Cellular mechanisms of signaling evolve from available components

Phospholipids constitute a readily available reservoir that regulates many intracellular events

H2O2

Hydrogen peroxide

PtdIns in yeast

Phospholipase C activation initiates the IP3 pathway

Cellular mechanisms of signaling evolve from available components

Phospholipids act as a readily available reservoir that regulate many intracellular events

The phosphatidylinositol (PI) includes inositol 1-phosphate bound via its phosphate group to 1-stearoyl,2-arachidoyl diacylglycerol, prevalent in mammal cells favouring exposure of the inositol ring and its interaction with PIBMs.The 7 known PIs in eukaryotes are PI4P,PI5P,PI3P,PI45P2, PI35P2,

PI34P2 and PI345P3. Each PI is indicated according to colour codes. Blue and arrows indicate routes of PI phosphorylation and dephosphorylation, respectively. Unlike phosphoinositides, the soluble inositol phosphates (IPs) can be phosphorylated in all of the six positions,giving rise to more than 60 soluble species.This is because other IP-specific enzymes are present in the cell as well as the kinases/phosphatases acting on the phosphoinositides and IPs.The PIs can be hydrolysed by PLC to generate inositol 1,4,5-trisphosphate and diacylglycerol from PI45P2; by PLA2 to LPIs; PLA/lysophospholipases (LPLA1) to form the GPIs;and by PLD to form phosphatidic acid. PLC acts preferentially on PI45P2, whereas the other phospholipases may act on the different PIs (for simplicity in the figure,all the phospholipases are shown acting only on PI45P2).

Pathways of phosphoinositide synthesis and degradation.

Proposed functions of PX-domain proteins. a, Recruitment of the NADPH oxidase complex. Upon neutrophil activation, PI3K-I converts PI(4,5)P2 into PI(3,4,5)P3. The cytosolic subunits of the NADPH oxidase complex (p40, p47, p67) are recruited to developing phagosome at the plasma membrane by binding of the PX domain of p47phox to PI(3,4)P2 (green), generated upon dephosphorylation of PI(3,4,5)P3 by the 5-phosphatase SHIP-1. PI(3,4)P2 is then dephosphorylated by a PI(3,4)P2 4-phosphatase to generate PI(3)P (red), which binds to the PX domain of p40phox. The correct assembly of p40phox, p47phox and p67phox with the membrane-bound (cytb558) components of the complex results in a functional Phox complex that produces O2

-

b, Membrane trafficking. PI3K-II is recruited to the plasma membrane through binding of its PX domain to PI(4,5)P2 and may promote the formation of clathrin-coated vesicles. Snx3 binds to PI(3)P (red) in the sorting early endosome and augments transport of transferrin (Tf) from the sorting to the recycling endosome. The yeast SNARE Vam7p is recruited by PI(3)P on multivesicular bodies (MVBs) and vacuoles to complex with other SNAREs and thereby promote vacuolar membrane docking and fusion.

Simplified overview of the main synthetic pathways involved in the formation of polyphosphoinositides in higher plant cells

The two kinases, phosphatidylinositol 3-kinase (PtdIns 3K) and PtdIns(3)phosphate [PtdIns(3)P] 5-kinase (Fab1) are shown. Routes of synthesis that are established are shown by unbroken arrows, whereas steps that still need confirmation or are less well defined in vivo are indicated by broken arrows. Abbreviations: PtdIns(4)P, PtdIns(4)phosphate; PtdIns(5)P, PtdIns(5)phosphate; PtdIns(4,5)P2, PtdIns(4,5)bisphosphate; PtdIns(3,4)P2,

PtdIns(3,4)bisphosphate; PtdIns(3,5)P2, PtdIns(3,5)bisphosphate

CH

2

O P OH

OH

O

HC OC R2

O

CH2 OC R1

O

Phosphatidic Acid serves as the precursor from whichmany of these second messenger lipids are derived →

Lipid substrates and messengers produced by phospholipids- and/or galactolipid-hydrolyzing

enzymes, and their downstream physiological effects

Note that the substrate lipids can be located on the plasma membrane or other membranes, depending on the nature of a specific enzyme and its intracellular location

PtdIns(4,5)P2 accumulates in pseudopods during extension of the phagocytic cup. As the phagosome seals, PtdIns(4,5)P2 disappears. This could be explained in part by its catabolism by phospholipases but also by its conversion into PtdIns(3,4,5)P3, and indeed, PtdIns(3,4,5)P3 appearance coincides with PtdIns(4,5)P2 clearance. PtdIns(3,4,5)P3 accumulates transiently in the phagocytic cup and is required for its closure. Once the phagosome is formed, PtdIns(3)P is produced on its surface and recruits proteins that control phagosome fusion and maturation. Other phosphoinositide species are present in the trans-Golgi complex (PtdIns(4)P) or in the nucleus (PtdIns(5)P), leading to the proposal that membrane identity can be mediated by compartmentalization of specific phosphoinositides

Phosphoinositides involved in classical phagocytosis

Phospholipid signaling under salt stress, drought, cold, or ABA. Osmotic stress, cold, and ABA activate several types of phospholipases that cleave phospholipids to generate lipid messengers (e.g., PA, DAG, and IP3), which regulate stress tolerance partly through modulation of gene expression. FRY1 (a 1-phosphatase) and 5-phosphatase-mediated IP3 degradation attenuates the stress gene regulation by helping to control cellular IP3 levels.

PLD and PA in response to H2O2PLD , is activated in response to H2O2 and the resulting PA functions in

amplification of H2O2 -promoting PCD

Stress stimulates production of H2O2 that activates PLD associated with the plasma membrane.

Potential activators: Ca2+ and oleic acid. This increases PLD affinity to its substrates, stimulating lipid hydrolysis and PA production. PA may bind to target proteins, such as Raf-like MAPKK, that contain a PA binding moti, leading to the activation of MAPK cascades. PA may also function by modulating membrane trafficking and remodeling. These interactions modulate the cell's ability to respond to oxidative stress and decrease cell death. Dashed lines - hypothetical interactions.

PLD & PA

• Knockout of PLD renders Arabidopsis plants more sensitive to the reactive oxygen species H2O2 and to stresses

• H2O2 activates PLD , and PLD -derived PA functions to

decrease the promotion of cell death by H2O2. These results

suggest that both PLD and its product PA play a positive role in signaling stress responses

• PLD and its derivative PA provide a link between phospholipid signaling and H2O2-promoted cell death. PLD

and PA positively regulate plant cell survival and stress responses.

The role of PLD in vesicular trafficking & signal transduction

A) PLD catalytic activity. In the first step of the reaction (left panel), PLD removes the head group of a structural phospholipid, such as PC, forming covalent bond with the resulting phosphatidyl moiety, the PLD-PA intermediate (middle panel). In the second step (right panel), PLD transfers the phosphatidyl moiety to a nucleophile. Under physiological conditions, this is water, representing the hydrolysis of PC to generate PA. Primary alcohols, such as 1-butanol, can also be used as acceptors, resulting in the formation of PBut, a reaction that is used to measure PLD activity in vivo and in vitro (3, 8, 32). (B) Cytokinesis in plant cells. (C) Model of PLD binding to microtubules and membranes. PLD binds vesicular and plasma membranes through its covalent PLD-PA intermediate (Fig. 1A, middle panel). (D) PLD's contribution in PA signaling. A summary of factors activating PLD in plants and the role of PA in signaling

Phospholipid signalling pathways that are involved in plant defence responses.

PLA2 generates lyso-phospholipids (LPL) and FFAs that stimulate the plasma membrane H+-ATPase, and free fatty acids can be metabolised via octadecanoid pathway to JA. PLC hydrolyses PIP2 into IP3 and DAG. IP3 diffuses into the cytosol, where it could release Ca2+ from intracellular stores, or is metabolised further to IP6. DAG remains in the membrane to be phosphorylated by DGK to PA. Activation of PLD generates PA directly by hydrolysing structural phospholipids such as PC. PA can activate MAPK, CDPK, ion channels, and NADPH oxidase , all of which are involved in typical defence-

related responses. PA signalling is attenuated by its conversion to DGPP by PA kinase. All lipids or their derivatives that are involved in signalling are shown in red. Solid arrows indicate metabolic conversion; dashed arrows indicate activation (directly or indirectly) of downstream targets.

PI metabolism in Arabidopsis

The different steps in the synthesis of PIs and the lipid kinases catalyzing the different reactions are indicated. PtdIns(3,4,5)P3 is present in animal cells but has not been detected in plant

tissues, so far. In animal cells, PtdIns(3,4)P2 can be generated from PtdIns4P by a PtdIns 3-

kinase or by an as-yet-unidentified PIPkin from PtdIns3P. Plant cells do not contain any homolog of the heterodimeric inositol lipid 3-kinases that are able to phosphorylate PtdIns4P to PtdIns(3,4)P2 and PtdIns(4,5)P2 to PtdIns(3,4,5)P3. PtdIns(4,5)P2 can be synthesized by type I

and type II PIPkins from PtdIns4P and PtdIns5P, respectively. On the basis of sequence comparison, plants cells do not possess type II PIPkins. PtdIns5P is present in plants, but an enzyme capable of producing it has not been identified.

PLD is involved in O2.- production in Arabidopsis

PLD suppression decreases Phosphatidic acid (PA) production

PA-stimulated production of superoxide in PLD -deficient and wt leaves

PA levels increase during various stress conditions.

Plant Physiol. 126 (2001) 1449-1Plant Physiology, 2004, Vol. 134, pp. 129

PA specifically induces leaf cell death in Arabidopsis

A) WT plants were infiltrated with PA or PC and photographed 24 h after treatment with the lipids. Arrows indicate the area of liposome infiltration.

B) Leaves of WT plants were floated on phospholipid liposomes

C) Trypan blue staining was used to visualize dying cells in areas of turgor loss in PA-treated leaves. Leaves of WT plants were detached, floated on PA (left), or PC (right) suspensions for 2 h, and stained with Trypan blue

Phospholipase C ActivityPhospholipase C Activity

O

O

O

Phosphatidylinositol –4,5-bisphosphate (PIP2)

PLA2

PLC

PLD

Diacylglycerol (DAG)

O

O

PO- O

O

HOOHOH

O-

O

P O-O

O

O-

P O-O

4

5

1

Inositol-1,4,5-triphosphate(IP3)

Phospholipase C (PLC) hydrolysis PIP2 to yield two second messengers

These phospholipases are involved in second messenger generation from membrane phosphoinositides

N.B. Different phospholipid specificities (releases different PIs)

The receptor for inositol 1,4,5-triphosphate (IP 3 )is located on the tonoplast and ER membranes

Conformational changes in this receptor transduce subsequent signaling. Certain ion channel receptors,including the IP3 receptor,are composed of four subunits. Each subunit contains four membrane-spanning domains (not shown). When IP3 binds to the receptor,conformational changes result in movement of two of the subunits.The distribution of positive and negative charges stabilizes the open conformation of the channel and allows the entry of Ca2+ into the cytoplasm.

Domain structures of PLD, PLD, and PLD in Arabidopsis

XX in the PLD C2 marks the loss of two acidic residues potentially involved in Ca2+ binding; XX in the PPI-binding motifs marks the loss of the number of basic residues potentially required for PPI binding.

Direct and derived products of PLD activation

LysoPA and free fatty acid (FA) can be formed from PA by nonspecific acyl hydrolase or by PLA. PA is dephosphorylated to DAG by PA phosphatase. CDP-DAG is the precursor for the synthesis of PS, PI, and PG. XOH, Primary alcohol used for transphosphatidylation; Ptd, phosphatidyl; NAE, N-acylethanolamine.

PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; ARF, ADP-ribosylation factor; LPA, lysophosphatidic acid; PLA, phospholipase A; PA, phosphatidic acid; DGK, diacylglycerol kinase; PAP, PA phosphohydrolase; PIP5K, phosphatidylinositol 4-phosphate 5-kinase; MAPK, mitogen-activated protein kinase; MEK, MAP kinase kinase; ERK, extracellular signal-regulated kinase; SPHK, sphingosine kinase; Edg, endothelial differentiation gene

Classes of Phospholipase CClasses of Phospholipase C

900-1315

1220-1285

600-870

Four main isoforms (+variants)

Animals only

G protein activated

Two main isoforms

Animals only

Tyrosine kinase activated

Four mammalian isoforms + four splice variants

All non-animal PLCs are in this class

Ca2+ activated?

The Structure of PLCThe Structure of PLC

P

P P

EF hand-like Ca2+ binding?EF hand-like Ca2+ binding?

Catalytic X and Y domainsCatalytic X and Y domains

Pleckstrin homology – phosphoinositide bindingPleckstrin homology – phosphoinositide binding

SH2 – phosphotyrosine bindingSH2 – phosphotyrosine binding

SH3 – interaction with cytoskeleton?

C3 – part of catalytic domain? Phospholipid interaction?

Phosphotyrosines

PLC phosphorylation may release it from interaction with inhibitor

Receptor phosphorylated

P

P

PLC PIP2

IP3

DAG

Activation of PLCActivation of PLC by EGFR complex by EGFR complex

Inactive PLC

EGFEGF binds to receptor

P

PLC phosphorylated

P

PLC hydrolyses PIP2 to yield IP3 and DAG

O

O

PO- O

HOOHOH

O-

O

P O-O

O

O-

P O-O

4

5

1

Inositol-1,4,5-triphosphate (IPInositol-1,4,5-triphosphate (IP33))

O

O

O

O

Diacyleglycerol (DAG)Diacyleglycerol (DAG)

PIPPIP22-derived Second Messengers-derived Second Messengers

Hydrophilic

Binds to receptor on ER

IP3 Receptor is Ca2+ channel

Hydrophobic

Remains in plasmalemma

Activates Protein Kinase C (PKC)

AMPLIFICATION – many IP3/DAG per bound ligand

SummarySummary

Phosphatidylinositol-specific PLC hydrolyses membrane PIP2

PLC associates with activated receptor tyrosine kinases

PLC is activated by tyrosine phosphorylation

PLC has domains that allow binding to phosphotyrosine (SH2)

IP3 – soluble, induces Ca2+ releaseDAG – hydrophobic, activates protein kinase C

Loewen, et al (2004). Phospholipid Metabolism Regulated by a Transcription Factor Sensing Phosphatidic Acid. Science 304, 1644-1647.

Inositol-induced alteration in phospholipid synthesis.

Phosphatidylinositol 3’-Kinase (PI3K) ActivityPhosphatidylinositol 3’-Kinase (PI3K) Activity

O

O

PO- O

HOOHOH

4

5

1

O

O

PO- O

HOOHOH

4

5

1

O

O-

P O-O

O

O

PO- O

HOOHOH

4

5

1

O-

O

P O-O

OH

OH

OH

O

O

PO- O

OHOH

4

5

1

O

O

PO- O

OHOH

4

5

1

O

O

PO- O

OHOH

4

5

1

OH

OHOH

PI4K PI5K

PI3K

PI3K

PI3K

PI5Ptase

PTE

N

PTE

N

O

O-

P O-O

O-

O

P O-O

O

O-

P O-O

O

O-

P O-O

O

O-

P O-OO

O-

P O-O

O

O-

P O-O

Headgroup of PIP2

PI3K phosphorylates inositol on the 3 positionPTEN dephosphorylates inositol on 3 position

Class I PI3KClass I PI3K

p110, , kinases

p85

p85

p55,

p50

p110 kinase

p101 adapter

adapters

CLASS IA

CLASS IB

Catalyticras bindingp85 binding

SH3

p110-binding

Proline-Rich

SH2

Class I PI3K RegulationClass I PI3K Regulation

regulation by p21ras

p110 autophosphorylationinhibits PI3K activity

p110 phosphorylation of p85 (S608) inhibits PI3K activityp110 phosphorylation of p85 (S608) inhibits PI3K activity

SH2 bind pY-X-X-M

Inter-SH binds PI(4)P and PI(4,5)P2SH2 also binds PI(3,4,5)P3 this binding competes with pY binding

Proline-rich repeats bind SH3 domains of e.g. src, fyn or lck

Proline-rich repeats bind SH3 domains of e.g. src, fyn or lck

Subunit interaction

Protein Kinase C (PKC)Protein Kinase C (PKC)

Three Classes

Novel nPKC

, , and

Activated by DAG but donot require Ca2+

Classical cPKC

, 1, 2 and

Activated by DAG and Ca2+

Atypical aPKC

, and

Do not require DAG or Ca2+

All forms require phosphatidylserine (PS) for activity

cPKC have two zinc finger domainsC1 – binds PS and Ca2+

C2 – binds DAG

Pro

life

ratio

nPKC SubstratesPKC Substrates

P P P

MARCKS

Calmodulin (CAM) binding

PKC phosphorylation sites – release from membrane

Myristoylation site – membrane association

MARCKS Protein

MARCKS Phosphorylation

Associated with

• Decreased MARCKS- F actin association

• Actin polymerisation

• Decrease CAM-dependent mlc phosphorylation

VEGF

Effects mediated by PKCEffects mediated by PKC

Proliferation - insulin

Differentiation – wnt pathway

+Apoptosis – UV-B, neutrophils (PKC activated by caspase 3)

-Apoptosis – suppresses Fas-induced PCD (PKC?)

Cell Polarity – atypical PKC and interacting protein

Feedback Inhibition of IP3/Ca2+

Receptor Downregulation - e.g. EGF

MAP Kinase Pathway – ras independent

Inhibition of PLC - -ve feedback

STAT inhibition – PKC blocks STAT DNA association

SummarySummary

Three classes of PKC

Pre-activation of PKC requires PDK-1 phosphorylation

Activation completed by DAG (except aPKC class)

All require phosphatidylserine for activity

MARCKS – major substrate for PKC

MARCKS role in proliferation and cell morphology

PKCs many roles in proliferation,differentiation and death

SummarySummary

PI3 kinases phosphorylate phosphoinositides at position 3

p110 contains catalytic activity

p85 responsible for recruiting enzyme to RTK

PI-3,4,5-P3 recruits PDK1, PDK2 and Akt

PI-3,4,5-P3 recruits other proteins and regulates cytoskeleton and transport

PTEN dephosphorylates phosphoinositides at position 3

PI(4,5)P2

S124

T308

S473

T450Akt

Pleckstrin homology domain

Kinase Domain

pS124

pT308

pS473

pT450

Fully-activated Akt

Pre-activation of AktT450 phosphorylation

Kinase?

PI3K and Akt ActivationPI3K and Akt Activation

p110

p85

p85 binds to activated RTK

Ligand-activated RTK

PI(3,4,5)P3

P110 phosphorylates PIP2

PDK1 PDK2

PDK1 phosphorylates Akt T308 (activation loop)

PDK2 phosphorylates Akt S473

Akt, PDK1 and PDK2 all bind PIP3 (plekstrin homology domains)

S473

pT450

pS124

T308

Other PI-3,4,5-POther PI-3,4,5-P33 Functions Functions

Regulation of Vesicle Transport either…

• Binding to FYVE domain proteins

• Regulating small GTP-binding protein Arf

Rearrangement of actin cytoskeleton (rac)

Recruitment of Tyrosine kinases – PH domains in Btk

Enhancement of PLC – direct interaction