Sequence analysis of a human RhoGAP domain-containing gene and characterization of its expression in human multiple tissues.

Rho GTPase activating proteins (GAPs) stimulate the intrinsic GTP hydrolysis activity of Rho family proteins. Here we isolated a rhoGAP domain-containing protein gene with the same reading frame with ARHGAP19 gene, which has an ORF of 1485 bp encoding a putative protein of 494 amino acid residues with a predicted molecular mass of 55.806 kDa. Protein pattern analysis shows that it contains a bipartite nuclear localization signal (NLS) besides the rhoGAP domain, and it is consistent with the result of sub-cellular localization. ARHGAP19 is located in chromosome 10q24.1 and consists of 12 exons according to the Blastn result. Weak expression was detected in adult pancreas, spleen, thymus and ovary of the 16 adult tissues examined, while it had a more abundant expression pattern in eight important human fetal tissues. The expression pattern of ARHGAP19 shows it may have functions related to fetus development and gives us some clues on its probable functions in adult tissues.     

ARHGAP21 is a RhoGAP for RhoA and RhoC with a role in proliferation and migration of prostate adenocarcinoma cells

Background: Several Rho GTPase-activating proteins (RhoGAPs) are implicated in tumor progression through their effects on Rho GTPase activity. ARHGAP21 is a RhoGAP with increased expression in head and neck squamous cell carcinoma and with a possible role in glioblastoma tumor progression, yet little is known about the function of ARHGAP21 in cancer cells. Here we studied the role of ARHGAP21 in two prostate adenocarcinoma cell lines, LNCaP and PC3, which respectively represent initial and advanced stages of prostate carcinogenesis. Results: ARHGAP21 is located in the nucleus and cytoplasm of both cell lines and its depletion resulted in decreased proliferation and increased migration of PC3 cells but not LNCaP cells. In PC3 cells, ARHGAP21 presented GAP activity for RhoA and RhoC and induced changes in cell morphology. Moreover, its silencing altered the expression of genes involved in cell proliferation and cytoskeleton organization, as well as the endothelin-1 canonical pathway. Conclusions: Our results reveal new functions and signaling pathways regulated by ARHGAP21, and indicate that it could contribute to prostate cancer progression.     

ARHGAP18, a GTPase-activating protein for RhoA, controls cell shape, spreading, and motility

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.     

Identification and characterization of a novel Rho GTPase activating protein implicated in receptor-mediated endocytosis

Cbl-interacting protein of 85 kDa (CIN85) is a recently identified adaptor protein involved in the endocytic process of several receptor tyrosine kinases. Here we have identified a novel RhoGAP, CIN85 associated multi-domain containing Rho1 (CAMGAP1) as a binding protein for CIN85. CAMGAP1 is composed of an Src homology 3 (SH3) domain, multiple WW domains, a proline-rich region, a PH domain and a RhoGAP domain, and has the domain architecture similar to ARHGAP9 and ARHGAP12. CAMGAP1 mRNA is widely distributed in murine tissues. Biochemical assays showed its GAP activity toward Rac1 and Cdc42. Protein binding and expression studies indicated that the second SH3 domain of CIN85 binds to a proline-rich region of CAMGAP1. Overexpression of a truncated form of CAMGAP1 interferes with the internalization of transferrin receptors, suggesting that CAMGAP1 may play a role in clathrin-mediated endocytosis.     

ADAR1 regulates ARHGAP26 gene expression through RNA editing by disrupting miR-30b-3p and miR-573 binding

Rho GTPase activating protein 26 (ARHGAP26) is a negative regulator of the Rho family that converts the small G proteins RhoA and Cdc42 to their inactive GDP-bound forms. It is essential for the CLIC/GEEC endocytic pathway, cell spreading, and muscle development. The present study shows that ARHGAP26 mRNA undergoes extensive A-to-I RNA editing in the 3′ UTR that is specifically catalyzed by ADAR1. Furthermore, the mRNA and protein levels of ARHGAP26 were decreased in cells in which ADAR1 was knocked down. Conversely, ADAR1 overexpression increased the abundance of ARHGAP26 mRNA and protein. In addition, we found that both miR-30b-3p and miR-573 target the ARHGAP26 gene and that RNA editing of ARHGAP26 mediated by ADAR1 abolished the repression of its expression by miR-30b-3p or miR-573. When ADAR1 was overexpressed, the reduced abundance of ARHGAP26 protein mediated by miR-30b-3p or miR-573 was rescued. Importantly, we also found that knocking down ADAR1 elevated RhoA activity, which was consistent with the reduced level of ARHGAP26. Conversely, when ADAR1 was overexpressed, the amount of RhoA-GTP decreased. The similar expression patterns of ARHGAP26 and ADAR1 in human tissue samples further confirmed our findings. Taken together, our results suggest that ADAR1 regulates the expression of ARHGAP26 through A-to-I RNA editing by disrupting the binding of miR-30b-3p and miR-573 within the 3′ UTR of ARHGAP26. This study provides a novel insight into the mechanism by which ADAR1 and its RNA editing function regulate microRNA-mediated modulation of target genes.     

ARHGAP25 Inhibits Pancreatic Adenocarcinoma Growth by Suppressing Glycolysis via AKT/mTOR Pathway

Increasing evidence reveals that the Rho GTPase-activating protein is a crucial negative regulator of Rho family GTPase involved in tumorigenesis. The Rho GTPase-activating protein 25 (ARHGAP25) has been shown to specifically inactivate the Rho family GTPase Rac1, which plays an important role in pancreatic adenocarcinoma (PAAD) progression. Therefore, here we aimed to clarify the expression and functional role of ARHGAP25 in PAAD. The ARHGAP25 expression was lower in PAAD tissues than that in normal pancreatic tissues based on bioinformatics analysis and immunohistochemistry staining. Overexpression of ARHGAP25 inhibited cell growth of AsPC-1 human pancreatic cancer cells in vitro, while opposite results were observed in BxPC-3 human pancreatic cancer cells with ARHGAP25 knockdown. Consistently, in vivo tumorigenicity assays also confirmed that ARHGAP25 overexpression suppressed tumor growth. Mechanically, overexpression of ARHGAP25 inactivated AKT/mTOR signaling pathway by regulating Rac1/PAK1 signaling, which was in line with the results from the Gene set enrichment analysis on The Cancer Genome Atlas dataset. Furthermore, we found that ARHGAP25 reduced HIF-1α-mediated glycolysis in PAAD cells. Treatment with PF-04691502, a dual PI3K/mTOR inhibitor, hampered the increased cell growth and glycolysis due to ARHGAP25 knockdown in PAAD cells. Altogether, these results conclude that ARHGAP25 acts as a tumor suppressor by inhibiting the AKT/mTOR signaling pathway, which might provide a therapeutic target for PAAD.     

Hypoxia-induced ARHGAP26 deficiency inhibits the proliferation and migration of human ductus arteriosus smooth muscle cell through activating RhoA-ROCK-PTEN pathway

The Rho family plays crucial roles in O₂-induced vasoconstriction, cell proliferation, and migration. Rho GTPase-activating protein 26 (ARHGAP26) is a GTPase-activating protein for the small GTPases of the Rho family. Our previous studies have demonstrated that ARHGAP26 expression was significantly downregulated in patent human ductus arteriosus (DA) tissue. However, its role underlying the maintenance of DA patency is unclear. In this study, patent (fetal) and constricted (newborn) mouse DA tissues were harvested to confirm the differences in the levels of expression of ARHGAP26. Human DA smooth muscle cells (DASMCs) were isolated and cultured in vitro and used to test the function of ARHGAP26. The expression of ARHGAP26 was significantly lower in patent (fetal) than constricted (newborn) mouse DA. ARHGAP26-knocked-down human DASMCs showed reduced proliferation and migration, which are both crucial to anatomic closure of DA. Moreover, after culturing under hypoxic conditions, the expression of ARHGAP26 in human DASMCs was significantly lower and hypoxia-induced ARHGAP26 deficiency activated the phosphorylation level of phosphatase and tensin homolog (PTEN) in DASMCs by mediating the activity of RhoA and RhoA-associated kinase 1 (ROCK1). Use of Y27632, an inhibitor of ROCK which further reduces the phospholipid activity of PTEN can reverse the inhibitory effect of PTEN on the proliferation and migration of human DASMCs. This provides insight into the molecular regulation of the RhoA-ROCK-PTEN pathway in DA smooth muscle cells, which may be a suitable therapeutic target or diagnostic biomarker for perinatal DA tone management.     

The role of GTPase-activating protein ARHGAP26 in human cancers

Rho GTPases are molecular switches that play an important role in regulating the behavior of a variety of tumor cells. RhoA GTPase-activating protein 26 (ARHGAP26) is a GTPase-activating protein and inhibits the activity of Rho GTPases by promoting the hydrolytic ability of Rho GTPases. It also affects tumorigenesis and progression of various tumors through several methods, including formation of abnormal fusion genes and circular RNA. This review summarizes the biological functions and molecular mechanisms of ARHGAP26 in different tumors, proposes the potential clinical value of ARHGAP26 in cancer treatment, and discusses current issues that need to be addressed.

     

ARHGAP21 as a master regulator of multiple cellular processes

The cellular cytoskeleton is involved with multiple biological processes and is tightly regulated by multiple proteins and effectors. Among these, the RhoGTPases family is one of the most important players. RhoGTPAses are, in turn, regulated by many other elements. In the past decade, one of those regulators, the RhoGAP Rho GTPase Activating Protein 21 (ARHGAP21), has been overlooked, despite being implied as having an important role on many of those processes. In this paper, we aimed to review the available literature regarding ARHGAP21 to highlight its importance and the mechanisms of action that have been found so far for this still unknown protein involved with cell adhesion, migration, Golgi regulation, cell trafficking, and even insulin secretion.     

ARHGAP4-SEPT2-SEPT9 complex enables both up- and down-modulation of integrin-mediated focal adhesions,cell migration,and invasion

The Rho family of GTPases are inactivated in a cell context–dependent manner by Rho-GTPase-activating proteins (Rho-GAPs), but their signaling mechanisms are poorly understood. Here we demonstrate that ARHGAP4, one of the Rho-GAPs, forms a complex with SEPT2 and SEPT9 via its Rho-GAP domain and SH3 domain to enable both up- and down-modulation of integrin-mediated focal adhesions (FAs). We show that silencing ARHGAP4 and overexpressing its two mutually independent upstream regulators, SEPT2 and SEPT9, all induce reorganization of FAs to newly express Integrin Beta 1 and also enhance both cell migration and invasion. Interestingly, even if these cell migration/invasion–associated phenotypic changes are induced upon perturbations to the complex, it does not necessarily cause enhanced clustering of FAs. Instead, its extent depends on whether the microenvironment contains ligands suitable for the up-regulated Integrin Beta 1. These results provide novel insights into cell migration, invasion, and microenvironment-dependent phenotypic changes regulated by the newly identified complex.     

Golgi-localized GAP for Cdc42 functions downstream of ARF1 to control Arp2/3 complex and F-actin dynamics

The small GTP-binding ADP-ribosylation factor 1 (ARF1) acts as a master regulator of Golgi structure and function through the recruitment and activation of various downstream effectors. It has been proposed that members of the Rho family of small GTPases also control Golgi function in coordination with ARF1, possibly through the regulation of Arp2/3 complex and actin polymerization on Golgi membranes. Here, we identify ARHGAP10--a novel Rho GTPase-activating protein (Rho-GAP) that is recruited to Golgi membranes through binding to GTP-ARF1. We show that ARHGAP10 functions preferentially as a GAP for Cdc42 and regulates the Arp2/3 complex and F-actin dynamics at the Golgi through the control of Cdc42 activity. Our results establish a role for ARHGAP10 in Golgi structure and function at the crossroads between ARF1 and Cdc42 signalling pathways.     

Direct activation of phospholipase C-epsilon by Rho

Unique among the phospholipase C isozymes, the recently identified phospholipase C-epsilon (PLC-epsilon) contains an amino-terminal CDC25 domain capable of catalyzing nucleotide exchange on Ras family GTPases as well as a tandem array of Ras-associating (RA) domains near its carboxyl terminus that are effector binding sites for activated H-Ras and Rap. To determine whether other small GTPases activate PLC-epsilon, we measured inositol phosphate accumulation in COS-7 cells expressing a broad range of GTPase-deficient mutants of Ras superfamily proteins. RhoA, RhoB, and RhoC all markedly stimulated inositol phosphate accumulation in PLC-epsilon-expressing cells. This stimulation matched or exceeded phospholipase activation promoted by co-expression of PLC-epsilon with the known regulators Ras, Galpha12/13, or Gbeta1gamma2. In contrast, little effect was observed with the other Rho family members Rac1, Rac2, Rac3, and Cdc42. Truncation of the two carboxyl-terminal RA domains caused loss of responsiveness to H-Ras but not to Rho. Truncation of PLC-epsilon to remove the CDC25 and pleckstrin homology (PH) domains also did not cause loss of responsiveness to Rho, Galpha12/13, or Gbeta1gamma2. Comparative sequence analysis of mammalian phospholipase C isozymes revealed a unique approximately 65 amino acid insert within the catalytic core of PLC-epsilon not present in PLC-beta, gamma, delta, or zeta. A PLC-epsilon construct lacking this region was no longer activated by Rho or Galpha12/13 but retained regulation by Gbetagamma and H-Ras. GTP-dependent interaction of Rho with PLC-epsilon was illustrated in pull-down experiments with GST-Rho, and this interaction was retained in the PLC-epsilon construct lacking the unique insert within the catalytic core. These results are consistent with the conclusion that Rho family GTPases directly interact with PLC-epsilon by a mechanism independent of the CDC25 or RA domains. A unique insert within the catalytic core of PLC-epsilon imparts responsiveness to Rho, which may signal downstream of Galpha12/13 in the regulation of PLC-epsilon, because activation by both Rho and Galpha12/13 is lost in the absence of this sequence.     

Structural basis for ARF1-mediated recruitment of ARHGAP21 to Golgi membranes

ARHGAP21 is a Rho family GTPase-activating protein (RhoGAP) that controls the Arp2/3 complex and F-actin dynamics at the Golgi complex by regulating the activity of the small GTPase Cdc42. ARHGAP21 is recruited to the Golgi by binding to another small GTPase, ARF1. Here, we present the crystal structure of the activated GTP-bound form of ARF1 in a complex with the Arf-binding domain (ArfBD) of ARHGAP21 at 2.1 A resolution. We show that ArfBD comprises a PH domain adjoining a C-terminal alpha helix, and that ARF1 interacts with both of these structural motifs through its switch regions and triggers structural rearrangement of the PH domain. We used site-directed mutagenesis to confirm that both the PH domain and the helical motif are essential for the binding of ArfBD to ARF1 and for its recruitment to the Golgi. Our data demonstrate that two well-known small GTPase-binding motifs, the PH domain and the alpha helical motif, can combine to create a novel mode of binding to Arfs.     

ARHGAP30 is a Wrch-1-interacting protein involved in actin dynamics and cell adhesion

The atypical Rho GTPase Wrch-1 has been proposed roles in cell migration, focal adhesion dissolution, stress fibre break down and tight junction heterogeneity. A screen for Wrch-1 binding-partners identified the novel RhoGAP protein, ARHGAP30, as a Wrch-1 interactor. ARHGAP30 is related to the Cdc42- and Rac1-specific RhoGAP CdGAP, which was likewise found to bind Wrch-1. In contrast to CdGAP, ARHGAP30 serves as a Rac1- and RhoA-specific RhoGAP. Ectopic expression of ARHGAP30 results in membrane blebbing and dissolution of stress-fibres and focal adhesions. Our data suggest roles for ARHGAP30 and CdGAP in regulation of cell adhesion downstream of Wrch-1.     

Chimeric G alpha i2/G alpha 13 proteins reveal the structural requirements for the binding and activation of the RGS-like (RGL)-containing Rho guanine nucleotide exchange factors (GEFs) by G alpha 13

The alpha-subunit of G proteins of the G(12/13) family stimulate Rho by their direct binding to the RGS-like (RGL) domain of a family of Rho guanine nucleotide exchange factors (RGL-RhoGEFs) that includes PDZ-RhoGEF (PRG), p115RhoGEF, and LARG, thereby regulating cellular functions as diverse as shape and movement, gene expression, and normal and aberrant cell growth. The structural features determining the ability of G alpha(12/13) to bind RGL domains and the mechanism by which this association results in the activation of RGL-RhoGEFs are still poorly understood. Here, we explored the structural requirements for the functional interaction between G alpha(13) and RGL-RhoGEFs based on the structure of RGL domains and their similarity with the area by which RGS4 binds the switch region of G alpha(i) proteins. Using G alpha(i2), which does not bind RGL domains, as the backbone in which G alpha(13) sequences were swapped or mutated, we observed that the switch region of G alpha(13) is strictly necessary to bind PRG, and specific residues were identified that are critical for this association, likely by contributing to the binding surface. Surprisingly, the switch region of G alpha(13) was not sufficient to bind RGL domains, but instead most of its GTPase domain is required. Furthermore, membrane localization of G alpha(13) and chimeric G alpha(i2) proteins was also necessary for Rho activation. These findings revealed the structural features by which G alpha(13) interacts with RGL domains and suggest that molecular interactions occurring at the level of the plasma membrane are required for the functional activation of the RGL-containing family of RhoGEFs.     

Blockade of ARHGAP11A reverses malignant progress via inactivating Rac1B in hepatocellular carcinoma

Background

The molecular signaling events involving in high malignancy and poor prognosis of hepatocellular carcinoma (HCC) are extremely complicated. Blockade of currently known targets has not yet led to successful clinical outcome. More understanding about the regulatory mechanisms in HCC is necessary for developing new effective therapeutic strategies for HCC patients.

Methods

The expression of Rho GTPase-activating protein 11A (ARHGAP11A) was examined in human normal liver and HCC tissues. The correlations between ARHGAP11A expression and clinicopathological stage or prognosis in HCC patients were analyzed. ARHGAP11A was downregulated to determine its role in the proliferation, invasion, migration, epithelial-to-mesenchymal transition (EMT) development, and regulatory signaling of HCC cells in vitro and in vivo.

Results

ARHGAP11A exhibited high expression in HCC, and was significantly correlated with clinicopathological stage and prognosis in HCC patients. Moreover, ARHGAP11A facilitated Hep3B and MHCC97-H cell proliferation, invasion, migration and EMT development in vitro. ARHGAP11A knockdown significantly inhibited the in vivo growth and metastasis of HCC cells. Furthermore, ARHGAP11A directly interacted with Rac1B independent of Rho GTPase- activating activity. Rac1B blockade effectively interrupted ARHGAP11A-elicited HCC malignant phenotype. Meanwhile, upregulation of Rac1B reversed ARHGAP11A knockdown mediated mesenchymal-to-epithelial transition (MET) development in HCC cells.

Conclusion

ARHGAP11A facilitates malignant progression in HCC patients via ARHGAP11A-Rac1B interaction. The ARHGAP11A/Rac1B signaling could be a potential therapeutic target in the clinical treatment of HCC.

     

β-Arrestin 1 inhibits the GTPase-activating protein function of ARHGAP21, promoting activation of RhoA following angiotensin II type 1A receptor stimulation

Activation of the small GTPase RhoA following angiotensin II stimulation is known to result in actin reorganization and stress fiber formation. Full activation of RhoA, by angiotensin II, depends on the scaffolding protein β-arrestin 1, although the mechanism behind its involvement remains elusive. Here we uncover a novel partner and function for β-arrestin 1, namely, in binding to ARHGAP21 (also known as ARHGAP10), a known effector of RhoA activity, whose GTPase-activating protein (GAP) function it inhibits. Using yeast two-hybrid screening, a peptide array, in vitro binding studies, truncation analyses, and coimmunoprecipitation techniques, we show that β-arrestin 1 binds directly to ARHGAP21 in a region that transects the RhoA effector GAP domain. Moreover, we show that the level of a complex containing β-arrestin 1 and ARHGAP21 is dynamically increased following angiotensin stimulation and that the kinetics of this interaction modulates the temporal activation of RhoA. Using information gleaned from a peptide array, we developed a cell-permeant peptide that serves to inhibit the interaction of these proteins. Using this peptide, we demonstrate that disruption of the β-arrestin 1/ARHGAP21 complex results in a more active ARHGAP21, leading to less-efficient signaling via the angiotensin II type 1A receptor and, thereby, attenuation of stimulated stress fiber formation.