ARHGAP18, a GTPase-activating protein for RhoA, controls cell shape, spreading, and motility.
ABSTRACT: Rho GTPases are molecular switches that transmit biochemical signals in response to extracellular stimuli to elicit changes in the actin cytoskeleton. Rho GTPases cycle between an active, GTP-bound state and an inactive, GDP-bound state. These states are regulated by two distinct families of proteins-guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We studied the role of a previously uncharacterized GAP, ARHGAP18 (MacGAP). Overexpression of ARHGAP18 suppressed the activity of RhoA and disrupted stress fiber formation. Conversely, silencing of ARHGAP18 by small interfering RNA transfection-enhanced stress fiber formation and induced rounding of cells. We examined the role of ARHGAP18 in cell spreading and migration. Immunofluorescence analysis revealed that ARHGAP18 was localized to the leading edge during cell spreading and migration. ARHGAP18-knockdown cells showed impaired spreading, premature formation of stress fibers, and sustained activation of RhoA upon cell attachment. In addition, knockdown and overexpression of ARHGAP18 resulted in the inhibition and promotion of cell migration, respectively. Furthermore, ARHGAP18 was required for the polarization of cells for migration. Our results define ARHGAP18 as one of the crucial factors for the regulation of RhoA for the control of cell shape, spreading, and migration.
Project description:Protein kinase N3 (PKN3) is a serine/threonine kinase implicated in tumor progression of multiple cancer types, however, its substrates and effector proteins still remain largely understudied. In the present work we aimed to identify novel PKN3 substrates in a phosphoproteomic screen using analog sensitive PKN3. Among the identified putative substrates we selected ARHGAP18, a protein from RhoGAP family, for validation of the screen and further study. We confirmed that PKN3 can phosphorylate ARHGAP18 in vitro and we also characterized the interaction of the two proteins, which is mediated via the N-terminal part of ARHGAP18. We present strong evidence that PKN3-ARHGAP18 interaction is increased upon ARHGAP18 phosphorylation and that the phosphorylation of ARHGAP18 by PKN3 enhances its GAP domain activity and contributes to negative regulation of active RhoA. Taken together, we identified new set of potential PKN3 substrates and revealed a new negative feedback regulatory mechanism of Rho signaling mediated by PKN3-induced ARHGAP18 activation.
Project description:RhoGTPases are important regulators of the cell cytoskeleton, controlling cell shape, migration and proliferation. Previously we showed that ARHGAP18 in endothelial cells is important in cell junctions. Here we show, using structured illumination microscopy (SIM), ground-state depletion (GSD), and total internal reflection fluorescence microscopy (TIRF) that a proportion of ARHGAP18 localizes to microtubules in endothelial cells, as well as in nonendothelial cells, an association confirmed biochemically. In endothelial cells, some ARHGAP18 puncta also colocalized to Weibel-Palade bodies on the microtubules. Depletion of ARHGAP18 by small interfering RNA or analysis of endothelial cells isolated from ARHGAP18-knockout mice showed microtubule destabilization, as evidenced by altered morphology and decreased acetylated ?-tubulin and glu-tubulin. The destabilization was rescued by inhibition of ROCK and histone deacetylase 6 but not by a GAP-mutant form of ARHGAP18. Depletion of ARHGAP18 resulted in a failure to secrete endothelin-1 and a reduction in neutrophil transmigration, both known to be microtubule dependent. Thrombin, a critical regulator of the Rho-mediated barrier function of endothelial cells through microtubule destabilization, enhanced the plasma membrane-bound fraction of ARHGAP18. Thus, in endothelial cells, ARHGAP18 may act as a significant regulator of vascular homeostasis.
Project description:RhoA is a small guanosine-5'-triphosphatase (GTPase) suggested to be essential for cytokinesis, stress fiber formation, and epithelial cell-cell contacts. In skin, loss of RhoA was suggested to underlie pemphigus skin blistering. To analyze RhoA function in vivo, we generated mice with a keratinocyte-restricted deletion of the RhoA gene. Despite a severe reduction of cofilin and myosin light chain (MLC) phosphorylation, these mice showed normal skin development. Primary RhoA-null keratinocytes, however, displayed an increased percentage of multinucleated cells, defective maturation of cell-cell contacts. Furthermore we observed increased cell spreading due to impaired RhoA-ROCK (Rho-associated protein kinase)-MLC phosphatase-MLC-mediated cell contraction, independent of Rac1. Rho-inhibiting toxins further increased multinucleation of RhoA-null cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA. Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence. These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA-null keratinocytes. Our data indicate a crucial role for RhoA and contraction in regulating cell spreading and a contraction-independent function of RhoA in keratinocyte migration. In addition, our data show that RhoA is dispensable for skin development.
Project description:BACKGROUND:Rho family GTPases are critical regulators of the cytoskeleton and affect cell migration, cell-cell adhesion, and cell-matrix adhesion. As with all GTPases, their activity is determined by their guanine nucleotide-bound state. Understanding how Rho proteins are activated and inactivated has largely focused on regulatory proteins such as guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). However, recent in vitro studies have indicated that GTPases may also be directly regulated by redox agents. We hypothesized that this redox-based mechanism occurs in cells and affects cytoskeletal dynamics, and in this report we conclude this is indeed a novel mechanism of regulating the GTPase RhoA. METHODOLOGY/PRINCIPAL FINDINGS:In this report, we show that RhoA can be directly activated by reactive oxygen species (ROS) in cells, and that this requires two critical cysteine residues located in a unique redox-sensitive motif within the phosphoryl binding loop. First, we show that ROS can reversibly activate RhoA and induce stress fiber formation, a well characterized readout of RhoA activity. To determine the role of cysteine residues in this mechanism of regulation, we generated cysteine to alanine RhoA mutants. Mutation of these cysteines abolishes ROS-mediated activation and stress fiber formation, indicating that these residues are critical for redox-regulation of RhoA. Importantly, these mutants maintain the ability to be activated by GEFs. CONCLUSIONS/SIGNIFICANCE:Our findings identify a novel mechanism for the regulation of RhoA in cells by ROS, which is independent of classical regulatory proteins. This mechanism of regulation may be particularly relevant in pathological conditions where ROS are generated and the cellular redox-balance altered, such as in asthma and ischemia-reperfusion injury.
Project description:The Rho GTPases comprise a subfamily of the Ras superfamily of small GTPases. Their importance in regulation of cell morphology and cell migration is well characterized. According to the prevailing paradigm, Cdc42 regulates the formation of filopodia, Rac1 regulates the formation of lamellipodia, and RhoA triggers the assembly of focal adhesions. However, this scheme is clearly an oversimplification, as the Rho subfamily encompasses 20 members with diverse effects on a number of vital cellular processes, including cytoskeletal dynamics and cell proliferation, migration, and invasion. This article highlights the importance of the catalytic activities of the classical Rho GTPases Cdc42 and Rac1, in terms of their specific effects on the dynamic reorganization of the actin filament system. GTPase-deficient mutants of Cdc42 and Rac1 trigger the formation of broad lamellipodia and stress fibers, and fast-cycling mutations trigger filopodia formation and stress fiber dissolution. The filopodia response requires the involvement of the formin family of actin nucleation promotors. In contrast, the formation of broad lamellipodia induced by GTPase-deficient Cdc42 and Rac1 is mediated through Arp2/3-dependent actin nucleation.
Project description:Cell migration involves the cooperative reorganization of the actin and microtubule cytoskeletons, as well as the turnover of cell-substrate adhesions, under the control of Rho family GTPases. RhoA is activated at the leading edge of motile cells by unknown mechanisms to control actin stress fiber assembly, contractility, and focal adhesion dynamics. The microtubule-associated guanine nucleotide exchange factor (GEF)-H1 activates RhoA when released from microtubules to initiate a RhoA/Rho kinase/myosin light chain signaling pathway that regulates cellular contractility. However, the contributions of activated GEF-H1 to coordination of cytoskeletal dynamics during cell migration are unknown. We show that small interfering RNA-induced GEF-H1 depletion leads to decreased HeLa cell directional migration due to the loss of the Rho exchange activity of GEF-H1. Analysis of RhoA activity by using a live cell biosensor revealed that GEF-H1 controls localized activation of RhoA at the leading edge. The loss of GEF-H1 is associated with altered leading edge actin dynamics, as well as increased focal adhesion lifetimes. Tyrosine phosphorylation of focal adhesion kinase and paxillin at residues critical for the regulation of focal adhesion dynamics was diminished in the absence of GEF-H1/RhoA signaling. This study establishes GEF-H1 as a critical organizer of key structural and signaling components of cell migration through the localized regulation of RhoA activity at the cell leading edge.
Project description:Shear stress induces endothelial polarization and migration in the direction of flow accompanied by extensive remodeling of the actin cytoskeleton. The GTPases RhoA, Rac1, and Cdc42 are known to regulate cell shape changes through effects on the cytoskeleton and cell adhesion. We show here that all three GTPases become rapidly activated by shear stress, and that each is important for different aspects of the endothelial response. RhoA was activated within 5 min after stimulation with shear stress and led to cell rounding via Rho-kinase. Subsequently, the cells respread and elongated within the direction of shear stress as RhoA activity returned to baseline and Rac1 and Cdc42 reached peak activation. Cell elongation required Rac1 and Cdc42 but not phosphatidylinositide 3-kinases. Cdc42 and PI3Ks were not required to establish shear stress-induced polarity although they contributed to optimal migration speed. Instead, Rho and Rac1 regulated directionality of cell movement. Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement. Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.
Project description:The GTPases RhoA and Rac1 are key regulators of cell spreading, adhesion, and migration, and they exert distinct effects on the actin cytoskeleton. While RhoA classically stimulates stress fiber assembly and contraction, Rac1 promotes branched actin polymerization and membrane protrusion. These competing influences are reinforced by antagonistic crosstalk between RhoA and Rac1, which has complicated efforts to identify the specific mechanisms by which each GTPase regulates cell behavior. We therefore wondered whether RhoA and Rac1 are intrinsically coupled or whether they can be manipulated independently. To address this question, we placed constitutively active (CA) RhoA under a doxycycline-inducible promoter and CA Rac1 under an orthogonal cumate-inducible promoter, and we stably introduced both constructs into glioblastoma cells. We found that doxycycline addition increased RhoA activity without altering Rac1, and similarly cumate addition increased Rac1 activity without altering RhoA. Furthermore, co-expression of both mutants enabled high activation of RhoA and Rac1 simultaneously. When cells were cultured on collagen hydrogels, RhoA activation prevented cell spreading and motility, whereas Rac1 activation stimulated migration and dynamic cell protrusions. Interestingly, high activation of both GTPases induced a third phenotype, in which cells migrated at intermediate speeds similar to control cells but also aggregated into large, contractile clusters. In addition, we demonstrate dynamic and reversible switching between high RhoA and high Rac1 phenotypes. Overall, this approach represents a unique way to access different combinations of RhoA and Rac1 activity levels in a single cell and may serve as a valuable tool for multiplexed dissection and control of mechanobiological signals.
Project description:RhoA, the founding member of mammalian Rho GTPase family, is thought to be essential for actomyosin regulation. To date, the physiologic function of RhoA in mammalian cell regulation has yet to be determined genetically. Here we have created RhoA conditional knock-out mice. Mouse embryonic fibroblasts deleted of RhoA showed no significant change in actin stress fiber or focal adhesion complex formation in response to serum or LPA, nor any detectable change in Rho-kinase signaling activity. Concomitant knock-out or knockdown of RhoB and RhoC in the RhoA(-/-) cells resulted in a loss of actin stress fiber and focal adhesion similar to that of C3 toxin treatment. Proliferation of RhoA(-/-) cells was impaired due to a complete cell cycle block during mitosis, an effect that is associated with defective cytokinesis and chromosome segregation and can be readily rescued by exogenous expression of RhoA. Furthermore, RhoA deletion did not affect the transcriptional activity of Stat3, NF?B, or serum response factor, nor the expression of the cell division kinase inhibitor p21(Cip)1 or p27(Kip1). These genetic results demonstrate that in primary mouse embryonic fibroblasts, RhoA is uniquely required for cell mitosis but is redundant with related RhoB and RhoC GTPases in actomyosin regulation.
Project description:The Dishevelled (Dvl) and Dishevelled-associated activator of morphogenesis 1 (Daam1) pathway triggered by Wnt5a regulates cellular polarity during development and tissue homoeostasis. However, Wnt5a signaling in breast cancer progression remains poorly defined.We showed here that Wnt5a activated Dvl2, Daam1 and RhoA, and promoted migration of breast cancer cells, which was, however, abolished by Secreted Frizzled-related protein 2 (sFRP2) pretreatment. Dominant negative Dvl2 mutants or Dvl2 siRNA significantly decreased Wnt5a-induced Daam1/RhoA activation and cell migration. Ectopic expression of N-Daam1, a dominant negative mutant, or Daam1 siRNA remarkably inhibited Wnt5a-induced RhoA activation, stress fiber formation and cell migration. Ectopic expression of dominant negative RhoA (N19) or C3 exoenzyme transferase, a Rho inhibitor, decreased Wnt5a-induced stress fiber formation and cell migration.Taken together, we demonstrated for the first time that Wnt5a promotes breast cancer cell migration via Dvl2/Daam1/RhoA.