Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Enzyme-linked Cell Surface Receptors 30 April 2007 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 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 Non-Receptor (Cytoplasmic) Protein Tyrosine Kinases From Hunter (2001) Nature 411,355. 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 Receptor and Non-receptor tyrosine (cytokine) kinases 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 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 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 Erythropoietin: proliferation and differentiation of erythroid progenitor cells Progenitors cells are saved from death, each generating more than 50 red blood cells All cytokines are structurally similar: All cytokine receptors are also similar Four helices Two subdomains, each consisting of seven ß strands Both cytokines and their receptors are thougt to be derived from a common ancestor 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 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. SIGNALING RECEPTOR-ASSOCIATED STATS ACTIVATED SOME RESPONSES LIGAND JAKS g -interferon Jak1 and Jak2 STAT1 a -interferon Tyk2 and Jak2 STAT1 and STAT2 increases cell resistance to viral infection Erythropoietin Prolactin Jak2 Jak1 and Jak2 activates macrophages; increases MHC protein STAT5 stimulates production of erythrocytes 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 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 JAK-STAT activated by alfa interferon 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 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 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 Protein Tyrosine Phosphatases (PTPs) Protein Tyrosine Kinase Substrate + ATP Substrate-P + ADP Protein Tyrosine Phosphatase (PTP) Protein Tyrosine Phosphatases (PTPs) Receptor-like or Transmembrane PTPs CD45 PTP LAR Non-receptor or Cytoplasmic PTPs PTP1B SHP1 SHP2 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 PTP, CD45 Oxidation of essential Cys PTP1B with H2O2 or ROS Association with cell matrix PTP(DEP-1) Ligand interactions ? LAR, PTP 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 TGF-ß Pathway 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 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 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 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 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. 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 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 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 Activated RI P* Smad TFs Three types of Smad proteins Receptor-regulated Smads (R-Smads) Co-Smads Smad2, Smad3 Smad4 Inhibitory Smads (I-Smads) Smad7 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 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 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