TI - Characterization of c-src tyrosine kinase activities using a continuous assay : autoactivation of the enzyme is an intermolecular autophosphorylation process . AB - A continuous assay for pp60c-src tyrosine kinase ( srcTK ) was developed . A lag in phosphorylation of the peptide RRLIEDAEYAARG was observed that could be eliminated by preincubation with MgATP . The induction time for this lag was dependent upon MgATP and srcTK concentrations . When autophosphorylation was monitored by 32P incorporation from [gamma-32P] ATP , a lag in the time course was also observed . These results demonstrate that autoactivation is an intermolecular process . The electrospray ionization mass spectrum of the enzyme before and after activation demonstrated an increase in the phosphorylation state of the enzyme after incubation with MgATP . The delta 85-N-terminal mutant protein and a full-length G2A c-src mutant , which removes the myristylation site , used in these studies were partially phosphorylated on Y338 and Y530 as isolated . This is the first report of phosphorylation on Y338 , but the significance of this site of phosphorylation is unknown . These phosphorylations were insufficient to active the enzyme for transfer of the gamma-phosphoryl of MgATP to the peptides . The unphosphorylated enzyme initially present was converted to a monophosphorylated species upon treatment with MgATP . Y-419 phosphorylation was evident only after treatment with MgATP . These data are consistent with autophosphorylation on Y-419 as predicted . Intermolecular autophosphorylation is consistent with the ability of srcTK to dimerize , which is analogous to activation of receptor tyrosine kinases such as the EGF receptor kinase in response to growth factors . These results indicate that dimerization leading to activation does not require binding to the membrane or a hydrophobic N-terminus in the case of srcTK .