Structural Requirements for Enhancement of T-Cell Responsiveness by the Lymphocyte-Specific Tyrosine Protein Kinase p56^lck

Abstract

To understand the mechanism(s) by which p56^lck participates in T-cell receptor (TCR) signalling, we have examined the effects of mutations in known regulatory domains of p56^lck on the ability of F505 p56^lck to enhance the responsiveness of an antigen-specific murine T-cell hybridoma. A mutation of the amino-terminal site of myristylation (glycine 2), which prevents stable association of p56^lck with the plasma membrane, completely abolished the ability of F505 p56^lck to enhance TCR-induced tyrosine protein phosphorylation. Alteration of the major site of in vitro autophosphorylation, tyrosine 394, to phenylalanine diminished the enhancement of TCR-induced tyrosine protein phosphorylation by F505 p56^lck. Such a finding is consistent with the previous demonstration that this site is required for full activation of p56^lck by mutation of tyrosine 505. Strikingly, deletion of the noncatalytic Src homology domain 2, but not of the Src homology domain 3, markedly reduced the improvement of TCR-induced tyrosine protein phosphorylation by F505 Lck. Additional studies revealed that all the mutations tested, including deletion of the Src homology 3 region, abrogated the enhancement of antigen-triggered interleukin-2 production by F505 p56^lck, thus implying more stringent requirements for augmentation of antigen responsiveness by F505 Lck. Finally, it was also observed that expression of F505 p56^lck greatly increased TCR-induced tyrosine phosphorylation of phospholipase C-γ₁, raising the possibility that phospholipase C-γ₁ may be a substrate for p56^lck in Τ lymphocytes. Our results indicate that p56^lck regulates T-cell antigen receptor signalling through a complex process requiring multiple distinct structural domains of the protein.