CHARACTERIZATION OF PP60(C-SRC) TYROSINE KINASE-ACTIVITIES USING A CONTINUOUS ASSAY - AUTOACTIVATION OF THE ENZYME IS AN INTERMOLECULAR AUTOPHOSPHORYLATION PROCESS

A continuous assay for pp60(c-src) tyrosine kinase (srcTK) was develop ed. A lag in phosphorylation of the peptide RRLIEDAEYAARG was observed that could be eliminated by preincubation with MgATP. The induction t ime for this lag was dependent upon MgATP and srcTK concentrations. Wh en autophosphorylation was monitored by P-32 incorporation from [gamma -P-32]ATP, a lag in the time course was also observed. These results d emonstrate that autoactivation is an intermolecular process. The elect rospray ionization mass spectrum of the enzyme before and after activa tion demonstrated an increase in the phosphorylation state of the enzy me after incubation with MgATP. The Delta 85-N-terminal mutant protein and a full-length G2A pp60(c-src) mutant, which removes the myristyla tion 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 activate the enzyme for t ransfer of the gamma-phosphoryl of MgATP to the peptides. The unphosph orylated enzyme initially present was converted to a monophosphorylate d species upon treatment with MgATP. Y-419 phosphorylation was evident only after treatment with MgATP. These data are consistent with autop hosphorylation on Y-419 as predicted. Intermolecular autophosphorylati on is consistent with the; ability of srcTK to dimerize, which is anal ogous to activation of receptor tyrosine kinases such as the EGF recep tor kinase in response to growth factors. These results indicate that dimerization leading to activation does not require binding to the mem brane or a hydrophobic N-terminus in the case of srcTK.