CD28 signal transduction: tyrosine phosphorylation and receptor association of phosphoinositide‐3 kinase correlate with Ca2+‐independent costimulatory activity

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Abstract
The interaction of CD28 with its counter‐receptor, B7, induces a cosignal in T cells required to prevent clonal anergy and to promote antigen‐dependent interleukin‐2 production. The molecular basis of the CD28 cosignal is not well understood but involves the activation of protein tyrosine kinase(s) (PTK). In this report we demonstrate that CD28 cross‐linking on Jurkat T leukemic cells causes the activation of at least two PTK pathways. A CD28‐induced, p56lck kinase‐independent pathway causes tyrosine‐phosphorylation of a 110‐kDa substrate while recruitment of p56lck kinase activity is apparently required for CD28‐induced tyrosine‐phosphorylation of 97‐ and 68‐kDa substrates as well as CD28‐induced increases in intracellular calcium. The tyrosine phosphorylation of p110, but not p97 or p68, correlated with CD28 calcium‐independent costimulatory activity. The pp110 molecule was tentatively identified as the catalytic subunit of phosphoinositide (PI)‐3 kinase based upon its coimmunoprecipitation with the p85 regulatory subunit of PI‐3 kinase. PI‐3 kinase protein and catalytic activity were found complexed with the CD28 receptor if the receptor was “activated” by cross‐linking on the surface of intact cells prior to detergent solubilization. The kinetics of association of PI‐3 kinase with the “activated” CD28 receptor was rapid, occurring within 30 s of receptor cross‐linking and was stable for at least 30 min. Analysis of the CD28 cytoplasmic peptide sequence revealed a putative PI‐3 kinase src homology 2 binding motif and CD28 tyrosine phosphorylation site, DYMNM. Tyrosine phosphorylation of CD28 was detected in pervanadate‐treated Jurkat B2.7 cells, but not untreated cells. Pervanadate‐induced tyrosine phosphorylation of CD28 correlated with receptor association of PI‐3 kinase in the absence of CD28 cross‐linking, suggesting that CD28 association with PI‐3 kinase uses a tyrosine phosphorylation‐dependent mechanism. These data provide a model for CD28 signal transduction and support a role for PI‐3 kinase in mediating the CD28 calcium‐independent, cyclosporin A‐insensitive costimulatory signal.