P56(LCK) AND P59(FYN) REGULATE CD28 BINDING TO PHOSPHATIDYLINOSITOL 3-KINASE, GROWTH-FACTOR RECEPTOR-BOUND PROTEIN GRB-2, AND T-CELL-SPECIFIC PROTEIN-TYROSINE KINASE-ITK - IMPLICATIONS FOR T-CELL COSTIMULATION

T-cell activation requires cooperative signals generated by the T-cell antigen receptor zeta-chain complex (TCR zeta-CD3) and the costimulatory antigen CD28, CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS), PI 3-kinase End GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains, The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28, Candidate kinases include p56(Lck), p59(Fyn), zeta-chain-associated 70-kDa protein (ZAP-70), and ITK, In this study, we demonstrate in coexpression studies that p56(Lck) and p59(Fyn) phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28, Phosphatase digestion of CD28 eliminated binding, In contrast to Src kinases, ZAP-70 and ITK failed to induce these events, Further, ITK binding to CD28 was dependent on the presence of p56(Lck) and is thus likely to act downstream of p56(Lck)/p59(Fyn) in a signaling Cascade, p56(Lck) is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.