Deletions within the amino-terminal half of the c-src gene product that alter the functional activity of the protein.

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.

Blood platelets express high levels of the pp60c-src-specific tyrosine kinase activity.

We have examined human and rabbit blood platelets for expression of pp60c-src, the normal cellular homolog of the transforming protein of Rous sarcoma virus. pp60c-src kinase activity was determined by an immune-complex kinase assay that uses enolase as the substrate, and pp60c-src protein levels were determined by an immunoblot assay. Lysates from platelets expressed high levels of pp60c-src-specific kinase activity and pp60c-src protein compared to the levels found in other tissues. pp60c-src was also found to be one of the major proteins phosphorylated in vitro in membranes isolated from platelets. Multiple protein species other than pp60c-src were also phosphorylated on tyrosine in the membrane phosphorylation reactions, and phosphotyrosine represented approximately equal to 80% of the total phosphoamino acid residues phosphorylated in the membranes. These results indicate that tyrosine kinases represent the major protein phosphorylating enzymes detected in isolated platelet membranes. Although the association of tyrosine kinase activity with many viral oncogene products and cellular growth hormone receptors has suggested a role for these enzymes in the regulation of cell proliferation, these results indicate that the expression of high levels of tyrosine kinase activity is not exclusively associated with proliferating cells.