Lidder, Sukhwinderjit; (2004) Involvement of cortactin, Crk and Eps8 as intracellular signal transducers of the hepatocyte growth factor signal. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Hepatocyte growth factor/ scatter factor (HGF/SF) is a glycoprotein that induces a variety of cellular responses including proliferation, migration, the formation of branching epithelial tubules and in the case of tumour cells, invasion and metastasis. HGF transduces its activity through the HGF receptor (Met receptor) tyrosine kinase. The Met receptor is coupled to a number of signal transduction molecules that transmit the HGF signal through the cytoplasm to the nucleus. The different downstream signals transducers participate in overlapping pathways to determine the cellular responses. In this thesis, potential effects of HGF on cortactin, an Filamentous actin (F-actin) binding protein, Crk, a 40-kDa adapter protein possessing Src homology-2 (SH2) and two SH3 domains, and Eps8, a SH3-containing epidermal growth factor receptor (EGF-R) substrate were investigated. Cortactin, Crk and Eps8 became tyrosine phosphorylated in response to HGF in A431 human epidermoid carcinoma cells expressing the Met receptor. The Met receptor was enriched in the detergent-insoluble fraction and was found to co-precipitate with cortactin and Eps8, whereas Crk was enriched in the detergent-soluble fraction. The small adapter protein Grb2, which is known to associate with Met, was also associated with cortactin and Eps8. Transient transfection of A431 cells with dominant- negative Grb2 constructs revealed that Grb2-C-SH3 domain possesses a central role in coractin phosphorylation in response to HGF. Expression of Myc-cortactin in transfected A431 revealed that cortactin with mutations at Tyr421, Tyr466, Tyr482 significantly reduced cell migration. Cortactin phosphorylation was found to be uncoupled of endogenous Src kinase activity, suggesting that cortactin is directly phosphorylated by the Met receptor and may constitute a docking protein for Met-derived signals leading to cell migration. CrkII was found to associate with Met via its SH3 domain. Grb2 was also shown to co-immunoprecipitate with CrkII in an HGF-dependent manner, suggesting that Crkll can associate with Met indirectly through multi-protein complexes of Grb2-Sos-Gab1-CrkII. CrkII-SH3-C and -δSH2 were found to associate Grb2, implying that Grb2 may contribute to the association of CrkII to Met. Over expression of wild type CrkII enhanced HGF-induced cell proliferation whereas CrkII-δSH2 failed to produce a similar proliferative response to HGF. These data suggest that cortactin and possibly Eps8 (previously shown to interact with the cytoskeleton and F-actin binding proteins (Provenzano et al., 1998; Scita et al., 2001)) contribute to HGF signalling to the cytoskeleton, and that cortactin has a role in cell migration, whereas Crk contributes to HGF-induced DNA synthesis.

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