Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p. This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity. The C-terminal 254 amino acid region of Spa2p, Spa2p(1213–1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826–987). Genetic analyses revealed that both thebni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.

Proteins containing the formin homology (FH) domains FH1 and FH2 are involved in cytokinesis or establishment of cell polarity in a variety of organisms. We have shown that the FH proteins Bni1p and Bnr1p are potential targets of the Rho family small GTP-binding proteins and bind to an actin-binding protein, profilin, at their proline-rich FH1 domains to regulate reorganization of the actin cytoskeleton in the yeast Saccharomyces cerevisiae. We found here that a novel Src homology 3 (SH3) domain-containing protein, encoded by YMR032w, interacted with Bnr1p in a GTP-Rho4p-dependent manner through the FH1 domain of Bnr1p and the SH3 domain of Ymr032wp. Ymr032wp weakly bound to Bni1p. Ymr032wp was homologous to cdc15p, which is involved in cytokinesis in Schizosaccharomyces pombe, and we named this gene HOF1 (homolog of cdc 15). Both Bnr1p and Hof1p were localized at the bud neck, and both the bnr1 and hof1 mutations showed synthetic lethal interactions with the bni1 mutation. The hof1 mutant cells showed phenotypes similar to those of the septin mutants, indicating that HOF1 is involved in cytokinesis. These results indicate that Bnr1p directly interacts with Hof1p as well as with profilin to regulate cytoskeletal functions in S. cerevisiae.The Rho family belongs to the small G protein superfamily and regulates various cell functions through reorganization of the actin cytoskeleton (for reviews, see Refs. 1 and 2). Many potential targets of Rho have been identified (for a review, see Ref.3), but it has not yet been thoroughly clarified how Rho regulates reorganization of the actin cytoskeleton through these targets.The actin cytoskeleton plays a pivotal role in the budding processes in the yeast Saccharomyces cerevisiae (for a review, see Ref. 4). This yeast has the Rho family members, including RHO1, RHO2, RHO3, RHO4, and CDC42, which are involved in the budding processes (for reviews, see Refs. 4 and 5). We have isolated BNI1 as a potential target of RHO1, which links RHO1 with the actin cytoskeleton (6). BNI1 has subsequently been shown to be a potential target of CDC42, RHO3, and RHO4 (7). BNR1 is a BNI1-related gene and is a potential target of RHO4 (8). Bni1p and Bnr1p are members of the FH 1 family of proteins, which are defined by the presence of two formin homology domains, the proline-rich FH1 domain and the FH2 domain. The FH proteins play an important role in the actin cytoskeleton-dependent processes, including cytokinesis and establishment of cell polarity (for reviews, see Refs. 9 and 10). We have recently shown that Bni1p interacts with elongation factor 1␣, which binds to and bundles actin filaments (11), and that Spa2p is required for the localization of Bni1p at the bud tip (12). Bni1p and Bnr1p, at their FH1 domains, bind to an actin monomer-binding protein, profilin, which is implicated in actin polymerization (7,8). A proline-rich sequence also interacts with an SH3 domain, which is found in a wide variety of proteins, ranging from cytoskeletal components to signal transducing enzymes (for a ...

Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering through the tyrosine kinase-type HGF/SF receptor c-Met. We have previously shown that Rho small G protein (Rho) is involved in the HGF/SF-induced scattering of Madin-Darby canine kidney (MDCK) cells by regulating at least the assembly and disassembly of stress fibers and focal adhesions, but it remains unknown how c-Met regulates Rho activity. We have found here a novel signaling pathway of c-Met consisting of SHP-2-Rho that regulates the assembly and disassembly of stress fibers and focal adhesions in MDCK cells. SHP-2 is a protein-tyrosine phosphatase that contains src homology-2 domains. Expression of a dominant negative mutant of SHP-2 (SHP-2-C/S) markedly increased the formation of stress fibers and focal adhesions in MDCK cells and inhibited their scattering. C3, a Clostridium botulinum ADP-ribosyltransferase, and Y-27632, a specific inhibitor for ROCK, reversed the stimulatory effect of SHP-2-C/S on stress fiber formation and the inhibitory effect on cell scattering. Vav2 is a GDP/GTP exchange protein for Rho. Expression of a dominant negative mutant of Vav2 blocked the stimulatory effect of SHP-2-C/S on stress fiber formation. Conversely, expression of mutants of Vav2 that increased stress fiber formation inhibited HGF/SF-induced cell scattering. These results indicate that SHP-2 physiologically modulates the activity of Rho to form stress fibers and focal adhesions and thereby regulates HGF/SF-induced cell scattering. In addition, Vav2 may be involved in the SHP-2-Rho pathway. INTRODUCTIONCell migration is a crucial process required during a variety of biological phenomena, including normal embryonic development, wound healing, inflammatory responses, and metastasis. Hepatocyte growth factor/ scatter factor (HGF/SF) is known to induce cell migration in many types of cultured cells, including Madin-Darby canine kidney (MDCK) cells (Stoker and Gherardi, 1991). These stimulatory effects of HGF/SF are mediated through the HGF/SF receptor c-Met, which possesses tyrosine kinase activity (Bottaro et al., 1991;Naldini et al., 1991;Schlessinger, 1994). The tyrosine kinase activity of c-Met is essential for cellular responses to HGF/SF (Weidner et al., 1993;Zhu et al., 1994). Activation of c-Met tyrosine kinase induces the rapid autophosphorylation of c-Met itself as well as tyrosine phosphorylation of several cytoplasmic proteins (Rodrigues and Park, 1994;Nguyen et al., 1997).Recent studies have shown that cyclical inactivation and activation of the Rho small G protein (Rho) are involved in HGF/SF-induced cell scattering. Expression of a dominant active mutant of Rho inhibits HGF/SF-induced cell scattering (Ridley et al., 1995;Imamura et al., 1998;Kamei et al., 1999), whereas C3, a Clostridium botulinum ADP-ribosyltransferase that inhibits Rho function, or Rho GDP dissociation inhibitor, which inhibits Rho activation, blocks HGF/ SF-induced cell scattering (Takaishi et al., 1993(Takaishi et al., , 1994. The mode of action of Rho in cell sca...

The molecular interaction involved in the ligand binding of the rat angiotensin II receptor (AT1A) was studied by site-directed mutagenesis and receptor model building. The three-dimensional structure of AT1A was constructed on the basis of a multiple amino acid sequence alignment of seven transmembrane domain receptors and angiotensin II receptors and after the beta 2 adrenergic receptor model built on the template of the bacteriorhodopsin structure. These data indicated that there are conserved residues that are actively involved in the receptor-ligand interaction. Eleven conserved residues in AT1, His166, Arg167, Glu173, His183, Glu185, Lys199, Trp253, His256, Phe259, Thr260, and Asp263, were targeted individually for site-directed mutation to Ala. Using COS-7 cells transiently expressing these mutated receptors, we found that the binding of angiotensin II was not affected in three of the mutations in the second extracellular loop, whereas the ligand binding affinity was greatly reduced in mutants Lys199-->Ala, Trp253-->Ala, Phe259-->Ala, Asp263-->Ala, and Arg167-->Ala. These amino acid residues appeared to provide binding sites for Ang II. The molecular modeling provided useful structural information for the peptide hormone receptor AT1A. Binding of EXP985, a nonpeptide angiotensin II antagonist, was found to be involved with Arg167 but not Lys199.

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