Phospholipase C γ Prof. Graham Carpenter

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1 Graham Carpenter, h.d. rofessor of Biochemistry Cornelia Crooke Department of Biochemistry Vanderbilt University School of Medicine, Nashville, TN 1 Receptor Tyrosine Kinases GF Extracellular M Intracellular A B C D Integration of Biochemical Signals (Time, Space, Concentration) Cellular Responses 2 Signal Transduction athways GF Extracellular M Intracellular Stat I-3Kinase Ras/ Map Kinase LC-γ Cellular Responses 3 The screen versions of these slides have full details of copyright and acknowledgements 1

2 - LC-γ What does it do? ow is it activated? ow does it effect cellular responses? 4 LC-γ is an I Specific ydrolytic Enzyme Diacylglycerol () C C 2 C C C 2 C C LC-γ + C C C 2 hosphotidlyinositol 4,5-bisphosphate (I2) 3 Inositol 1,4,5-trisphosphate (I3) What is I 2? C C C 2 C C 2 3 hosphotidlyinositol 4,5-bisphosphate Minor membrane component Located at cytoplasmic leaflet of plasma membrane No structural role in membrane integrity Co-factor for cellular proteins Substrate for enzymes i. LC-γ ii. I-3 Kinase 6 The screen versions of these slides have full details of copyright and acknowledgements 2

3 What is? C C 2 C C C 2 Diacylglycerol Activation of rotein Kinase 1A C 1B( KC) 2 KC rotein X Signaling 7 Diacylglycerol roduction Extracellular Kinase hosphatidic Acid Transmembrane Intracellular A hosphatase 3 2- hosphatidic acid can be a signal transduction element is also produced by LCs that act on other phospholipds such as phosphatidylcholine 8 What is I 3? I3 inositol 1,4,5 - trisphosphate = I3 Nucleus ER 9 The screen versions of these slides have full details of copyright and acknowledgements 3

4 ow is LC-γ activated? 10 The I-Specific LC Family Activators GCRs RTK s? Ras Fertilization? Families β γ δ ε ζ η Isozymes β1, β2, β3, β4 γ1, γ 2 δ1, δ2, δ3, δ4 2 splice variants ne Isozyme η1, η2 *LC α was misidentified as a LC 11 What Defines a hospholipase C? X X X+= Catalytic Active Site 12 The screen versions of these slides have full details of copyright and acknowledgements 4

5 Domains in LC-γ1 ½ ½ EF X S2 S2 S3 C2 I2 and Binding ther roteins Catalysis Interaction with RTK ther roteins Catalysis Membrane 13 GF Association of LC-γ1 with the Receptor GF Nucleus 14 Membrane Association X I 2 I 3 Activation of signaling pathways by KC and hosphatase 15 The screen versions of these slides have full details of copyright and acknowledgements 5

6 Tyrosine hosphorylation of LC-γ1 p771 p783 p1254 X S2 S2 S3 1) hosphorylation of 783 is essential for LC-γ1 function 2) Roles of the other phosphotyrosine sites has yet to be determined 3) Roles of serine and threonine phosphorylation has not been determined 16 LC-γ1 Verses LC-γ2 γ1 is widely expressed; γ2 expression is limited to hematopoetic cells γ1 knockout in mice is embryonic lethal at E9.0; γ2 knockout is viable with minor defects in certain hematopoetic lineages 17 Measurement of LC-γ1 Tyrosine phosphorylation of LC-γ1 In vivo i. measurement of I 3 generation ii. measurement of intracellular release In vitro measurement of I 3 generation 18 The screen versions of these slides have full details of copyright and acknowledgements 6

7 Measurement of LC-γ Tyrosine hosphorylation 1) Treat cells with EGF (5 min) 2) Lysis and immunoprecipitation with anti-lc-γ1 3) Western blot with anti-phosphotyrosine 4) Strip and re-blot with anti- LC-γ EGF EGFR LC-γ1 anti-p IgG heavy chain anti-lc-γ1 19 In Vivo Measurement of I 3 1) re-label cells with 3 -inositol (24-48 hours) 2) Growth factor treatment (5 minutes) 3) Lyse cells 4) Isolate I 3 using dowex chromatography, LC, or I 3 binding protein 20 In Vivo Measurement of Intracellular Release reload cells with fluorescent chelator Treat cells with growth factor Measure fluorescence in cell suspension or in attached single cells Repeat using calcium-free media release of intracellular influx of extracellular EGF 21 The screen versions of these slides have full details of copyright and acknowledgements 7

8 Measurement of LC-γ1 Activity in vitro 1) Treat cells +/- growth factor 2) Lyse cells and immunoprecipitate LC-γ1 3) Add 3 -I 2 to immunoprecipitate, add to start reaction 4) Measure water soluble 3 -I 3 22 Ag T-Cell roliferation T-Cell IL2 IL2 IL2 IL2 T-cell roliferation Immunologic Response 23 Role of Immediate Early Genes in Cell roliferation 1) Growth Factors/ Antigens Activate Signaling athway 2) Signals Activate the Expression of Genes termed Immediate Early Genes (IEG) 3) IEG mrnas 1. Induced by growth factors without the need for synthesis of new proteins, i.e. in the presence of cyclohexamide 2. ccurs rapidly (15 min- 1 hr) 3. Number of IEGs induced is Many IEGs code for secreted cytokines or transcription factors which participate in additional rounds of signaling and the induction of later genes, i.e. early(2-4 hrs) or late (6-12 hrs) genes. 24 The screen versions of these slides have full details of copyright and acknowledgements 8

9 Downstream Effects of LC Signaling I 2 ydrolysis I 3 KC Response A Response B Response C 25 Role of LC-γ in IL-2 Expression Ag I 3 RTK Ras KC-θ NFAT Erk 1,2 NF-kB A-1 26 IL-2 mrna T Cell Receptor Activation Growth Factor Antigen Ligand dependant tyrosine kinase activation TK TK (LCK) TK (Zap-70) Receptor Tyrosine Kinase T Cell Receptor 27 The screen versions of these slides have full details of copyright and acknowledgements 9

10 Tyrosine Kinase hosphorylation of LC-γ1 in T-Cells Antigen I(3,4,5)3 LCK ITK Zap-70 L A T S L Calcium Regulation of IL-2 Transcription rotein hosphatase 2 B Apocalmodulin Calmodulin Cam Kinase Immunosuppressants (cyclosporin A) Calcineurin NFAT Nuclear Factor of Activated T Cells IL-2 mrna 29 Control of IL-2 Transcription Ras GD GT RasGR Raf Mek Erk 1,2 ELK-1 A-1 IL-2 mrna 30 The screen versions of these slides have full details of copyright and acknowledgements 10

11 Control of IL-2 Transcription KC-θ? NF-kB IL-2 mrna 31 Role of LC-γ in IL-2 Expression Ag I 3 RTK Ras KC-θ Calmodulin NFAT A-1 NF-kB 32 IL-2 mrna Cell Morphogenesis Adhesion Spreading Migration ECM 33 The screen versions of these slides have full details of copyright and acknowledgements 11

12 Role of Integrins in Cell Morphogenesis Fibronectin Actin reorganization Adhesion and Spreading 34 Role of LC-γ1 in Cell Morphogenesis Src FAK? Actin reorganization Adhesion and Spreading 35 LC-γ Biologic Roles Cell roliferation Immune Responses Embryogenesis Formation of erythrocytes, endothelial cells Cardiac contractility 36 The screen versions of these slides have full details of copyright and acknowledgements 12

13 37 The screen versions of these slides have full details of copyright and acknowledgements 13