Control of intramolecular interactions between the pleckstrin homology andDb1 homology domains of Vav and Sos1 regulates Rac binding

Vav and Sos1 are Dbl family guanine nucleotide exchange factors, which acti vate Rho family GTPases in response to phosphatidylinositol 3-kinase produc ts. A pleckstrin homology domain adjacent to the catalytic Dbl homology dom ain via an unknown mechanism mediates the effects of phosphoinositides on g uanine nucleotide exchange activity. Here we tested the possibility that ph osphatidylinositol 3-kinase substrates and products control an interaction between the pleckstrin homology domain and the Dbl homology domain, thereby explaining the inhibitory effects of phosphatidylinositol 3-kinase substra tes and stimulatory effects of the products. Binding studies using isolated fragments of Vav and Sos indicate phosphatidylinositol 3-kinase substrate promotes the binding of the pleckstrin homology domain to the Dbl homology domain and blocks Rac binding to the DH domain, whereas phosphatidylinosito l 3-kinase products disrupt the Dbl homology/pleckstrin homology interactio ns and permit Rac binding. Additionally, Lck phosphorylation of Vav, a know n activating event, reduces the affinities between the Vav Dbl homology and pleckstrin homology domains and permits Rac binding, We also show Vav acti vation in cells, as monitored by phosphorylation of Vav, Vav association wi th phosphatidylinositol 3,4,5-trisphosphate, and Vav guanine nucleotide exc hange activity, is blocked by the phosphatidylinositol 3-kinase inhibitor w ortmannin, These results suggest the molecular mechanisms for activation of Vav and Sos1 require disruption of inhibitory intramolecular interactions involving the pleckstrin homology and Dbl homology domains.