THE SH2 DOMAIN IS REQUIRED FOR STABLE PHOSPHORYLATION OF P56(LCK) AT TYROSINE-505, THE NEGATIVE REGULATORY SITE

The catalytic function of Src-related tyrosine protein kinases is repr essed by phosphorylation of a conserve carboxy-terminal tyrosine resid ue. Recent studies suggest that this inhibitory event is not the resul t of autophosphorylation but that it is mediated by another cytoplasmi c tyrosine protein kinase, termed p50csk. In this report, we have eval uated the processes regulating the extent of phosphorylation of the in hibitory carboxy-terminal tyrosine residue of p56lck, a lymphocyte-spe cific member of the Src family. By analyzing kinase-defective variants of p56lck expressed in mouse NIH 3T3 cells, we have found that the no ncatalytic Src homology 2 (SH2) domain, but not the SH3 sequence or th e sites of Lck myristylation and autophosphorylation, is necessary for stable phosphorylation at the carboxy-terminal tyrosine 505. Further studies in which Lck and Csk were coexpressed in S. cerevisiae indicat ed that the absence of the SH2 domain did not affect the ability of Cs k to phosphorylate p56lck at tyrosine 505. However, we observed that i ncubation of cells with the tyrosine phosphatase inhibitor pervanadate restored the tyrosine 505 phosphorylation of Lck polypeptides devoid of the SH2 motif. Additionally, the presence of the SH2 sequence prote cted tyrosine 505 from in vitro dephosphorylation by the hemopoietic t yrosine protein phosphatase CD45. Taken together, these findings raise d the possibility that the SH2 motif contributes to the physiological suppression of the catalytic function of p56lck at least in part throu gh its ability to stabilize phosphorylation at the inhibitory site.