ROLE OF THE LCK SRC HOMOLOGY-2 AND HOMOLOGY-3 DOMAINS IN PROTEIN-TYROSINE PHOSPHORYLATION

Many protein tyrosine phosphorylation events that occur as a result of T cell receptor (TCR) stimulation are enhanced when CD4 is co-cross-l inked with the TCR, and this increased phosphorylation is thought to b e a mechanism by which T cell functions are augmented by CD4. Such enh anced tyrosine phosphorylation was originally attributed to the kinase activity of the CD4-associated tyrosine kinase Lck. However, it has b een shown that CD4-associated Lck lacking the catalytic domain can enh ance T cell functions, suggesting that the noncatalytic domains of Lck are also important in CD4 signaling. Using T cells expressing various CD4-Lck chimeric molecules, we assessed the role of different Lck dom ains in early T cell signaling, Following TCR-CD4 co-cross-linking, ce lls expressing a CD4-Lck full-length chimera showed enhanced tyrosine phosphorylation of many cellular proteins in a CD4-dependent manner. S urprisingly, cells expressing a CD4 Lck chimera lacking the catalytic domain (termed CD4-N32) also showed enhanced phosphorylation. This enh ancement of phosphorylation required both the Src homology 2 (SH2) and SH3 domains of Lck, Lck has been postulated to dimerize through the S H2 and SH3 domains. In this way CD4-N32 may interact with endogenous L ck, and although it lacks intrinsic kinase activity, it may be capable of enhancing phosphorylation through the associated full-length Lck. Consistent with this model, when CD4-Lck chimeric molecules were expre ssed in J.CaM1.6 cells lacking endogenous Lck, CD4-N32 failed to enhan ce tyrosine phosphorylation. Moreover, a Lck SH2 and SH3 domain fragme nt expressed as a glutathione S-transferase fusion protein associated with Lck when incubated with activated Jurkat T cell lysates, suggesti ng that the SH2 and SH3 domains of Lck can associate with endogenous f ull-length Lck upon activation, Thus, our data suggest that dimerizati on is an important mechanism of Lck function in T cell activation.