MUTATIONAL ANALYSIS OF LCK IN CD45-NEGATIVE T-CELLS - DOMINANT ROLE OF TYROSINE-394 PHOSPHORYLATION IN KINASE-ACTIVITY

The CD45 tyrosine phosphatase has been reported to activate the src fa mily tyrosine kinases Lck and Fyn by dephosphorylating regulatory COOH -terminal tyrosine residues 505 and 528, respectively, However, recent studies with CD45(-) T-cell lines have found that despite the fact th at Lck and Fyn were constitutively hyperphosphorylated, the tyrosine k inase activity of both enzymes was actually increased, In the present study, phosphoamino acid analysis revealed that the increased phosphor ylation of Lck in CD45(-) YAC-1 T cells was restricted to tyrosine res idues, To understand the relationship between tyrosine phosphorylation and Lck kinase activity, CD45(-) YAC-1 cells were transfected with fo rms of Lck in which tyrosines whose phosphorylation is thought to regu late enzyme activity (Tyr-192, Tyr-394, Tyr-505, or both Tyr-394 and T yr-505) were replaced with phenylalanine. While the Y-to-F mutation at position 192 (192-Y-->F) had little effect, the 505-Y-->F mutation in creased enzymatic activity, In contrast, the 394-Y-->F mutation decrea sed the kinase activity to very low levels, an effect that the double mutation, 394-Y-->F and 505Y-->F, could not reverse. Phosphopeptide an alysis of tryptic digests of Lck from CD45(-) YAC-1 cells revealed tha t it is hyperphosphorylated on two tyrosine residues, Tyr-505 and, to a lesser extent, Tyr-394. The purified and enzymatically active intrac ellular portion of CD45 dephosphorylated Lck Tyr-394 in vitro, These r esults demonstrate that in addition to Tyr-505, CD45 can dephosphoryla te Tyr-394, and that in the absence of CD45 the hyperphosphorylation o f Tyr-394 can cause an increase in the kinase activity of Lck despite the inhibitory hyperphosphorylation of Tyr-505, Therefore, Lck kinase activity is determined by the balance of activating and inhibitory tyr osine phosphorylations that are, in turn, regulated by CD45.