p56 super(Lck) and p59 super(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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 92; no. 19; pp. 8891 - 8895
• Main Authors: Raab, M, Cai, Yun-Cai, Bunnell, S C, Heyeck, S D, Berg, L J, Rudd, CE
• Format: Journal Article
• Language: English
• Published: 01.01.1995
• ISSN: 0027-8424

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 and 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 or 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 super(Lck), p59 super(Fyn), zeta -chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56 super(Lck) and p59 super(Fyn) phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducting 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 super(Lck) and is thus likely to act downstream of p56 super(Lck)/p59 super(Fyn) in a signaling cascade. p56 super(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.