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 p56lck participates in T-cell receptor (TCR) signalling, we have examined the effects of mutations in known regulatory domains of p56lck on the ability of F505 p56lck 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 p56lck with the plasma membrane, completely abolished the ability of F505 p56lck 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 p56lck. Such a finding is consistent with the previous demonstration that this site is required for full activation of p56lck 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 p56lck, thus implying more stringent requirements for augmentation of antigen responsiveness by F505 Lck. Finally, it was also observed that expression of F505 p56lck greatly increased TCR-induced tyrosine phosphorylation of phospholipase C-gamma 1, raising the possibility that phospholipase C-gamma 1 may be a substrate for p56lck in T lymphocytes. Our results indicate that p56lck regulates T-cell antigen receptor signalling through a complex process requiring multiple distinct structural domains of the protein.

The SH2 Domain is Required for Stable Phosphorylation of p561ck at Tyrosine 505, the Negative Regulatory Site

“…As previously reported (2,8), the electrophoretic mobility of the myristylation-defective mutant (A2R273 Lck; Fig. 3A, lane 2) was slower than that of R273 Lck (lane 1).…”

“…In contrast, ASH2.1R273 Lck (Fig. 3A, lane 3) and ASH3R273 Lck (lane 5) migrated faster, at approximately 42 and 51 kDa, respectively (8,46). The mobility of ASH2.3R273 Lck (Fig.…”

“…Mutation of lysine 273 within the putative phosphotransfer motif to arginine 273 (R273 Lck) was accomplished by using the oligonucleotide 5' TCAGACTCCTCACCGCC 3'. Lck variants carrying complete deletions of the SH3 (ASH3 Lck) and SH2 (ASH2.1 Lck) motifs were described previously (8,46). These deletions removed amino acids 67 to 122 and 122 to 234, respectively, of p56lck.…”