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1.  GAP1 Family Members Constitute Bifunctional Ras and Rap GTPase-activating Proteins* 
The Journal of biological chemistry  2006;281(15):9891-9900.
GAP1IP4BP is a member of the GAP1 family of Ras GTPase-activating proteins (Ras GAPs) that includes GAP1m, CAPRI, and RASAL. Composed of a central Ras GAP domain, surrounded by amino-terminal C2 domains and a carboxyl-terminal pleckstrin homology/Bruton’s tyrosine kinase domain, GAP1IP4BP has previously been shown to possess an unexpected GAP activity on the Ras-related protein Rap, besides the predicted Ras GAP activity (Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P., and Irvine, R. F. (1995) Nature 376, 527–530). Here we have shown that GAP1IP4BP is indeed an efficient Ras/Rap GAP, having Kms of 213 and 42 μM and estimated kcats of 48 and 16 s−1 for Ras and Rap, respectively. For this dual activity, regions outside the Ras GAP domain are required, as the isolated domain (residues 291–569) retains a pronounced Ras GAP activity yet has very low activity toward Rap. Interestingly, mutagenesis of the Ras GAP argi-nine finger, and surrounding residues important in Ras binding, inhibit both Ras and Rap GAP activity of GAP1IP4BP. Although the precise details by which GAP1IP4BP can function as a Rap GAP remain to be determined, these data are consistent with Rap associating with GAP1IP4BP through the Ras-binding site within the Ras GAP domain. Finally, we have established that such dual Ras/Rap GAP activity is not restricted to GAP1IP4BP. Although GAP1m appears to constitute a specific Ras GAP, CAPRI and RASAL display dual activity. For CAPRI, its Rap GAP activity is modulated upon its Ca2+-induced association with the plasma membrane.
doi:10.1074/jbc.M512802200
PMCID: PMC1904491  PMID: 16431904
2.  A new role of multi scaffold protein Liprin-α 
Bioarchitecture  2012;2(2):43-49.
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.
PMCID: PMC3383721  PMID: 22754629
Liprin; Rho; actin cytoskeleton; formin; mDia
3.  Deficiency of mDia, an Actin Nucleator, Disrupts Integrity of Neuroepithelium and Causes Periventricular Dysplasia 
PLoS ONE  2011;6(9):e25465.
During development of the central nervous system, the apical-basal polarity of neuroepithelial cells is critical for homeostasis of proliferation and differentiation of neural stem cells. While adherens junctions at the apical surface of neuroepithelial cells are important for maintaining the polarity, the molecular mechanism regulating integrity of these adherens junctions remains largely unknown. Given the importance of actin cytoskeleton in adherens junctions, we have analyzed the role of mDia, an actin nucleator and a Rho effector, in the integrity of the apical adherens junction. Here we show that mDia1 and mDia3 are expressed in the developing brain, and that mDia3 is concentrated in the apical surface of neuroepithelium. Mice deficient in both mDia1 and mDia3 develop periventricular dysplastic mass widespread throughout the developing brain, where neuroepithelial cell polarity is impaired with attenuated apical actin belts and loss of apical adherens junctions. In addition, electron microscopic analysis revealed abnormal shrinkage and apical membrane bulging of neuroepithelial cells in the remaining areas. Furthermore, perturbation of Rho, but not that of ROCK, causes loss of the apical actin belt and adherens junctions similarly to mDia-deficient mice. These results suggest that actin cytoskeleton regulated by Rho-mDia pathway is critical for the integrity of the adherens junctions and the polarity of neuroepithelial cells, and that loss of this signaling induces aberrant, ectopic proliferation and differentiation of neural stem cells.
doi:10.1371/journal.pone.0025465
PMCID: PMC3182227  PMID: 21980468
4.  mDia1 Targets v-Src to the Cell Periphery and Facilitates Cell Transformation, Tumorigenesis, and Invasion ▿ †  
Molecular and Cellular Biology  2010;30(19):4604-4615.
The small GTPase Rho regulates cell morphogenesis through remodeling of the actin cytoskeleton. While Rho is overexpressed in many clinical cancers, the role of Rho signaling in oncogenesis remains unknown. mDia1 is a Rho effector producing straight actin filaments. Here we transduced mouse embryonic fibroblasts from mDia1-deficient mice with temperature-sensitive v-Src and examined the involvement and mechanism of the Rho-mDia1 pathway in Src-induced oncogenesis. We showed that in v-Src-transduced mDia1-deficient cells, formation of actin filaments is suppressed, and v-Src in the perinuclear region does not move to focal adhesions upon a temperature shift. Consequently, membrane translocation of v-Src, v-Src-induced morphological transformation, and podosome formation are all suppressed in mDia1-deficient cells with impaired tyrosine phosphorylation. mDia1-deficient cells show reduced transformation in vitro as examined by focus formation and colony formation in soft agar and exhibit suppressed tumorigenesis and invasion when implanted in nude mice in vivo. Given overexpression of c-Src in various cancers, these findings suggest that Rho-mDia1 signaling facilitates malignant transformation and invasion by manipulating the actin cytoskeleton and targeting Src to the cell periphery.
doi:10.1128/MCB.00197-10
PMCID: PMC2950524  PMID: 20679479
5.  Rho and Anillin-dependent Control of mDia2 Localization and Function in Cytokinesis 
Molecular Biology of the Cell  2010;21(18):3193-3204.
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.
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.
doi:10.1091/mbc.E10-04-0324
PMCID: PMC2938385  PMID: 20660154
6.  mDia2 Induces the Actin Scaffold for the Contractile Ring and Stabilizes Its Position during Cytokinesis in NIH 3T3 Cells 
Molecular Biology of the Cell  2008;19(5):2328-2338.
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.
doi:10.1091/mbc.E07-10-1086
PMCID: PMC2366861  PMID: 18287523
7.  GAP1 Family Members Constitute Bifunctional Ras and Rap GTPase-activating Proteins* 
The Journal of biological chemistry  2006;281(15):9891-9900.
GAP1IP4BP is a member of the GAP1 family of Ras GTPase-activating proteins (Ras GAPs) that includes GAP1m, CAPRI, and RASAL. Composed of a central Ras GAP domain, surrounded by amino-terminal C2 domains and a carboxyl-terminal pleckstrin homology/Bruton’s tyrosine kinase domain, GAP1IP4BP has previously been shown to possess an unexpected GAP activity on the Ras-related protein Rap, besides the predicted Ras GAP activity (Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P., and Irvine, R. F. (1995) Nature 376, 527–530). Here we have shown that GAP1IP4BP is indeed an efficient Ras/Rap GAP, having Kms of 213 and 42 μM and estimated kcats of 48 and 16 s−1 for Ras and Rap, respectively. For this dual activity, regions outside the Ras GAP domain are required, as the isolated domain (residues 291–569) retains a pronounced Ras GAP activity yet has very low activity toward Rap. Interestingly, mutagenesis of the Ras GAP argi-nine finger, and surrounding residues important in Ras binding, inhibit both Ras and Rap GAP activity of GAP1IP4BP. Although the precise details by which GAP1IP4BP can function as a Rap GAP remain to be determined, these data are consistent with Rap associating with GAP1IP4BP through the Ras-binding site within the Ras GAP domain. Finally, we have established that such dual Ras/Rap GAP activity is not restricted to GAP1IP4BP. Although GAP1m appears to constitute a specific Ras GAP, CAPRI and RASAL display dual activity. For CAPRI, its Rap GAP activity is modulated upon its Ca2+-induced association with the plasma membrane.
doi:10.1074/jbc.M512802200
PMCID: PMC1904491  PMID: 16431904
8.  The Rho-mDia1 Pathway Regulates Cell Polarity and Focal Adhesion Turnover in Migrating Cells through Mobilizing Apc and c-Src†  
Molecular and Cellular Biology  2006;26(18):6844-6858.
Directed cell migration requires cell polarization and adhesion turnover, in which the actin cytoskeleton and microtubules work critically. The Rho GTPases induce specific types of actin cytoskeleton and regulate microtubule dynamics. In migrating cells, Cdc42 regulates cell polarity and Rac works in membrane protrusion. However, the role of Rho in migration is little known. Rho acts on two major effectors, ROCK and mDia1, among which mDia1 produces straight actin filaments and aligns microtubules. Here we depleted mDia1 by RNA interference and found that mDia1 depletion impaired directed migration of rat C6 glioma cells by inhibiting both cell polarization and adhesion turnover. Apc and active Cdc42, which work together for cell polarization, localized in the front of migrating cells, while active c-Src, which regulates adhesion turnover, localized in focal adhesions. mDia1 depletion impaired localization of these molecules at their respective sites. Conversely, expression of active mDia1 facilitated microtubule-dependent accumulation of Apc and active Cdc42 in the polar ends of the cells and actin-dependent recruitment of c-Src in adhesions. Thus, the Rho-mDia1 pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and delivering Apc/Cdc42 and c-Src to their respective sites of action.
doi:10.1128/MCB.00283-06
PMCID: PMC1592856  PMID: 16943426
9.  ROCK-I regulates closure of the eyelids and ventral body wall by inducing assembly of actomyosin bundles 
The Journal of Cell Biology  2005;168(6):941-953.
Rho-associated kinase (ROCK) I mediates signaling from Rho to the actin cytoskeleton. To investigate the in vivo functions of ROCK-I, we generated ROCK-I–deficient mice. Loss of ROCK-I resulted in failure of eyelid closure and closure of the ventral body wall, which gave rise to the eyes open at birth and omphalocele phenotypes in neonates. Most ROCK-I−/− mice died soon after birth as a result of cannibalization of the omphalocele by the mother. Actin cables that encircle the eye in the epithelial cells of the eyelid were disorganized and accumulation of filamentous actin at the umbilical ring was impaired, with loss of phosphorylation of the myosin regulatory light chain (MLC) at both sites, in ROCK-I−/− embryos. Stress fiber formation and MLC phosphorylation induced by EGF were also attenuated in primary keratinocytes from ROCK-I−/− mice. These results suggest that ROCK-I regulates closure of the eyelids and ventral body wall through organization of actomyosin bundles.
doi:10.1083/jcb.200411179
PMCID: PMC2171774  PMID: 15753128
10.  Targeted Disruption of the Mouse Rho-Associated Kinase 2 Gene Results in Intrauterine Growth Retardation and Fetal Death 
Molecular and Cellular Biology  2003;23(14):5043-5055.
Rho-associated kinase (ROCK), including the ROCK-I and ROCK-II isoforms, is a protein kinase involved in signaling from Rho to actin cytoskeleton. However, in vivo functions of each ROCK isoform remain largely unknown. We generated mice deficient in ROCK-II by gene targeting. ROCK-II−/− embryos were found at the expected Mendelian frequency until 13.5 days postcoitum, but approximately 90% died thereafter in utero. ROCK-II−/− mice of both genders that survived were born runts, subsequently developed without gross abnormality, and were fertile. Whole-mount staining for a knocked-in lacZ reporter gene revealed that ROCK-II was highly expressed in the labyrinth layer of the placenta. Disruption of architecture and extensive thrombus formation were found in the labyrinth layer of ROCK-II−/− mice. While no obvious alteration in actin filament structures was found in the labyrinth layer of ROCK-II−/− placenta and stress fibers were formed in cultured ROCK-II−/− trophoblasts, elevated expression of plasminogen activator inhibitor 1 was found in ROCK-II−/− placenta. These results suggest that ROCK-II is essential in inhibiting blood coagulation and maintaining blood flow in the endothelium-free labyrinth layer and that loss of ROCK-II leads to thrombus formation, placental dysfunction, intrauterine growth retardation, and fetal death.
doi:10.1128/MCB.23.14.5043-5055.2003
PMCID: PMC162229  PMID: 12832488
11.  ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts 
The Journal of Cell Biology  2002;157(5):819-830.
The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632–induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632–induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.
doi:10.1083/jcb.200112107
PMCID: PMC2173402  PMID: 12021256
Rho; mDia; Rac; membrane ruffles; Y-27632
12.  Focal Contacts as Mechanosensors 
The Journal of Cell Biology  2001;153(6):1175-1186.
The transition of cell–matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II–driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein–tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136–143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.
PMCID: PMC2192034  PMID: 11402062
adhesion-dependent signaling; cell contractility; GFP–vinculin; myosin II; Rho
13.  Molecular Dissection of the Rho-associated Protein Kinase (p160ROCK)-regulated Neurite Remodeling in Neuroblastoma N1E-115 Cells  
The Journal of Cell Biology  1998;141(7):1625-1636.
A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho–ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells.
PMCID: PMC2133015  PMID: 9647654
14.  Activation of Rho-Dependent Cell Spreading and Focal Adhesion Biogenesis by the v-Crk Adaptor Protein 
Molecular and Cellular Biology  1998;18(5):3044-3058.
The 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.
PMCID: PMC110683  PMID: 9566923
15.  Inhibition of Rho Is Required for cAMP-induced Melanoma Cell Differentiation 
Molecular Biology of the Cell  1998;9(6):1367-1378.
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 botulinum C3 exotransferase, which inhibits Rho, mimicked the effect of forskolin in promoting dendricity and stress fiber disruption, while the Escherichia coli toxin 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 p160ROCK are 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.
PMCID: PMC25356  PMID: 9614180

Results 1-15 (15)