The Aggregation of the T Cell Costimulatory Molecule CD28 Induces Tyrosine Phosphorylation of the Protein Tyrosine Kinase Pyk2.

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890 CD28 is a transmembrane protein found on most mature T cells. CD28 is critical for T cell activation; thus, mice lacking CD28 because of targeted gene disruption show significant immune system defects consistent with disrupted T cell function. Furthermore, CD28-initiated signals regulate T cell receptor (TCR)-mediated activation, as TCR-induced T cell proliferation and cytokine release are markedly reduced in the absence of CD28 ligation. Protein tyrosine kinases are critical for the function of CD28 in T cells. We examined whether the tyrosine kinases Pyk2 and Fak (members of the focal adhesion kinase family) are involved in CD28 signaling. Ligating CD28 with anti-CD28 mAb in Jurkat T cells rapidly increased the tyrosine phosphorylation of Pyk2 but not of Fak. The cytoskeletal protein paxillin, which is a substrate for Pyk2 and Fak, was not tyrosine phosphorylated after CD28 ligation. CD28-induced tyrosine phosphorylation of Pyk2 was markedly reduced in the absence of external Ca2+. T cell antigen receptor (TCR) has previously been shown to induce tyrosine phosphorylation of Pyk2. Here, the concurrent ligation of CD28 and TCR induced an additive increase in the phosphorylation of Pyk2. The Syk/Zap inhibitor piceatannol blocked CD28 and TCR induced tyrosine phosphorylation of Pyk2, suggesting that Syk/Zap are involved in Pyk2 phosphorylation. In contrast, the PI 3-kinase inhibitor wortmannin blocked TCR- but not CD28-induced phosphorylation of Pyk2; thus, CD28 and TCR activate distinct pathways to induce tyrosine phosphorylation of Pyk2. Notably, depleting PMA-sensitive PKC did not block CD28- or CD3-induced tyrosine phosphorylation of Pyk2. These data provide evidence for the involvement of Pyk2 in the CD28 signaling cascade and suggest that Pyk2 may be important for CD28-induced enhancement of TCR-mediated T cell activation.