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Lecture 11

Lecture 11 - Receptor Tyrosine Kinase (RTK).docx

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Western University
Biology 2382B
Cumming/ Damjanovski

Lecture 11: Receptor Tyrosine Kinase Learning Objectives • Receptor tyrosine kinases (RTK) – structure – dimerization – autophosphorylation • Adapter proteins, GRB2 – PTB, SH2, SH3 • Ras and its regulation (GEF, GAP, Sos) • HERs and human breast cancer • Ras/MAP kinase pathway operation (Raf, MEK, MAPK), EGF Receptor tyrosine kinases (RTKs) • Extracellular (ligand-binding) domain – Ligands include growth factors and insulin – Growth can entail cell getting bigger or mitogenic factor that induce a cell to divide. – Insulin can be considered as a mitogenic factor • Single transmembrane α-helix (as opposed to GPCR with 7) • Cytoplasmic domain with intrinsic tyrosine kinase activity which is stimulated by ligand binding due to receptor dimerization • Adapter proteins are required • Ras (downstream effector) acts as a GTPase switch protein to signal further “downstream” kinases • Aberrant signaling is at root of many human cancers Activation of RTKs 1. Growth factor binds extracellular domain of 2 independent RTKs. 2. Each of the transmembrane domains undergo conformational change that allows dimerization. 3. Dimerization  partial activation of the kinase domain in the cytosolic portion of the RTK. 4. Trans-activation: one kinase domain phosphorylates and activates the activation lip of the other RTK. 5. Fully active kinase domain: can self- phosphorylate the lower tyrosine residues on the RTK. • Dimerization allows for auto-phosphorylation of the cytoplasmic domain. – This is important for the recruitment of the Phosphotyrosine. Phosphotyrosines: serve as docking sites for signal transduction proteins that have SH2 or PTB domains (adapter proteins) Page 1 of5 Adapter proteins Adapter proteins contain unique domains that recognize specific sequences. Common adapter domains: • SH2 –src (sarcoma) homology 2 domain – found in at least 100 human proteins – binding recognition domain for phosphorylated tyrosines • PTB – Phosphotyrosine-binding domain. – Found on multi-docking proteins, serve as docking sites for other signal transduction or adapter proteins – Binding recognition domain for phosphorylated tyrosines • IRS-1- Insulin receptor substrate protein Recruitment of adapter proteins • Growth factor binding to extracellular domain of the protein à trans-activation: one domain activated first to activate the other à autophosphorylation moving down on tyrosine residues. – This is a signal of the proteins that cause recruitment of proteins that recognize the phosphotyrosines. Those proteins do so through their SH2 and PTB domains. • When the two proteins with the SH2 and PTB domains get recruited to the RTK kinase domain and bind to the phosphotyrosines, they may get phosphorylated in some cases. – Proteins that have PTB domains usually have multi-docking capabilities. • Multi-docking: IRS-1 when it binds to phosphorylated tyrosine on the RTK, and it is phosphorylated it acts as a signal for the recruitment of other adapter proteins many of which will also have SH2 domains. . Discovery of First Oncogene or why is SH2 named like that? • In 1911 Rous injected a cell free extract from a chicken tumor (sarcoma) into healthy chickens - all developed tumours – If you take the sarcoma and grind it all up to kill all the cells and inject it into healthy chickens, the chickens got tumours. – This proved that the tumours were not cell-based, it was actually from a virus. • The transmissible agent was determined to be a retrovirus (Rous Sarcoma Virus) • In 1980s Bishop-Varmus isolated virus and sequenced it. – it was found that one of the viral genes encodes a tyrosine kinase (src) protein. • Sarcomology domains are highly conserved and found in humans (SH2 and SH3) – The SH2 domain was found in both the viral and human genes. • Normal version is cellular src (c-Src) • Viral version is viral src (v-Src) – constitutively active V-Src The Rous Sarcoma virus encoded sarc protein (v-Src) has no regulatory region: It’s missing the tyrosine residue 527 that is inhibited by phosphorylation. Page 2 of 5 The kinase encoded is involved in the mutagen activated signaling pathway. So now you don’t need the mitogen to turn it on the signaling pathway that leads to cell division, v-SRC can just phosphorylate everything because it is constitutively active. This is what was causing cancer in the chickens. If you express v-Src at high levels in a normal human cell, you can make it cancerous. Ras, a GTPase switch protein Ras: Monomeric G protein, GTPase superfamily, lipid-anchored protein • Downstream effector of RTK signaling • Similar to Gα but smaller, poor GTPase activity(requires GAP) • Not linked directly to cell-surface receptors • Ras activity is regulated by GEFs and GAPs. • Ras protein: 20-30% Human cancers: Ras binds GTP but no hydrolysis, mutation of glycine- 12 which prevents binding GAPs By itself, Ras has poor GTPase activity. Its regulation is created by GEF (activation) and GAP (inactivation) RasD is a constitutively active Ras protein, mutant. • Mutation in cancers where Ras can no longer bind GAP and is constitutively active because it is permanently hydrolyze to GTP. EGF-induced Ras activation: step 1 Epidermal Growth Factor (EGF) 1. Ras is inactive (anchored to the membrane by prenylation) 2. EGF binds EGFR as monomers – EGF binding to each EGFR causes conformational change that leads to dimerization. 3. Trans-activation is prom
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