CHARACTERIZATION OF P60C-SRC BINDING TO MEMBRANES

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Human colon carcinoma cell lines have been shown by our own studies (S.I. Rayter, unpublished results) and by the report of Bolen et al., 1987 (1) to possess elevated levels of specific p60c-src protein-tyrosine kinase activity when compared to control colon lines. Two parameters appear to be essential for transformation by p60src; 1) the presence of an active protein tyrosine kinase and 2) the correct localization of the p60src protein at the plasma membrane. Recent studies have suggested the presence of a specific, high affinity p60src binding protein at the internal face of red cell ghost membranes which recognizes peptide sequences located within the 15 N-terminal residues of p60src (Goddard, manuscript submitted to J. Biol. Chem). The aims of this Phase I proposal are 1) to demonstrate specific high affinity binding of an N-terminal 19-mer p60src peptide to red cell ghost membranes and to colon carcinoma and normal colon plasma membranes, (ii) to demonstrate high affinity binding of purified human p60c-src to these membranes, and (iii) to cross-link purified p60c-src to the red cell membrane acceptor protein. In Phase II we propose to purify the specific plasma membrane p60c-src binding protein(s) from colon carcinoma and normal cell lines and develop assays for detection of agents enabling the therapeutic manipulation of this essential process in p60c-src mediated transformation.