2 DISTINCT INTRACYTOPLASMIC REGIONS OF THE T-CELL ADHESION MOLECULE CD28 PARTICIPATE IN PHOSPHATIDYLINOSITOL 3-KINASE ASSOCIATION

Through the interaction with its ligands, CD80/B7-1 and CD86/B7-2 or B 70, the human CD28 molecule plays a major functional role as a costimu lator of T cells along with the CD3-TcR complex, We and others have pr eviously reported that phosphatidylinositol 3-kinase inducibly associa tes with CD28. This association is mediated by the SH2 domains of the p85 adaptor subunit interacting with a cytoplasmic YMNM consensus moti f present in CD28 at position 173-176. Disruption of this binding site by site-directed mutagenesis abolishes CD28-induced activation events in a murine T-cell hybridoma transfected with human CD28 gene. Here w e show that the last 10 residues of the intracytoplasmic domain of CD2 8 (residues 193-202) are required for its costimulatory function, Thes e residues are involved in interleukin-2 secretion, p85 binding, and C D28-associated phosphatidylinositol 3-kinase activity. In contrast, th e CD28/CD80 interaction is unaffected by this deletion, as is the indu ction of other second messengers such as the rise in intracellular cal cium and tyrosine phosphorylation of CD28-specific substrates, Further more, we also demonstrate that, within these residues, the tyrosine at position 200 is involved in p85 binding, probably together with the s hort proline-rich motif present between residues 190 and 194 (PYAPP).