MCB Cell Signaling Lectures 1 & 2 Ken Blumer Dept

MCB Cell Signaling Lectures 1 & 2

Ken Blumer

Dept. of Cell Biology & Physiology

506 McDonnell Sciences

[email protected]

362-1668

Lecture 1

General Concepts of Signal TransductionCell CommunicationTypes of ReceptorsMolecular Signaling

Receptor BindingScatchard AnalysisCompetitive Binding

Second Messengers

G proteins

Modes of cell communication

Lodish, 20-1

Four classes of cell-surface receptors

Lodish, 20-3

Transmitting/transducing signals within cells:

3 basic modes (may be combined)

1. Allostery

2. Covalent modification

3. Proximity (= regulated recruitment)

P

Shape change, often induced by binding a protein or small moleculeSwitching can be very rapid

Modification itself changes molecule’s shapeMemory device; may be reversible (or not)

Regulated molecule may already be in “signaling mode;” induced proximity to a target promotes transmission of the signal

P P

Finding and analyzing receptors:Ligand binding assays

Saturation Binding studiesCan be performed in intact cells, membranes, or purified receptors1. Add various amounts of labeled ligand (drug, hormone, growth factor)2. To determine specific binding, add an excess of unlabeled ligand to compete for specific binding sites.QU: Why is there non-specific binding?3. Bind until at equilibrium4. Separate bound from unbound ligand5. Count labeled ligand

[Adapted from A. Ciechanover et al., 1983, Cell 32:267.]

Receptor: ligand binding must be specific, saturable, and of high affinity

Receptor abundance, affinity, cooperativity:Scatchard plots

Slope = - 1/Kd

X intercept = # rec

(Bound Lig)

(Bound Lig)(Free)

For an excellent discussion of principles of receptor binding, andpractical considerations, see http://www.graphpad.com; also posted on MCB website.

Cooperativity indicated by non-linearScatchard plots

(Bound Lig)

(Bound Lig)(Free)

Negative cooperativity: binding of ligand to first subunit decreases affinity of subsequent binding events.

Positive cooperativity: binding of ligand to first subunit increasesAffinity of subsequent binding events. Example: hemoglobin binding O2

What receptors do:Generate second messengers

• Cyclic nucleotides: cAMP, cGMP• Inositol phosphate (IP)• Diacylglycerol (DAG)• Calcium• Nitric oxide (NO)• Reactive oxygen species (ROS)

Molecular mediators of signal transduction. Cells carefully, and rapidly, regulate the intracellular concentrations. Second messengers can be used by multiple signaling networks (at the same time).

cAMP regulates protein kinase (PKA) activity

Alberts 15-31,32

Positive cooperativity--binding of increases affinity for second cAMP

PKA targets include Phosphorylase kinase and the transcription regulator, cAMP response element binding (CREB) protein

Lipid-derived second messengers:Diacylglycerol and inositol phosphates

Alberts, 15-35

IP3 evokes calcium release as third messenger

Lodish, 20-39

A key effector of Ca2+-CaM:CaM-kinase II

Alberts, 15-41

NO signaling

Lodish, 20-42

NO effects are local, since it has half-life of 5-10 seconds (paracrine).NO activates guanylate cyclase by binding heme ring (allosteric mechanism)

Gases can act as second messengers!

Discovery of NO signaling

Robert F Furchgott showed that acetylcholine-induced relaxation of blood vessels was dependent on the endothelium. His "sandwich" experiment set the stage for future scientific development. He used two different pieces of the aorta; one had the endothelial layer intact, in the other it had been removed.

Louis Ignarro reported that EDRF relaxed blood vessels. He also identified EDRF as a molecule by using spectral analysis of hemoglobin. When hemoglobin was exposed to EDRF, maximum absorbance moved to a new wave-length; and exposed to NO, exactly the same shift in absorbance occurred! EDRF was identical with NO.

Furchgott, Ignarro, Murad, Nobel Prize 1998

http://www.nobel.se/medicine/laureates/1998/illpres/index.html

G proteins:Switches linking receptors & 2nd messengers

• Discovery and Structure of Heterotrimeric G proteins

• Signaling pathways of G proteins• Receptors that activate G proteins• Small G proteins-discovery and structure• Activation and inactivation mechanisms• Alliance for Cell Signaling (AfCS)

Signal Transduction by G proteins



G protein signal transduction

Neves, Ram, Iyengar, Science 2002

Hydrolysis of GTP

• Arg & Gln stabilize the b and g phospates of GTP molecule in correct orientation for hydrolysis by H2O

• Hydrolysis leads to major conformation change in Gs a

• Mutations in the Gln or Arg (or ADP ribosylation by cholera toxin) blocks the ability to stabilize transition state, and therefore locks G protein in the “on” position.

• Examples include adenomas of pituitary and thyroid glands (GH secreting tumors, acromegaly), and McCune-Albright syndrome.

Iiri, et al. NEJM (1999)




G protein-coupled receptors (GPCRs)• Many ligands

• Robust switches• Multiple effectors• Conserved 7 TM

architecture• More than 50% of

drugs target GPCRs

Bockaert & Pin, EMBO J (1999)2012 Nobel Prize

Lefkowitz Kobilka

GPCR desensitization mechanisms

Arrestins act as scaffolds for ERK and JNK signaling pathways

Lefkowitz reviews




Reverse genetics: express one or two mutant versions of the protein of interest

Depends on understanding how the machines work

1. Inhibit activity of the protein with a “dominant-negative” interfering mutant of that protein

2. Increase activity of the protein with a “dominant-positive” or “constitutively active” interfering mutant of the protein

The mutant titrates (binds up) a limiting component to block the normal protein’s signal

The mutant exerts the same effect as the normal protein would, if it were activated in the cell

Reverse genetics: small GTPases as examplesDepends on understanding how the machines work

“Dominant-negative” mutation “Dominant-positive”

mutation

The mutant titrates (binds up) a limiting component to block the normal protein’s signal

The mutant exerts the same effect as the normal protein would, if it were activated

GAP

GTP

Pi

GDPGEF

GEFGDP

Binds GEF but cannot replace GDP by GTP; so GEF not available for activating normal protein

Cannot hydrolyze GTP, so remains always active



• Signaling pathways of G proteins• Receptors that activate G proteins• Small G proteins-discovery and structure• Activation and inactivation mechanisms

Small G protein “turn on” mechanisms

First mammalian GEF, Dbl, isolated in 1985 as an oncogene in NIH 3T3 focus forming assay. It had an 180 amino acid domain with homology to yeast CDC24. This domain, named DH (Dbl homology) is necessary for GEF activity.

In 1991, Dbl shown to catalyze nucleotide exchange on Cdc42.

Schmidt & Hall, Genes & Dev. (2002)Dbl= Diffuse B-cell lymphoma

Many RhoGAPsRhoGAPs outnumber the small G proteins Rho/Rac/Cdc42 by nearly 5-fold.Why so much redundancy?Luo group did RNAi against 17 of the 20 RhoGAPs in fly.

Six caused lethality when expressed ubiquitously. Tissue specific expression of RNAi revealed unique phenotypes.

P190RhoGAP implicated in axon withdrawal. Increasing amounts of RNAi caused more axon withdrawal (panels C-G).

Why so many RhoGAPs?Billuart, et al. Cell (2001)

The GTPase switch

Schmidt & Hall, Genes & Dev. (2002)

Growth Factors and Receptor Tyrosine Kinases

• RTK’s--How do they work?• EGFR signaling and ras• MAP kinase cascades• PI3K, PKB, PLCg• PTPs (Protein Tyrosine Phosphatases)

How RTKs (& TK-linked Rs) work

1. Ligand promotes formation of RTK dimers, by different mechanisms:

Ligand itself is a dimer (PDGF)

One ligand binds both monomers (GH)

2. Dimerization allows trans-phosphorylation of catalytic domains, which induces activation of catalytic (Y-kinase) activity

3. Activated TK domains phosphorylate each other and proteins nearby, sometimes on multiple tyrosines

4. Y~P residues recruit other signaling proteins, generate multiple signals

EGF receptor as a model1st RTK to be characterized

v-erbB oncogene = truncated EGFR

How do we know that the EGFR auto- phosphorylates in trans?

Experiment: test WT and short EGFRs, each with or without a kin- mutation

Honneger et al. (in vitro) PNAS 1989; (in vivo) MCB 1999

wt +

Does this result rule out phosphorylation in cis as well?

If not, how can you find out?

PS: What do trans and cis mean?

kin- + +

short kin+ + +short kin- +

How can we know that the EGFR does not autophosphorylate in cis?

Need an EGFR that cannot homodimerize

EGFR family is huge, with many RTK members and many EGF-like ligands

Such receptors often form obligatory heterodimers with a similar but different partnerIf A can dimerize only with A’, then we can inactivate the kinase domain of A’ and ask whether A phosphorylates itself

Answer: NO

QED


• RTK’s--How do they work?• EGFR signaling and ras• MAP Kinase Cascades• PI3K, PKB, PLCg• PTPs (Protein Tyrosine Phosphatases)

.P

.P .PPP

.P

P PP

Signals generated by the EGFR

The activated dimer phosphorylates itself

T-loop only Multiple sites

Individual Y~P residues recruit specific proteins, generate different signals

SOS, a Ras GEFDocks via intermediate adapters to activate Ras

Ras activates multiple targets (MAPK)

PLC-gDocking of Y-kinases allows Tyr-phos’n of PLC-g, which activates it

PI3-kinaseAdapters again

Docking allosterically activates PI3K

Each signal, in turn, activates a different set of pathways, which cooperate to produce the overall response

Adapters connect A with B, B with C . . . to create complex, localized assemblies of signaling proteins

Each adapter has at least 2 interaction domains, and may have other functions as well

Types of adapter interactions

Y~P sequence motifs allow regulatable adapter functionsSH2 Tyrosine phosphatesPTB Tyrosine phosphates

AlsoSH3 Polyproline-containing sequences

PDZ Specific 4-residue sequences at C-terminiPleckstrin homol. (PH) Phosphoinositides

Many others

A CBP

Adapter 1

Adapter 2

EGF

EGFR

EGFR~P

Grb2

SOS

Ras

Raf

Mek

ERKs

C-Jun

Extracellular GF

RTK

Phospho-RTK

Adapter

Ras-GEF

Small GTPase

Ser kinase

Tyr/thr kinase

Ser kinase

Transcription factor

MechanismProximity

Allostery

Covalent modification

EGF activates the MAPK pathway in multiple steps, with multiple mechanisms

.PPP

.P

PP

SH2SH3

SH3

Grb2SOS

EGFR Activation of Ras: Proximity & Allostery

The PlayersRTK = EGFR

“Rat Sarcoma”Small GTPase, attached to PM by prenyl group

“GF receptor binding 2”Adapter, found in screen for binders to EGFR~P

“Son of Sevenless”GEF, converts Ras-GDP to Ras-GTPFound in Drosophila, homol. To S.c. Cdc25

RasGDP

. .

SH2SH3

SH3

Grb2 SOS


SOS is “ready to go”: already (mostly) associated with Grb2 in cytoplasm, in the resting state

Even before EGF arrives . . .

RasGDP


Then . . . Covalent modification

RasGDP

.PPP

.P

PP

EGF-bound dimers trigger phosphorylation, in trans SH2

SH3

SH3

Grb2 SOS

SH2SH3

SH3

Grb2

.PPP

.P

PP

SOS

RasGDP

Grb2’s SH2 domain binds Y~P on EGFR, bringing SOS to the plasma membrane


Then . . . Proximity

SH2SH3

SH3

Grb2

.PPP

.P

PP

SOS


RasGDP

GDP

SOS now binds Ras-GDP, causing GDP to dissociate, and . . .

Then . . . Allostery

SH2SH3

SH3

Grb2

.PPP

.P

PP

SOS


Ras

GTP

GTP enters empty pocket on Ras, which dissociates from SOS and converts into its active conformation

Then . . . Allostery continues

GTP

Raf

SH2SH3

SH3

Grb2

.PPP

.P

PP

SOS


Ras

GTP

Ras-GTP brings Raf to the PM for activation, and the MAPK cascade is initiated

Finally . . . Proximity again!

GTP

MAPKCascade

Raf



.

The best understood MAPK cascadeMAPK = Mitogen-activated protein kinase

Raf-1A-rafB-raf

MEK1MEK2

ERK1ERK2

C-Jun

Altered gene expression

Phos’n of T-loop Ser residues

.PP

.PP Phos’n of Ser/Thr

Phos’n of T-loop Thr and Tyr

MAPKKK

MAPKK

MAPK

Res

pons

e 1.0

0.5

00 1 5

Stimulus (multiples of EC50)

MAPKKK

MAPKK

MAPK

Frogoocyte

Progesterone

G2-M transition

Switch-like behavior*

*JE Ferrell, Tr Bioch Sci 22:288, 1997

Responses are not always graded

Amplified sensitivity: reduces noise @ low stimulus; reversible

Bistable responses: off or on, often via positive feedback & used for irreversible responses (e.g., cell cycle)

Other examples?

Instead . . .

All or nothing response in Xenopus oocytesProgesterone, or fertilization, induces germinal vesicle breakdown of Xenopus oocytes--a process mediated by the MAPK cascade.

Question: At a concentration of progesterone that half-maximally activates MAPK (0.01 uM, panel A), are all the oocytes activated halfway (panel B), or are half of the oocytes activated fully (panel C)?

Since Xenopus oocytes are HUGE, one can look at MAPK on a cell by cell basis.

Answer: All or nothing.Ferrell, et al., Science (1998)

Dhanasekaran (2007) Oncogene

Scaffold proteins involved in ERK-signaling pathways


• RTK’s--How do they work?• EGFR signaling and ras• MAP Kinase Cascades• PI3K, PKB, PLCg• PTPs (Protein Tyrosine Phosphatases

SH2

SH2 p85

.P

P

.P

Pp110

EGFR Activation of PI3K combines Proximity & Allostery

How do we know proximity is not enough?

SH2

SH2 p85 p110

PIP2 PIP3

Recruitment from cytoplasm to PM, via SH2 domains

Activated by EGFR/p85

Can also be activated by Rac or Ras!

1. p85 mutants that activate without binding to RTKs2. Tethering to membrane does not activate

P

PP

P

PIP3 targets include many GEFs, many tyrosine kinases, and others, including . . .

PKB (aka Akt) = ser/thr kinase that promotes cell survival

PIP3(= membrane lipid)

PH K

PKB . . . is inactive in cytoplasm

. . . contains a PH (pleckstrin homology) domain & a kinase domain

P

PP

P PH K

Multi-step activation of PKB: proximity

PIP3

PH K

PH domain recognizes 3’- phosphate of PIP3, bringing kinase domain to the PMProximity to PM

alone does not activate the kinase

P

PP

P PH K P

PP

P PH KP

PPIP3PDK1*

Multi-step activation of PKB: covalent modification

Inactive PKB Active (phos’d) PKB

*PDK1 is also recruited to the membrane via a PIP3-binding PH domain

Overall, two proximity steps plus (at least) one phosphorylation step

.P

P

.P

P

EGFR Activation of PLCg combines THREE inputs

PIP2

1. PROXIMITY: Recruitment from cytoplasm to PM, via SH2 domains

P

PP

P

SH2

SH2PH

Cata- lytic

P

P

PLCg (Inactive, in cytoplasm)

PIP3

.P

P

.P

P

EGFR Activation of PLCg combines THREE inputs

PIP2 DAG

2. COVALENT: Activated by EGFR phosph’n

3. PROXIMITY: Binds to PIP3 via PH domain

P

PP

P

SH2

SH2PH

Cata- lyticP

P

P

InsP3



PTEN opposes PI3K by removing PI3-phosphatePTEN discovered as a tumor suppressor gene.

Mutated in brain, breast and prostate cancers.

Has homology to dual specificity phosphates, but shows little activity toward phosphoproteins.

Was discovered to remove phosphates from PIPs; thereby providing likely mechanism for tumor suppression.

Cantley & Neel, PNAS (1999)

Gleevec--proof that you can target kinases for drug therapy

Documents

MCB Cell Signaling Lectures 1 & 2 Ken Blumer Dept