The kinase-dependent function of Lck in T-cell activation requires an intact site for tyrosine autophosphorylation.
The cytoplasmic protein tyrosine kinase p56lck (Lck) has important signaling roles in T-cell development and activation. We have mutated the two known regulatory tyrosine residues of CD4-associated Lck and examined the effects on its kinase-dependent function in an antigen-specific CD4-dependent T-cell hybridoma. Substitution of phenylalanine for the negative regulatory tyrosine-505 within a CD4/Lck chimera resulted in a slightly increased response to antigen, whereas mutation of the major in vitro autophosphorylation site (tyrosine-394) completely abolished the kinase-dependent function of Lck. Even though its kinase activity was only slightly affected, the F394 mutant behaved similarly to a catalytically inactive chimeric protein. Cross-linking of the F505 mutant, but not of wild-type Lck or F394 mutants, resulted in tyrosine phosphorylation of multiple cellular proteins. Although the pattern of tyrosine phosphorylation resembled that observed upon T-cell receptor cross-linking, there was no induction of interleukin-2 synthesis upon cross-linking of the chimeric protein. These results suggest that the activity of the Lck kinase domain in vivo is controlled by dephosphorylation at the negative regulatory site and phosphorylation at the positive regulatory (autophosphorylation) site. Additionally, our data show that the specific kinase activity of Lck towards an artificial substrate need not correlate with its ability to phosphorylate cellular proteins or its biological function.
D000818:Animals; D015704:CD4 Antigens / Q000096:biosynthesis / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D007700:Kinetics; D008213:Lymphocyte Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000201:enzymology / Q000276:immunology*; D014162:Transfection; D014443:Tyrosine; D019061:src-Family Kinases / Q000096:biosynthesis / Q000378:metabolism*
D015704:CD4 Antigens; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019061:src-Family Kinases