DIMERIZATION OF NATIVE AND C-TERMINALLY PROTEOLYZED P56(LCK) TYROSINEKINASE

Recombinant p56(lck) tyrosine kinase was purified to near homogeneity from a baculovirus/insect cell expression system. Treatment with throm bin proteolytically removed the C-terminal 54 amino acids from p56(lck ). Processed enzyme migrated on sodium dodecyl sulfate (SDS) gels with a M(r) approximate to 6,000 lower than intact enzyme. Analytical ultr acentrifugation of intact and processed p56(lck) gave M(r)'s of 62,600 and 56,200, respectively, confirming that the thrombin treated enzyme existed in solution as a processed polypeptide and that there was no anomalous migration in SDS gels due to thrombin treatment. Simultaneou s multispeed analysis of sedimentation equilibrium data demonstrated t hat both intact and processed enzyme can dimerize with a weak binding constant in the range of 200-300 mu M. Purified intact p56(lck) incorp orated 2 mol of [P-32]P-i per mole of enzyme. Purified processed p56(l ck) incorporated only 1 mol of [P-32]P-i per mole of enzyme. The loss of 1 mol of [P-32]P-i per mole of enzyme after thrombin deletion of th e C-terminus demonstrates that p56(lck) undergoes autophosphorylation at the C-terminus. The data are consistent with autophosphorylation at tyrosine 505, which has previously been thought to be a regulatory ph osphorylation site, but which now must also be considered as an autoph osphorylation site. (C) 1995 Academic Press, Inc.