Many cellular functions are regulated by integrins, which transduce signals from the extracellular environment to the interior of the cell. The manner by which integrin activation is coupled to phenotypic effects is not fully understood. Our laboratory has identified an integrin-induced pathway in hematopoietic cells which leads to activation of Vav, a guanine nucleotide exchange factor (GEF). Vav activates the smGTPase Rac, which induces lamellipodia formation and cell spreading and strongly enhances activation of many downstream signaling proteins like ERK and the P1-3 kinase target Akt. Vav is composed of multiple structural domains that mediate its GEF function and its binding interactions with many cellular proteins. Thus, Vav is able to both activate Rac and recruit signaling proteins to receptor complexes. Vav serves as a prototype to understand how a single protein can coordinate signals to proteins that regulate changes in the actin cytoskeleton and activate important intracellular signaling pathways. The purpose of this proposal is to determine which domains are required for linking Vav to integrins and its upstream kinase, Syk and which are required for activation of different downstream effectors. I propose to initially characterize mutants of Vav for the ability to transduce signals from integrins in a Chinese hamster ovary (CHO) cell model system. This model system will be used to identify the requirements for interaction of Vav with Syk, activation of Rac, MAP kinases, and Akt, phosphorylation of Cbl, and stimulation of cytoskeletal rearrangement. Wild type and mutant Vav proteins will also be tested for the ability to rescue defects in cell morphology, adhesion, migration, and phagocytosis in macrophages from Vav knockout mice. Studying the role of different Vav domains in signaling from integrin receptors will allow us to investigate how Vav is able to integrate signals from receptors to the actin cytoskeleton as well as many key cellular signaling pathways.
