TARGETING OF TIAM1 TO THE PLASMA-MEMBRANE REQUIRES THE COOPERATIVE FUNCTION OF THE N-TERMINAL PLECKSTRIN HOMOLOGY DOMAIN AND AN ADJACENT PROTEIN-INTERACTION DOMAIN

The Rho-like GTPases Cdc42, Rac, and Rho play key roles in the regulat ion of the actin cytoskeleton and are implicated in transcriptional ac tivation and cell transformation. We have previously identified the in vasion-inducing Tiam1 gene, which encodes an activator of Pac, In fibr oblasts, Tiam1 induces Rac-mediated membrane ruffling, which requires the N-terminal pleckstrin homology (PHn) domain. Here we show that thi s PHn domain is part of a protein interaction domain, which mediates m embrane localization of Tiam1. After subcellular fractionation, up to 50% of Tiam1 is recovered in the Triton X-100-insoluble high speed pel let that contains small protein complexes. The regions in Tiam1 that a re responsible for these protein interactions comprise the PHn domain, an adjacent putative coiled coil region (CC), and an additional flank ing region (Ex). Deletions in each of these regions abolish membrane l ocalization of Tiam1 and membrane ruffling, suggesting that they funct ion cooperatively. Indeed, only polypeptides encompassing the PHn-CC-E x region, and not the PHn-CC or the Ex region, localize at the membran e. These results indicate that the N-terminal PH domain is part of a l arger functional Tiam1 domain that mediates protein complex formation and membrane localization of Tiam1.