p56lck interactions accelerate autophosphorylation and enhance activity toward exogenous substrates.
p56lck, which is a lymphoid-restricted tyrosine kinase, plays a significant role both in activation of mature T lymphocytes and in T cell development. Recombinant human p56lck was expressed in a baculovirus system and purified to > 95% purity. Purified recombinant p56lck was found to have enzymatic characteristics indistinguishable from those of the native enzyme expressed in the Jurkat human T lymphocytic cell line. The comparisons of recombinant and native p56lck were performed using an assay in which the enzyme was immobilized with antibodies onto the wells of a microtiter plate, enabling in situ purification of the native enzyme. Further characterization of the purified recombinant p56lck revealed significantly different enzyme concentration curves for peptide phosphorylation by immobilized and soluble p56lck. In particular, there was very low activity at low concentrations of enzyme assayed in solution. The activity at low enzyme concentrations could be substantially increased by preincubation of high concentrations of enzyme with ATP prior to dilution for the peptide phosphorylation reaction. We examined the relationship between autophosphorylation and activation for peptide phosphorylation. As for peptide phosphorylation, nonlinear enzyme concentration curves were also observed for autophosphorylation in solution, with very low levels of autophosphorylation at low enzyme concentrations. There was a correlation between the enzyme concentration dependency for autophosphorylation and for preincubation with ATP for maximal enhancement of subsequent peptide phosphorylation. The results suggest that autophosphorylation activates p56lck for phosphorylation of exogenous substrates and that high enzyme concentrations accelerate intermolecular autophosphorylation and enzyme activation.
Department of Immunology, Sterling Winthrop, Inc., Collegeville, Pennsylvania 19426.
S
S
Wang
R
R
Lehr
P
P
Stevis
X M
XM
Wang
J
J
Trevillyan
C R
CR
Faltynek
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D004789:Enzyme Activation; D006801:Humans; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008214:Lymphocytes / Q000201:enzymology; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)