Role of the Lck Src homology 2 and 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-crosslinked with the TCR, and this increased phosphorylation is thought to he a mechanism by which T cell functions are augmented by CD4. Such enhanced tyrosine phosphorylation was originally attributed to the kinase activity of the CD4- associated tyrosine kinase Lck. However, it has been shown that CD4- associated Lck lacking the catalytic domain can enhance 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 domains in early T cell signaling. Following TCR-CD4 co-cross-linking, cells expressing a CD4-Lck full-length chimera showed enhanced tyrosine phosphorylation of many cellular proteins in a CD4-dependent manner. Surprisingly, cells expressing a CD4-Lck chimera lacking the catalytic domain (termed CD4-N32) also showed enhanced phosphorylation. This enhancement of phosphorylation required both the Src homology 2 (SH2) and SH3 domains of Lck. Lck has been postulated to dimerize through the SH2 and SH3 domains. In this way CD4-N32 may interact with endogenous Lck, 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 expressed in J.CaM1.6 cells lacking endogenous Lck, CD4-N32 failed to enhance tyrosine phosphorylation. Moreover, a Lck SH2 and SH3 domain fragment expressed as a glutathione S-transferase fusion protein associated with Lck when incubated with activated Jurkat T cell lysates, suggesting that the SH2 and SH3 domains of Lck can associate with endogenous full-length Lck upon activation. Thus, our data suggest that dimerization is an important mechanism of Lck function in T cell activation.