Dimerization of native and C-terminally proteolyzed p56lck tyrosine kinase.
Recombinant p56lck 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 p56lck. Processed enzyme migrated on sodium dodecyl sulfate (SDS) gels with a M(r) approximately 6,000 lower than intact enzyme. Analytical ultracentrifugation of intact and processed p56lck 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 microM. Purified intact p56lck incorporated 2 mol of [32P]P(i) per mole of enzyme. Purified processed p56lck 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 p56lck 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.
Robertson
J G
JG
Enzymology Laboratory, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543.
Yanchunas
J
J
Villafranca
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D007313:Insecta; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D013917:Thrombin / Q000737:chemistry / Q000494:pharmacology; D014461:Ultracentrifugation
D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D013917:Thrombin