We have examined human and rabbit blood platelets for expression of pp6oc-,rc, the normal cellular homolog of the transforming protein of Rous sarcoma virus. pp6Oc8rc kinase activity was determined by an immunecomplex kinase assay that uses enolase as the substrate, and pp6srC protein levels were determined by an immunoblot assay. Lysates from platelets expressed high levels of pp60c-rc-specific kinase activity and pp6O4' protein compared to the levels found in other tissues. pp6Ocsrc was also found to be one of the major proteins phosphorylated in vitro in membranes isolated from platelets. Multiple with the addition of NaDodSO4 sample buffer and then subjected to electrophoresis on 7.5% NaDodSO4/polyacrylamide gels as described by Laemmli (19). The dried gels were Abbreviations: RSV, Rous sarcoma virus; PBL, peripheral blood lymphocyte(s); mAb, monoclonal antibody. 852The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

To examine how amino acid sequences outside of the catalytic domain of pp6O-src influence the functional activity of this protein, we have introduced deletion mutations within the amino-terminal half of pp60cs. These mutations caused distinct changes in the biochemical properties of the c-src gene products and in the properties of cells infected with retroviruses carrying these mutant c-src genes. Cells expressing the c-srcNX protein, which contains a deletion of amino acids 15 to 89, displayed a refractile, spindle-shaped morphology, formed intermediate-sized, tightly packed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Thus, deletion of amino acids 15 to 89 can activate the kinase activity and transforming potential of the c-src gene product. Deletion of amino acids 112 to 225, however, did not increase the kinase activity or transforming ability of pp60csrc; indeed, deletion of these sequences in c-srcHP suppressed phenotypic alterations induced by pp60.csrc* Cells expressing the c-srcNP or c-srcBS gene products (containing deletions of amino acids 15 to 225 and 55 to 169, respectively) displayed a fusiform, refractile morphology and formed diffuse colonies in soft agar; the mutant proteins displayed an increased in vitro protein-tyrosine kinase activity. However, only a few cellular proteins contained elevated levels of phosphotyrosine in vivo. Thus, deletions downstream of amino acid 89 severely restricted the ability of c-src to phosphorylate cellular substrates in vivo without affecting the intrinsic tyrosine kinase activity of the c-src gene product. These results suggest the existence of at least two modulatory regions within the amino-terminal half of pp6Ocsrc that are important for the regulation of tyrosine kinase activity and for the interaction of pp60"csrc with cellular substrates.Rous sarcoma virus (RSV) encodes a 60-kilodalton (kDa) tyrosine protein kinase, pp6Ov-src, that is both necessary and sufficient for the induction of oncogenic transformation by RSV (for reviews, see references 2 and 3). This protein represents a mutated variant of the normal cell protein, pp60csr, which is unable to induce oncogenic transformation even when expressed at high levels (for a review, see reference 26). There are multiple amino acid differences that distinguish the viral and cellular src gene products (60). Whereas many of the mutations in pp6Ov-sr' contribute to the transforming potential of this protein, it has been shown that single-amino-acid substitutions can activate the transforming potential of pp60c-src (26). Thus, the c-src gene product is highly sensitive to alterations in structure that affect the functional activity of this protein.pp6Osrc can be divided into distinct functional domains. The catalytic domain is contained within the carboxy-terminal half of the protein (5, 39). The greatest degree of amino acid sequence homology among all cellular and viral tyrosine kinases is found within this region (for a review, see reference 27). Muta...

To examine how amino acid sequences outside of the catalytic domain of pp60c-src influence the functional activity of this protein, we have introduced deletion mutations within the amino-terminal half of pp60c-src. These mutations caused distinct changes in the biochemical properties of the c-src gene products and in the properties of cells infected with retroviruses carrying these mutant c-src genes. Cells expressing the c-srcNX protein, which contains a deletion of amino acids 15 to 89, displayed a refractile, spindle-shaped morphology, formed intermediate-sized, tightly packed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Thus, deletion of amino acids 15 to 89 can activate the kinase activity and transforming potential of the c-src gene product. Deletion of amino acids 112 to 225, however, did not increase the kinase activity or transforming ability of pp60c-src; indeed, deletion of these sequences in c-srcHP suppressed phenotypic alterations induced by pp60c-src. Cells expressing the c-srcNP or c-srcBS gene products (containing deletions of amino acids 15 to 225 and 55 to 169, respectively) displayed a fusiform, refractile morphology and formed diffuse colonies in soft agar; the mutant proteins displayed an increased in vitro protein-tyrosine kinase activity. However, only a few cellular proteins contained elevated levels of phosphotyrosine in vivo. Thus, deletions downstream of amino acid 89 severely restricted the ability of c-src to phosphorylate cellular substrates in vivo without affecting the intrinsic tyrosine kinase activity of the c-src gene product. These results suggest the existence of at least two modulatory regions within the amino-terminal half of pp60c-src that are important for the regulation of tyrosine kinase activity and for the interaction of pp60c-src with cellular substrates.