MgcRacGAP regulates cortical activity through RhoA during cytokinesis

Although Rho GTPases regulate multiple cellular events, their role in cell division is still obscure. Here we show that expression of a GTPase-activating protein (GAP)-deficient mutant (R386A) of the Rho regulator MgcRacGAP induces abnormal cortical activity during cytokinesis in U2OS cells. Multiple large blebs were observed in cells expressing MgcRacGAP R386A from the onset of anaphase to the late stage of cell division. When mitotic blebbing was excessive, cytokinesis was inhibited, and cells with micronuclei were generated. It has been reported that blebbing is caused by abnormal cortical activity. The MgcRacGAP R386A-induced abnormal cortical activity was inhibited by the dominant negative form of RhoA, but not Rac1 or Cdc42. Moreover, expression of constitutively active RhoA also induced drastic cortical activity during cytokinesis. Unlike apoptotic blebbing, MgcRacGAP R386A-induced blebbing was not inhibited by the ROCK inhibitor Y-27632, suggesting that MgcRacGAP regulates cortical activity during cytokinesis through a novel signaling pathway. We propose that MgcRacGAP plays a pivotal role in cytokinesis by regulating cortical movement through RhoA.     

Identification of a mitotic Rac-GEF,Trio, that counteracts MgcRacGAP function during cytokinesis

The Rho GTPases RhoA and Rac1 function as master regulators of cytokinesis by controlling the actomyosin cytoskeleton. RhoA and Rac1 have to be respectively activated and inactivated at the division plane for cytokinesis to occur properly. The inactivation of Rac1 at the cleavage furrow is controlled by MgcRacGAP. However, the guanine-nucleotide exchange factor (GEF) that activates Rac1 during cell division remains unknown. Here, using a siRNA screening approach in HeLa cells, we identify Trio as a mitotic GEF of Rac1. We demonstrate that Trio controls Rac1 activation and subsequent F-actin remodeling in dividing cells. Moreover, Trio depletion specifically rescues the cytokinesis failure induced by MgcRacGAP depletion. Of importance, we demonstrate that this rescue is mediated by the Trio-Rac1 pathway, using GEF-dead mutants of Trio and a specific inhibitor of Rac1 activation by Trio. Overall this work identifies for the first time a GEF controlling Rac1 activation in dividing cells that counteracts MgcRacGAP function in cytokinesis.     

Phosphorylation by aurora B converts MgcRacGAP to a RhoGAP during cytokinesis

Cell division is finely controlled by various molecules including small G proteins and kinases/phosphatases. Among these, Aurora B, RhoA, and the GAP MgcRacGAP have been implicated in cytokinesis, but their underlying mechanisms of action have remained unclear. Here, we show that MgcRacGAP colocalizes with Aurora B and RhoA, but not Rac1/Cdc42, at the midbody. We also report that Aurora B phosphorylates MgcRacGAP on serine residues and that this modification induces latent GAP activity toward RhoA in vitro. Expression of a kinase-defective mutant of Aurora B disrupts cytokinesis and inhibits phosphorylation of MgcRacGAP at Ser387, but not its localization to the midbody. Overexpression of a phosphorylation-deficient MgcRacGAP-S387A mutant, but not phosphorylation-mimic MgcRacGAP-S387D mutant, arrests cytokinesis at a late stage and induces polyploidy. Together, these findings indicate that during cytokinesis, MgcRacGAP, previously known as a GAP for Rac/Cdc42, is functionally converted to a RhoGAP through phosphorylation by Aurora B.     

MgcRacGAP is involved in cytokinesis through associating with mitotic spindle and midbody

We have recently cloned a cDNA for a full-length form of MgcRacGAP. Here we show using anti-MgcRacGAP antibodies that, unlike other known GAPs for Rho family, MgcRacGAP localized to the nucleus in interphase, accumulated to the mitotic spindle in metaphase, and was condensed in the midbody during cytokinesis. Overexpression of an N-terminal deletion mutant resulted in the production of multinucleated cells in HeLa cells. This mutant lost the ability to localize in the mitotic spindle and midbody. MgcRacGAP was also found to bind alpha-, beta-, and gamma-tubulins through its N-terminal myosin-like domain. These results indicate that MgcRacGAP dynamically moves during cell cycle progression probably through binding to tubulins and plays critical roles in cytokinesis. Furthermore, using a GAP-inactive mutant, we have shown that the GAP activity of MgcRacGAP is required for cytokinesis, suggesting that inactivation of the Rho family of GTPases may be required for normal progression of cytokinesis.     

The RhoGAP domain of CYK-4 has an essential role in RhoA activation

Cytokinesis in animal cells is mediated by a cortical actomyosin-based contractile ring. The GTPase RhoA is a critical regulator of this process as it activates both nonmuscle myosin and a nucleator of actin filaments [1]. The site at which active RhoA and its effectors accumulate is controlled by the microtubule-based spindle during anaphase [2]. ECT-2, the guanine nucleotide exchange factor (GEF) that activates RhoA during cytokinesis, is regulated by phosphorylation and subcellular localization [3-5]. ECT2 localization depends on interactions with CYK-4/MgcRacGAP, a Rho GTPase-activating protein (GAP) domain containing protein [5, 6]. Here we show that, contrary to expectations, the Rho GTPase-activating protein (GAP) domain of CYK-4 promotes activation of RhoA during cytokinesis. Furthermore, we show that the primary phenotype caused by mutations in the GAP domain of CYK-4 is not caused by ectopic activation of CED-10/Rac1 and ARX-2/Arp2. However, inhibition of CED-10/Rac1 and ARX-2/Arp2 facilitates ingression of weak cleavage furrows. These results demonstrate that?a GAP domain can contribute to activation of a small GTPase. Furthermore, cleavage furrow ingression is sensitive to the balance of contractile forces and cortical tension.     

Collapsin response mediator protein switches RhoA and Rac1 morphology in N1E-115 neuroblastoma cells and is regulated by Rho kinase

The formation and directional guidance of neurites involves dynamic regulation of Rho family GTPases. Rac and Cdc42 promote neurite outgrowth, whereas Rho activation causes neurite retraction. Here we describe a role for collapsin response mediator protein (Crmp-2), a neuronal protein implicated in axonal outgrowth and a component of the semaphorin 3A pathway, in switching GTPase signaling when expressed in combination with either dominant active Rac or Rho. In neuroblastoma N1E-115 cells, co-expression of Crmp-2 with dominant active RhoA V14 induced Rac morphology, cell spreading and ruffling (and the formation of neurites). Conversely, co-expression of Crmp-2 with dominant active Rac1 V12 inhibited Rac morphology, and in cells already expressing Rac1 V12, Crmp-2 caused localized peripheral collapse, involving Rho (and Cdc42) activation. Rho kinase was a pivotal regulator of Crmp-2; Crmp-2 phosphorylation was required for Crmp-2/Rac1 V12 inhibition, but not Crmp-2/RhoA V14 induction, of Rac morphology. Thus Crmp-2, regulated by Rho kinase, promotes outgrowth and collapse in response to active Rho and Rac, respectively, reversing their usual morphological effects and providing a mechanism for dynamic modulation of growth cone guidance.     

RhoA inhibits the nerve growth factor-induced Rac1 activation through Rho-associated kinase-dependent pathway

The Rho family of small GTPases has been shown to be involved in the regulation of neuronal morphology, and Rac and Rho exert antagonistic actions in neurite formation. In this study, we have examined the cross-talk between Rac and Rho in relation to the nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. NGF induced a rapid activation of Rac1 and suppression of RhoA activity. Constitutively active RhoA, RhoA(V14), or constitutively active Galpha(12)-induced endogenous RhoA activation inhibited the NGF-induced Rac1 activation without any effect on the NGF-induced extracellular signal-regulated kinase activation. Moreover, Y-27632, an inhibitor of Rho-associated kinase, completely abolished the RhoA-induced down-regulation of the NGF-induced Rac1 activation. We also revealed that NGF induced a rapid recruitment of Rac1 to the cell surface protrusion sites and formed filamentous actin-rich protrusions. Activation of RhoA and Rho-associated kinase formed a thick ringlike structure of cortical actin filaments at the cell periphery and then inhibited the NGF-induced recruitment of Rac1 to protrusions. These results indicate that RhoA down-regulates the NGF- induced Rac1 activation through Rho-associated kinase, inhibiting the neurite formation.     

A Highlights from MBoC Selection: MgcRacGAP restricts active RhoA at the cytokinetic furrow and both RhoA and Rac1 at cell–cell junctions in epithelial cells

Localized activation of Rho GTPases is essential for multiple cellular functions, including cytokinesis and formation and maintenance of cell–cell junctions. Although MgcRacGAP (Mgc) is required for spatially confined RhoA-GTP at the equatorial cortex of dividing cells, both the target specificity of Mgc''s GAP activity and the involvement of phosphorylation of Mgc at Ser-386 are controversial. In addition, Mgc''s function at cell–cell junctions remains unclear. Here, using gastrula-stage Xenopus laevis embryos as a model system, we examine Mgc''s role in regulating localized RhoA-GTP and Rac1-GTP in the intact vertebrate epithelium. We show that Mgc''s GAP activity spatially restricts accumulation of both RhoA-GTP and Rac1-GTP in epithelial cells—RhoA at the cleavage furrow and RhoA and Rac1 at cell–cell junctions. Phosphorylation at Ser-386 does not switch the specificity of Mgc''s GAP activity and is not required for successful cytokinesis. Furthermore, Mgc regulates adherens junction but not tight junction structure, and the ability to regulate adherens junctions is dependent on GAP activity and signaling via the RhoA pathway. Together these results indicate that Mgc''s GAP activity down-regulates the active populations of RhoA and Rac1 at localized regions of epithelial cells and is necessary for successful cytokinesis and cell–cell junction structure.     

Role of Rho, Rac, and Rho-kinase in phosphorylation of myosin light chain, development of polarity, and spontaneous migration of Walker 256 carcinosarcoma cells

As previously shown, constitutive activation of the small GTPase Rho and its downstream target Rho-kinase is crucial for spontaneous migration of Walker carcinosarcoma cells. We now show that after treatment of cells with either the Rho inhibitor C3 exoenzyme or the Rho-kinase inhibitor Y-27632, constitutive myosin light chain (MLC) phosphorylation is significantly decreased, correlating with inhibition of cell polarization and migration. Transfection with a dominant-negative Rho-kinase mutant similarly inhibits cell polarization and MLC phosphorylation. Transfection with a dominant-active Rho-kinase mutant leads to significantly increased MLC phosphorylation, membrane blebbing, and inhibition of cell polarization. This Rho-kinase-induced membrane blebbing can be inhibited by Y-27632, ML-7, and blebbistatin. Unexpectedly, overactivation of RhoA has similar effects as its inhibition. Introduction of a bacterially expressed constitutively activated mutant protein (but not of wild-type RhoA) into the cells or transfection of cells with a constitutively active RhoA mutant both inhibit polarization and decrease MLC phosphorylation. Transfection of cells with constitutively active or dominant-negative Rac both abrogate polarity, and the latter inhibits MLC phosphorylation. Our findings suggest an important role of Rac, Rho/Rho-kinase, and MLCK in controlling myosin activity in Walker carcinosarcoma cells and show that an appropriate level of RhoA, Rac, and Rho-kinase activity is required to regulate cell polarity and migration.     

Human mitotic spindle-associated protein PRC1 inhibits MgcRacGAP activity toward Cdc42 during the metaphase

Although many proteins have been shown to participate in mitotic events, including cytokinesis, their specific roles and interactions remain unclear. A novel interaction of proteins is demonstrated in this report. Yeast two-hybrid screening using PRC1 (protein-regulating cytokinesis 1) cDNA, a human mitotic spindle-associated cyclin-dependent kinase (CDK) substrate, which is involved in cytokinesis, as bait was performed. Data show that the PRC1 bait bound to MgcRacGAP, which is a GTPase-activating protein (GAP) for the Rho family GTPases also involved in cytokinesis. In addition, the two proteins showed similar localization during the M phase. PRC1 was shown to bind to the COOH-terminal GAP-conserved domain of MgcRacGAP and to inhibit its GAP activity toward Cdc42. This binding and/or inhibition of MgcRacGAP GAP activity was found to depend on further binding of PRC1 to the basic region (125-285 amino acids) of MgcRacGAP. Furthermore, the basic region was phosphorylated with Aurora B kinase, and this phosphorylation prevented the inhibition of GAP activity by PRC1. Cells overexpressing a phosphorylation mimic mutant of MgcRacGAP exhibited an abnormality of spindle morphology in the metaphase. Cdc42 showed high activity and was localized to the mitotic spindles and centrosomes during the metaphase. We propose that PRC1 down-regulates the GAP activity of MgcRac-GAP during the metaphase and thereby contributes to the correct formation of the spindle.     

Aurora B but Not Rho/MLCK Signaling Is Required for Localization of Diphosphorylated Myosin II Regulatory Light Chain to the Midzone in Cytokinesis

Non-muscle myosin II is stimulated by monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC) further enhances the ATPase activity of myosin II. Phosphorylated MRLCs localize to the contractile ring and regulate cytokinesis as subunits of activated myosin II. Recently, we reported that 2P-MRLC, but not 1P-MRLC, localizes to the midzone independently of myosin II heavy chain during cytokinesis in cultured mammalian cells. However, the mechanism underlying the distinct localization of 1P- and 2P-MRLC during cytokinesis is unknown. Here, we showed that depletion of the Rho signaling proteins MKLP1, MgcRacGAP, or ECT2 inhibited the localization of 1P-MRLC to the contractile ring but not the localization of 2P-MRLC to the midzone. In contrast, depleting or inhibiting a midzone-localizing kinase, Aurora B, perturbed the localization of 2P-MRLC to the midzone but not the localization of 1P-MRLC to the contractile ring. We did not observe any change in the localization of phosphorylated MRLC in myosin light-chain kinase (MLCK)-inhibited cells. Furrow regression was observed in Aurora B- and 2P-MRLC-inhibited cells but not in 1P-MRLC-perturbed dividing cells. Furthermore, Aurora B bound to 2P-MRLC in vitro and in vivo. These results suggest that Aurora B, but not Rho/MLCK signaling, is essential for the localization of 2P-MRLC to the midzone in dividing HeLa cells.     

Regulation of Rho family GTPases by cell-cell and cell-matrix adhesion

Integrins and cadherins are transmembrane adhesion receptors that are necessary for cells to interact with the extracellular matrix or adjacent cells, respectively. Integrins and cadherins initiate signaling pathways that modulate the activity of Rho family GTPases. The Rho proteins Cdc42, Rac1, and RhoA regulate the actin cytoskeleton. Cdc42 and Rac1 are primarily involved in the formation of protrusive structures, while RhoA generates myosin-based contractility. Here we examine the differential regulation of RhoA, Cdc42, and Rac1 by integrin and cadherin signaling. Integrin and cadherin signaling leads to a decrease in RhoA activity and activation of Cdc42 and Rac1. When the normal RhoA suppression is antagonized or RhoA signaling is increased, cells exhibited impaired spreading on the matrix protein fibronectin and decreased cell-cell adhesion. Spreading on fibronectin and the formation of cell-cell adhesions is decreased in cells expressing dominant negative forms of Cdc42 or Rac1. These data demonstrate that integrins and cadherins regulate Rho proteins in a comparable manner and lead us to speculate that these changes in Rho protein activity participate in a feedback mechanism that promotes further cell-matrix or cell-cell interaction, respectively.     

Monocyte adhesion and spreading on human endothelial cells is dependent on Rho-regulated receptor clustering.

The GTPase Rho is known to mediate the assembly of integrin-containing focal adhesions and actin stress fibers. Here, we investigate the role of Rho in regulating the distribution of the monocyte-binding receptors E-selectin, ICAM-1, and VCAM-1 in human endothelial cells. Inhibition of Rho activity with C3 transferase or N19RhoA, a dominant negative RhoA mutant, reduced the adhesion of monocytes to activated endothelial cells and inhibited their spreading. Similar effects were observed after pretreatment of endothelial cells with cytochalasin D. In contrast, dominant negative Rac and Cdc42 proteins did not affect monocyte adhesion or spreading. C3 transferase and cytochalasin D did not alter the expression levels of monocyte-binding receptors on endothelial cells, but did inhibit clustering of E-selectin, ICAM-1, and VCAM-1 on the cell surface induced by monocyte adhesion or cross-linking antibodies. Similarly, N19RhoA inhibited receptor clustering. Monocyte adhesion and receptor cross-linking induced stress fiber assembly, and inhibitors of myosin light chain kinase prevented this response but did not affect receptor clustering. Finally, receptor clusters colocalized with ezrin/moesin/ radixin proteins. These results suggest that Rho is required in endothelial cells for the assembly of stable adhesions with monocytes via the clustering of monocyte-binding receptors and their association with the actin cytoskeleton, independent of stress fiber formation.     

MgcRacGAP interacts with cingulin and paracingulin to regulate Rac1 activation and development of the tight junction barrier during epithelial junction assembly

The regulation of Rho-family GTPases is crucial to direct the formation of cell–cell junctions and tissue barriers. Cingulin (CGN) and paracingulin (CGNL1) control RhoA activation in epithelial cells by interacting with RhoA guanidine exchange factors. CGNL1 depletion also inhibits Rac1 activation during junction assembly. Here we show that, unexpectedly, Madin–Darby canine kidney epithelial cells depleted of both CGN and CGNL1 (double-KD cells) display normal Rac1 activation and tight junction (TJ) formation, despite decreased junctional recruitment of the Rac1 activator Tiam1. The expression of the Rac1 inhibitor MgcRacGAP is decreased in double-KD cells, and the barrier development and Rac1 activation phenotypes are rescued by exogenous expression of MgcRacGAP. MgcRacGAP colocalizes with CGN and CGNL1 at TJs and forms a complex and interacts directly in vitro with CGN and CGNL1. Depletion of either CGN or CGNL1 in epithelial cells results in decreased junctional localization of MgcRacGAP but not of ECT2, a centralspindlin-interacting Rho GEF. These results provide new insight into coordination of Rho-family GTPase activities at junctions, since apical accumulation of CGN and CGNL1 at TJs during junction maturation provides a mechanism to spatially restrict down-regulation of Rac1 activation through the recruitment of MgcRacGAP.     

Regulation of cytokinesis by mgcRacGAP in B lymphocytes is independent of GAP activity

In cytokinesis, several molecules including small G proteins and their regulators are known to have important roles. One of these regulators, mgcRacGAP has GTPase activating protein (GAP) activity for Rac, Cdc42 and Rho. MgcRacGAP has also been shown to be involved in cytokinesis using various cell types. However, the requirement of mgcRacGAP for cytokinesis and survival in B lymphocytes has not been fully examined. Here, we demonstrate that normal cytokinesis in B lymphocytes requires the GAP and NH2 terminal domains but not GAP activity of mgcRacGAP. In addition, we report that apoptosis induced by conditional ablation of mgcRacGAP in the B cell line is fully rescued by the introduction of a GAP-inactive mutant, suggesting that the survival defect in mgcRacGAP-deficient B cells is also independent of GAP activity.     

Coxiella burnetii Phagocytosis Is Regulated by GTPases of the Rho Family and the RhoA Effectors mDia1 and ROCK

The GTPases belonging to the Rho family control the actin cytoskeleton rearrangements needed for particle internalization during phagocytosis. ROCK and mDia1 are downstream effectors of RhoA, a GTPase involved in that process. Coxiella burnetii, the etiologic agent of Q fever, is internalized by the host´s cells in an actin-dependent manner. Nevertheless, the molecular mechanism involved in this process has been poorly characterized. This work analyzes the role of different GTPases of the Rho family and some downstream effectors in the internalization of C. burnetii by phagocytic and non-phagocytic cells. The internalization of C. burnetii into HeLa and RAW cells was significantly inhibited when the cells were treated with Clostridium difficile Toxin B which irreversibly inactivates members of the Rho family. In addition, the internalization was reduced in HeLa cells that overexpressed the dominant negative mutants of RhoA, Rac1 or Cdc42 or that were knocked down for the Rho GTPases. The pharmacological inhibition or the knocking down of ROCK diminished bacterium internalization. Moreover, C. burnetii was less efficiently internalized in HeLa cells overexpressing mDia1-N1, a dominant negative mutant of mDia1, while the overexpression of the constitutively active mutant mDia1-ΔN3 increased bacteria uptake. Interestingly, when HeLa and RAW cells were infected, RhoA, Rac1 and mDia1 were recruited to membrane cell fractions. Our results suggest that the GTPases of the Rho family play an important role in C. burnetii phagocytosis in both HeLa and RAW cells. Additionally, we present evidence that ROCK and mDia1, which are downstream effectors of RhoA, are involved in that process.     

Endomitotic Megakaryocytes form a Midzone in Anaphase but Have a Deficiency in Cleavage Furrow Formation

Megakaryocyte differentiation is marked by development of progressive polyploidy and accumulation of large nuclear mass and cytoplasmic volume. During differentiation, megakaryocytes undergo repeated incomplete cell cycles in which mitosis is aborted in late anaphase with failure of cytokinesis, termed endomitosis. Recent studies have postulated that failure of Aurora-B kinase to localize to the spindle midzone is responsible for endomitosis in megakaryocytes. In diploid cells, the translocation of Aurora-B kinase is critical for positioning of the cleavage furrow, in part through its phosphorylation of the Rho family GTPase activating protein MgcRacGAP which in turn alters activity of RhoA. However, we have previously demonstrated that Aurora-B kinase localizes to centromeres and is functional in endomitotic megakaryocytes. Here, we show that endomitotic megakaryocytes form midzone structures that recruit Aurora-B kinase and its substrate MgcRacGAP. Although many cells with polyploid anaphases showed cortical localization of Aurora-B kinase, we did not observe accumulation of RhoA in furrows or formation of an actin ring. When mitotic exit was induced by inhibition of cdk1, diploid control cells formed furrows exhibiting cortical RhoA but megakaryocytes exited endomitosis without evidence of furrowing. Therefore, localization of Aurora-B kinase to the midzone is normal in endomitotic megakaryocytes but furrowing is abnormal. These data suggest that endomitotic MKs fail to complete cytokinesis due to aberrant regulation of furrowing at a step subsequent to the localization of Aurora-B kinase, possibly involving the activation or localization of RhoA. This work explores the mechanism of a normally occurring furrowing defect in a non-malignant primary cell.     

GDI-1 phosphorylation switch at serine 96 induces RhoA activation and increased endothelial permeability

We identified the GDI-1-regulated mechanism of RhoA activation from the Rho-GDI-1 complex and its role in mediating increased endothelial permeability. Thrombin stimulation failed to induce RhoA activation and actin stress fiber formation in human pulmonary arterial endothelial cells transduced with full-length GDI-1. Expression of a GDI-1 mutant form (C-GDI) containing the C terminus (aa 69 to 204) also prevented RhoA activation, whereas further deletions failed to alter RhoA activation. We observed that protein kinase Calpha-mediated phosphorylation of the C terminus of GDI-1 at Ser96 reduced the affinity of GDI-1 for RhoA and thereby enabled RhoA activation. Rendering GDI-1 phosphodefective with a Ser96 --> Ala substitution rescued the inhibitory activity of GDI-1 toward RhoA but did not alter the thrombin-induced activation of other Rho GTPases, i.e., Rac1 and Cdc42. Phosphodefective mutant GDI-1 also suppressed myosin light chain phosphorylation, actin stress fiber formation, and the increased endothelial permeability induced by thrombin. In contrast, expressing the phospho-mimicking mutant S96D-GDI-1 protein induced RhoA activity and increased endothelial permeability independently of thrombin stimulation. These results demonstrate the crucial role of the phosphorylation of the C terminus of GDI-1 at S96 in selectively activating RhoA. Inhibiting GDI-1 phosphorylation at S96 is a potential therapeutic target for modulating RhoA activity and thus preventing the increase in endothelial permeability associated with vascular inflammation.