Vav proteins are GDP/GTP exchange factors for Rho/Rac GTPases that are activated by tyrosine phosphorylation. These proteins activate Rac1, RhoG, and RhoA but not the highly related Cdc42 protein. At present, there is no available information to explain this substrate selectivity at the structural level. Here we show that the selection of Vav proteins substrates is achieved at two different levels. On one hand, Var proteins utilize some residues of the β2/β3 region of Rho/Rac GTPases (D49 and E54) to assure the specific binding to its substrate. In addition, these exchange factors need a second structural signal located in the β5 region of Rho/Rac proteins (residue K118) to promote proper GDP/GTP exchange. These results identify the amino acid residues that allow the discrimination of the Var family substrates from Cdc42 and, in addition, demonstrate that the activation of specific Rho/Rac GTPases by these GEFs requires two concatenated events, binding and subsequent enzyme reaction, whose specificities are determined by two separate regions of Rho proteins.

This work was supported by grants given to XR Bustelo by the National Cancer Institute (CA7373501), the Programa General del Conocimiento (PM99-0093; Spanish Ministry for Science and Technology), and the Programa FEDER (1FD97-2116; Spanish Ministry for Education).

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