CHARACTERIZATION OF P60C-SRC BINDING TO MEMBRANES

HUMAN COLON CARCINOMA CELL LINES HAVE BEEN SHOWN TO POSSES ELEVATED LEVELS OF SPECIFIC P60C-SRC PROTEIN-TYROSINE KINASEACTIVITY WHEN COMPARED TO CONTROL COLON LINES. TWO PARAMETERS APPEAR TO BE ESSENTIAL FOR TRANSFORMATION BY P60C-SRC: (1) THE PRESENCE OF AN ACTIVE PROTEIN TYROSINE KINASE, AND (2) THE CORRECT LOCALIZATION OF THE P60C-SRC PROTEIN AT THE PLASMA MEMBRANE. RECENT STUDIES HAVE SUGGESTED THE PRESENCE OF A SPECIFIC, HIGH-AFFINITY P60C-SRC-BINDING PROTEIN AT THE INTERNAL FACE OF RED CELL GHOST MEMBRANES THAT RECOGNIZES PEPTIDE SEQUENCES LOCATED WITHIN THE 15 N-TERMINAL RESIDUES OF P60C-SRC. THE AIMS OF THIS PHASE I PROJECT ARE (1) TO DEMONSTRATE SPECIFIC HIGH-AFFINITY BINDING OF A N-TERMINAL 19-MER P60C-SRC PEPTIDE TO RED CELL GHOST MEMBRANES AND TO COLON CARCINOMA AND NORMAL COLON PLASMA MEMBRANES, (2) TO DEMONSTRATE HIGH-AFFINITY BINDING OF PURIFIED HUMAN P60C-SRC TO THESE MEMBRANES, AND (3) TO CROSS-LINK PURIFIED P60C-SRC TO THE RED CELL MEMBRANE ACCEPTOR PROTEIN. IN PHASE II, IT IS PLANNED TO PURIFY THE SPECIFIC PLASMA MEMBRANE P60C-SRC-BINDING PROTEINS FROM COLON CARCINOMA AND NORMAL CELL LINES AND TO DEVELOP ASSAYS FOR DETECTION OF AGENTS ENABLING THE THERAPEUTIC MANIPULATION OF THIS ESSENTIAL PROCESS IN P60C-SRC-MEDIATED TRANSFORMATION.