ANTI-CD3 AND PHORBOL ESTER INDUCE DISTINCT PHOSPHORYLATED SITES IN THE SH2 DOMAIN OF P56(LCK)

p56lck is a protein tyrosine kinase of the Src family specifically exp ressed in T lymphocytes. Triggering of T cells with anti-CD3 or with p horbol 12-myristate 13-acetate (PMA) results in the appearance of slow er migrating forms (shift) of p56lck. To investigate the phosphorylati on sites on the shifted forms of p56lck and to assess the role of prot ein kinase C in this phosphorylation, Jurkat cells were treated with a selective inhibitor of this kinase (GF 109203X). This inhibitor compl etely reversed the shift induced by PMA but only partially reversed th e one induced after triggering with anti-CD3. To analyze the shift fur ther, p56lck was immunoprecipitated from in vivo labeled cells treated either with anti-CD3 or with PMA. Tryptic phosphopeptides were genera ted and analyzed by using a combination of thin layer chromatography, high reticulation polyacrylamide gel electrophoresis, reverse phase ch romatography, and phosphopeptide sequencing. We identified serine 158 as a newly phosphorylated site after PMA treatment and tyrosine 192 an d serine 194 in the major tryptic phosphopeptide obtained after anti-C D3 triggering. The three sites identified are located in the SH2 domai n of p56lck; this suggests that their phosphorylation may regulate the interaction with other proteins or with other internal domains in p56 lck.