CHARACTERIZATION, PARTIAL-PURIFICATION, AND PEPTIDE SEQUENCING OF P130, THE MAIN PHOSPHOPROTEIN ASSOCIATED WITH V-CRK ONCOPROTEIN

The transforming gene v-crk found in CT10 and ASV-1 avian sarcoma viru ses induces marked phosphorylation of several proteins in cells expres sing p47(v-crk) (v-Crk), In this work, the main tyrosine-phosphorylate d proteins in ASV-1-infected chicken cells and v-crk transfected rat c ells were characterized biochemically, Both these proteins have a mole cular mass of about 130 kDa and are tightly associated with v-Crk in v ivo, Two-dimensional gel electrophoresis revealed that they are both e ssentially single proteins (p130) with modifications that result in a broad spot in an acidic region, The broad band of semi-purified p130 b ecame sharp at an elevated position in the gel upon treatment with ort hovanadate in vivo or with c-Src kinase produced using a baculovirus v ector in vitro, whereas it shifted at a lower position upon treatment with alkaline phosphatase in vitro, These results suggest multiple pho sphorylation states of p130, which result in a broad band of p130. Two procedures of immunoaffinity purification were used to purify p130 fr om SY1 cells transfected with v-crk. Approximately 30 pmol of purified p130 was obtained in an immobilized form on a filter starting from 3 x 10(10) cells, Peptide mapping of p130 digested in situ by peptidase revealed that the purity and quantity of the final material were enoug h for peptide sequencing, Several stretches of partial amino acid sequ ences were determined, and they indicated that p130 is a novel protein .