To proliferate in response to foreign antigens T cells must receive both TCR signals and co-stimulatory signals. An important T cell co-stimulatory receptor is the CD28 molecule that additionally functions to protect T cells from antigen induced anergy and apoptosis. In addition to responses to foreign antigens T cells require CD28 signals to initiate harmful responses to self antigens in autoimmune disease. Therefore, an ability to specifically manipulate CD28 signals would represent an effective means of controlling these aberrant immune responses. To identify potential molecular targets for therapy the long-term aims of this proposal are to understand the nature of CD28 intracellular signaling pathways. Previous studies have determined that CD28 signaling involves recruitment of PI3-kinase and Grb-2 to the tyrosine phosphorylated CD28 cytoplasmic tail. However, there is evidence that other signaling molecules are recruited to the CD28 tail and that these molecules play important roles in CD28 signal transduction. Recently, additional CD28 binding proteins were identified. One is a previously un-described dual- specific phosphatase of the MAP kinase phosphatase family, MKP5, which binds the CD28 tail independent of tyrosine phosphorylation. Another is the GRAP adaptor protein, which is related to but distinct from Grb-2, and also binds only the phosphorylated CD28 tail. Preliminary evidence indicates that MKP5 functions as a negative regulator of CD28 signal transduction. In contrast, GRAP likely performs a positive role in CD28 signaling. Specifically, here it is proposed to use a variety of molecular genetic, biochemical and cellular techniques to 1) Confirm negative and positive signaling roles for MKP5 and GRAP respectively, and 2) Determine the precise function of these molecules in the CD28 signal transduction pathway. In addition, using a novel yeast-based genetic assay that has been developed in the laboratory, it is proposed to characterize other signaling intermediates that bind the phosphorylated CD28 cytoplasmic tail.
