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.
Yang
X M
XM
Department of Pathology, University of California, San Diego, La Jolla 92093.
Walter
G
G
D000818:Animals; D005347:Fibroblasts / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D051379:Mice; D009363:Neoplasm Proteins / Q000378:metabolism; D009447:Neuroblastoma / Q000201:enzymology*; D015688:Oncogene Protein pp60(v-src); D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src); D012191:Retroviridae Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000201:enzymology
D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D012191:Retroviridae Proteins; D010768:Phosphoserine; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src); D016392:Proto-Oncogene Proteins pp60(c-src)