/2

Ras

Proteinsynthesis

DNAsynthesis

Ca2+

Ca2+

NF- B

NF- B

I B

NFATCaN

AP1SRFTCFEts1/2

STAT

STAT FOXO

DAG

NFAT

PI 3-K

mRNA

PKC

PDGFEGF

BombesinAngiotensin IIEndothelin

IGF-IInterleukin-2

PKB

PKB

TOR S6K

Transcription factors

PtdIns3,4,5P3

InsP3

CBPCREB

JAK1/2PDK

GLI1 CaM

CaM

GLI1

HhTGF- Wnt

Fz

SMO

1 2 3 4 5 76 8 9

P P

PP

PP

Ndr

S100

Growth factors use a large number of signalling pathways to activate cell proliferation.A number of signalling pathways co-operate with each other to mediate the action of growth factors. Those components coloured in red are responsiblefor carrying information from the cell surface into the nucleus. Some of these components are transcription factors, whereas others are signallingcomponents such as Ca2 + and protein kinases such as extracellular-signal-regulated protein kinase 1/2 (ERK1/2) and protein kinase B (PKB) thattarget transcription factors resident within the nucleus. All of these transcription factors control the expression of the cell cycle components that arerequired to induce DNA synthesis. Further details of these signalling pathways are given in the text.

pathways that are constitutively active, and thus appear tobe independent of Ca2 + . Some evidence for this is that alarge number of transformed cells can continue to prolif-erate at low levels of external Ca2 + that completely inhibitthe growth of normal cells.

6. One of the functions of Ca2 + is to activate the NFAT(Module 4: Figure NFAT activation). In its inactivestate, it is phosphorylated and located in the cytoplasm.When Ca2 + rises, it activates the enzyme calcineurin(CaN) to remove the phosphate to release the activeform of NFAT, which diffuses into the nucleus to initi-ate gene transcription (Module 9: Figure growth factorsignalling).

7. Ca2 + is one of the messengers responsible for activ-ating the nuclear translocation of NF-κB. The latteris retained in the cytoplasm through its attachment toIκB, which is hydrolysed following its phosphoryla-tion through various signalling pathways, including theCa2 + and the DAG/PKC pathways.

8. The Janus kinase (JAK)/signal transducer and activatorof transcription (STAT) signalling pathway functionsby activating the STATs (Module 2: Figure JAK/STATfunction), which then translocate into the nucleus toactivate gene transcription.

9. The PtdIns 3-kinase has a number of functions in regu-lating cell proliferation (Module 9: Figure growth factorsignalling). The lipid second messenger PtdIns3,4,5P3

activates both phosphoinositide-dependent kinase 1(PDK1) and protein kinase B (PKB). The latter translo-

cates into the nucleus, where it inactivates the forkheadbox O (FOXO) transcription factors (Module 4: Fig-ure FOXO control mechanisms). FOXO is respons-ible for driving the expression of various inhibitors ofproliferation, such as p27 and Rb. Inhibition of thistranscriptional activity accounts for the rapid declinein the level of p27 that occurs when cells are stimu-lated to enter the cell cycle (Module 9: Figure cell cycledynamics). PDK1/2 acts through ribosomal S6 pro-tein kinase (S6K) to stimulate protein synthesis. PKBalso contributes to the target of rapamycin (TOR) sig-nalling pathway that functions in the control of proteinsynthesis (Module 9: Figure target of rapamycin sig-nalling). Through this control of protein synthesis, thePtdIns 3-kinase signalling pathway plays a key role incell growth control.

The signalling mechanisms summarized in Module 9:Figure growth factor signalling represent a proliferativesignalling toolkit from which specific systems are as-sembled to control the proliferation of specific cell types.

K+ channels and cell proliferationThere are many examples of K+ channels playing a rolein controlling cell proliferation. The primary candidatefor carrying out this role is the intermediate-conductance(IK) channel KCa3.1 (Module 3: Table properties ofCa2 + -sensitive K+ channels), which is one of theCa2 + -sensitive K+ channels. The primary action ofKCa3.1 is to hyperpolarize the membrane and thereby

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �24

maintain the driving force for the entry of external Ca2 + ,as has been demonstrated for lymphocytes (Module 9: Fig-ure T cell Ca2 + signalling).

Proliferation is reduced by toxins (charybdotoxin andiberiotoxin) or inhibitors that block these K+ channels.Overexpression of these channels has been found in manytumours.

Cell growth controlThe growth factors that control cell proliferation have twomajor actions. They must exert both cell cycle control andcell growth control. The latter is concerned with the con-trol of the macromolecular synthetic events that are ne-cessary for cells to double their size prior to mitosis. Thiscontrol of growth is complex, because not only is it stim-ulated by growth factors, but also it is regulated by nutri-ents such as amino acids and by the energy state of the cell.What is remarkable about these different regulatory inputsis that they all impinge upon a common regulatory sys-tem based on the mammalian target of rapamycin (TOR),which then controls the target of rapamycin (TOR), whichhas a major role in regulating protein synthesis by phos-phorylating both ribosomal S6 protein kinase 1 (S6K1)and eukaryotic initiation factor 4E (eIF4E)-binding pro-tein (eIF4E-BP1). TOR is a protein kinase that forms twocomplexes: TOR complex 1 and TOR complex 2. Most in-formation is available for TOR complex 1, which consistsof TOR, regulatory-associated protein of TOR (raptor),G protein-like protein (GβL) and proline-rich PKB/Aktsubstrate 40 kDa (PRAS40). This TOR complex 1 is regu-lated through two separate signalling mechanisms (Module9: Figure target of rapamycin signalling). One pathway isactivated by hormones and growth factors and uses sig-nalling components such as the tumour suppressor pro-teins tuberous sclerosis complex 1 and 2 (TSC1/2) and theRas-homologue enriched in brain (Rheb). The other path-way is regulated by and increase in circulating amino acidsthat activate a Ca2 + signalling mechanism. The followingsequence of events indicates how these two pathways func-tion to activate TOR to regulate protein synthesis (Module9: Figure target of rapamycin signalling):

1. Growth factors operating through the PtdIns 3-kinase (PI 3-K) signalling cassette generate the lipidmessenger PtdIns3,4,5P3 (PIP3) (Module 2: Fig-ure PtdIns 3-kinase signalling), which then func-tions to activate both protein kinase B (PKB) andphosphoinositide-dependent kinase 1 (PDK1). One ofthe functions of PKB is to inhibit tuberous sclerosiscomplex 1 and 2 (TSC1/2). TSC2 is phosphorylated onSer-969 and Thr-1462.

2. TSC1/2 also plays a critical role in co-ordinating sig-nal inputs relating to energy state and the level of aminoacids (Module 9: Figure target of rapamycin signalling).TSC1/2 relays information to TOR concerning the nu-tritional status of the cell. For example, if the level ofamino acids falls, their inhibitory effect on TSC1/2 is re-moved, enabling the latter to shut off protein synthesisby inhibiting Rheb. Likewise, a decrease in the energy

state activates AMP-activated protein kinase (AMPK),which then phosphorylates TSC2 to enhance its inhib-itory action on Rheb.

3. As noted above, TSC1/2 acts by inhibiting Rheb, whichis a major activator of TOR. This inhibitory action ismediated through TSC2, which is a GTPase-activatingprotein capable of stimulating Rheb. Rheb acts by stim-ulating TOR, which exerts a major influence over pro-tein synthesis by regulation both the initiation andelongation phases.

4. The activity of TOR is also activated by an increasein the concentration of circulating amino acids, whichbring about an elevation of intracellular Ca2 + througha mechanism that depends on both the entry of externalCa2 + and the release of Ca2 + from internal stores. TheCa2 + response often appears as Ca2 + oscillations withrelatively long periodicities (3--5 mins). These aminoacid-induced oscillations appear to be driven by themechanism of Ca2 + oscillations found in many othercell types.

5. The increase in Ca2 + then binds to calmodulin (CaM)that then activates the human vacuolar protein sorting34 (hVps34), which is a Class III PtdIns 3-kinase thatfunctions to generate PtdIns3P (Module 2: Figure PI3-K family).

6. Control of initiation depends upon the ability of TORto phosphorylate the eIF4E-BP1. When eIF4E-BP1 isphosphorylated, eIF4E is released to contribute to theformation of the initiation complex, together with otherinitiation factors such as eIF4G.

7. TOR can regulate the translocation step of elongationby altering the activity of eukaryotic elongation factor2 (eEF2). This factor is inactivated when it is phos-phorylated on Thr-56 by an eEF2 kinase. The activityof this eEF2 kinase is inhibited by TOR.

8. Control of translation depends upon TOR phos-phorylating ribosomal S6 protein kinase 1 (S6K1),which then phosphorylates the S6 protein componentof the 40S ribosome subunit.

Tuberous sclerosis complex 1 and 2(TSC1/2)The tuberous sclerosis complex 1 and 2 (TSC1/2) playsa critical role in regulating cell growth control becauseit co-ordinates many of the inputs that regulate the con-trol of protein synthesis by the protein kinase target ofrapamycin (TOR) (Module 9: Figure target of rapamy-cin signalling). TSC is a heterodimer composed of TSC1(also known as hamartin), which contains a coiled-coilstructure, and TCS2 (also known as tuberin), which is aGTPase-activating protein (GAP). TSC1 and 2 are phos-phorylated by protein kinase B (PKB) during the action ofgrowth factors.

TSC1 and TSC2 are considered to be tumour suppressorgenes because they act by inhibiting the growth promotingsignals from nutrients. The genes encoding TSC1/2 areinactivated in the autosomal dominant disorder tuberoussclerosis.

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �25

Module 9: Figure target of rapamycin signalling

mTORComplex 1

FKBP12

RapamycinRheb

hVps34

TSC1/2PKB

mRNA

PDK1

Aminoacids

PI3KAMPK

LKB1

Met4E-BP1 4E-BP1

S6K1

S6K1P P PP P

PP P

P

P

P

PPP

S6

eIF-4E

eIF-4E

eEF-2

eEF-2 kinase

Energy state

Autophagy

Aminoacids

Growthfactors

eIF-4G

40S

__

__

____

__

__

PIP3PIP2

1

3

5

4

21

6

8

7

+

+

InsP R3

Ca2+

Ca2+

?

CaM

++

Integration of growth factor and nutrient signalling by the target of rapamycin (TOR).The target of rapamycin (TOR) is a major integrative serine/threonine protein kinase that responds to information coming in from growth factor receptorsand from metabolic signalling pathways that assess the nutritional status of the cell. All this information is integrated by the tuberous sclerosis complex1 and 2 (TSC1 and TSC2), which then relay signals to TOR through the Ras homologue enriched in brain (Rheb) protein. TOR acts through a numberof targets to control the rate of protein synthesis. TOR also has an important role to inhibit autophagy (Module 11: Figure autophagy).

Target of rapamycin (TOR)The target of rapamycin (TOR) derives its name from thefact that it is inhibited by the drug rapamycin, which is apotent inhibitor of cell proliferation. Some analogues ofrapamycin are being tested as anti-tumour agents. Rapa-mycin exerts its inhibitory action by first binding to theimmunophilin FK506-binding protein (FKBP) of 12 kDa(FKBP12) (Module 9: Figure target of rapamycin sig-nalling).

TOR is a serine/threonine protein kinase, which op-erates as a nutrient-sensitive cell cycle checkpoint. Thehighly conserved kinase domain is located at the C-terminus close to an FRB domain that binds the FKBP12.The activity of TOR is switched off, and cell proliferationceases under conditions when amino acid levels are lowor when energy is limiting. This inhibition of TOR is alsoresponsible for the onset of autophagy (Module 11: Figureautophagy)

The N-terminal region of TOR contains a large num-ber of HEAT repeats. These helical repeat units foldinto an extended superhelical scaffold-like structure thatmay provide interfaces for interactions with other mo-lecules. TOR is organized into two complexes. In theTOR complex 1, TOR is associated with a regulatory-associated protein of TOR (raptor), G protein-like pro-tein (GβL) and a proline-rich PKB/Akt substrate 40 kDa(PRAS40). Raptor is a scaffolding protein that providesa bridge between TOR and its downstream targets ofeIF4E-BP1 and ribosomal S6 protein kinase 1 (S6K1).The TOR complex 2 contains TOR together with GβL,

rapamycin-insensitive companion of TOR (rictor), mam-malian stress-activated protein kinase (SAPK)-interactingprotein 1 (mSin1) and protein observed with rictor (pro-tor).

TOR is also sensitive to the phospholipase D (PLD)signalling pathway (Module 2: Figure PLD signalling).

Proliferation of specific cell typesCell proliferation is evident in many different cell types.Stem cells are specialized for cell proliferation. For ex-ample, in the intestine, there are nests of undifferentiatedstem cells that are constantly proliferating to replace ab-sorptive cells that are sloughed off from the ends of thevilli. In the immune system, lymphocyte activation plays avital role during clonal expansion of unique lymphocytesprogrammed to fight off specific infections. There also aremany differentiated primary cells that perform their spe-cific functions while retaining the option of returning tothe cell cycle (Module 8: Figure cellular life history). Inall of these examples, it is clear that cell proliferation iscarefully orchestrated to maintain cellular homoeostasis.

The following are some examples of in vivo cell pro-liferation events that can be induced with the appropriatestimuli:

• Lymphocyte activation• Smooth muscle cell proliferation• Insulin-secreting β-cell proliferation• Astrocyte proliferation

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �26

Module 9: Figure T cell chemotaxis

Ant

igen

pre

sent

ing

cell

TCRMHCII

Immunologicalsynapse

Chemokinegradient

T Cell

T cell migration towards antigen-presenting cells through a process of chemotaxis.Antigen-presenting cells release cytokines that attract T cells. When they reach the antigen-presenting cell, they form the immunological synapsewhere the T cell receptor (TCR) on the T cell binds to the major histocompatibility complex II (MHCII) molecules on the antigen-presenting cell. Theantigenic peptide (shown in red) is held on the surface of the MHCII molecules. Details of the synapse are shown in Module 9: Figure immunologicalsynapse.

• Intestinal stem cell proliferation (Module 12: Figurecolon cancer)

• Neuronal stem cell proliferation during neurogenesis(Module 10: Figure granule cell neurogenesis)

• Endothelial cell angiogenesis (Module 9: Figure an-giogenesis)

• Parathyroid gland hyperplasia• Mesangial cell proliferation (Module 7: Figure mesangial

cell). Excessive mesangial cell proliferation is one of themajor causes of diabetic nephropathy.

• Hepatic stellate cell stimulation is induced during liverinjury (Module 7: Figure hepatic stellate cell).

Lymphocyte activationThe immune system employs a highly sophisticated chem-ical surveillance system to recognize and combat foreignsubstances. The foot soldiers of this defence force are thelymphocytes, which are separated into thymus-derivedcells (T cells) and bone marrow-derived cells (B cells). TheT cells act against intracellular pathogens (cell-mediatedimmunity), whereas the B cells deal with extracellularpathogens (humoral immunity). There are two main typesof T-cell: the cytotoxic cells, which can kill infected cells,and the helper cells, which, as their name implies, help Bcells to fight infections by providing specific cytokines.From a signalling point of view, T cell activation and B cellactivation are of interest because they are examples of howcells are stimulated to proliferate in response to a specificstimulus, in this case antigen. In addition, these cells canalso respond to antigen either by dying through apoptosisor by entering an unresponsive state known as anergy. Ex-actly which course they take depends on the nature of the

signalling information they receive through a variety ofreceptor pathways.

T cell activationT cell activation depends upon a sequential series of eventsthat begin when the circulating T cell responds to thecytokines emanating from the source of an infection.Through a process of T cell chemotaxis, the T cell homesin on the infected area, where it makes intimate contactwith the antigen-presenting cell to form an immunologicalsynapse. The chemotactic response and the formation ofthe synapse are very dependent upon T cell cytoskeletalreorganization. As the two cells come into contact, themajor histocompatibility complex II (MHCII) moleculeson the antigen-presenting cell present the antigenic pep-tides to the T cell receptors to initiate the T cell receptor(TCR) signalling responsible for T cell activation. Activa-tion through the TCR is facilitated by the CD28 co-stim-ulatory pathway.

T cell chemotaxisCirculating T cells detect and move towards sites of infec-tion by a process of chemotaxis. This chemotactic responseis usually driven by a gradient of chemokines (Module 9:Figure T cell chemotaxis). Resting T cells have a roundshape with numerous small microvilli. This uniform shape,which is maintained by a cortical web of microfilaments,undergoes a rapid change when the T cell begins to de-tect chemokines. This T cell cytoskeletal reorganizationinduces a clearly defined polarity that contributes to theprocess of chemotaxis.

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �27

Module 9: Figure T cell actin scaffold

Formation of an actin scaffold at an immunological synapse.At stage a, the T cell has made contact with the antigen-presenting cells, and the T cell receptor has begun to assemble the signalling components[e.g. Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76), Nck, Vav, Wiskott–Aldrich syndrome protein (WASP) and actin-relatedprotein 2/3 (Arp2/3) complex] necessary to form actin filaments, as shown in b. In c, myosin II begins a contraction process that pulls together theindividual actin scaffolds associated with each receptor complex to produce the mature synapse shown in the panel at the bottom. The signallingcomponents of the T cell receptor complex are shown in Module 9: Figure TCR signalling. Reproduced by permission from Macmillan Publishers Ltd:Nat. Immunol. Dustin, M.L. and Cooper, J.A. (2000) The immunological synapse and the actin cytoskeleton: molecular hardware for T cell signalling.1:23–29. Copyright (2000); http://www.nature.com/ni; see Dustin and Cooper 2000.

T cell cytoskeletal reorganizationRemodelling of the actin cytoskeleton plays a key rolein T cell activation. It is responsible for the dramaticchanges in cell shape that occur during T cell chemotaxis,and it comes into play at a later stage to help assemblethe immunological synapse. These cytoskeletal rearrange-ments are controlled by an interplay between the PtdIns3-kinase signalling pathway and the PtdIns4,5P2 signallingcassette. The reorganization of the actin cytoskeleton thatoccurs during T cell chemotaxis is very similar to that seenin neutrophil chemotaxis.

As chemotaxis proceeds and the two cells begin to makecontact with each other, there is another cytoskeletal reor-ganization that forms the immunological synapse (Module9: Figure T cell actin scaffold).

When the T cell makes contact with potential antigen-presenting cells, it carries out an initial surveillance thatcauses a change in shape (see Sequence a--d in Module 9:Figure T cell shape change). If this target is a naıve cell,there is no further response, and the T cell moves away.However, if the T cell recognizes an antigen-presentingcell, there is a dramatic change in shape as it throws outpseudopodia, almost totally engulfing its target (see Se-quence e--h in Module 9: Figure T cell shape change). This

rapid change in shape is accompanied by a large Ca2 + tran-sient, which is thought to stabilize the interaction with theantigen-presenting cell.

Immunological synapseThe antigenic peptides responsible for activating the T cellare not free in solution, but are held by the major histocom-patibility complex II (MHCII) molecules embedded on thesurface of the antigen-presenting cell (Module 9: FigureT cell chemotaxis). As a result of the process of chemo-taxis, the two cells come together to form an immunolo-gical synapse made up of highly organized adhesive andsignalling components (Module 9: Figure immunologicalsynapse structure). These adhesive components, such aslymphocyte function-associated antigen (LFA-1), form aprotective outer ring surrounding an inner core contain-ing the signalling components. Within this synapse, there-fore, molecules appear to be segregated into ‘supramolecu-lar activation clusters’ (SMACs). The peripheral SMAC(pSMAC) contains the adhesion molecules, whereas thecentral SMAC (cSMAC) contains the signalling compon-ents.

An important aspect of the immunological synapse isthe polymerization of actin to provide an ‘actin-scaffold

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �28

Module 9: Figure T cell shape change

T cell shape change and Ca2 + signals triggered by contact with an antigen-presenting cell.A. Images of fura-2-loaded T cells approaching either a naıve B cell (a–d) or a B cell coated with an antigen (e–h). In the latter case, there wasa dramatic increase in the level of Ca2 + accompanying the change in shape. B and C. These traces illustrate the time course for the change inshape and intracellular Ca2 + taken during the responses shown in A. Reproduced from Immunity, Vol. 4, Negulescu, P.A., Krasieva, T.B., Khan, A.,Kerschbaum, H.H. and Cahalan, M.D., Polarity of T cell shape, motility, and sensitivity to antigen, pp. 421–430. Copyright (1994), with permission fromElsevier; see Negulescu et al. 1996.

signalling highway’ that helps in the relay of informa-tion from the cSMAC into the cell. The organization ofthe actin cytoskeleton depends upon the recruitment ofthe Wiskott--Aldrich syndrome protein (WASP) and theWASP-interacting protein (WIP) into the immunologicalsynapse. This translocation may be assisted by a Crk-like(CrkL) adaptor protein. The ability of WIP to inhibit theactivity of WASP may be alleviated by phosphorylation ofthe former by protein kinase Cθ (PKCθ), which is knownto play a role in T cell activation. This actin scaffold mayalso act to reorientate the microtubule organizing centre(MTOC) so that it faces towards the antigen-presentingcell. This actin remodelling was also found to be necessaryfor the activation of Oria1, which is essential for lymph-ocyte activation (Module 3: Figure STIM-induced Ca2 +

entry).The important principle is that the efficient functioning

of the signalling system depends upon the construction ofan elaborate scaffold made up of both the cytoskeleton anda range of scaffolding proteins that contributes to the mo-lecular organization of a functional T cell receptor (TCR)(Module 9: Figure TCR signalling). One of the importantsignalling events regulated by the cytoskeleton is Ca2 +

entry, which seems to be controlled by actin remodellingcarried out by a signalling pathway that includes Vav, Racand WASP verprolin homologous 2 (WAVE2) (Module 3:Figure STIM-induced Ca2 + entry).

There is a disruption in lymphocyte signalling inWiskott-Aldrich syndrome, which results from mutations

of WASP that result in a disruption in the formation of theimmunological synapse.

T cell receptor (TCR) signallingThe T cell receptor (TCR) signalling processes respons-ible for the activation of proliferation contain a bewilder-ing array of components. The main components of the Tcell signalling toolkit are collected together into functionalgroups (Module 9: Table T cell signalling toolkit). Manyof these components are collated together on a spatiotem-poral map, which provides a visual framework to illustratehow they interact with each other to drive T cells intoDNA synthesis (Module 9: Figure T cell signalling map).In effect, this is a spatiotemporal flow diagram that be-gins at the top left with the arrival of the antigen and endswith DNA synthesis in the bottom right-hand corner. Us-ing such a map, it is possible to determine approximatelywhere and when different signalling components functionduring the activation process.

The signalling events proceed through a conventionalsequence, as shown in the green panel on the left ofthe map. The activation process begins with the ligands[e.g. antigen held on the major histocompatibility com-plex II (MHCII)] that bind to the large TCR complexthat is coupled to transducers to create the messengersthat relay information through various signalling cassettesto activate the cytosolic and nuclear effectors that initi-ate the transcriptional cascade that finally results in DNA

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �29

Module 9: Figure immunological synapse structure

An immunological synapse formed between a T cell and an antigen-presenting cell.The cells have been stained with an antibody directed against talin(green), which highlights the outer ring of adhesive elements that con-stitute the peripheral supramolecular activation cluster (pSMAC). An an-tibody against protein kinase Cθ (PKCθ) has stained the inner core ofsignalling components (red), which make up the central SMAC (cSMAC),as illustrated in Module 9: Figure immunological synapse. The synapse isviewed in two orientations. In the top panels (a and b), it is seen from theside (i.e. the x–y axis), whereas in panels d–f, it is seen from the top (i.e.the x–z axis), which clearly shows the bull’s-eye appearance of the syn-apse. Reproduced by permission from Macmillan Publishers Ltd: Nature,Monks, C.R.F., Freiberg, B.A., Kupfer, H., Sciaky, N. and Kupfer, A. (1998)Three-dimensional segregation of supramolecular activation clusters inT-cells. 395:82–86; http://www.nature.com; see Monks et al. 1998.

synthesis. Some of the main components of T cell signallingare outlined below.

T cell ligandsT cells respond to a number of ligands, most of which areheld on the surface of the antigen-presenting cells (Module9: Figure T cell signalling map). The primary ligand for Tcell activation is the antigen itself. The antigen is takenup by the antigen-presenting cell, where it is chopped upinto short fragments (8--15 amino acids) that are boundto the major histocompatibility complex II (MHCII). TheMHCII molecules then display this antigen fragment onthe cell surface, where it is recognized by the T cell re-ceptor (TCR). In effect, this peptide fragment acts as agrowth factor to initiate cell proliferation. For a successfulresponse, however, the T cell must be stimulated by addi-tional ligands, such as CD86, that act through the CD28receptor.

T cell receptors, precursors and transducersThe T cell receptor (TCR) is a complicated multimolecu-lar complex. It begins with a few fixed components, but assoon as it binds antigen, it draws in a large number of ad-ditional transducing and scaffolding components to builda huge macromolecular complex, which has also been re-ferred to as a ‘transducisome’, which functions to relay in-formation into the cell (Module 9: Figure TCR signalling).The TCR consists of transmembrane proteins (α, β and ζ)that are associated with the CD3 components (CD3γ, δ

and ε). This TCR complex is associated with the accessoryco-receptor CD4 that binds to the major histocompatibil-ity complex II (MHCII). The TCRα and TCRβ subunitsform an immunoglobulin-like receptor that is responsiblefor recognizing the antigen fragment held in the jaws ofthe MHC. Activation of the TCR creates a large macro-molecular complex responsible for relaying informationthrough a number of signalling cassettes.

Since the α and β subunits of the TCR have very shortcytoplasmic tails, they are incapable of engaging the signaltransducing elements, and this function is carried out byother members of the receptor. The CD3 subunits (γ, δ

and ε) and the ζ subunits have long cytoplasmic chains thatcontain immunoreceptor tyrosine-based activation motifs(ITAMs) (red bars), which provide the docking sites toassemble the receptor scaffold. The ‘glue’ that directs andholds the scaffold together is provided by the phosphategroups that are added sequentially to the various ITAMs.The assembly of this signalling scaffold follows an orderlysequence of events:

Positioning the tyrosine kinases (Module 9: Figure TCRsignalling). Since the TCR has no enzymatic activity, thisis assembled by recruiting various non-receptor proteintyrosine kinases such as Lck and ζ-associated protein of70 kDa (ZAP70). An important early event is the recruit-ment of Lck, which is drawn in to the TCR through itsattachment to CD4, the co-receptor that interacts withMHCII.

Activation of the tyrosine kinases. Lck is kept inactive byphosphorylation of a tyrosine residue in its C-terminalregion by a C-terminal Src kinase (CSK). To activatethese kinases, this inhibitory phosphate group must beremoved by the transmembrane tyrosine phosphataseCD45.

Phosphorylation of the ITAM docking sites. Lck phos-phorylates tyrosine residues on the ITAMs located onthe CD3 and ζ chains.

Recruitment of ZAP70 and scaffolding elements. One ofthe key components of the developing scaffold is thetyrosine kinase ζ-associated protein of 70 kDa (ZAP70)that uses its Src homology 2 (SH2) domains to bind tothe phosphorylated ITAMs. Once in place, ZAP70 thenphosphorylates scaffolding components such as a linkerfor activation of T cells (LAT) and an SH2-domain-containing leukocyte protein of 76 kDa (SLP-76), whichare drawn into the growing central supramolecular ac-tivation cluster (cSMAC) and function as docking sitesto communicate to the different signalling cassettes.An important function of both SLP-76 and LAT isto provide a scaffold, which includes Vav1. This scaf-folding complex then assembles the transducers, such asphospholipase Cγ1 (PLCγ1), Ras and PtdIns 3-kinasethat are responsible for activating the TCR signallingcassettes that relay information into the nucleus.

T cell receptor (TCR) signalling cassettesThe T cell receptor (TCR) activates cell proliferation byrelaying information to the nucleus using many of the

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �30

Module 9: Figure immunological synapse

See :Figure TCR signalling

Module 9

InsP3 3 CytoskeletonERK1/2 DAG PIP

T Cell

ANTIGEN PRESENTING CELL

The immunological synapse.When a T cell makes contact with an antigen-presenting cell, the resulting signalling events are localized to an immunological synapse, where T cellreceptor complexes are concentrated in a two-dimensional array. The region illustrated here is the central supramolecular activation cluster (cSMAC)shown in Module 9: Figure immunological synapse structure. For sake of clarity, the peripheral SMAC (pSMAC) containing the adhesion moleculesand the actin scaffold has not been included. The signalling complexes, which co-operate with each other to generate the signalling information thatinduces the T cell to proliferate, are shown in more detail in Module 9: Figure T cell Ca2 + signalling.

conventional growth factor signalling pathways (Module9: Figure T cell signalling map):

T cell receptor (TCR) Ca2 + signallingOne of the major signals for activating lymphocyte pro-liferation is Ca2 + , which is generated by the inositol1,4,5-trisphosphate (InsP3)/Ca2 + signalling cassette. Thephosphate group of LAT binds to the Src homology 2(SH2) domain of phospholipase Cγ1 (PLCγ1) throughthe conventional mechanism for activating the γ isoformof PLC (Module 2: Figure PLC structure and function).The PLCγ1 is thus positioned close to the membrane,where it can begin to initiate the following sequence ofevents (Module 9: Figure T cell Ca2 + signalling):

1. Antigen initiates the process by binding to the T cellreceptor (TCR), which activates phospholipase Cγ1(PLCγ1) to hydrolyse PtdIns4,5P2 to generate inos-itol 1,4,5-trisphosphate (InsP3). This process is en-hanced by PtdIns 3-kinase that forms the lipid mes-senger PtdIns3,4,5P3, which acts through inducible Tcell kinase (Itk) to enhance PLCγ1.

2. The InsP3 acts through the InsP3 receptor (InsP3R) torelease Ca2 + from the internal store.

3. As the ER empties, it sends a signal to the Ca2 + re-lease-activated Ca2 + (CRAC) channel responsible formaintaining the Ca2 + entry current ICRAC for the 2 hperiod required to induce lymphocytes to grow. TheOrai1 protein has been identified as the channel re-sponsible for carrying ICRAC. Mutation of the Orai1has been linked to severe combined immune deficiency(SCID) (Module 3: Figure Ca2 + signalling in SCID).

The coupling between store emptying and activation ofthe CRAC channel is still unclear. A stromal interac-tion molecule (STIM) in the ER may function as thesensor of store emptying (Module 3: Figure STIM-in-duced Ca2 + entry). In addition, there is a role forWiskott--Aldrich syndrome protein (WASP) verprolinhomologous 2 (WAVE2), which may function to sta-bilize the coupling complex responsible for Ca2 + entryby remodelling the actin cytoskeleton.

4. Mitochondria play an important role in enhancing theentry process by removing Ca2 + from the vicinityof the ICRAC channel, thereby negating the inhibit-ory effect of Ca2 + . The membrane hyperpolarization,which is necessary to maintain Ca2 + entry, dependson both a voltage-dependent potassium (Kv) channel(KV1.3) and the intermediate-conductance (IK) channel(KCa3.1).

5. Ca2 + acts to stimulate the transcription factor nuclearfactor of activated T cells (NFAT) (see Module 4: FigureNFAT activation for details).

6. NFAT enters the nucleus where it switches on the genesfor the IL-2 signalling pathway. In addition, NFAT ac-tivates the expression of Down’s syndrome critical re-gion 1 (DSCR1) and carabin, which inhibit calcineurinto set up a negative-feedback loop (Module 9: Figure Tcell Ca2 + signalling).

An important point to stress is that the Ca2 + signallingpathway has to remain active for at least 2 h in order tostimulate lymphocytes to proliferate. During this activa-tion period, the Ca2 + signals are presented as prolonged

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �31

Module 9: Figure T cell Ca2 + signalling

1

2

6

3

4

5

InsPInsP R3

3

+ -

+

+

PLC 1

Ca 2+

2PtdIns4,5P 3PtdIns3,4,5P

NFAT3

NFAT3

K 3.1K 1.3 CaV

Hyperpolarization

Mitochondrion

CRACI

GSK-3

Orai1

IL-2IL-2RCarabinDSCR1

P

PP

CaN

CaM

STIM1

Endoplasmicreticulum

PI 3-K

Itk

TCR

KK ++Antigen

+

Carabin

DSCR1

The role of Ca2 + signalling in lymphocyte proliferation.Antigen initiates the proliferative process by binding to the T cell receptor (TCR), which activates phospholipase Cγ1 (PLCγ1) to hydrolyse PtdIns4,5P2to generate inositol 1,4,5-trisphosphate (InsP3). InsP3 binds to the InsP3 receptor (InsP3R) to release Ca2 + from the endoplasmic reticulum (ER) andthis initiates the signalling cascade described in the text. The way in which Ca2 + acts to stimulate the transcription factor nuclear factor of activated Tcells (NFAT) is described in Module 4: Figure NFAT activation.

Module 9: Figure T cell Ca2 + oscillations

Oscillations in activated T cells.Response of single human peripheral lymphocytes to phytohaemagglutinin (PHA; open bars) or to PHA plus charybdotoxin (PHA + CTX; closed bar).The traces on the bottom represent the average response from 200 cells. Reproduced from Cell Calcium, Vol. 21, Verheugen, J.A.H., Le Deist, F.,Devignot, V. and Korn, H., Enhancement of calcium signaling and proliferation responses in activated human T lymphocytes: inhibitory effects of K+

channel block by charybdotoxin depend on the T cell activation state, pp. 1--17. Copyright (1997), with permission from Elsevier; see Verheugen et al.1997.

oscillations (Module 9: Figure T cell Ca2 + oscillations).An increase in Ca2 + is thus a key regulator of cell pro-liferation. Lymphocyte activation and smooth muscle cellproliferation provide paradigms for such a role for Ca2 +

in controlling cell proliferation.T cell activation provides a good example of the link

between K+ channels and cell proliferation. At least twoK+ channels are responsible for hyperpolarizing the mem-brane to maintain the electrical gradient necessary for theprolonged influx of external Ca2 + through the Oria1 chan-nels (Module 9: Figure T cell Ca2 + signalling). Thereis KV1.3 and the intermediate-conductance (IK) channel(KCa3.1). Hypoxia results in a decrease in the expressionof the KV1.3 channel and this can lead to a decrease in cellproliferation.

The Ras/mitogen-activated protein kinase (MAPK)cassette(Module 9: Figure T cell signalling map). The mitogen-activated protein kinase (MAPK) signalling pathwayplays an important role in lymphocyte activation. Thegrowth-factor-receptor-bound protein 2 (Grb2) sub-unit is drawn into the complex where, together withSon-of-sevenless (SoS), it helps to assemble the membranecomplex that initiates the extracellular-signal-regulatedkinase (ERK) signalling pathway (Module 2: Figure ERKsignalling).

Small GTP-binding proteins and the cytoskeleton(Module 9: Figure T cell signalling map) Thescaffold protein Src homology 2 domain-containing

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �32

leukocyte protein of 76 kDa (SLP-76) binds Rac, Vavand Nck, which function to organize the cytoskeleton.For example, Vav is a guanine-nucleotide exchange factor(GEF) for the Rho family of monomeric G proteinssuch as Rac, which function by organizing the cyto-skeleton (Module 2: Figure Rac signalling). Modificationof the cytoskeleton may also depend on the ability of Vavto bind talin and vinculin, which function to anchor thecytoskeleton to the plasma membrane. In addition, Vavmay influence Ca2 + signalling by acting through Rho tostimulate the PtdIns4P 5-kinase (PtdIns4P 5-K) to providethe precursor lipid PtdIns4,5P2 used by the PLCγ1 andPtdIns 3-kinase signalling cassettes (Module 9: Figure TCRsignalling).

T cell PtdIns 3-kinase signalling cassette(Module 9: Figure T cell signalling map). The regulatorysubunit of the PtdIns 3-kinase has Src homology 3 (SH3)and two Src homology 2 (SH2) domains that enable it tobind to the activated receptor, thus enabling its associatedcatalytic subunit to begin to phosphorylate PtdIns4,5P2

to the second messenger PtdIns3,4,5P3 (PIP3) (Module 2:Figure PtdIns 3-kinase signalling). One of the functionsof PIP3 is to facilitate the activation of phospholipase Cγ1(PLCγ1) by binding directly to the enzyme, and also byactivating the protein inducible T cell kinase (Itk) (Module9: Figure TCR signalling).

CD28 co-stimulatory pathwayActivation of the T cell receptor (TCR) by itself is notsufficient to induce cell proliferation, even though it iscapable of sending information down a number of prom-inent signalling cassettes. To complete its role in initiatingevents early in G1, the TCR complex requires assistancefrom the co-stimulatory pathway (Module 9: Figure T cellsignalling map). In addition to facilitating the proliferat-ive signal, the CD28 pathway also prevents the TCR fromentering into an unresponsive state that has been referredto as clonal anergy. Just how the CD28 pathway acts toprevent the TCR from becoming unresponsive is still verymuch a mystery.

CD28 is a transmembrane glycoprotein that functions asa homodimer linked together through disulphide bonds inthe extracellular domain. The ligands for CD28 are CD80and CD86, which are members of an immunoglobulin fam-ily located in the membrane of the antigen-presenting cell(Module 9: Figure TCR signalling). The cytoplasmic do-main of CD28 lacks enzymatic activity, but it does containtyrosine residues that become phosphorylated followingstimulation, and which may then recruit signalling ele-ments such as PtdIns 3-kinase. There also is an indicationthat CD28 may activate the sphingomyelin signalling path-way, resulting in the activation of a number of pathwaysincluding the JNK pathway.

The inducible costimulatory molecule (ICOS), whichis also known as CD278, is related to CD28 and alsoacts by providing positive signals during T cell activation.CD28 and ICOS have unique and overlapping functionsand act synergistically to promote cell activation. CD28is expressed in resting T cells, whereas the expression of

ICOS is induced once T cells are activated. For example,ICOS is induced in follicular helper T cells (TFH cells)during B cell differentiation in the lymph node (Module 8:Figure B cell maturation signalling). ICOS is activated bythe ICOS ligand (ICOSL), also known as B7RP-1, whichacts by stimulating the PtdIns 3-kinase signalling path-way to induce the production of effector cytokines suchas interleukin 4 (IL-4) and interleukin 21 (IL-21).

Signalling lymphocyte activation molecule (SLAM)There are nine genes in the signalling lymphocyte activ-ation molecule (SLAM) family (SLAM1--9) that play animportant role in controlling the various interactions thatlymphocytes make with different cells. They belong to animmunoglobulin superfamily that is located on the cellsurface of haematopoietic cells. Most of the family aretype I transmembrane glycoproteins whereas SLAMF2is attached to the membrane through a glycosylphos-phatidylinositol anchor and functions as a receptor forSLAM4. All of the other SLAMs interact in a homophilicmechanism in that their ectodomains can interact witheach other. The cytoplasmic tails of six of these receptors(SLAM1, SLAM3--7) carry unusual intracellular tyrosine-based switch motifs (ITSMs). When the SLAMs inter-act with each other, these ITSMs are phosphorylated andrecruit various SH2-domain-containing adaptor proteinssuch as the SLAM-associated protein (SAP) and EWS/FLIactivated transcript-2 (EAT-2).

The SLAM/SAP signalling pathway functions in a largenumber of lymphocyte interactions to control processessuch as the development of T cell lineages, natural killerT cells (NKT cells) and the affinity maturation of B cells.For example, during B cell differentiation in the lymphnode, there is an increase in the expression of SAP andSLAM in the TFH cells that are responsible for the B cellselection within the germinal centre (Module 8: Figure Bcell maturation signalling).

SLAM-associated protein (SAP)SLAM-associated protein (SAP) and the related EWS/FLIactivated transcript-2 (EAT-2), which is expressed mainlyin antigen-presenting cells, are SH2-domain adaptorproteins that bind to the phosphotyrosine motifs on thecytoplasmic tails of some of the signalling lymphocyte ac-tivation molecule (SLAM) family (SLAM1, SLAM3--7).Activated SAP is known to relay information to other sig-nalling components such as Fyn.

Mutations in the gene Src homology 2 (SH2) domainprotein 1A (SH2D1A) causes X-linked lymphoproliferat-ive syndrome (XLP).

T cell gene transcriptionThe signalling cassettes activated by the T cell receptor(TCR) produce various cytosolic factors (signalling com-ponents and activated transcription factors) that enter thenucleus to initiate a programme of early gene transcription(Module 9: Figure T cell signalling map). Some of the keyearly genes that are activated code for the interleukin-2(IL-2) signalling pathway, which is a classical autocrinemechanism that is critical for completing the signalling

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �33

events that end with the onset of DNA synthesis (Module9: Figure G1 proliferative signalling). The promoter regionof the IL-2 gene is thus an example of one of the earlygenes. It has an array of response elements that bind dif-ferent transcription factors activated by the different sig-nalling cassettes. Such promoter regions integrate informa-tion relayed to them by a number of different transcriptionfactors:

• Nuclear factor of activated T cells (NFAT). One of thekey transcription factors is NFAT, which mediates theaction of Ca2 + . It has two components: one is loc-ated in the nucleus (NFATn) and is rapidly inducedby the diacylglycerol (DAG)/protein kinase C (PKC)signalling cassette; the second component is cytosolicNFAT (NFATc), which resides in the cytoplasm as an in-active phosphorylated intermediate. Ca2 + acts by stim-ulating the enzyme calcineurin (CaN), which dephos-phorylates NFATc, thus enabling it to enter the nucleusto initiate gene transcription (Module 4: Figure NFATactivation). Once it enters the nucleus, NFATc combineswith NFATn to form a dimer that binds to two separ-ate sites. At one of these sites, NFAT co-operates withanother factor, activating protein 1 (AP-1) (see below),thus illustrating the combinatorial aspect of gene tran-scription. One of the genes activated by NFAT codesfor carabin, which is an inhibitor of calcineurin, andthus constitutes a negative-feedback loop to limit T cellsignalling.

• This Ca2 + -dependent gene transcription mediatedthrough NFAT is of particular significance, because itrepresents the site of action of a potent class of im-munosuppressants, such as cyclosporin A (CsA) andFK506. These drugs bind to the immunophilins to blocklymphocyte proliferation by inhibiting the calcineurin-dependent dephosphorylation of NFATc. These drugsinhibit calcineurin by binding to cyclophilin A or aFK506-binding protein (FKBP).

• Activating protein 1 (AP-1) (Fos/Jun). The transcrip-tion factor activating protein 1 (AP-1) is a heterodi-mer of c-Fos and c-Jun. This AP-1 (Fos/Jun) dimer alsobinds to NFAT to form a transcriptional complex. Theactivation of AP-1 is complicated in that it requires aninduction of the genes that code for the two compon-ents, and, once they have formed the AP-1 complex, thishas to be activated through phosphorylation by kinaseslinked to different signalling pathways (Module 4: Fig-ure SRF and AP-1).

• Nuclear factor κB (NF-κB). IL-2 gene transcriptionalso depends upon activation of the nuclear factor κB(NF-κB) signalling pathway. In the case of T cells, theformation of DAG and its activation of protein kinaseCθ (PKCθ) plays a role in stimulating the transcrip-tion factor NF-κB (Module 9: Figure TCR signalling).The PKCθ acts by assembling a scaffolding complexby phosphorylating CARMA1, which is then recruitedinto the immunological synapse. The CARMA1 thenassociates with a pre-existing complex that consists ofthe CARD protein Bcl10 and the mucosa-associated

lymphoid tissue protein-1 (MALT1 also known asparacaspase) to form the CARMA--Bcl10--Malt1 scaf-folding complex. This complex then brings about theUBC13-dependent ubiquitination of IκB kinase (IKK)resulting in activation of the NF-κB signalling pathway.See Module 2: Figure NF-κB activation for details ofhow IKK phosphorylates the IκB that is targeted fordestruction by the ubiquitin--proteasome system. OnceIκB is destroyed, the NF-κB dimer can enter the nuc-leus, where it helps to switch on the genes for IL-2 andthe IL-2 receptor.

As a result of these early transcriptional events, genessuch as those for IL-2 and the IL-2 receptor initiatethe autocrine loop that results in the activation of theinterleukin-2 (IL-2) signalling pathway that functions laterin G1 to send signals to the nucleus to interact with thecell cycle control elements to switch on DNA synthesis(Module 9: Figure T cell signalling map).

Interleukin-2 (IL-2) signalling pathwayInterleukin-2 (IL-2) is a cytokine that functions throughnon-enzyme-containing receptors (Module 1: Figure cy-tokines). IL-2 provides the link between the earlier eventsinitiated by the T cell receptor (TCR) to the cell cycle com-ponents necessary to initiate DNA synthesis (Module 9:Figure T cell signalling map). The IL-2 receptor (IL-2R)has three subunits (α, β and γ), which have to come to-gether to form a functional unit. The IL-2 signalling path-way transfers information to the cell cycle componentsusing the Janus kinase (JAK)/signal transducer and activ-ator of transcription (STAT) signalling pathway (Module2: Figure JAK/STAT heterogeneity). In addition, it mayalso use the PtdIns 3-kinase and mitogen-activated pro-tein kinase (MAPK) signalling pathways.

B cell activationB cell activation depends on a number of receptor mechan-isms. The primary process is B cell antigen receptor (BCR)activation, but this response is modulated by informationcoming from both B cell stimulatory co-receptors (CD19)and B cell inhibitory co-receptors (CD22 and FcγRIIB).

B cell antigen receptor (BCR) activationThe B cell antigen receptor (BCR) appears early during thedifferentiation of B cells (Module 8: Figure B cell differen-tiation). One of the earliest steps in differentiation, whichoccurs in the pro-B cell stage, is the expression of Igα

(CD79a) and Igβ (CD79b) that are key components of theBCR. These two signalling components begin to transmitinformation to control differentiation and continue to be amajor component of the BCR in mature B cells. The BCRis a hetero-oligomeric complex consisting of these two Igα

and Igβ signalling proteins associated with the antigen-binding immunoglobulin IgM receptor, which consists oftwo heavy chains and two light chains (Module 9: FigureB cell activation). When this IgM component detects anti-gen, a non-receptor tyrosine kinase called Lyn brings aboutthe tyrosine phosphorylation of their immunoreceptortyrosine-based activation motifs (ITAMs) located on the

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �34

Module 9: Figure T cell signalling map

SERCA

IKCa1

STIMOrai 1

PMCA

Ca2+ CaM

CaN

CARMA1Bcl10MALT1IKK

NFAT-P

LIGANDS

RECEPTORS

and

PRECURSORS

TRANSDUCERS

SIGNALL

ING

CASSETTES

NUCLEAR

FACTORS

CYTOSOLIC

FACTORS

DAG

PS

PKC

PKC

PKC

raf-1

S1P

MEKERK1/2

PIP3 PIP3

PDK1/2 PDK1/2

PKB PKB

PKB-P PKB-P

FRAP FRAP

P70rsk

p70rsk

5-Pase 5-Pase

PIP2

PI

PIP

PIP2

PI 4-KPIP5-K

CD4TCR Cd3TCR Cd3

CD3

lck fyn Zap70

CD45CSK

MHCII CD86CD86(CD80)(CD80)

CD28ICOS (CTLA-4)

Lck/itk

IL-2

IL-2

IL2-R

IL2-R

IL2-RIL2-R

lck Sykfyn

raf-1

MEK

ERK1/2

ERK1/2

STAT3

STAT3-P

STAT5

STAT5-PERK1/2-P

ERK1/2-P

NFAT

PITP

PI3-K SMase PI3-KPLC 1

SHCmSOSGRB2

I BUb/proteasome

ref-1NF B

FOXO

JNK

Jun/Fos

JAK1JAK3

ras

Bcl-2

E2F Myc

p21

CAK

p27kip1cyclin D Cdc25

ceramide

CAPKSPHK

SPPCAPP

SMSHC

DAGkinase

CaMK IVBcl-x

P85rsk

IP R1

IP

3

3

DNA Synthesis

PKC

JNK

JNK

G1G0

SHIP SHIPPTEN PTEN

SLP-76 LAT

Rac

VavNck

SHP-1

Cdc42WASPWAVE 2Arp2/3Actin

Rb

GADS

PAK1

DOCK2

ras

Antigen

A spatiotemporal map of T cell activation.Signalling begins at the top left with the major histocompatibility complex II (MHCII) presenting the antigen (red) to the T cell receptor (TCR) complex,which then initiates a sequence of events summarized in the green panel on the left of the figure. The sequence begins with the TCR interactingthrough various transducers to activate signalling cassettes, which relay information into the nucleus through cytosolic messengers to induce genetranscription. Activation of these early gene products sets up an autocrine loop to put in place the interleukin-2 (IL-2) signalling system that activatesadditional signalling cassettes to complete the signalling cascade by inducing DNA synthesis. The names and abbreviations of all the componentsof this signalling map are described in Module 9: Table T cell signalling toolkit.

Module 9: Figure TCR signalling

PIP

PIP

PIP2 PIP2

3

PI 3-K

ANTIGEN PRESENTING CELL

T Cell

PLC 1

Rac

Nck Pak1

InsP3

CytoskeletonERK1/2 Ca2+

Grb2

Sos

Ras

MekRaf

DAG

PKC

Lck

LAT

CD

4 PIP5K

MHCIICD28

CD45

GADS

SLP-76

Vav

Itk

ZA

P-7

0

PP

PPP

P

P

P

CARMA1

Bcl10 MALT1

NF- B

IKK

Molecular organization of a functional T cell receptor (TCR).Details of the signalling complexes located within the immunological synapse (Module 9: Figure immunological synapse). Signalling begins when theα and β subunits of the T cell receptor (TCR) detect antigen (red dot) held in the jaws of the major histocompatibility complex II (MHCII) embedded inthe surface of the antigen-presenting cell. The sequence begins with CD4 associating with MHCII, thereby bringing Lck into the complex to begin thephosphorylation cascade that also depends upon ζ-associated protein of 70 kDa (ZAP70). The phosphates on the scaffolding protein then draw in thesignal transducers, such as phospholipase Cγ1 (PLCγ1), PtdIns 3-kinase (PI 3-K) and the Ras complex, to activate the various signalling pathwaysas described in more detail in the text. The activation of Ca2 + signalling by inositol 1,4,5-trisphosphate (InsP3) is described in Module 9: Figure T cellCa2 + signalling.

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �35

Module 9: Table T cell signalling toolkitAn inventory of the major signalling components used to stimulate Tcell proliferation.Component CommentsLigands

MHC class II Major histocompatibility complex IICD86 Transmembrane glycoproteins (on

antigen-presenting cells) responsible forstimulating CD28

CD80 Transmembrane glycoproteins (onantigen-presenting cells) responsible forstimulating CTLA4

IL-2 Interleukin-2Receptors

TCRα T cell receptor α subunitTCRβ T cell receptor β subunitCD3γ The γ chain of the CD3 component of the TCRCD3δ The δ chain of the CD3 component of the TCRCD3ε The ε chain of the CD3 component of the TCRTCRζ The ζ chain of the TCRTCRη The η chain of the TCRCD4 An accessory co-receptor that binds to MHC class

IICD8 An accessory co-receptor that binds to MHC class

ICD28 A co-stimulatory receptor that responds to CD86ICOS Inducible co-stimulatory molecule that acts

synergistically with CD28IL-2Rα IL-2 receptor α subunitIL-2Rβ IL-2 receptor β subunitIL-2Rγ IL-2 receptor γ subunit

PrecursorsPITP Phosphatidylinositol transfer proteinPI PhosphatidylinositolPIP Phosphatidylinositol 4-phosphatePIP2 Phosphatidylinositol 4,5-bisphosphatePI 4-K Phosphatidylinositol 4-kinasePIP 5-K Phosphatidylinositol 4-phosphate 5-kinaseSM Sphingomyelin

TransducersEnzymaticcomponents

Lck A non-receptor tyrosine kinase (a member of theSrc family)

Fyn A non-receptor tyrosine kinase (a member of theSrc family)

Itk Inducible T cell kinase, T cell-specific tyrosinekinase

ZAP70 ζ-Associated protein of 70 kDa, a tyrosine kinaseclosely related to ZAP70

Syk Spleen tyrosine kinase closely related to ZAP70CD45 A transmembrane tyrosine protein phosphataseCSK C-terminal Src kinasePLCγ1 Phospholipase Cγ1SHP Src homology 2 (SH2) domain-containing protein

tyrosine phosphataseG proteincomponents

Rac A monomeric G proteinCdc42 A monomeric G proteinVav A guanine nucleotide exchange factor (GEF) for

Rho family GTPasesScaffoldingcomponents

LAT Linker for activation of T cellsSLP-76 Src homology 2 (SH2)-domain containing

leukocyte protein of 76 kDaGADS Grb2-related adaptor proteinCrkL Crk-like scaffolding proteinNck A scaffolding proteinCARMA1 A scaffolding protein that links PKCθ to the

activation of the NF-κB signalling pathway(Module 9: Figure TCR signalling)

Bcl10 A scaffolding protein that links CARMA1 to theactivation of the NF-κB signalling pathway

MALT1 Mucosa-associated lymphoid tissue protein-1 thatfunctions with CARMA1 and Bcl10 to theactivation of the NF-κB signalling pathway(Module 9: Figure TCR signalling)

Module 9: Table continuedComponent CommentsSignallingcassettes

InsP3/Ca2 +

pathwayInsP3 Inositol 1,4,5-trisphosphateInsP3R1 Inositol 1,4,5-trisphosphate receptor type IInsP3R2 Inositol 1,4,5-trisphosphate receptor type IIInsP3R3 Inositol 1,4,5-trisphosphate receptor type IIIIP3 3-K InsP3 3-kinaseIP3 5-Pase InsP3 5-phosphataseKCa3.1 Ca2 + -activated intermediate-conductance (IK) K+

channelSERCA Sarco/endo-plasmic reticulum Ca2 + -ATPasePMCA Plasma membrane Ca2 + -ATPaseSTIM1 Stromal interaction molecule 1 (Module 3: Figure

SOC signalling components)Orai1 The main component of the Ca2 +

release-activated Ca2 + (CRAC) channel(Module 3: Figure SOC signalling components)

DAG/PKCpathway

DAG DiacylglycerolDAG kinase Diacylglycerol kinasePS PhosphatidylserinePKCα Protein kinase Cα

PKCε Protein kinase Cε

PKCζ Protein kinase Cζ

PKCθ Protein kinase Cθ

PI 3-kinasepathway

PIP3 Phosphatidylinositol 3,4,5-trisphosphatePI 3-K Phosphatidylinositol 3-kinasePTEN Phosphatase and tensin homologue deleted on

chromosome 10SHIP Src homology 2 (SH2) domain-containing inositol

polyphosphate 3-phosphatasePKB Protein kinase B (also known as Akt)FRAP FK506-binding protein 12 (FKBP12)

rapamycin-associated proteinp70S6K 70 kDa ribosomal S6 kinasep85S6K 85 kDa ribosomal S6 kinase

MAPK pathwayRas A small GTP-binding proteinmSoS Mammalian Son-of-sevenlessShc Src homology 2 (SH2)-domain-containing protein

(an adaptor protein for Ras activation)Grb2 Growth factor receptor-bound protein 2Raf-1 Cellular homologue of the v-Raf oncogene (a

serine/threonine protein kinase)MEK MAPK/extracellular-signal-regulated kinase (ERK)

kinaseMAPK Mitogen-activated protein kinase

JAK/STATpathway

JAK1 Janus kinase 1JAK3 Janus kinase 3STAT3 Signal transducer and activator of transcription 3STAT5 Signal transducer and activator of transcription 5

Sphingomyelinpathway

S1P Sphingosine 1-phosphateCeramide A second messenger released from

sphingomyelinSMase SphingomyelinaseCAPK Ceramide-activating protein kinaseCAPP Ceramide-activated protein phosphataseSPHK Sphingosine kinaseSPP S1P phosphatase

Actinremodelling

WASP Wiskott--Aldrich syndrome proteinWAVE2 WASP verprolin homologous; related to WASP

and regulates actin cytoskeletonWIP WASP-interacting proteinArp2/3 Actin-related proteins 2 and 3

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �36

Module 9: Table continuedComponent Comments

Cytosolicfactors

CaM CalmodulinCaN CalcineurinCaMKIV Ca2 + /calmodulin-dependent protein kinase IVUb UbiquitinProteasome A multisubunit complex responsible for degrading

ubiquitin/rotein conjugatesNFAT Nuclear factor of activated T cells (cytoplasmic

component)IκB Inhibitor of NF-κBNF-κB Nuclear factor-κB

Nuclear factorsE2F E2 promotor binding factorJun A nuclear transcription factor (a component of

activating protein 1)Fos A nuclear transcription factor (a component of

activating protein 1)Ref-1 Redox factor-1p21 A 21 kDa inhibitor of cyclin-dependent kinasesp27kip1 A 27 kDa inhibitor of cyclin-dependent kinasesCyclin E A cell cycle protein that functions late in G1 to

control onset of DNA synthesisCDK2 Cyclin-dependent kinase 2 (a threonine/serine

protein kinase)CAK CDK-activating kinaseRb Retinoblastoma gene product

The purpose of this T cell signalling toolkit, which is by no meanscomplete, is to highlight the fact that cell proliferation dependsupon the co-operation of a large number of signallingcomponents. The description of the abbreviations often helps todescribe their different functions. The way in which many ofthese components contribute to T cell activation to transferinformation from the cell surface into the cytoplasm and nucleusis summarized in Module 9: Figure T cell signalling map.

cytoplasmic regions of Igα and Igβ. The transmembraneprotein tyrosine phosphatase CD45 is responsible for ac-tivating Lyn to initiate the following cascade of signallingevents that emanate from the activated BCR.

The phosphorylated ITAMs on Igα and Igβ recruitand activate the non-receptor spleen tyrosine kinase Syk,which then recruits adaptors and various signalling trans-ducers to form a large macromolecular signalling complex.A key function of Syk is to phosphorylate the B cell linkerprotein (BLNK) at several sites to provide docking sites torecruit various key signalling components such as Bruton’styrosine kinase (Btk) and phospholipase Cγ2 (PLCγ2).The Btk activates PLCγ2 by phosphorylating Tyr753 andTyr759. The enzymatic activity of both Btk and PLCγ2is stimulated by PtdIns3,4,5P3, the lipid messenger ofthe PtdIns 3-kinase signalling pathway. The PtdIns3,4,5P3

stimulates Btk to phosphorylate PLCγ2 and the latter isalso directly activated by this highly charged lipid. Theincrease in PtdIns3,4,5P3 formation is described in moredetail in the section on the B cell stimulatory co-receptors.

Once PLCγ2 is activated, it hydrolyses PtdIns4,5P2

to release diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP3). The DAG activates protein kinaseCβ (PKCβ) to contribute to the onset of proliferation. TheInsP3 plays a primary role in activating proliferation by in-creasing the intracellular level of Ca2 + (Module 9: FigureB cell activation). The InsP3 binds to the InsP3 receptorto release Ca2 + from the endoplasmic reticulum (ER) andonce this internal store is depleted the store-operated chan-

nels (SOCs) are activated to enhance the entry of externalCa2 + . The mechanism of store-operated channels (SOCs)activation depends on an interaction between STIM pro-teins in the ER-sensing store depletion to send informationto open the Orai channels in the plasma membrane. Oneof the actions of Ca2 + is to activate NFAT that leads on tothe increase in proliferation.

The phosphorylated BLNK adaptor is also connectedto other signalling pathways such as the MAP kinasesignalling pathway and the Rac signalling mechanism(Module 9: Figure B cell activation). The adaptor growthfactor receptor-bound protein 2 (Grb2) binds to one of thephosphorylated tyrosine residues and then assembles thesignalling components of the MAP kinase signalling path-way that results in the activation of ERK1/2 that contrib-utes to the onset of proliferation (Module 2: Figure ERKsignalling). Another output signal from the BLNK sig-nalling complex is the Rac signalling mechanism (Module2: Figure Rac signalling), which is initiated by the guan-ine nucleotide-exchange factor Vav that then stimulatesRac to promote the membrane localization and activationof PtdIns4P 5-kinase (PIP5K in Module 9: Figure B cellactivation). Following activation of PIP5K, the resultinglocal formation of PtdIns4,5P2 provides the substrate usedby both PLCγ2 and the PtdIns 3-kinase. The latter is a keysignalling component of the B cell stimulatory co-recept-ors.

The BCR is of interest because it appears early during Bcell differentiation in the bone marrow (Module 8: Figure Bcell differentiation) and plays a critical role in providing theproliferative and survival signals to drive B cells throughtheir programme of differentiation. It is then retained inmature B cells to enable them to function as memory cellsto respond to foreign antigens.

B cell linker protein (BLNK)The B cell linker protein (BLNK), which is also known asSrc homology 2 (SH2) domain-containing leukocyte pro-tein of 65 kDa (SLP-65), is phosphorylated during B cellantigen receptor (BCR) activation to provide binding sitesfor both Bruton’s tyrosine kinase (Btk) and phospholipaseCγ2 (PLCγ2) (Module 9: Figure B cell activation).

B cell stimulatory co-receptorsThe B cell stimulatory co-receptors can fine tune the B cellantigen receptor (BCR) response by activating PtdIns 3-k-inase signalling to provide the PtdIns3,4,5P3 to enhancethe activity of both Bruton’s tyrosine kinase (Btk) andphospholipase Cγ2 (PLCγ2) (Module 9: Figure B cell ac-tivation). The trimolecular complex consisting of CD19,CD21 and CD81 is recruited into the vicinity of the BCRcomplex when CD21 binds to the C3d complement com-ponent. CD19, which is a B cell-specific surface protein,first appears in the pre-B cell stage during B cell differenti-ation in the bone marrow (Module 8: Figure B cell differ-entiation). The non-receptor tyrosine kinase Lyn, whichinitiated the BCR activation process, is also responsiblefor phosphorylating the cytoplasmic domain of CD19 toprovide a binding site for the Class 1A PtdIns 3-kinase.The regulatory subunit p85 attaches the catalytic p110

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �37

Module 9: Figure B cell activation

PI 3-K

PLC

Rac

PKBERK1/2

Ca

Ca

2+

2+

RasMek

RafDAGInsP3

NFAT

Light chainHeavy chain

PKC

Ig IgLyn

CD19C3d

CD81CD21

IgG

CD45 BCRIgM

AntigenCD22

Inhibitoryco-receptors

Stimulatoryco-receptors

BLNK(SLP-65)

Vav

Btk

P

PP

P PP P

P

P

P

P

P

P

P

Grb2

Syk

Sos

Proliferation Survival

Fc RIIB

SHIP SHIP

SHP-1

SHP-1

STIM

Orai

ER

InsP R3

+

+

+

+

++

PIP5K

PIP2

PIP2

PIP2 PIP3PIP3

PIP

B cell activation.B cells have complex activation processes driven by information from the primary B cell receptor (BCR), which detects antigen, and a number ofstimulatory co-receptors (CD19) and inhibitory co-receptors (CD22 and FcγRIIB), which function to modulate the activity of the BCR. See the text forfurther details.

subunits to the phosphorylated tyrosine residue on CD19,thus positioning the enzyme close to the membrane whereit can begin to generate the lipid messenger PtdIns3,4,5P3

(Module 9: Figure B cell activation).

B cell inhibitory co-receptorsThe B cell inhibitory co-receptors regulate the amplitudeof the B cell antigen receptor (BCR) response througha number of inhibitory mechanisms. One such inhibit-ory process depends on CD22, which is a type I mem-brane protein that first appears in the pre-B cell stage dur-ing B cell differentiation in the bone marrow (Module 8:Figure B cell differentiation). CD22 has seven immuno-globulin (Ig)-like extracellular domains and it is the V-setN-terminal Ig-like domain that binds to its ligand, whichis the α2,6-linked sialic acid (2,6Sia) ligand found on thesurface of many membrane proteins such as the IgM ofthe BCR or CD45. When it is cross-linked to such pro-teins, the immunoreceptor tyrosine-based inhibition mo-tifs (ITIMs) on its cytoplasmic tail are phosphorylated toprovide docking sites for both Src homology 2 (SH2) do-main-containing inositol phosphatase 1 (SHIP1) and Srchomology 2 (SH2) domain-containing protein tyrosinephosphatase 1 (SHP1) (Module 9: Figure B cell activa-tion). SHIP-1 hydrolyses Ins1,3,4,5P4 and PtdIns3,4,5P3

and thus reduces the stimulatory effect of this lipid onboth Bruton’s tyrosine kinase (Btk) and phospholipaseCγ2 (PLCγ2). The primary function of SHP-1, which is atyrosine phosphatase, is to inhibit the BCR signalling path-ways by dephosphorylating the phosphotyrosine residueson components such as Syk, BLNK and CD19.

Another inhibitory co-receptor FcγRIIB, which alsohas ITIMs capable of recruiting both SHIP-1 and SHP1,

carry out lipid and protein dephosphorylation reactionsrespectively, similar to those described for CD22.

Smooth muscle cell proliferationThe proliferation of smooth muscle cells plays an import-ant role in wound healing, but this also occurs during vas-cular remodelling, which can have serious pathophysiolo-gical consequences when it leads to pulmonary vasocon-striction and hypertension.

A central role of Ca2 + in the control of cell prolifera-tion (Module 9: Figure growth factor signalling) is nicelyexemplified in the case of smooth muscle cell prolifera-tion. A smooth muscle cell provides a good example ofa proliferation/differentiation switch whereby a fully dif-ferentiated cell, which is carrying out its function of con-traction, returns to the cell cycle and begins to proliferate.What is so interesting about this dual role of contractionand proliferation is that Ca2 + plays a role in controllingboth processes. Proliferation is also regulated throughgrowth factors acting through transcription factors such asETS-like transcription factor-1 (Elk-1) and Kruppel-likefactor 4 (KLF4) (Module 8: Figure smooth muscle celldifferentiation). The microRNAs miR-143 and miR-145play an important role in converting such proliferatingcells back into differentiated smooth muscle cells.

This dual action of Ca2 + in smooth muscle cells is sim-ilar to what occurs in cardiac cells, where Ca2 + controlsboth contraction and the transcriptional events responsiblefor cardiac hypertrophy (Module 12: Figure hypertrophysignalling mechanisms). As for cardiac hypertrophy, it isreasonable to argue that normal levels of Ca2 + signallingregulate contraction, but when this becomes excessive, cellproliferation is induced. As for other examples of cell pro-liferation, it is likely that a prolonged period of Ca2 +

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �38

Module 9: Figure SMC proliferation

L-Type Cachannel

2+

PLC

Ca2+

BK channel

G q/11

Store-operatedchannel

K+

VASOCONSTRICTION PROLIFERATION

HYPERTENSION

INCREASED VASCULARRESISTANCE

RYR

RPsnI

InsP

3

3

Agonis

ts

Role of Ca2 + in the regulation of smooth muscle cell (SMC) contraction and proliferation.Both contraction and proliferation of smooth muscle cells (SMCs) are regulated by Ca2 + . The role of different ion channels in regulating Ca2 +

signalling is described in Module 7: Figure smooth muscle cell E-C coupling. High levels of such signalling contribute to both vasoconstriction andproliferation.

signalling might be necessary to stimulate smooth musclecell proliferation (Module 9: Figure SMC proliferation). Inthe case of pulmonary artery smooth muscle cells, this pro-longed signalling seems to depend upon a constant influxof Ca2 + through canonical transient receptor potential 1(TRPC1), i.e. one of the store-operated channels (SOCs)that carry out capacitative Ca2 + entry, similarly to theICRAC used for lymphocyte activation (Module 9: FigureT cell Ca2 + signalling).

AngiogenesisA process of angiogenesis carries out the growth and repairof blood vessels, during which pre-existing blood vesselsgive rise to new vessels (Module 9: Figure angiogenesis).Differentiated endothelial cells that line the inner surface ofblood vessels are normally in a quiescent state (G0) in thatthey have a very low turnover, of the order of hundreds ofdays. During the process of angiogenesis, however, they re-turn to the cell cycle in response to various growth factors.Angiogenesis is usually driven by local tissue demandsfor oxygen and glucose, or by various mechanical stresses.An important component of this angiogenic response isthe activation of hypoxia-inducible factor (HIF), whichis a transcription factor that increases the expression of alarge number of components such as vascular endothelialgrowth factor (VEGF) that function to regulate cell prolif-eration (Module 4: Figure HIF functions). One of the mainangiogenic mechanisms is the sprouting and branching ofpre-existing vessels, a process triggered by VEGF, whichacts on endothelial cells to induce them to proliferate andto migrate to form the new vessels as shown in the follow-ing sequence of events (Module 9: Figure angiogenesis):

A. A typical example of angiogenesis is found near tu-mours, where the growing cell mass begins to runout of oxygen and this triggers the activation ofhypoxia-inducible factor (HIF) that switches on theexpression and release of vascular endothelial growthfactor A (VEGF-A) (Module 4: Figure HIF functions).The VEGF-A (yellow hallow) diffuses out towardsneighbouring blood vessels where it acts on the VEGFreceptor 2 (VEGFR2) to induce one of the en-dothelial cells (coloured red) to begin to differ-entiate into a tip cell). VEGF is also releasedduring wounding and inflammation (Module 11: Fig-ure inflammation). Not only does VEGF activate an-giogenesis, but it also contributes to the endothelialregulation of paracellular permeability.

B. Despite the fact that a number of endothelial cellsbegin to sense the VEGF-A, only one turns into atip cell and this rather precise specification is enabledthrough a process of lateral inhibition whereby the tipcell switches off the ability of neighbouring cells tobecome tip cells. This inhibition depends on the tipcell activating the Notch signalling pathway to inhibitthe conversion into tip cells. The extracellular matrix(ECM) is degraded to provide an opening for the out-ward growth of the sprout.

C. The tip cell releases platelet-derived growth factor(PDGF) that then acts locally to induce endothelial cellproliferation to initiate sprouting through the growthof stalk cells.

D. As the endothelial cells proliferate to form stalk cells,the sprout grows out towards the tumour led by thetip cells that have filopodia that appear to providethe guidance system that senses the source of the

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �39

VEGF-A gradient. The PDGF also stimulates the pro-liferation of the pericytes. As the sprout lengthens,branching can occur through a process that recapit-ulates that of sprouting. One cell (shown in red) be-comes a tip cell and this then leads the way for thedeveloping branch. As the new vessel develops, de-position of a new ECM begins to restore the externalcovering of the new blood vessel.

E. As the sprout and branch develops, the pericytes beginto migrate up the new vessel.

F. As the new vessel reaches its destination and invadesthe tumour, the pericytes assist in the maturation pro-cess by stabilizing the endothelial cells by flatteningout over their surface and by developing various cellsurface junctions some of which are concentrated inthe peg--socket junctional complexes where the N-cadherin complexes are located (Module 9: Figure an-giogenesis signalling).

The sequence of events that occur during angiogen-esis are orchestrated by a number of cell--cell sig-nalling mechanisms (Module 9: Figure angiogenesissignalling):

1. A gradient of vascular endothelial growth factor A(VEGF-A), which is produced either at a site of in-flammation, damage or by tumour cells, initiates an-giogenesis by acting on the VEGFR2 on an endothelialcell to induce it to transform in to a tip cell (see stagesA and B in Module 9: Figure angiogenesis).

2. As part of this differentiation, the tip cell increases theexpression of the Delta-like-4 (DLL4) ligand that activ-ates Notch receptors on the neighbouring endothelialcells to prevent them from becoming tip cells- a pro-cess known as lateral inhibition (Module 9: Figure an-giogenesis signalling). Activation of this Notch sig-nalling pathway in the endothelial cells appears to actby reducing the expression of VEGFR2 thus prevent-ing the induction of tip cells but the endothelial cellsthat retain the ability to respond to VEGF-A by pro-liferating.

3. The endothelial cells located close to the tip cells are in-duced to proliferate by VEGF-A to produce the stalkcells of the sprout. Activation of the VEGFR2 on en-dothelial cells recruits a number of signalling pathwaysassociated with the onset of proliferation (Module 9:Figure VEGF-induced proliferation).

4. The tip cells release platelet-derived growth factor B(PDGF-B) that acts on the PDGF receptor β (PDGFR-β) to stimulate proliferation of the pericytes using thesignalling pathways activated by this growth factor(Module 1: Figure PDGFR activation). The PDGF-Bbinds to heparan sulphate proteoglycans (HSPGs) andthis prevents it from diffusing away and thus restrictsits action to a local region around the tip cell. Indeed,the HSPGs not only restrict PDGF-B diffusion butit facilitates its binding to the PDGFR-β (Module 9:Figure angiogenesis signalling).

5. The endothelial cells that have been induced to prolifer-ate by VEGF-B, release sphingosine 1-phosphate (S1P),

which is a component of the sphingomyelin signallingpathway (Module 2: Figure sphingomyelin signalling).

6. The pericytes respond to the S1P using EDG receptorsthat can activate a number of signalling pathways thatappear to target the N-cadherin complex that functionsin the peg--socket junctional complex (Module 9: Fig-ure angiogenesis signalling). The S1P signalling path-ways may interact with the signalling pathways that areassociated with such N-cadherin complexes (Module 6:Figure classical cadherin signalling). Since S1P is knownto activate cell proliferation in other cell types, it is pos-sible that it may act together with PDGF-β to drive theproliferation of the pericytes.

7. In addition to the N-cadherin complex, the peri-cytes communicate with the endothelial cells throughan angiopoietin-TIE2 signalling pathway (Module 9:Figure angiogenesis signalling). The pericytes releaseangiopoietin 1 (Ang1) that normally acts through TIE2receptors to control the maturation and stability of theendothelial cells.

8. At the time of angiogenesis, such local cell--cell contactsare reduced to enable cells to proliferate and migrate toform a sprout. The proliferating endothelial cells facilit-ate this loosening of cell contacts by releasing angiopoi-etin 2 (Ang2), which acts by inhibiting the activation ofthe TIE2 receptors by Ang1.

Alterations in angiogenesis have a number of severepathological consequences, such as diabetic retino-pathy and age-related macular degeneration. Tumourangiogenesis plays a vital role in tumour growth andmetastasis.

Vascular endothelial growth factor (VEGF)Vascular endothelial growth factor (VEGF) is one ofthe main growth factors responsible for regulating bothangiogenesis (Module 9: Figure angiogenesis) and theendothelial regulation of paracellular permeability. VEGFis also released during wounding and inflammation(Module 11: Figure inflammation).

The VEGF family, which are glycoproteins secretedin the vicinity of blood vessels to induce an-giogenesis, has five members: VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor(PLGF).

Abnormal activation of choroidal neovascularization byVEGF may be responsible for age-related macular degen-eration (AMD).

Vascular endothelial growth factor receptors (VEGFR)These VEGFs act on three VEGF receptors (VEGFRs),which are typical protein tyrosine kinase-linked receptors(Module 1: Figure stimuli for enzyme-linked receptors)that have slightly different functions. VEGFR1, which re-sponds to VEGF-A, VEGF-B and placental growth factor(PLGF), functions in monocyte migration and the organ-ization of proper vessels. VEGFR2, which is activatedmainly by VEGF-A, plays a major role in stimulating theendothelial cells to proliferate, to migrate and to differ-entiate into new vessels during angiogenesis (Module 9:Figure angiogenesis). It is also responsible for stimulating

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �40

Module 9: Figure angiogenesis

Filopodia

Lateralinhibition

Pericyteproliferation

Pericytesstabilize the

ECsECM

deposition

Pericytemigration

Tumour

Sprouting

Branching

ECM

A. B. C.

D. E. F.

Blood vessel

VEGF-A

PDGF

Pericyte Tipcell

Endothelialcell (EC)

ECproliferation

ECMdegradation

Angiogenesis.This figure depicts the growth of new vessels responding to vascular endothelial growth factor A (VEGF-A) released from a tumour. In the initialresponse (stages A–C), a single endothelial cell differentiates into a tip cell to initiate the growth of a sprout. As the endothelial cells (ECs) proliferate,this new sprout grows and new tip cells appear (stages D and E) to form a branch. The newly formed vessels then invade the tumour providing a newblood supply. See text for further details.

Module 9: Figure angiogenesis signalling

PDGF-BHSPG

EDG

PericytePeg-socketjunctionalcomplex

S1P

Ang-1Ang-2 Tie-2

Proliferation

N-cadherin

Endothelial cell stabilityEndothelial

cell

VEGFR-2

VEGFR-2

VEGFFilopodia

Tip cell

Proliferation

Tip cell differentiation

Lateralinhibition

DLL4

PDGFR-

Notch

1

2

3

4

5

6

78

Angiogenesis signalling mechanisms.Angiogenesis, begins when one of the endothelial cells responds to vascular endothelial growth factor (VEGF) by differentiating into a tip cell. Thelatter then releases platelet-derived growth factor B (PDGF-B) to stimulate the proliferation of neighbouring endothelial cells and pericytes and itrecruits the Notch signalling pathway to inhibit these other cells from becoming tip cells. This signalling is restricted to a local region around the tipcell because the PDGF is prevented from diffusing away by being bound to heparan sulphate proteoglycans (HSPGs). The activated endothelial cellsrelease sphingosine 1-phosphate (S1P) and angiopoietin 2 (Ang-2). See text for further details.

the formation of nitric oxide (NO). VEGFR3, which isactivated by VEGF-C and VEGF-D, is mainly located onlymphatic vessels, where it functions to stimulate lymph-angiogenesis. Physical interactions between endothelialcells and the extracellular matrix (ECM) are responsiblefor the mechanosensitive control of VEGF receptor ex-pression.

These VEGFRs are capable of activating a number ofsignalling pathways (Module 9: Figure VEGF-inducedproliferation):

• The extracellular-signal-regulated kinase (ERK) path-way (Module 2: Figure ERK signalling) that controlsproliferation.

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �41

Module 9: Figure VEGF-induced proliferation

VEGF

VEGFR2

Ras

KS

R1

Raf-1

MEK1/2

ERK1/2 eNOSInsP

ERK1/2ERK1/2

Ca

PtdIns4,5P

MCP-3

PP

PP

P

P

P

P

3

2+

Grb2

SOS

DAG

PKC

PKB

BAD

2 PtdIns3,4,5P3

PLC

PI 3-KNO

production

Survival

Proliferation DSCR1

DSCR1

NFAT

NFAT

CaN

+

+

+

+

PP

P

Control of endothelial cell proliferation, NO production and survival by the vascular endothelial growth factor (VEGF).Endothelial cell activation by vascular endothelial growth factor (VEGF) depends upon its binding and dimerization of two VEGF receptor 2 (VEGFR2)monomers. The latter are tyrosine kinase-linked receptors that have an extracellular domain composed of seven immunoglobulin homology domainsand an intracellular tyrosine kinase domain split by a kinase insert sequence. Following receptor dimerization, these kinase regions phosphorylateeach other to provide the phosphotyrosine docking sites responsible for initiating the activation of a number of signalling pathways.

• The PtdIns 3-kinase signalling cassette (Module 2: Fig-ure PtdIns 3-kinase signalling) that promotes survivalby inhibiting the pro-apoptotic factor Bad.

• The inositol 1,4,5-trisphosphate (InsP3)/Ca2 + sig-nalling cassette that acts on endothelial nitric oxide syn-thase (eNOS) and co-operates with the diacylglycerol(DAG)/protein kinase C (PKC) signalling cassette tohelp activate the ERK pathway. The elevation of Ca2 +

also acts on calcineurin (CaN) to dephosphorylate thetranscription factor NFAT thus enabling it to enter thenucleus to activate proliferation. NFAT also induces thetranscription of the Down’s syndrome critical region 1(DSCR1) gene, which sets up a negative-feedback loopbecause DSCR1 is a potent inhibitor of CaN. The in-creased expression of DSCR1 in a Down’s syndromeindividual can protect against cancer by suppressing theactivity of VEGF and thus reducing the flow of bloodto tumours.

The miR-126 plays a role in maintaining angiogenesisby reducing the expression of SPRED1, which inhibits theMAP kinase signalling pathway, and the PtdIns 3-kinaseregulatory p85β subunit that inhibits the PtdIns 3-kinasesignalling pathway.

Mechanosensitive control of VEGF receptor expressionThe physical interaction between endothelial cells and theunderlying extracellular matrix (ECM) may be respons-ible for regulating transcription of the vascular endothelialgrowth factor receptor 2 (VEGFR2). This mechanical sig-nal appears to be detected by p190RhoGAP, which nor-mally acts to inhibit the Rho signalling mechanism by en-

hancing the hydrolysis of GTP (Module 2: Figure Rhosignalling). Rho appears to influence the transcription ofVEGFR2 by altering the balance between two competingtranscription factors: TFII-I and GATA2

Astrocyte proliferationAstrocytes are induced to grow at the sites of brain injury.

Insulin-secreting β-cell proliferationFully differentiated insulin-secreting β-cells have beenshown to proliferate in response to pancreatectomy inmice, which raises the possibility of devising strategies toreplace damaged cells in diabetics. The survival and pro-liferation of β-cells is regulated by glucose and by vari-ous hormones such as glucagon-like peptide-1 (GLP-1),which appear to act synergistically to activate gene tran-scription by stimulating the transducers of regulated cyclicAMP response element-binding protein (CREB) (TORC)(Module 7: Figure β-cell signalling). β-Cell proliferation isalso controlled by Ca2 + acting through the nuclear factorof activated T cells (NFAT) transcriptional cascade.

Parathyroid gland hyperplasiaHyperplasia of the parathyroid gland occurs when thegland is being stimulated excessively due either to hypo-calcaemia or to a decrease in the level of vitamin D. Undernormal conditions, there is little proliferation of the chiefcells, but this is rapidly reversed during intense stimula-tion as occurs during the fall in plasma Ca2 + that occursduring the secondary hyperparathyroidism brought on bykidney disease.

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 9 � Cell Cycle and Proliferation 9 �42

The hyperplasia induced by a fall in level of vitaminD provides some clues as to the mechanisms respons-ible for controlling proliferation of the chief cells. Oneof the actions of the hormone 1,25-dihydroxyvitaminD3 [1,25(OH)2D3] is to promote the expression ofcyclin-dependent kinase (CDK) inhibitors such as p21,which inhibit cell proliferation (Module 7: Figure PTHsecretion). The onset of hyperplasia can be rapid and maybe accelerated by expression of both the epidermal growthfactor (EGF) receptor and its normal ligand TGFα.

ReferencesCell cycleBarr, F.A., Sillje, H.H.W. and Nigg, E.A. (2004) Polo-like kinases and the

orchestration of cell division. Nat. Rev. Mol. Cell Biol. 5:429–440.Cobrinik, D. (2005) Pocket proteins and cell cycle control. Oncogene

24:2796–2809.Meyer, D.L., Bahassi, E.M. and Stambrook, P.J. (2005) The Plk3--Cdc25

circuit. Oncogene 24:299–305.Dimova, D.K. and Dyson, N.J. (2005) The E2F transcription network: old

acquaintances with new faces. Oncogene 24:2810–2826.Glotzer, M. (2005) The molecular requirements for cytokinesis. Science

307:1735–1739.Hwang, H.C. and Clurman, B.E. (2005) Cyclin E in normal and

neoplastic cell cycles. Oncogene 24:2776–2786.Li, C.J., Heim, R., Lu, P., Tsien, R.Y. and Chang, D.C. (1999) Dynamic

redistribution of calmodulin in HeLa cells during cell division asrevealed by a GFP--calmodulin fusion protein technique. J. Cell Sci.112:1567–1577.

Matsumura, F. (2005) Regulation of myosin II during cytokinesis inhigher eukaryotes. Trends Cell Biol. 15:371–377.

McCollum, D. (2004) Cytokinesis: the central spindle takes center stage.Curr. Biol. 14:R953–R955.

Mitsuyama, F., Sawai, T., Carafoli, E., Furuichi, T. and Mikoshiba, K.(1999) Microinjection of Ca2 + store-enriched microsome fractions todividing newt eggs induces extra-cleavage furrows via inositol1,4,5-trisphosphate-induced Ca2 + release. Dev. Biol. 214:160–167.

Pei, X.-H. and Xiong, Y. (2005) Biochemical and cellular mechanisms ofmammalian CDK inhibitors: a few unresolved issues. Oncogene24:2787–2795.

Stegmeier, F., Rape, M., Draviam, V.M., Nalepa, G., Sowa, M.E., Ang,X.L., McDonald, E.R., Li, M.Z., Hannon, G.J., Sorger, P.K., Kirschner,M.W., Harper, J.W. and Elledge, S.J. (2007) Anaphase initiation isregulated by antagonistic ubiquitination and deubiquitinationactivities. Nature 446:876–881.

Takizawa, C.G. and Morgan, D.O. (2000) Control of mitosis by changesin the subcellular location of cyclin-B1--Cdk1 and Cdc25C. Curr. Opin.Cell Biol. 12:658–665.

Van Vugt, M.A. and Medema, R.H. (2005) Getting in and out of mitosiswith Polo-like kinase-1. Oncogene 24:2844–2859.

Checkpoint signallingBartek, J. and Lukas, J. (2007) DNA damage checkpoints: from initiation

to recovery or adaptation. Curr. Opin. Cell Biol. 19:238–245.Branzei, D. and Foiani, M. (2008) Regulation of DNA repair throughout

the cell cycle. Nat. Rev. Mol. Cell Biol. 9:297–308.Enders, G.H. (2008) Expanded roles for Chk1 in genome maintenance.

J. Biol. Chem. 283:17749–17752.Kennedy, RD. and D’Andrea, A.D. (2009) The Fanconi anemia/BRCA

pathway: new faces in the crowd. Genes Develop. 19:2925–2940.Song, L. and Rape, M. (2008) Reverse the curse- the role of

deubiquitination in cell cycle control. Curr. Opin. Cell Biol. 20:156–163.

Lymphocyte activationDonnadieu, E., Bismuth, G. and Trautmann, A. (1994) Antigen

recognition by helper T cells elicits a sequence of distinct changes oftheir shape and intracellular calcium. Curr. Biol. 4:584–595.

Dustin, M.L. and Cooper, J.A. (2000) The immunological synapse andthe actin cytoskeleton: molecular hardware for T cell signalling. Nat.Immunol. 1:23–29.

Feske, S. (2007) Calcium signalling in lymphocyte activation anddisease. Nat. Rev. Immunol. 7:690–702.

Monks, C.R.F., Freiberg, B.A., Kupfer, H., Sciaky, N. and Kupfer, A.(1998) Three-dimensional segregation of supramolecular activationclusters in T-cells. Nature 395:82–86.

Negulescu, P.A., Krasieva, T.B., Khan, A., Kerschbaum, H.H. andCahalan, M.D. (1994) Polarity of T cell shape, motility, and sensitivityto antigen. Immunity 4:421–430.

Verheugen, J.A.H., Le Deist, F., Devignot, V. and Korn, H. (1997)Enhancement of calcium signaling and proliferation responses inactivated human T lymphocytes: inhibitory effects of K+ channelblock by charybdotoxin depend on the T cell activation state. CellCalcium 21:1–17.

AngiogenesisAdams, R.H. and Alitalo, K. (2007) Molecular regulation of angiogenesis

and lymphogenesis. Nat. Rev. Mol. Cell Biol. 8:464–478.Armulik, A., Abrahamsson, A. and Betsholtz, C. (2005)

Endothelial/pericyte interactions. Circ. Res. 97:512–523.Olsson, A-K., Dimberg, A., Kreuger, J. and Claesson-Welsh, L. (2006)

VEGF receptor signalling in control of vascular function. Nat. Rev. Mol.Cell Biol. 8:359–371.

Insulin-secreting β-cell proliferationHeit, J.J., Apelqvist, ı.A., Gu, X., Winslow, M.M., Neilson, J.R., Crabtree,

G.R. and Kim, S.K. (2006) Calcineurin/NFAT signalling regulatepancreatic β-cell growth and function. Nature 443:345–349.

C©2012 Portland Press Limited www.cellsignallingbiology.org