En z yme- linked Cell Surface Receptors

1. Enzyme-linked Cell Surface Receptors 30 April 2007

2. Non-Receptor Tyrosine Kinases • NRTK’s associate with membrane receptors or multiprotein complexes which regulate their activity. • Activation involves both conformational changes and tyrosine phosphorylation of activation loop residues by heterologous kinases or autophosphorylation. • NRTK’s contain domains that mediate binding to proteins, lipids, or DNA. • proteins: SH2, SH3, FERM • lipids: Pleckstrin homology (PH) • DNA: example, Abl

3. Nonreceptor tyrosine kinases • This kind of receptors lack intrinsic enzymatic activity. Instead they are non- covalently associated with intracellular protein tyrosine kinases. • N-terminal extracellular ligand binding domain • single transmembrane  helix • cytosolic C-terminal without tyrosine kinase activity

4. Non-Receptor (Cytoplasmic) Protein Tyrosine Kinases From Hunter (2001) Nature 411,355.

6. Signalling Pathways Involving src Kinases • Broadly expressed: fyn, c-src, c-yes, and yrk • hematopoietic lineages: blk, c-fgr, hck, lck, and lyn. • C-src associates with receptor and non-receptor tyrosine kinases via its SH2 domain: • PDGFR, EGFR, IGF-1R, FAK, CSF-1 etc. • C-src associates with transmembrane proteins that are not kinases: • interleukin receptors, T and B cell receptors

7. Receptor and Non-receptor tyrosine (cytokine) kinases

8. For both receptor types, dimerization brings two intrinsic kinases, which then phosphorylate each other on a Tyr residue in the activation lip • Catalytic site is then exposed to ATP or protein substrate • P* of other Tyr residues, which become docking sites for signaling proteins • SH2, PTB domains

9. Some cytokine receptors (IL-4R) and some RTKs bind multidocking proteins (i.e IRS1) via PTB domain • P* of docking protein recruits SH2-domain containing signaling proteins • These proteins too are P* by activated receptor

10. Cytokines control many aspects of cell growth and differentiation: • Prolactin: differentiation of ductular epithelial cells into milk secreting acinar cells • IL-2: T Cell proliferation • IL-4: B Cell antibody production • Interferon-: resistance to viral infection • Differentiation of blood cells • G-CSF, Thrombopoietin

11. Erythropoietin: proliferation and differentiation of erythroid progenitor cells • Progenitors cells are saved from death, each generating more than 50 red blood cells

12. All cytokines are structurally similar: • Four  helices • All cytokine receptors are also similar • Two subdomains, each consisting of seven ß strands • Both cytokines and their receptors are thougt to be derived from a common ancestor

13. All cytokine receptors activate similar signaling pathway; BUT different cellular responses arise depending on the TFs sets, chromatin structures • For instance, prolactin expression in erythroid progenitors results in cell division and differentiation but not milk secretion

14. JAK-STAT Signaling Pathway • Cytoplasmic tyrosine kinases: Jaks (Janus activated kinases) • Latent gene regulatory protein: STAT(Signal Transducer and activator of transcription) • Ligand receptor interaction leads to phosphorylation of Jaks. • Jaks, then phosphorylate and activate STATs, which are normally inactive located on the plasma membrane. • Activated STATs then migrate to nucleus and activate gene transcription.

15. SIGNALING RECEPTOR-ASSOCIATED STATS ACTIVATED SOME RESPONSES LIGAND JAKS g -interferon Jak1 and Jak2 STAT1 activates macrophages; increases MHC protein a -interferon Tyk2 and Jak2 STAT1 and STAT2 increases cell resistance to viral infection Erythropoietin Jak2 STAT5 stimulates production of erythrocytes Prolactin Jak1 and Jak2 STAT5 stimulates milk production Growth hormone Jak2 STAT1 and STAT5 stimulates growth by inducing IGF-1 production GM-CSF Jak2 STAT5 stimulates production of granulocytes and macrophages IL-3 Jak2 STAT5 stimulates early blood cell production

16. JAK-STAT Signalling Pathway • Activated JAKs P* Tyr residues on the receptor, which then become docking sites for STATs • STATs contains: • N-terminal SH2 domain, which binds to Tyr-P* on the receptor • Central DNA binding domain • C-terminal Tyr residue, which is P* by JAK

17. JAK-STAT activated by alfa interferon

18. Modulation of Signaling • As in other signaling pathways, cells must turn off signals generated by JAK-STAT pathway • Two classes of proteins dampen signaling • One over the short term (minutes) • The other over longer time

19. SHP1 phosphatase: short term modulation • Removes P* from a particular P*-Tyr residue on JAK and inactivates it, unless another cytokine binds to cell surface receptor

20. Long term regulation: SOCS (CIS) proteins • Their transcription is induced by STATs • Act in two ways: • 1. SH2 domains in several SOCSs bind to P*-Tyr on receptor and prevent the binding of other signaling proteins. SOCS-1 binds to P*-Tyr on the activation lip of JAK • 2. All SOCSs contain a SOCS box domain that can recruit E3 ubiquitn ligases

21. Protein Tyrosine Phosphatases (PTPs)

22. Protein Tyrosine Kinase Substrate + ATP Substrate-P + ADP Protein Tyrosine Phosphatase (PTP)

23. Protein Tyrosine Phosphatases (PTPs) Receptor-like or Transmembrane PTPs • CD45 • PTPa • LAR Non-receptor or Cytoplasmic PTPs • PTP1B • SHP1 • SHP2

24. Regulation of PTP Activity Mechanism Effect Example Regulated expression  DEP-1, LAR, PTP1B etc Tyrosine phosphorylation  SHP1,SHP2, PTP1B Association with substrates  SHP1, SHP2 via SH2 domains Dimerization  PTPa, CD45 Oxidation of essential Cys  PTP1B with H2O2 or ROS Association with cell matrix  PTP(DEP-1) Ligand interactions ? LAR, PTP

25. Selected Functions of PTPs • The obvious - dephosphorylation of phosphotyrosine residues • counter-regulates TK-dependent reactions • suppresses growth factor, cytokine, integrin receptor pathways • essential for mitogenic effects of growth factor receptors (eg.PDGFR, EGFR) • tumor suppressors

26. TGF-ß Pathway

27. Overview • Play widespread roles in the development • Bone morhogenetic protein (BMP7) induce bone formation in cultured cells • Many others BMP contribute to the formation of mesoderm and earliest blood-forming cells • TGFß1 stimulates the transformation of some cells in culture

28. Other isoforms of TGFß have antiproliferative effects on mammalian cells • Loss of TGFß receptors, thereby induce tumor formation by releasing inhibitory pressure of these isoforms • BUT, TGFß proteins also triggers the secretion of GFs from cells, counterbalancing their inhibitory effect

29. Drosophila homolog dpp controls dorso-ventral patterning • Other members activin and inhibin regulate early development of genital tract • Despite this diversity, the signaling pathway by TGFß superfamily proteins is simple • Direct P* and activation of transcription factors

30. In humans, there are 3 TGFß isoforms • TGFß1, TGFß2, TGFß3 • Each encoded by unique gene and expressed in tissue specific fashion • Synthesized as a large precursor with a prodomain • Although cleaved, this prodomain remains associated non-covalently with TGFs after secretion

31. TGFß is stored in the ECM as an inactive complex containing the cleaved prodomain and Latent TGFß Binding Protein (LTBP) • Binding of LTBP by thrombospondin or integrins affects its conformation and release matuer dimeric TGFß • Another way is the digestion of LTBP by matrix metalloproteases.

34. Intrachain S-S bonds render monomeric TGFß resistant to denaturation • Homo-, heterodimer formation occurs via S-S bonds between N-terminal Cys residues on both monomers • Sequence variation among TGFß isoforms is observed in the N-terminal region

35. Ser-Thr Kinase Activity of TGFß Receptors • Receptors were identified by 125I-labelled TGFß • Three receptors: • Rı, RII and RIII having MW 55, 85 and 280 kD, respectively • Most abundant RIII is a proteoglycan also named ß-glycan • Binds and concentrates TGFß near the cell surface

36. Type I and type II are dimeric receptors with cytosolic Ser-Thr kinase activity • RII is constitutively active and can P* itself • Upon TGFß binding, a complex consisting two copies each of RI and RII forms • RII then P* RI cytoplasmic subunit, activating its kinase activity

37. Activated RI P* Smad TFs • Three types of Smad proteins • Receptor-regulated Smads (R-Smads) • Smad2, Smad3 • Co-Smads • Smad4 • Inhibitory Smads (I-Smads) • Smad7

39. All mammalian cells secrete one TGFß isoform and most express TGFß-R • Why are cellular responses different? • Different cells have different sets of TFs. • Response diversity is also generated by binding of different TGFß isoforms to their related receptors and thereby activating different Smad proteins • i.e. BMP bind to a different receptor, activating Smad1

40. I-Smads and Negative Feedback • SnoN and Ski regulate Smad signaling • They relieve growth-inhibitory effects of TGFß signaling • They don’t affect DNA binding of Smad complexes, but rather they block the transcription activation effect of Smads • The expression of SnoN, Ski, as well as Smad-I are induced by TGFß stimulation • SMAD 7 (I-Smad) blocks the P of R-Smads by RI

42. Inactivation of TGFß receptors and Smad proteins is a common event in human cancer • Smad4 mutation in pancreatic cancer • Abrogated TGFß pathway is unable to induce transcription of growth-inhibitory genes such as p15 and myc