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Endocytosis after insulin secretion plays a pivotal role in the regulation of insulin secretion in pancreatic β-cells. Our recent study suggested that EPI64, a GTPase activating protein for Rab27a, contributes to the regulation of glucose-induced endocytosis, which is mediated by the GDP-bound form of Rab27a. Here, we identified insulin receptor-related receptor (IRR) as an EPI64-interacting protein. Knockdown of IRR inhibited glucose-induced uptake of transferrin, a marker of endocytosis, translocation of the guanine-nucleotide-exchange factor ARNO to the plasma membrane, and generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3). These results suggest that IRR functions upstream of PIP3generation and controls endocytosis after insulin secretion.

The small GTPase Rho and mDia2, a Rho-regulated actin nucleator, function as critical regulators of cytokinesis in cultured cells. However, their involvement in cytokinesis during mammalian development remains unknown. Here, we generated mice deficient in mDia2 and examined the role of Rho signaling in cytokinesis during development. mDia2-deficient mice survive until embryonic day 11.5 (E11.5), exhibit severe anemia with multinucleate erythroblasts, and die in utero by E12.5. mDia2-deficient erythroid cells differentiate normally, though in a delayed manner, but exhibit cytokinesis failure with decreased accumulation of F-actin in the cleavage furrow during late differentiation from proerythroblasts. On the other hand, inactivation of Rho induces cytokinesis failure from the earlier progenitor stage. mDia2-deficient erythroblasts, however, are able to enucleate their nuclei. Our findings have thus revealed that mDia2 functions critically in cytokinesis in vivo during erythropoiesis and further suggest that the cytokinesis mechanism in development diverges downstream of Rho. They also demonstrate that cytokinesis and enucleation utilize different mechanisms.

Regulation of the actin cytoskeleton is crucial for cell morphology and migration. One of the key molecules that regulates actin remodeling is the small GTPase Rho. Rho shuttles between the inactive GDP-bound form and the active GTP-bound form, and works as a molecular switch in actin remodeling in response to both extra- and intra-cellular stimuli. Mammalian homolog of Diaphanous (mDia) is one of the Rho effectors and produces unbranched actin filaments. While Rho GTPases activate mDia, the mechanisms of how the activity of mDia is downregulated in cells remains largely unknown. In our recent paper, we identified Liprin-α as an mDia interacting protein and found that Liprin-α negatively regulates the activity of mDia in the cell by displacing it from the plasma membrane through binding to the DID-DD region of mDia. Here, we review these findings and discuss how Liprin-α regulates the Rho-mDia pathway and how the mDia-Liprin-α complex functions in vivo.

Dendritic cells (DCs) are essential for the initiation of acquired immune responses through antigen acquisition, migration, maturation, and T-cell stimulation. One of the critical mechanisms in this response is the process actin nucleation and polymerization, which is mediated by several groups of proteins, including mammalian Diaphanous-related formins (mDia). However, the role of mDia in DCs remains unknown. Herein, we examined the role of mDia1 (one of the isoforms of mDia) in DCs. Although the proliferation and maturation of bone marrow-derived DCs were comparable between control C57BL/6 and mDia1-deficient (mDia1−/−) mice, adhesion and spreading to cellular matrix were impaired in mDia1−/− bone marrow–derived DCs. In addition, fluorescein isothiocyanate-induced cutaneous DC migration to draining lymph nodes in vivo and invasive migration and directional migration to CCL21 in vitro were suppressed in mDia1−/− DCs. Moreover, sustained T-cell interaction and T-cell stimulation in lymph nodes were impaired by mDia1 deficiency. Consistent with this, the DC-dependent delayed hypersensitivity response was attenuated by mDia1-deficient DCs. These results suggest that actin polymerization, which is mediated by mDia1, is essential for several aspects of DC-initiated acquired immune responses.

Dendritic cells (DCs) are essential for the initiation of acquired immune responses through antigen acquisition, migration, maturation, and T-cell stimulation. One of the critical mechanisms in this response is the process actin nucleation and polymerization, which is mediated by several groups of proteins, including mammalian Diaphanous-related formins (mDia). However, the role of mDia in DCs remains unknown. Herein, we examined the role of mDia1 (one of the isoforms of mDia) in DCs. Although the proliferation and maturation of bone marrow-derived DCs were comparable between control C57BL/6 and mDia1-deficient (mDia1−/−) mice, adhesion and spreading to cellular matrix were impaired in mDia1−/−bone marrow–derived DCs. In addition, fluorescein isothiocyanate-induced cutaneous DC migration to draining lymph nodes in vivo and invasive migration and directional migration to CCL21 in vitro were suppressed in mDia1−/−DCs. Moreover, sustained T-cell interaction and T-cell stimulation in lymph nodes were impaired by mDia1 deficiency. Consistent with this, the DC-dependent delayed hypersensitivity response was attenuated by mDia1-deficient DCs. These results suggest that actin polymerization, which is mediated by mDia1, is essential for several aspects of DC-initiated acquired immune responses.

Diaphanous-related formin, mDia, is an actin nucleation/polymerization factor functioning downstream of the small GTPase Rho. Although Rho is critically involved in cytokinesis, it remains elusive how Rho effectors and other regulators of cytoskeletons work together to accomplish this process. Here we focused on mDia2, an mDia isoform involved in cytokinesis of NIH 3T3 cells, and analyzed mechanisms of its localization in cytokinesis. We found that targeting of mDia2 to the cleavage furrow requires not only its binding to RhoA but also its diaphanous-inhibitory domain (DID). We then performed pulldown assays using a fragment containing the latter domain as a bait and identified anillin as a novel mDia2 interaction partner. The anillin-binding is competitive with the diaphanous autoregulatory domain (DAD) of mDia2 in its autoinhibitory interaction. A series of RNA interference and functional rescue experiments has revealed that, in addition to the Rho GTPase-mediated activation, the interaction between mDia2 and anillin is required for the localization and function of mDia2 in cytokinesis.

Diaphanous-related formin, mDia, is an actin nucleation/polymerization factor functioning downstream of the small GTPase Rho. We found that, in addition to the Rho GTPase-mediated activation, the interaction between mDia2 and anillin is required for the localization and function of mDia2 in cytokinesis.

mDia proteins are mammalian homologues of Drosophiladiaphanous and belong to the formin family proteins that catalyze actin nucleation and polymerization. Although formin family proteins of nonmammalian species such as Drosophiladiaphanous are essential in cytokinesis, whether and how mDia proteins function in cytokinesis remain unknown. Here we depleted each of the three mDia isoforms in NIH 3T3 cells by RNA interference and examined this issue. Depletion of mDia2 selectively increased the number of binucleate cells, which was corrected by coexpression of RNAi-resistant full-length mDia2. mDia2 accumulates in the cleavage furrow during anaphase to telophase, and concentrates in the midbody at the end of cytokinesis. Depletion of mDia2 induced contraction at aberrant sites of dividing cells, where contractile ring components such as RhoA, myosin, anillin, and phosphorylated ERM accumulated. Treatment with blebbistatin suppressed abnormal contraction, corrected localization of the above components, and revealed that the amount of F-actin at the equatorial region during anaphase/telophase was significantly decreased with mDia2 RNAi. These results demonstrate that mDia2 is essential in mammalian cell cytokinesis and that mDia2-induced F-actin forms a scaffold for the contractile ring and maintains its position in the middle of a dividing cell.

mDia proteins are mammalian homologues of Drosophila diaphanous and belong to the formin family proteins that catalyze actin nucleation and polymerization. Although formin family proteins of nonmammalian species such as Drosophila diaphanous are essential in cytokinesis, whether and how mDia proteins function in cytokinesis remain unknown. Here we depleted each of the three mDia isoforms in NIH 3T3 cells by RNA interference and examined this issue. Depletion of mDia2 selectively increased the number of binucleate cells, which was corrected by coexpression of RNAi-resistant full-length mDia2. mDia2 accumulates in the cleavage furrow during anaphase to telophase, and concentrates in the midbody at the end of cytokinesis. Depletion of mDia2 induced contraction at aberrant sites of dividing cells, where contractile ring components such as RhoA, myosin, anillin, and phosphorylated ERM accumulated. Treatment with blebbistatin suppressed abnormal contraction, corrected localization of the above components, and revealed that the amount of F-actin at the equatorial region during anaphase/telophase was significantly decreased with mDia2 RNAi. These results demonstrate that mDia2 is essential in mammalian cell cytokinesis and that mDia2-induced F-actin forms a scaffold for the contractile ring and maintains its position in the middle of a dividing cell.

Citron-kinase (Citron-K) is a Rho effector working in cytokinesis. It is enriched in cleavage furrow, but how Rho mobilizes Citron-K remains unknown. Using anti-Citron antibody and a Citron-K Green Fluorescence Protein (GFP)-fusion, we monitored its localization in cell cycle. We have found: (1) Citron-K is present as aggregates in interphase cells, disperses throughout the cytoplasm in prometaphase, translocates to cell cortex in anaphase and accumulates in cleavage furrow in telophase; (2) Rho colocalizes with Citron-K in the cortex of ana- to telophase cells and the two proteins are concentrated in the cleavage furrow and to the midbody; (3) inactivation of Rho by C3 exoenzyme does not affect the dispersion of Citron-K in prometaphase, but prevented its transfer to the cell cortex, and Citron-K stays in association with the midzone spindles of C3 exoenzyme-treated cells. To clarify further the mechanism of the Rho-mediated transfer and concentration of Citron-K in cleavage furrow, we expressed active Val14RhoA in interphase cells expressing GFP-Citron-K. Val14RhoA expression transferred Citron-K to the ventral cortex of interphase cells, where it formed band-like structures in a complex with Rho. This structure was localized at the same plane as actin stress fibers, and they exclude each other. Disruption of F-actin abolished the band and dispersed the Citron-K-Rho-containing patches throughout the cell cortex. Similarly, in dividing cells, a structure composed of Rho and Citron-K in cleavage furrow excludes cortical actin cytoskeleton, and disruption of F-actin disperses Citron-K throughout the cell cortex. These results suggest that Citron-K is a novel type of a passenger protein, which is dispersed to the cytoplasm in prometaphase and associated with midzone spindles by a Rho-independent signal. Rho is then activated, binds to Citron-K and translocates it to cell cortex, where the complex is then concentrated in the cleavage furrow by the action of actin cytoskeleton beneath the equator of dividing cells.

mDia1 is a mammalian homolog of Drosophila diaphanous and works as an effector of the small GTPase Rho. It is a member of the formin homology (FH) proteins and contains the Rho-binding domain and an FH3 region in its N terminus, an FH1 region containing polyproline stretches in the middle and an FH2 region in the C terminus. Several lines of evidence indicate that mDia1 and diaphanous are essential in cytokinesis. mDia1 is present in a large amount in the cytoplasm of both interphase and mitotic cells. Using the instantaneous fixation method that preferentially extracts soluble components, we have analyzed localization of mDia1 in mitotic HeLa cells. Immunocytochemistry using polyclonal anti-mDia1 antibody revealed specific immunofluorescence localized to the mitotic spindle. This localization was seen from prophase to telophase. Western blot analysis also detected anti-mDia1 immunoreactivity in the mitotic spindle fraction isolated from mitotic HeLa cells. Consistently, expression of full-length mDia1 as a fusion protein with green fluorescence protein (GFP) revealed the GFP fluorescence again in the mitotic spindle in HeLa cells. Expression of GFP fusions of various truncated mutants of mDia1 identified that this localization is determined by a 173 amino acid-long sequence between the Rho-binding domain and the FH1 region, which contains the C-terminal part of the FH3 region. Point mutation analysis revealed that Leu434 and Leu455 in the FH3 region are essential in localization to the mitotic spindle. Neither electroporation of botulinum C3 exoenzyme nor microinjection of Val14RhoA into mitotic cells affected the localization of endogenous mDia1 to the mitotic spindle, suggesting that mDia1 localizes to the mitotic spindle independent of Rho activity. The present study has thus established the mDia1 localization in the mitotic spindle. This localization suggests a role of mDia1 in the spindle-cleavage furrow interaction during cell division.

The small GTPase Rho, which regulates cell shape, is thought to contribute to cytokinesis. Recently, Citron was characterized as a Rho target. This large protein contains a Ser/Thr kinase domain related to that of ROCK, another Rho effector. Both endogenous Citron and recombinant Citron localize to the cleavage furrow in dividing cells and to the midbody in post‐mitotic cells. Moreover, overexpression of Citron deleted from its C‐terminal sequence caused abnormal contractions specifically during cytokinesis, resulting in the formation of multinucleated cells. Cell shape, F‐actin, intermediate filaments, and microtubules appeared essentially normal in these cells during interphase. Thus, Citron is a Rho effector that appears to function during cytokinesis, modulating its contractile process. In brain, however, Citron is highly expressed in a subset of neurons as a brain‐specific isoform that lacks a kinase domain, Citron‐N. This protein accumulates in synapses and associates to the NMDA receptor via interaction with the adaptor protein PSD95, suggesting that the function of Citron is specialized in the neurons. Microsc. Res. Tech. 49:123–126, 2000. © 2000 Wiley‐Liss, Inc.

The small GTPase Rho, which regulates cell shape, is thought to contribute to cytokinesis. Recently, Citron was characterized as a Rho target. This large protein contains a Ser/Thr kinase domain related to that of ROCK, another Rho effector. Both endogenous Citron and recombinant Citron localize to the cleavage furrow in dividing cells and to the midbody in post-mitotic cells. Moreover, overexpression of Citron deleted from its C-terminal sequence caused abnormal contractions specifically during cytokinesis, resulting in the formation of multinucleated cells. Cell shape, F-actin, intermediate filaments, and microtubules appeared essentially normal in these cells during interphase. Thus, Citron is a Rho effector that appears to function during cytokinesis, modulating its contractile process. In brain, however, Citron is highly expressed in a subset of neurons as a brain-specific isoform that lacks a kinase domain, Citron-N. This protein accumulates in synapses and associates to the NMDA receptor via interaction with the adaptor protein PSD95, suggesting that the function of Citron is specialized in the neurons. Microsc. Res. Tech. 49:123–126, 2000. © 2000 Wiley-Liss, Inc.

LIM-kinase 1 (LIMK1) phosphorylates cofilin, an actin-depolymerizing factor, and regulates actin cytoskeletal reorganization. LIMK1 is activated by the small GTPase Rho and its downstream protein kinase ROCK. We now report the site of phosphorylation of LIMK1 by ROCK. In vitrokinase reaction revealed that the active forms of ROCK phosphorylated LIMK1 on the threonine residue and markedly increased its cofilin-phosphorylating activity. A LIMK1 mutant (T508A) with replacement of Thr-508 within the activation loop of the kinase domain by alanine was neither phosphorylated nor activated by ROCK. Replacement of Thr-508 by serine changed the ROCK-catalyzed phosphorylation residue from threonine to serine. A LIMK1 mutant with replacement of Thr-508 by two glutamates increased the kinase activity about 2-fold but was not further activated by ROCK. In addition, wild-type LIMK1, but not its T508A mutant, was activated by co-expression with ROCK in cultured cells. These results suggest that ROCK activates LIMK1 in vitroand in vivoby phosphorylation at Thr-508. Together with the recent finding that PAK1, a downstream effector of Rac, also activates LIMK1 by phosphorylation at Thr-508, these results suggest that activation of LIMK1 is one of the common targets for Rho and Rac to reorganize the actin cytoskeleton.

Tissue distribution and cellular localization of rhophilin, a 71 kDa Rho‐binding protein, were examined in mice. Rhophilin mRNA was highly expressed in adult testis, but was absent in the testis of W/WVmice deficient in germ cells. An anti‐rhophilin antibody detected a band of an expected size in sperm extracts, which was enriched in the tail fraction. Immunofluorescence analysis revealed two lines of striated staining running in parallel in the principal piece of the sperm tail. These results suggest that rhophilin is expressed in germ cells and localized in the fibrous sheath of the sperm tail.

Background:The Ras-related GTPase RhoA controls signalling processes required for cytoskeletal reorganisation, transcriptional regulation, and transformation. The ability of RhoA mutants to transform cells correlates not with transcription but with their ability to bind ROCK-I, an effector kinase involved in cytoskeletal reorganisation. We used a recently developed specific ROCK inhibitor, Y-27632, and ROCK truncation mutants to investigate the role of ROCK kinases in transcriptional activation and transformation.

p160ROCK is a protein serine/threonine kinase that binds to GTP-Rho and is activated by this binding. We have recently found that the expression of p160ROCK induces focal adhesions and stress fibres in HeLa cells, whereas a dominant-negative form of this kinase suppresses Rho-induced formation of these structures, suggesting that this kinase is a downstream target of Rho in this process [Ishizaki, Naito, Fujisawa, Maekawa, Watanabe, Saito and Narumiya (1997) FEBS Lett. 404, 118-124]. To find out the mode of action of p160ROCK, we developed immunoblotting with an anti-p160ROCK antibody and investigated the subcellular localization of p160ROCK during platelet aggregation. In resting human platelets, more than 90% of p160ROCK was present in the Triton X-100-soluble fraction. When platelets were stimulated with thrombin, approx. 10% of p160ROCK was translocated to the Triton X-100-insoluble fraction. This translocation was detected as early as 20 s after stimulation and reached a maximum at 5 min; it was suppressed by the addition of EDTA or an Arg-Gly-Asp-Ser peptide (RGDS), both of which inhibit integrin αIIbβ3-mediated platelet aggregation. Using [32P]Pi-loaded platelets, we found that p160ROCK was phosphorylated in response to stimulation by thrombin. This phosphorylation, however, was not affected by the addition of EDTA and RGDS. These results suggest that p160ROCK translocates to cytoskeleton in a manner dependent on integrin ligation and works in an early stage of cytoskeletal reorganization in thrombin-stimulated platelets.

Renaturation kinase assay was used to detect protein kinases activated by lysophosphatidic acid (LPA) in cultured rat 3Y1 fibroblasts. LPA activated several Ser/Thr protein kinases with apparent molecular weights of 145K, 85K, 64–65K (a doublet), and 60K (each named p145, p85, p64165 and p60, respectively) in addition to p43 mitogen activated protein (MAP)‐kinase. Experiments using pertussis toxin and botulinum C3 exoenzyme showed that p145, p85, and p64165 kinases were activated by a pertussis toxin‐insensitive rhop21‐dependent pathway and that the activation of MAP‐kinase was mediated by both the pertussis toxin‐sensitive rhop21‐independent and the pertussis toxin‐insensitive rhop21‐dependent pathways.

Up-regulation of the cAMP pathway by forskolin or α-melanocyte stimulating hormone induces melanocyte and melanoma cell differentiation characterized by stimulation of melanin synthesis and dendrite development. Here we show that forskolin-induced dendricity is associated to a disassembly of actin stress fibers. Since Rho controls actin organization, we studied the role of this guanosine triphosphate (GTP)-binding protein in cAMP-induced dendrite formation.Clostridium botulinumC3 exotransferase, which inhibits Rho, mimicked the effect of forskolin in promoting dendricity and stress fiber disruption, while the Escherichia colitoxin cytotoxic necrotizing factor-1 (CNF-1), which activates Rho and the expression of a constitutively active Rho mutant, blocked forskolin-induced dendrite outgrowth. In addition, overexpression of a constitutively active form of the Rho target p160 Rho-kinase (P160ROCK) prevented the dendritogenic effects of cAMP. Our results suggest that inhibition of Rho and of its target p160ROCKare required events for cAMP-induced dendrite outgrowth in B16 cells. Furthermore, we present evidence that Rho is involved in the regulation of melanogenesis. Indeed, Rho inactivation enhanced the cAMP stimulation of tyrosinase gene transcription and protein expression, while Rho constitutive activation impaired these cAMP-induced effects. This reveals that, in addition to controlling dendricity, Rho also participates in the regulation of melanin synthesis by cAMP.

© 1997 Federation of European Biochemical Societies.

ABSTRACTThe small GTPase RhoA plays a critical role in signaling pathways activated by serum-derived factors, such as lysophosphatidic acid (LPA), including the formation of stress fibers in fibroblasts and neurite retraction and rounding of soma in neuronal cells. Previously, we have shown that ectopic expression of v-Crk, an SH2/SH3 domain-containing adapter proteins, in PC12 cells potentiates nerve growth factor (NGF)-induced neurite outgrowth and promotes the survival of cells when NGF is withdrawn. In the present study we show that, when cultured in 15% serum or lysophosphatidic acid-containing medium, the majority of v-Crk-expressing PC12 cells (v-CrkPC12 cells) display a flattened phenotype with broad lamellipodia and are refractory to NGF-induced neurite outgrowth unless serum is withdrawn. v-Crk-mediated cell flattening is inhibited by treatment of cells with C3 toxin or by mutation in the Crk SH2 or SH3 domain. Transient cotransfection of 293T cells with expression plasmids for p160ROCK(Rho-associated coiled-coil-containing kinase) and v-Crk, but not SH2 or SH3 mutants of v-Crk, results in hyperactivation of p160ROCK. Moreover, the level of phosphatidylinositol-4,5-bisphosphate is increased in v-CrkPC12 cells compared to the levels in mutant v-Crk-expressing cells or wild-type cells, consistent with PI(4)P5 kinase being a downstream target for Rho. Expression of v-Crk in PC12 cells does not result in activation of Rac- or Cdc42-dependent kinases PAK and S6 kinase, demonstrating specificity for Rho. In contrast to native PC12 cells, in which focal adhesions and actin stress fibers are not observed, immunohistochemical analysis of v-CrkPC12 cells reveals focal adhesion complexes which are formed at the periphery of the cell and are connected to actin cables. The formation of focal adhesions correlates with a concomitant upregulation in the expression of focal adhesion proteins FAK, paxillin, α3-integrin, and a higher-molecular-weight form of β1-integrin. Our results indicate that v-Crk activates the Rho-signaling pathway and serves as a scaffolding protein during the assembly of focal adhesions in PC12 cells.

We recently identified a novel human protein kinase, p160 ROCK, as a putative downstream target of the small GTPase Rho. Using the human ROCK cDNA as a probe, we isolated cDNA of two distinct, highly related sequences from mouse libraries. One encoded a mouse counterpart of human ROCK (ROCK-I), and the other encoded a novel ROCK-related kinase (ROCK-II). Like ROCK/ROCK-I, ROCK-II also bound to GTP-Rho selectively. ROCK-I mRNA was ubiquitously expressed except in the brain and muscle, whereas ROCK-II mRNA was expressed abundantly in the brain, muscle, heart, lung and placenta. These results suggest that at least two ROCK isoforms are present in a single species and play distinct roles in Rho-mediated signalling pathways.

Clostridium botulinumC3 exoenzyme specifically ADP-ribosylates rho-p21in eukaryotic cells. Trp18and Glu173of this enzyme were substituted with other amino acids via site-directed mutagenesis. All substitutions at Glu173caused a significant reduction in affinity for NAD and diminished ADP-ribosyltransferase activity. On the other hand, the activity of enzymes with the substitution at Trp18remained intact. Swiss 3T3 cells treated with the enzyme with the Trp18substitution showed the typical morphologic changes of the C3 exoenzyme phenotype. In contrast, no changes were found in cells incubated with the Glu173-substituted enzyme. These results indicate that the Glu173residue of the C3 exoenzyme plays a key role in interacting with NAD and in expression of ADP-ribosyltransferase activity, which is essential for the phenotypic change by C3 exoenzyme treatment.

Using the yeast two hybrid system and overlay assays we identified a putative rho/raceffector, citron, which interacts with the GTP-bound forms of rhoand rac1, but not with cdc42. Extensive homologies to known proteins were not observed. This 183 kDa protein contains a C 6H 2zinc finger, a PH domain, and a long coiled-coil forming region including 4 leucine zippers and the rho/racbinding site. We recently identified three others putative rhoeffectors characterized by a common rhobinding motif. Citrondoes not share this motif and displays a distinctive protein organization, thus defining a separate class of rhopartners.

We recently identified a novel human protein kinase, p160 ROCK, as a putative downstream target of the small GTPase Rho. Using the human ROCK cDNA as a probe, we isolated cDNA of two distinct, highly related sequences from mouse libraries. One encoded a mouse counterpart of human ROCK (ROCK‐I), and the other encoded a novel ROCK‐related kinase (ROCK‐II). Like ROCK/ROCK‐I, ROCK‐II also bound to GTP‐Rho selectively. ROCK‐I mRNA was ubiquitously expressed except in the brain and muscle, whereas ROCK‐II mRNA was expressed abundantly in the brain, muscle, heart, lung and placenta. These results suggest that at least two ROCK isoforms are present in a single species and play distinct roles in Rho‐mediated signalling pathways.

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 vitrobinding 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 ~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) Science271, 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.

The small GTPase Rho regulates several actomyosin‐based cellular processes such as cell adhesion, cytokinesis and contraction. The biochemical mechanisms of these actions remain unknown. Recently, several GTP‐Rho binding proteins were isolated. Among them, p140mDia and p160ROCK appear to work as Rho effectors mediating its action on the cytoskeleton. p140mDia induces actin polymerization by recruiting an actin binding protein, profilin, to the site of Rho action. p160ROCK induces focal adhesions and stress fibers by activating integrin and clustering them by the use of myosin‐based contractility.

p160 ROCKis a serine/threonine protein kinase that binds selectively to GTP-Rho and is activated by this binding. To identify its function, we transfected HeLa cells with wild type and mutants of p160 ROCKand examined morphology of the transfected cells. Transfection with wild type and mutants containing the kinase domain and the coiled-coil forming region induced focal adhesions and stress fibers, while no induction was observed with a kinase-defective mutant or a mutant containing only the kinase domain. Furthermore, Rho-induced formation of focal adhesions and stress fibers was inhibited by co-expression of a mutant defective in both kinase and Rho-binding activities. Rho, however, still induced an increase in F-actin content in these cells. These results suggest that p160 ROCKworks downstream of Rho to induce formation of focal adhesions and that Rho-induced actin polymerization is mediated by other effector(s).

ABSTRACTThe small GTPase RhoA plays a critical role in signaling pathways activated by serum-derived factors, such as lysophosphatidic acid (LPA), including the formation of stress fibers in fibroblasts and neurite retraction and rounding of soma in neuronal cells. Previously, we have shown that ectopic expression of v-Crk, an SH2/SH3 domain-containing adapter proteins, in PC12 cells potentiates nerve growth factor (NGF)-induced neurite outgrowth and promotes the survival of cells when NGF is withdrawn. In the present study we show that, when cultured in 15% serum or lysophosphatidic acid-containing medium, the majority of v-Crk-expressing PC12 cells (v-CrkPC12 cells) display a flattened phenotype with broad lamellipodia and are refractory to NGF-induced neurite outgrowth unless serum is withdrawn. v-Crk-mediated cell flattening is inhibited by treatment of cells with C3 toxin or by mutation in the Crk SH2 or SH3 domain. Transient cotransfection of 293T cells with expression plasmids for p160ROCK(Rho-associated coiled-coil-containing kinase) and v-Crk, but not SH2 or SH3 mutants of v-Crk, results in hyperactivation of p160ROCK. Moreover, the level of phosphatidylinositol-4,5-bisphosphate is increased in v-CrkPC12 cells compared to the levels in mutant v-Crk-expressing cells or wild-type cells, consistent with PI(4)P5 kinase being a downstream target for Rho. Expression of v-Crk in PC12 cells does not result in activation of Rac- or Cdc42-dependent kinases PAK and S6 kinase, demonstrating specificity for Rho. In contrast to native PC12 cells, in which focal adhesions and actin stress fibers are not observed, immunohistochemical analysis of v-CrkPC12 cells reveals focal adhesion complexes which are formed at the periphery of the cell and are connected to actin cables. The formation of focal adhesions correlates with a concomitant upregulation in the expression of focal adhesion proteins FAK, paxillin, a3-integrin, and a higher-molecular-weight form of ß1-integrin. Our results indicate that v-Crk activates the Rho-signaling pathway and serves as a scaffolding protein during the assembly of focal adhesions in PC12 cells.

Using the yeast two hybrid system and overlay assays we identified a putative rho/raceffector, citron, which interacts with the GTP‐bound forms of rhoand rac1, but not with cdc42. Extensive homologies to known proteins were not observed. This 183 kDa protein contains a C6H2zinc finger, a PH domain, and a long coiled‐coil forming region including 4 leucine zippers and the rho/racbinding site. We recently identified three others putative rhoeffectors characterized by a common rhobinding motif. Citrondoes not share this motif and displays a distinctive protein organization, thus defining a separate class of rhopartners.

Clostridium botulinumC3 exoenzyme specifically ADP‐ribosylates rho‐p21in eukaryotic cells. Trp18and Glu173of this enzyme were substituted with other amino acids via site‐directed mutagenesis. All substitutions at Glu173caused a significant reduction in affinity for NAD and diminished ADP‐ribosyltransferase activity. On the other hand, the activity of enzymes with the substitution at Trp18remained intact. Swiss 3T3 cells treated with the enzyme with the Trp18substitution showed the typical morphologic changes of the C3 exoenzyme phenotype. In contrast, no changes were found in cells incubated with the Glu173‐substituted enzyme. These results indicate that the Glu173residue of the C3 exoenzyme plays a key role in interacting with NAD and in expression of ADP‐ribosyltransferase activity, which is essential for the phenotypic change by C3 exoenzyme treatment.