The T cell antigen CD28 provides a co-stimulatory signal that regulates the T cell response to foreign antigen. Understanding the intracellular signalling mechanisms that mediate CD28 function is an issue of great importance. We have shown that CD28 binds to two intracellular signalling molecules, phosphatidylinositol 3-kinase(Pl 3-kinase) and GRB-2/SOS. Each mediate distinct and major pathways of sign transduction. PI 3-kinase generates D-3 inositol lipids, and is an essential component in signalling by various receptors. Similarly, GRB-2/SOS activate p21ras, a central component in the growth control of mammalian cells. Site-directs mutagenesis, peptide competition and binding studies demonstrated that PI kinase and GRB-2 bind to the same site wit cytoplasmic tail of CD28 define by the motif pYMNM. Despite this, both CD28-PI 3-kinase and CD28-GRB-2/SC complexes can be found in T-cells. CD28 is therefore endowed with the capacity to engage multiple signalling pathways in T-cells. In this application, we plan further our analysis. of the nature of CD28pYMNM mediated signalling by assessing CD28 phosphorylation sites, the influence of TcR/CD3-C 4 ligation, and the identification of the kinase responsible for phosphorylating CD28. We will also examine CTLA-4 and CD7 binding to these intracellular proteins, and whether CD7 can function as an alternate co-stimulatory antigen (Aims 1-3). We propose assess the roles played by PI 3-kinase and GRB-2 in mediating co-stimulation ar IL-2 production by generating mutants of CD28 with altered abilities to bind PI kinase or GRB-2 for use in the transfection of T-cell hybridomas and clones (Aim 4). Lastly, we propose to express CD28 signalling mutants in CD28-/- gene knockout mice in an effort to dissect the role of the different CD28 signalling pathways in T-cell immunity (Aim 5).
| Kim, H H; Tharayil, M; Rudd, C E (1998) Growth factor receptor-bound protein 2 SH2/SH3 domain binding to CD28 and its role in co-signaling. J Biol Chem 273:296-301 |
