1. Cellular mechanisms of signaling evolve from available components Phospholipids constitute a readily available reservoir that regulates many intracellular events H 2 O 2 Hydrogen peroxide

2. PtdIns in yeast

3. Phospholipase C activation initiates the IP 3 pathway Cellular mechanisms of signaling evolve from available components Phospholipids act as a readily available reservoir that regulate many intracellular events

4. 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.

5. Proposed functions of PX-domain proteins. a, Recruitment of the NADPH oxidase complex. Upon neutrophil activation, PI3K-I converts PI(4,5)P 2 into PI(3,4,5)P 3. 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 p47 phox to PI(3,4)P 2 (green), generated upon dephosphorylation of PI(3,4,5)P 3 by the 5- phosphatase SHIP-1. PI(3,4)P 2 is then dephosphorylated by a PI(3,4)P 2 4-phosphatase to generate PI(3)P (red), which binds to the PX domain of p40 phox. The correct assembly of p40 phox, p47 phox and p67 phox with the membrane-bound (cytb 558 ) components of the complex results in a functional Phox complex that produces O 2 - b, Membrane trafficking. PI3K-II is recruited to the plasma membrane through binding of its PX domain to PI(4,5)P 2 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.

6. 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)P 2, PtdIns(4,5)bisphosphate; PtdIns(3,4)P 2, PtdIns(3,4)bisphosphate; PtdIns(3,5)P 2, PtdIns(3,5)bisphosphate CH 2 O POH O HCOCR2R2 O CH 2 OCR1R1 O Phosphatidic Acid serves as the precursor from which many of these second messenger lipids are derived →

7. 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

9. 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

10. 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.

11. PLD and PA in response to H 2 O 2 PLD, is activated in response to H 2 O 2 and the resulting PA functions in amplification of H 2 O 2 -promoting PCD Stress stimulates production of H 2 O 2 that activates PLD associated with the plasma membrane. Potential activators: Ca 2+ 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.

12. PLD & PA Knockout of PLD renders Arabidopsis plants more sensitive to the reactive oxygen species H 2 O 2 and to stresses H 2 O 2 activates PLD, and PLD -derived PA functions to decrease the promotion of cell death by H 2 O 2. 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 H 2 O 2 -promoted cell death. PLD and PA positively regulate plant cell survival and stress responses.

13. 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

14. 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 Ca 2+ 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.

15. 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)P 3 is present in animal cells but has not been detected in plant tissues, so far. In animal cells, PtdIns(3,4)P 2 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)P 2 and PtdIns(4,5)P 2 to PtdIns(3,4,5)P 3. PtdIns(4,5)P 2 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.

16. PLD is involved in O 2. - 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) 1449-1 Plant Physiology, 2004, Vol. 134, pp. 129

17. 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

18. Phospholipase C Activity O O O Phosphatidylinositol –4,5-bisphosphate (PIP 2 ) PLA 2 PLC PLD Diacylglycerol (DAG) O O PO-O- O O HO OHOH OH O-O- O P O-O- O O O-O- PO-O- O 4 5 1 Inositol-1,4,5-triphosphate (IP 3 ) Phospholipase C (PLC) hydrolysis PIP 2 to yield two second messengers These phospholipases are involved in second messenger generation from membrane phosphoinositides N.B. Different phospholipid specificities (releases different PIs)

19. 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 Ca 2+ into the cytoplasm.

22. Domain structures of PLD , PLD , and PLD  in Arabidopsis XX in the PLD C2 marks the loss of two acidic residues potentially involved in Ca 2+ binding; XX in the PPI-binding motifs marks the loss of the number of basic residues potentially required for PPI binding.

23. 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.

24. 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

25. Classes 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 Ca 2+ activated?

26. The Structure of PLC  P P P EF hand-like Ca 2+ binding? Catalytic X and Y domains Pleckstrin homology – phosphoinositide binding SH2 – phosphotyrosine binding SH3 – interaction with cytoskeleton? C3 – part of catalytic domain? Phospholipid interaction? Phosphotyrosines

27. PLC  phosphorylation may release it from interaction with inhibitor Receptor phosphorylated P P PLC  PIP 2 IP 3 DAG Activation of PLC  by EGFR complex Inactive PLC  EGF EGF binds to receptor P PLC  phosphorylated P PLC  hydrolyses PIP 2 to yield IP 3 and DAG

28. O O PO-O- O HO OH O-O- O P O-O- O O O-O- PO-O- O 4 5 1 Inositol-1,4,5-triphosphate (IP 3 ) O O O O Diacyleglycerol (DAG) PIP 2 -derived Second Messengers Hydrophilic Binds to receptor on ER IP 3 Receptor is Ca 2+ channel Hydrophobic Remains in plasmalemma Activates Protein Kinase C (PKC) AMPLIFICATION – many IP 3 /DAG per bound ligand

29. Summary Phosphatidylinositol-specific PLC hydrolyses membrane PIP 2 PLC  associates with activated receptor tyrosine kinases PLC  is activated by tyrosine phosphorylation PLC  has domains that allow binding to phosphotyrosine (SH2) IP 3 – soluble, induces Ca 2+ release DAG – 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.

30. Phosphatidylinositol 3’-Kinase (PI3K) Activity O O PO-O- O HO OH 4 5 1 O O PO-O- O HO OH 4 5 1 O O-O- PO-O- O O O PO-O- O HO OH 4 5 1 O-O- O PO-O- O O O PO-O- O 4 5 1 O O PO-O- O 4 5 1 O O PO-O- O 4 5 1 PI4K PI5K PI3K PI5Ptase PTEN O O-O- PO-O- O O-O- O PO-O- O O O-O- PO-O- O O O-O- PO-O- O O O-O- PO-O- O O O-O- PO-O- O O O-O- PO-O- O Headgroup of PIP 2 PI3K phosphorylates inositol on the 3 position PTEN dephosphorylates inositol on 3 position

31. Class I PI3K p110 , ,  kinases p85  p85  p55 ,  p50  p110  kinase p101 adapter adapters CLASS I A CLASS I B Catalyticras bindingp85 binding SH3 p110-binding Proline-Rich SH2

32. Class I PI3K Regulation regulation by p21 ras p110  autophosphorylation inhibits PI3K activity p110  phosphorylation of p85  (S 608 ) inhibits PI3K activity SH2 bind pY-X-X-M Inter-SH binds PI(4)P and PI(4,5)P 2 SH2 also binds PI(3,4,5)P 3 this binding competes with pY binding Proline-rich repeats bind SH3 domains of e.g. src, fyn or lck Subunit interaction

33. Protein Kinase C (PKC) Three Classes Novel nPKC , ,  and  Activated by DAG but do not require Ca 2+ Classical cPKC ,  1,  2 and  Activated by DAG and Ca 2+ Atypical aPKC ,  and Do not require DAG or Ca 2+ All forms require phosphatidylserine (PS) for activity cPKC have two zinc finger domains C1 – binds PS and Ca 2+ C2 – binds DAG

34. Proliferation PKC Substrates PPP 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

35. Effects 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 IP 3 /Ca 2+ Receptor Downregulation - e.g. EGF MAP Kinase Pathway – ras independent Inhibition of PLC  - -ve feedback STAT inhibition – PKC  blocks STAT DNA association

36. Summary 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

37. Summary PI3 kinases phosphorylate phosphoinositides at position 3 p110 contains catalytic activity p85 responsible for recruiting enzyme to RTK PI-3,4,5-P 3 recruits PDK1, PDK2 and Akt PI-3,4,5-P 3 recruits other proteins and regulates cytoskeleton and transport PTEN dephosphorylates phosphoinositides at position 3

38. PI(4,5)P 2 S 124 T 308 S 473 T 450 Akt Pleckstrin homology domain Kinase Domain pS 124 pT 308 pS 473 pT 450 Fully-activated Akt Pre-activation of Akt T 450 phosphorylation Kinase? PI3K and Akt Activation p110 p85 p85 binds to activated RTK Ligand-activated RTK PI(3,4,5)P 3 P110 phosphorylates PIP 2 PDK1PDK2 PDK1 phosphorylates Akt T 308 (activation loop) PDK2 phosphorylates Akt S 473 Akt, PDK1 and PDK2 all bind PIP 3 (plekstrin homology domains) S 473 pT 450 pS 124 T 308

39. Other PI-3,4,5-P 3 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