1. Michael Jeltsch Tyrosine kinases http://msbl.helsinki.fi/tkseminar

2. Producer cell Target cell Receptor binding Signaling molecules Signal trans- duction Activation of Gene expression proliferation, differentiation, migration, metabolism, etc. RNA Biological responses

3. Cell surface receptors

4. extracellular juxtamembrane transmembrane tyrosine kinase carboxy-terminal tail ligand binding NH 2 COOH ATP substrate binding Receptor Tyrosine Kinases

5. Michael Jeltsch VEGF x VEGF-R1

6. ss EGF-RIGF-1-RFGF-R PDGF-R  VEGF-R3 RTK subclasses IIIIIIIV

7. Modular structure of EC domains

8. RTK ligands Some RTKs need two ligands for activation Extracellular matrix components (HSPGs) Membrane-bound proteins (ephrins) Cytokines (EGF, FGF, CSF-1, insulin)

9. Signaling through an RTK

10. Dimerization or oligomerization Bivalent ligand (e.g. VEGF, PDGF) Ligand-induced conformational change (probably EGF) Intracomplex conformational change (e.g. insulin)

11. Transmembrane signal transfer Exact mechanism unknown Same or similar mechanism for all RTKs (evidence from hybrid receptors)

12. Autophosphorylation Purposes: - Regulation of tyrosine kinase activity - Recruitment of signaling molecules Specific Tyr residues, usually outside the tyrosine kinase domain (juxtamembrane domain, cytoplasmic tail, kinase insert)

13. SH2 (Src-homolgy 2) WW SH3 (Src-homolgy 3) PH (pleckstrin homology)/FYVE PTB (phosphotyrosine binding) Signaling proteins are modular molecules SH2PTBSH3PH/FYVEPZBWW PDZ

14. SH2 (Src-homology 2)/ PTB (phosphotyrosine binding) Bind to PY (and sometimes Y) Specificity is achieved through the context SH2: 1-6 amino acids C-terminal to PY PTB: 3-5 amino aids N-terminal to PY (or Y)

15. SH3 (Src-homology 3) Binds to proline motif PXXP WW Binds to proline motif PXPX SH3 & WW

16. PH (pleckstrin homology)/FYVE PH domain binds to phosphoinositides (membrane- localization) FYVE domain binds specifically PtdIns-3-P PDZ (PSD-95, DDLP, ZP1) Bind to hydrophobic C-terminal strtches of target proteins PH/FYVE/PDZ

17. SH2 PTB P P PH/FYVESH3WWPDZ Enzymatic activity Kinases Phosphatases PLC-  Ras-GAP Rho-GRF Cellular Targets membrane Assembly of a specific signaling complex

18. Myristyl.PTB PH TM PPPPP PPPPPP PPP Membrane localization Receptor binding Tyrosines PP P FRS2  Dos LAT Indirect recruitment is the main recruitment method for some receptors (insulin receptor, FGF receptors) Docking proteins

19. Topics in activation of effector proteins 1.Activation by Membrane Translocation 2.Activation by a Conformational Change 3.Activation by Tyrosine Phosphorylation

20. 1. Membrane translocation 2.Conformational Change 3. Tyrosine Phosphorylation Activation of signaling molecules: PDGF receptor

21. Intracellular Signaling Pathways

22. Signal Termination

23. Ligand-induced oligomerization or conformational change Autophosphorylation/crossphosphorylation Recruitment of signaling molecules to phosphorylated tyrosines (“signaling complexes”) Termination of Signaling (endocytosis) 3 Steps to activation

24. Intracellular vs. Transmembrane Receptor Tyrosine Kinases: Phylogenetic tree of tyrosine kinases

25. Cytoplasmic Tyrosine Kinases

26. Tyrosine kinase-derived oncogenes oncogeneretroviruscellular counterpart v-srcRous sarcomac-src v-erbBavian erythroblastosisEGF receptor v-fmsMcDonough feline sarcomaCSF-1 receptor v-kitfeline sarcomaSCF receptor v-ablAbelson murine leukemiac-abl v-sissimian sarcomaPDGF

27. ss VEGF-R3 Dominant Negative Receptors: Dominant Negative Receptors: VEGFR-3 mutations in hereditary lymphedema S641P P1114L

28. Further reading Schlessinger, J. (2000): Cell Signaling by Receptor Tyrosine Kinases. Cell, Vol. 103, 211-225.