T cell antigen CD28 binds to the GRB‐2/SOS complex, regulators of p21ras

Abstract

The T cell molecule CD28 provides a co‐stimulatory signal that is required for T cell proliferation, and has been implicated in the control of T cell anergy. An important clue to the signaling mechanism of CD28 is the finding that CD28 can bind to phosphatidylinositol 3‐kinase (PI 3‐kinase) by means of a cytoplasmic phospho‐YMNM (pYMNM) motif. A remaining issue concerns whether CD28 can recruit other intracellular signaling molecules. In this study, we show that CD28 uses the same pYMNM motif to recruit a second intracellular protein, GRB‐2. CD28‐associated GRB‐2, as detected by anti‐GRB‐2 immunoblotting, was found in human peripheral T cells, HPB‐ALL and Jurkat cells. As in the case of PI 3‐kinase, antibody‐induced cross‐linking of CD28 induces a time‐dependent recruitment of GRB‐2. Likewise, mutation of the pY‐191 residue within the pYMNM motif reduces GRB‐2 binding. Peptide binding studies show that the SH2 domain of GRB‐2 binds to the pYMNM motif with an affinity comparable to GRB‐2/SHC, but some 10‐ to 100‐fold lower than the CD28/PI 3‐kinase. Despite this, CD28/GRB‐2 and CD28/PI 3‐kinase complexes are found to co‐exist in peripheral T cells. Finally, immunoblotting shows that CD28 also associates with the gene product of the human homolog of the Drosophila Son of sevenless gene (SOS), a GRB‐2‐complexed guanine nucleotide exchange factor responsible for converting p21^ras to a GTP‐bound active state. CD28‐associated GRB2/SOS is likely to serve an important link in the regulation of p21^ras and lymphokine expression mediated by CD28.