Cell-cell adhesion-mediated tyrosine phosphorylation of nectin-2delta, an immunoglobulin-like cell adhesion molecule at adherens junctions.

We have recently found a novel functional unit of cell-cell adhesion at cadherin-based adherens junctions, consisting of at least nectin, an immunoglobulin-like cell adhesion molecule, and afadin, an actin filament-binding protein which connects nectin to the actin cytoskeleton. Among the members of the nectin family, we have found here that nectin-2delta is tyrosine-phosphorylated in response to cell-cell adhesion. Expression of E-cadherin induced tyrosine phosphorylation of nectin-2delta, while disruption of cell-cell adhesion by an anti-E-cadherin antibody reduced the tyrosine phosphorylation of nectin-2delta. An inhibitor specific for Src family kinase or expression of Csk reduced tyrosine phosphorylation of nectin-2delta. In addition, Src kinase tyrosine phosphorylates the recombinant cytoplasmic region of nectin-2delta in vitro. The major tyrosine phosphorylation site of nectin-2delta was Tyr505 in the cytoplasmic region, because the mutant nectin-2delta, of which Tyr505 was replaced by Phe, showed a loss of tyrosine phosphorylation in vivo and in vitro. These results, together with our recent observations, indicate that the cadherin-catenin system and the nectin-afadin system are closely connected to each other. The cadherin-mediated cell-cell adhesion system may link to the activation of a Src family kinase, that is, at least in part, responsible for the tyrosine phosphorylation of the cytoplasmic region of nectin-2delta. Oncogene (2000) 19, 4022 - 4028.

Involvement of an SHP-2-Rho small G protein pathway in hepatocyte growth factor/scatter factor-induced cell scattering.

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.

An FH domain-containing Bnr1p is a multifunctional protein interacting with a variety of cytoskeletal proteins in Saccharomyces cerevisiae.

Proteins containing formin homology domains, FH1 and FH2, are involved in cytokinesis or establishment of cell polarity in a variety of organisms. Bni1p and Bnr1p are FH proteins and potential targets of the Rho family small GTP-binding proteins in S. cerevisiae. We have shown that Bnr1p is localized at the bud neck to interact with Hof1p, involved in cytokinesis. We report here that the overexpression of BNR1 causes a cytokinesis deficiency which is similar to the phenotypes of the septin mutants, including cdc3, cdc10, cdc11, and cdc12. The region required for the septin mutant phenotypes was mapped to Bnr1p (35-500), which coincided with the region required for the bud-neck localization. To further isolate a gene interacting with BNI1 or BNR1, a multicopy suppressor of the bni1 bnr1 mutant was isolated. This gene encoded Smy1p, a kinesin-related protein. Bnr1p, but not Bni1p, directly interacted with the C-terminal region of Smy1p. The Smy1p-interacting region of Bnr1p was mapped to a region containing the FH2 domain. Bnr1p also directly interacted with Bud6p, a novel actin-binding protein. Bnr1p is thus a multifunctional protein which interacts with the septin system, a microtubule-dependent motor protein, and the actin system, to regulate cytoskeletal functions in S. cerevisiae.

Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton in Saccharomyces cerevisiae.

The RHO1 gene encodes a yeast homolog of the mammalian RhoA protein. Rho1p is localized to the growth sites and is required for bud formation. We have recently shown that Bni1p is one of the potential downstream target molecules of Rho1p. The BNI1 gene is implicated in cytokinesis and the establishment of cell polarity in Saccharomyces cerevisiae but is not essential for cell viability. In this study, we screened for mutations that were synthetically lethal in combination with a bni1 mutation and isolated two genes. They were the previously identified PAC1 and NIP100 genes, both of which are implicated in nuclear migration in S. cerevisiae. Pac1p is a homolog of human LIS1, which is required for brain development, whereas Nip100p is a homolog of rat p150(Glued), a component of the dynein-activated dynactin complex. Disruption of BNI1 in either the pac1 or nip100 mutant resulted in an enhanced defect in nuclear migration, leading to the formation of binucleate mother cells. The arp1 bni1 mutant showed a synthetic lethal phenotype while the cin8 bni1 mutant did not, suggesting that Bni1p functions in a kinesin pathway but not in the dynein pathway. Cells of the pac1 bni1 and nip100 bni1 mutants exhibited a random distribution of cortical actin patches. Cells of the pac1 act1-4 mutant showed temperature-sensitive growth and a nuclear migration defect. These results indicate that Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton. Bni1p lacking the Rho-binding region did not suppress the pac1 bni1 growth defect, suggesting a requirement for the Rho1p-Bni1p interaction in microtubule function.

Interaction of Bnr1p with a novel Src homology 3 domain-containing Hof1p. Implication in cytokinesis in Saccharomyces cerevisiae.

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.

Rho1p-Bni1p-Spa2p interactions: implication in localization of Bni1p at the bud site and regulation of the actin cytoskeleton in Saccharomyces cerevisiae.

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 the bni1 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.

A domain for G protein coupling in carboxyl-terminal tail of rat angiotensin II receptor type 1A.

To delineate domains essential for Gq protein coupling in the C-terminal region (C-tail) of rat angiotensin II (Ang II) receptor type 1A (AT1A), we modified the putative cytosolic regions of the receptor by truncation or alanine substitution and determined resultant changes in the guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) effect on Ang II binding and inositol trisphosphate production by the agonist. Independently, we studied the effect of synthetic C-tail peptides (P-5) and its alanine substitution analogs on [35S]GTPgammaS binding to Gq. Effects of GTPgammaS on Ang II binding (shift to a low affinity form) and inositol trisphosphate production in the deletional mutant receptor 1-317 AT1A was similar to wild type AT1A, whereas in shorter C-terminal deletion mutants 1-309, 1-311, 1-312, 1-313 AT1A, and substitutional mutants Y312A, F313A, and L314A these activities were markedly reduced. The binding of [35S]GTPgammaS to Gq was promoted by the synthetic C-terminal peptide P-5 but not when mutated at Tyr312, Phe313, or Leu314. Results indicate that Tyr312, Phe313, and Leu314 in cytosolic carboxyl-terminal region of rat AT1A are essential for coupling and activation of Gq.

Mutagenesis and the molecular modeling of the rat angiotensin II receptor (AT1).

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.