Receptor tyrosine kinases. Introduction: Protein phosphorylation Recruitment of kinases in signalling pathways Consequences of protein phosphorylation RTK family: Classification & structure/function RTK ligands Receptor dimerization & autotransphosphorylation. Receptor tyrosine kinases.
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Receptor tyrosine kinases • Introduction: • Protein phosphorylation • Recruitment of kinases in signalling pathways • Consequences of protein phosphorylation • RTK family: • Classification & structure/function • RTK ligands • Receptor dimerization & autotransphosphorylation
Receptor tyrosine kinases • RTK-mediated pathways: • Ras-Raf-MAP kinase pathway, use of dominant negative mutants to map pathway • R7 photoreceptor development pathway in drosophila
Introduction • Protein phosphorylation represents the most common form of posttranslational modification in nature • Protein function altered by addition of a negatively charged phosphate group to a Ser, Thr, or Tyr residue: • Binding properties • Enzymatic activity if a catalytic protein
Introduction • Cell surface receptors recruit activity of protein kinases in two general ways: • Non-receptor tyrosine kinases: Receptors lacking self-contained kinase function recruit activities of intracellular protein kinases to the plasma membrane • Receptor tyrosine kinases: Possess an intrinsic tyrosine kinase activity that is part of the receptor protein. Examples include receptors for growth factors (PDGF, EGF, insulin, etc.)
RTK family classification & structure/function • Implicated in diverse cellular responses: • Cell division • Differentiation • Motility • At least 50 RTKs identified: • Subdivided into 10 subclasses based on differences within extracellular, ligand-binding domain of receptor • “Oncogenic” RTK mutants exist: • erbB gene encodes an N-terminal truncated, constitutively active form of EGF receptor
RTK family classification & structure/function • Four common structural features shared among RTKs: • Extracellular ligand-binding domain • Single transmembrane domain • Cytoplasmic tyrosine kinase domain(s) • Regulatory domains
RTK structure/function Regulatory domains
RTK ligands • Typically small soluble proteins • Work in autocrine and paracrine manner • Dimerize (may aid in receptor dimerization) • Some RTK ligands membrane-bound
Receptor dimerization & autotransphosphorylation • Ligand-induced RTK activation induces Receptor dimerization, leading to activation of catalytic domains • Receptor autotransphosphorylation: • Further stimulates kinase activity • Leads to phosphorylation of additional proteins involved in receptor signalling pathway • Provides “docking sites” for downstream signalling proteins (Grb2, PI3-kinase, phospholipase Cg, etc.)
Src homology (SH) 2 & SH3 domains • SH2 domains: bind P-Tyr-containing sequences • SH3 domains: bind to pro-rich (PxxP) sequences
RTK-mediated pathways: one pathway with two very different functions • Ras-Raf-MAP kinase pathway • R7 photoreceptor development in drosophila (fruitfly)
GDP GTP DNA The Ras-Raf-MAP kinase pathway SH2 domain SH3 domains Proline-rich regions (-PXXP-) Tyr-P Raf Sos Grb2 Ras (inactive) Ras (active) Pi Nucleus P MEK P P fos jun P P P P MAP kinase MAP kinase Increase gene expression
Use of oncogenic & dominant negative mutants to map pathways • Oncogenic Ras (V12Ras): defective GTPase function. Always turned “on” (always GTP-bound) • Dominant negative Ras (N17Ras): can interact with its immediate upstream partner (Sos), but cannot become activated to transduce a downstream signal (i.e., to Raf). Effect is to “sequester” Sos to prevent it from activating endogenous Ras.
GDP DNA Dominant negative Ras (N17Ras) sequesters Sos & blocks pathway from Ras on down SH2 domain SH3 domains Proline-rich regions (-PXXP-) GDP Tyr-P Raf Sos Grb2 N17Ras Ras (inactive) Nucleus jun MEK fos MAP kinase gene expression blocked
GDP DNA Combine oncogenic and DN mutants to map position of pathway components SH2 domain SH3 domains Proline-rich regions (-PXXP-) GDP Tyr-P Raf Ras (inactive) Sos Grb2 N17Ras Nucleus P Oncogenic Raf MEK P P fos jun P P P P MAP kinase MAP kinase Increased gene expression
R7 photoreceptor development • Fruitfly (Drosophila melanogaster) • Compound eye (800 ommatidia) • Each ommatidium has 8 photoreceptor cells; each detects a different wavelength of light
R7 photoreceptor development • Photoreceptor cells “recruited” as an undifferentiated precursor from epithelial sheet of cells • Each photoreceptor develops in a specific order beginning with R8 & ending with R7 (responds to ultraviolet light)
The R7 photoreceptor developmental pathway is a RTK-MAP kinase cascade