Specific kinase activity and phosphorylation state of pp60(c-src) from neuroblastomas and fibroblasts

Several tumor lines of neuronal origin were assayed for the presence of two forms of pp60(c-src), designated pp60 and pp60⁺. To determine the specific kinase activity of pp60 and pp60⁺, an enzymatic analysis was carried out with neurblastoma LA-N-5, containing a high level of pp60⁺, neuroblastoma SK-N-SH, containing a low level of pp60⁺, fibroblast FSD, containing pp60 but no pp60⁺, and Rous sarcoma virus transformed 3T3 cells, SR-3T3, containing pp60(v-src). Km values for Mg²⁺-ATP of approximately 21, 8, 17 and 13 μM were determined for pp60(src) kinase from LA-N-5, SK-N-SH, FSD and SR-3T3 respectively, using enolase as a substrate. The Vmax values for pp60(c-src) kinase from LA-N-5, SK-N-SH and FSD were similar. The Vmax value of pp60(v-src) was about 50-fold higher. Thirteen distinct phosphopeptides were found in tryptic digests from pp60 and pp60⁺ labeled in vivo with [³²P]. The presence of one phosphopeptide, derived from the N-terminus, correlated with the level of pp60⁺ in neuroblastoma cells, but a small amount of this peptide was also detected in fibroblasts. Only phosphoserine was detected in the N-terminus of pp60 and pp60⁺. Our data strongly suggest that the six extra amino acids, present in pp60⁺ but not in pp60, do not significantly alter the specific kinase activity of pp60⁺, but might influence its phosphorylation state.