Triggering of the T cell antigen receptor (TCR)/CD3 complex initiates a complex signaling cascade in which obligatory activation of receptor- coupled protein tyrosine kinases (PTKs) is the earliest identifiable event. Ras is stimulated by GDP-releasing factors (GRFs) that catalyze GDP/GTP exchange, leading to activation of Ras-linked downstream signals. Little is known, however, about the function and physiologic regulation of mammalian GRFs. We recently found that the hematopoietically expressed 95-kDa Vav protein is a TCR/CD3-coupled, Ras-specific GRF activated by PTK-and/or diglycerides. This is the first evidence for a molecularly defined mammalian GRF that is regulated in such a manner during cellular activation. The underlying working hypotheses that will be examine in this proposal are, first, that Vav plays a critical role in T cell activation as a GRF that couples receptor-associated PTK(s) to Ras and, regulation of the GRF activity of Vav by PTKs will be studied by mapping its tyrosine phosphorylation site(s), assessing their function by site-directed mutagenesis, and identifying the regulatory PTK(s) by several approaches, i.e., analyzing the tyrosine phosphorylation and GRF activity of Vav in mice expressing activated fyn or lck transgenes or lacking fyn, studying the association of Vav with, and its phosphorylation by, Src-(p56lck or p59fyn) and/or p72syk/p70zap-family PTKs and, finally, assessing the role of SH2 domains in these interactions. Vav immunoprecipitates or in vitro-translated complete or truncated forms of wild-type or mutated Vav activation pathway mediated directly by diglycerides via their putative binding to a cysteine-rich Vav domain found in known diglyceride-activated enzymes, e.g., protein kinase C. The significance and characteristics of this activation pathway will be studied by determining whether Vav is a phorbol ester/diglyceride receptor, defining the structure/function characteristics of the cysteine-rich domain by deletions or site-directed mutagenesis, and evaluating the resulting mutants for constitutive or inducible Ras GRF and transforming activities. The physiological significance of this pathway will be assessed using PTK-independent receptor systems linked to diglyceride second messenger formation. Third, Ras activity and downstream signaling events will be analyzed in transfected T cells expressing wild-type or inactive mutants of proto- or onco-vav. The following questions will be addressed: Can vav overexpression cause constitutive activation of Ras and other downstream signaling events, and by pass proximal PTK-dependent signals? Can overexpressed, mutationally inactivated Vav act as a transdominant inhibitory protein? The proposed studies are likely to elucidate the function and regulation of Vav in hematopoietic cell activation and growth. They will fill a critical gap in our knowledge regarding the regulation of Ras activity by GRFs, and the mechanisms that couples Ras to hematopoietic cell receptors and their surrogate, signal-transducing PTKs.
Showing the most recent 10 out of 30 publications
