Dimerization of Native and C-Terminally Proteolyzed p56 lck Tyrosine Kinase

Abstract

Recombinant p56 lck tyrosine kinase was purified to near homogeneity from a baculovirus/insect cell expression system. Treatment with thrombin proteolytically removed the C-terminal 54 amino acids from p56 lck. Processed enzyme migrated on sodium dodecyl sulfate (SDS) gels with a M r ≍ 6,000 lower than intact enzyme. Analytical ultracentrifugation 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. Simultaneous multispeed analysis of sedimentation equilibrium data demonstrated that both intact and processed enzyme can dimerize with a weak binding constant in the range of 200-300 μM. Purified intact p56 lck incorporated 2 mol of [ 32P] P i per mole of enzyme. Purified processed p56 lck incorporated only 1 mol of [ 32P] P i per mole of enzyme. The loss of 1 mol of [ 32P] P i per mole of enzyme after thrombin deletion of the 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 phosphorylation site, but which now must also be considered as an autophosphorylation site.