ASSOCIATION OF P59(FYN) WITH THE T-LYMPHOCYTE COSTIMULATORY RECEPTOR CD2 - BINDING OF THE FYN SRC HOMOLOGY (SH)3 DOMAIN IS REGULATED BY THEFYN SH2 DOMAIN

Human CD2 is a 50-55-kDa cell surface receptor specifically expressed on the surface of T lymphocytes and NK cells. Stimulation of human per ipheral blood T cells with mitogenic pairs of anti-CD2 monoclonal anti bodies (mAbs) is sufficient to induce interleukin-a production and T c ell proliferation in the absence of an antigen-specific signal through the T cell receptor. CD2 has been shown previously to associate physi cally with the Src family protein-tyrosine kinases p56(lck) and p59(fy n). We now report that stimulation of T cells with mitogenic pairs of anti-CD2 mAbs enhanced the association of the Fyn polypeptide with the CD2 complex, whereas stimulation with single anti-CD2 mAb had minimal effect. Using glutathione S-transferase (GST) fusion proteins, we fou nd that CD2 bound to the Src homology (SH) 3 domain of Fyn. Interestin gly, the CD2-Fyn association was negatively regulated by the Fyn SH2 d omain; CD2 bound poorly to GST fusion proteins expressing both the SH2 and SH3 domains of Fyn. However, the inhibitory effect of the Fyn SH2 domain on binding of the Fyn SH3 domain to CD2 was relieved by peptid es containing a phosphorylated YEEI sequence that bound directly to th e Fyn SH2 domain. In addition, we found that the ability of the Fyn SH 2 domain to precipitate tyrosine-phosphorylated proteins, including th e CD3 zeta chain, was enhanced after T cell stimulation with mitogenic pairs of CD2 mAbs. Finally, overexpression of a mutated Fyn molecule, in which the ability of the Fyn SH2 domain to bind phosphotyrosine-co ntaining proteins was abrogated, inhibited CD2-induced transcriptional activation of the nuclear factor of activated T cells (NFAT), suggest ing a functional involvement of the Fyn SH2 domain in CD2-induced T ce ll signaling. We thus propose that stimulation through the CD2 recepto r leads to the tyrosine phosphorylation of intracellular proteins, inc luding CD3 zeta itself, which in turn bind to the Fyn-SH2 domain, allo wing the direct association of the Fyn SH3 domain with CD2 and the ini tiation of downstream signaling events.