Specific kinase activity and phosphorylation state of pp60c-src from neuroblastomas and fibroblasts

Several tumor lines of neuronal origin were assayed for the presence of two forms of pp60c-src, designated pp60 and pp60+. To determine the specific kinase activity of pp60 and pp60+, an enzymatic analysis was carried out with neuroblastoma 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 pp60v-src. Km values for Mg2+-ATP of approximately 21, 8, 17 and 13 microM were determined for pp60src kinase from LA-N-5, SK-N-SH, FSD and SR-3T3 respectively, using enolase as a substrate. The Vmax values for pp60c-src kinase from LA-N-5, SK-N-SH and FSD were similar. The Vmax value of pp60v-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.