MOLECULAR ANALYSIS OF RAS ACTIVATION BY TYROSINE-PHOSPHORYLATED VAV

Vav has been shown to activate Ras (1-3) and is regulated by tyrosine phosphorylation (1) or binding of diglycerides (3) to the cysteine ric h domain. In the present study employing different Ras activation assa y techniques using [H-3]GDP release or [P-32]alpha GTP-binding from me mbrane-bound or soluble recombinant Ras, we demonstrate that Ras activ ity can be increased by tyrosine phosphorylated Vav upon cellular stim ulation via the IL-2 receptor or the TCR/CD3-complex. Increase of [P-3 2]GTP-binding to Ras catalyzed by phosphorylated Vav is similar to the activity of immunoprecipitated Sos. The activity of Vav measured by b inding of [P-32]alpha GTP to Ras was linear with respect to the concen tration of Vav protein used. To study molecular characteristics of thi s Vav-Ras interaction, we used several Ras mutants and demonstrate tha t Vav activity towards Ras depends on the integrity of the same or sim ilar domains as Ras activation by SDC 25 or CDC 25. (C) 1995 Academic Academic Press, Inc.