Rho small GTPase regulates cell morphology, adhesion and cytokinesis through the actin cytoskeleton. We have identified a protein, p140mDia, as a downstream effector of Rho. It is a mammalian homolog of Drosophila diaphanous, a protein required for cytokinesis, and belongs to a family of formin‐related proteins containing repetitive polyproline stretches. p140mDia binds selectively to the GTP‐bound form of Rho and also binds to profilin. p140mDia, profilin and RhoA are co‐localized in the spreading lamellae of cultured fibroblasts. They are also co‐localized in membrane ruffles of phorbol ester‐stimulated sMDCK2 cells, which extend these structures in a Rho‐dependent manner. The three proteins are recruited around phagocytic cups induced by fibronectin‐coated beads. Their recruitment is not induced after Rho is inactivated by microinjection of botulinum C3 exoenzyme. Overexpression of p140mDia in COS‐7 cells induced homogeneous actin filament formation. These results suggest that Rho regulates actin polymerization by targeting profilin via p140mDia beneath the specific plasma membranes.
We previously demonstrated that both Tiam1, an activator of Rac, and constitutively active V12Rac promote E-cadherin–mediated cell–cell adhesion in epithelial Madin Darby canine kidney (MDCK) cells. Moreover, Tiam1 and V12Rac inhibit invasion of Ras-transformed, fibroblastoid MDCK-f3 cells by restoring E-cadherin–mediated cell–cell adhesion. Here we show that the Tiam1/Rac-induced cellular response is dependent on the cell substrate. On fibronectin and laminin 1, Tiam1/Rac signaling inhibits migration of MDCK-f3 cells by restoring E-cadherin–mediated cell– cell adhesion. On different collagens, however, expression of Tiam1 and V12Rac promotes motile behavior, under conditions that prevent formation of E-cadherin adhesions. In nonmotile cells, Tiam1 is present in adherens junctions, whereas Tiam1 localizes to lamellae of migrating cells. The level of Rac activation by Tiam1, as determined by binding to a glutathione-S-transferase– PAK protein, is similar on fibronectin or collagen I, suggesting that rather the localization of the Tiam1/Rac signaling complex determines the substrate-dependent cellular responses. Rac activation by Tiam1 requires PI3-kinase activity. Moreover, Tiam1- but not V12Rac-induced migration as well as E-cadherin–mediated cell– cell adhesion are dependent on PI3-kinase, indicating that PI3-kinase acts upstream of Tiam1 and Rac.
Using a mouse embryo cDNA library, we conducted a two-hybrid screening to identify new partners for the small GTPase Rho. One clone obtained by this procedure contained a novel cDNA of 291 base pairs and interacted strongly with RhoA and RhoC, weakly with RhoB, and not at all with Rac1 and Cdc42Hs. Full-length cDNAs were then isolated from a mouse brain library. While multiple splicing variants were common, we identified three cDNAs with an identical open reading frame encoding a 61-kDa protein that we named rhotekin (from the Japanese "teki," meaning target). The N-terminal part of rhotekin, encoded by the initial cDNA and produced in bacteria as a glutathione S-transferase fusion protein, exhibited in vitro binding to 35S-labeled guanosine 5'-3-O-(thio)triphosphate-bound Rho, but not to Rac1 or Cdc42Hs in ligand overlay assays. In addition, this peptide inhibited both endogenous and GTPase-activating protein-stimulated Rho GTPase activity. The amino acid sequence of this region shares approximately 30% identity with the Rho-binding domains of rhophilin and a serine/threonine kinase, PKN, two other Rho target proteins that we recently identified (Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y., Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648). Thus, not only is rhotekin a novel partner for Rho, but it also belongs to a wide family of proteins that bear a consensus Rho-binding sequence at the N terminus. To our knowledge, this is the first conserved sequence for Rho effectors, and we have termed this region Rho effector motif class 1.
Protein geranylgeranyltransferase type-I (GGTase-I), one of two CaaX prenyltransferases, is an essential enzyme in eukaryotes. GGTase-I catalyzes C-terminal lipidation of >100 proteins, including many GTPbinding regulatory proteins. We present the ®rst structural information for mammalian GGTase-I, including a series of substrate and product complexes that delineate the path of the chemical reaction. These structures reveal that all protein prenyltransferases share a common reaction mechanism and identify speci®c residues that play a dominant role in determining prenyl group speci®city. This hypothesis was con®rmed by converting farnesyltransferase (15-C prenyl substrate) into GGTase-I (20-C prenyl substrate) with a single point mutation. GGTase-I discriminates against farnesyl diphosphate (FPP) at the product turnover step through the inability of a 15-C FPP to displace the 20-C prenyl-peptide product. Understanding these key features of speci®city is expected to contribute to optimization of anti-cancer and anti-parasite drugs.
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