Revised Manuscript Received July 18, 1995® abstract: A continuous assay for pp60c ‘m 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. yrcTK concentrations. When autophosphorylation was monitored by 32P incorporation from [y-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 enzymebefore and after activation demonstrated an increase in the phosphorylation state of the enzyme after incubation with MgATP. The A85-N-terminal mutant protein and a full-length G2A pp60c'1"’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 activate the enzyme for transfer of the y-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 notrequire binding to the membrane or a hydrophobic N-terminus in the case of srcTK.

The balance of protein phosphorylation and dephos-phorylation is known to control many processes. This equilibrium, when applied to protein tyrosine residues, regulates celldifferentiation and proliferation. The roles that these equilibria play insignal transduction pathways are under intense study. The tyrosine kinase (TK) 1 pp60csrc2 is derived from the proto-oncogenewhich is the cellular equivalent of the Rous sarcoma virus transforming gene which encodes for pp60l>! rc (Bruggio & Erikson, 1977). The latter is required for transformation inanimals infected with the virus (Hanafusa, 1977; Vogt, 1977). Activated pp60 ‘'src has been linked to the pathogenesis of a number of