P190RhoGAPs, the ARHGAP35- and ARHGAP5-Encoded Proteins, in Health and Disease.
ABSTRACT: Small guanosine triphosphatases (GTPases) gathered in the Rat sarcoma (Ras) superfamily represent a large family of proteins involved in several key cellular mechanisms. Within the Ras superfamily, the Ras homolog (Rho) family is specialized in the regulation of actin cytoskeleton-based mechanisms. These proteins switch between an active and an inactive state, resulting in subsequent inhibiting or activating downstream signals, leading finally to regulation of actin-based processes. The On/Off status of Rho GTPases implicates two subsets of regulators: GEFs (guanine nucleotide exchange factors), which favor the active GTP (guanosine triphosphate) status of the GTPase and GAPs (GTPase activating proteins), which inhibit the GTPase by enhancing the GTP hydrolysis. In humans, the 20 identified Rho GTPases are regulated by over 70 GAP proteins suggesting a complex, but well-defined, spatio-temporal implication of these GAPs. Among the quite large number of RhoGAPs, we focus on p190RhoGAP, which is known as the main negative regulator of RhoA, but not exclusively. Two isoforms, p190A and p190B, are encoded by ARHGAP35 and ARHGAP5 genes, respectively. We describe here the function of each of these isoforms in physiological processes and sum up findings on their role in pathological conditions such as neurological disorders and cancers.
Project description:The Rho family GTPases are stringently regulated through the action of a large family of GTPase activating proteins (GAPs) that stimulate their relatively weak intrinsic GTP hydrolyzing activity. The p190RhoGAPs, which include the p190A and p190B proteins, are potent and widely expressed GAPs acting on both Rho and Rac GTPases. We have observed that several acidic phospholipids inhibit the RhoGAP activity and promote the RacGAP activity of p190 proteins. In liposome binding assays we have demonstrated that binding of p190A to phospholipids is controlled by electrostatic interactions. Using mapping techniques, we determined that a small polybasic peptide stretch within p190A is a common site for both the phospholipid binding and PKC phosphorylation. Moreover, PKC-mediated phosphorylation of two amino acids (serine-1221 and threonine-1226) within this region of p190A prevents the binding and substrate specificity regulation by phospholipids. Transfection of COS-7 cells with mutant forms of p190A either unable to bind to phospholipids or unable to become phosphorylated induced distinct morphological changes. Together, these findings reveal a novel GAP regulatory mechanism in which phosphorylation indirectly alters GTPase substrate preference by affecting the interaction with acidic phospholipids. Our observations provide a potential mechanism of Rac/Rho antagonism described in several cellular functions.
Project description:The Rho GTPases are critical regulators of the actin cytoskeleton and are required for cell adhesion, migration, and polarity. Among the key Rho regulatory proteins in the context of cell migration are the p190 RhoGAPs (p190A and p190B), which function to modulate Rho signaling in response to integrin engagement. The p190 RhoGAPs undergo complex regulation, including phosphorylation by several identified kinases, interactions with phospholipids, and association with a variety of cellular proteins. Here, we have identified an additional regulatory mechanism unique to p190A RhoGAP that involves priming-dependent phosphorylation by glycogen synthase-3-beta (GSK-3beta), a kinase previously implicated in establishing cell polarity. We found that p190A-deficient fibroblasts exhibit a defect in directional cell migration reflecting a requirement for GSK-3beta-mediated phosphorylation of amino acids in the C-terminal "tail" of p190A. This phosphorylation leads to inhibition of p190A RhoGAP activity in vitro and in vivo. These studies identify p190A as a novel GSK-3beta substrate and reveal a mechanism by which GSK-3beta contributes to cellular polarization in directionally migrating cells via effects on Rho GTPase activity.
Project description:Rho GTPases are overexpressed and hyperactivated in human breast cancers. Deficiency of p190B RhoGAP, a major inhibitor of the Rho GTPases, inhibits mouse mammary tumor virus long terminal repeat (MMTV)-Neu/ErbB2 mammary tumor formation and progression in part through effects within the stromal environment, suggesting that p190B function is pro-tumorigenic. To further investigate the potential pro-tumorigenic actions of p190B, we examined the effects of exogenous p190B expression within the mammary epithelium on MMTV-Neu tumor formation and progression.Tetracycline-regulatable p190B transgenic mice were bred to MMTV-Neu mice, and the effects of exogenous p190B expression on tumor latency, multiplicity, growth rates, angiogenesis, and metastasis were examined. The effects of exogenous p190B expression on cell-matrix adhesion and invasion were tested using non-transformed primary mammary epithelial cells (MECs). Rho GTPase activity, oxidative stress as an indicator of reactive oxygen species (ROS) production, and downstream signaling pathways were analyzed.Altered p190B expression resulted in a 2-fold increase in tumor multiplicity and a 3-fold increase in metastases compared to control mice indicating that exogenous p190B expression in the mammary epithelium promotes MMTV-Neu mammary tumor formation and progression. Interestingly, non-transformed primary MECs expressing exogenous p190B displayed increased adhesion to laminin and type IV collagen and formed invasive structures in a three-dimensional culture assay. Ras related C3 botulinum toxin 1 (Rac1)-GTP levels were elevated in p190B transgenic tumors whereas Ras homologous A (RhoA) and cell division cycle 42 (Cdc42)-GTP levels were not significantly altered. Rac1 activity affects production of ROS, which regulate transformation, metastasis, and oxidative stress. Protein carbonylation, which is indicative of oxidative stress, was elevated 1.75-fold in p190B transgenic tumors as compared to control tumors suggesting that exogenous p190B expression may affect Rac1-dependent ROS production.These studies indicate that paradoxically, p190B RhoGAP, a major inhibitor of the Rho GTPases in vitro, has pro-tumorigenic functions that enhance MMTV-Neu induced mammary tumor formation and metastasis. Furthermore, exogenous p190B expression enhances cell adhesion and invasion, which may facilitate metastasis. Rac1 activity and oxidative stress are elevated in tumors expressing exogenous p190B suggesting that p190B may promote tumorigenesis through a Rac1/ROS dependent mechanism.
Project description:The Rho family of GTPases is strictly regulated by a large family of GTPase-activating proteins (GAPs) that stimulate the relatively weak intrinsic GTP-hydrolyzing activity of Rho GTPases. p190A is a potent and widely expressed GAP that acts on RhoA GTPases. p190A is frequently mutated in endometrial cancer, but the contribution of p190A mutations to endometrial tumorigenesis remains unclear. Here we identified that p190A is an upstream regulator of the Hippo-YAP signaling pathway, which is a critical regulator of cell proliferation, apoptosis, and cell fate. p190A knockout in endometrial cancer cells promoted cell proliferation, migration, and epithelial-mesenchymal transition (EMT), which were partially dependent on YAP activation. Wild-type p190A, but not endometrial cancer-associated mutants, suppressed the nuclear localization, transcriptional activity, and malignant transformation function of YAP. Moreover, the nuclear localization of YAP was enhanced in p190A-mutated endometrial cancer. These findings reveal novel molecular mechanisms underlying Hippo-YAP pathway-driven endometrial tumorigenesis and elucidate the potential for therapy targeting the Hippo-YAP pathway in p190A-mutated endometrial cancer.
Project description:The Rac GTPase regulates Rho signaling in a broad range of physiological settings and in oncogenic transformation [1-3]. Here, we report a novel mechanism by which crosstalk between Rac and Rho GTPases is achieved. Activated Rac1 binds directly to p190B Rho GTPase-activating protein (RhoGAP), a major modulator of Rho signaling. p190B colocalizes with constitutively active Rac1 in membrane ruffles. Moreover, activated Rac1 is sufficient to recruit p190B into a detergent-insoluble membrane fraction, a process that is accompanied by a decrease in GTP-bound RhoA from membranes. p190B is recruited to the plasma membrane in response to integrin engagement . We demonstrate that collagen type I, a potent inducer of Rac1-dependent cell motility in HeLa cells, counteracts cytoskeletal collapse resulting from overexpression of wild-type p190B, but not that resulting from overexpression of a p190B mutant specifically lacking the Rac1-binding sequence. Furthermore, this p190B mutant exhibits dramatically enhanced RhoGAP activity, consistent with a model whereby binding of Rac1 relieves autoinhibition of p190B RhoGAP function. Collectively, these observations establish that activated Rac1, through direct interaction with p190B, modulates subcellular RhoGAP localization and activity, thereby providing a novel mechanism for Rac control of Rho signaling in a broad range of physiological processes.
Project description:Rho family GTPases act as molecular switches regulating actin cytoskeleton dynamics. Attenuation of their signaling capacity is provided by GTPase-activating proteins (GAPs), including p190A, that promote the intrinsic GTPase activity of Rho proteins. In the current study we have performed a small-scale ENU mutagenesis screen and identified a novel loss of function allele of the p190A gene Arhgap35, which introduces a Leu1396 to Gln substitution in the GAP domain. This results in decreased GAP activity for the prototypical Rho-family members, RhoA and Rac1, likely due to disrupted ordering of the Rho binding surface. Consequently, Arhgap35-deficient animals exhibit hypoplastic and glomerulocystic kidneys. Investigation into the cystic phenotype shows that p190A is required for appropriate primary cilium formation in renal nephrons. P190A specifically localizes to the base of the cilia to permit axoneme elongation, which requires a functional GAP domain. Pharmacological manipulations further reveal that inhibition of either Rho kinase (ROCK) or F-actin polymerization is able to rescue the ciliogenesis defects observed upon loss of p190A activity. We propose a model in which p190A acts as a modulator of Rho GTPases in a localized area around the cilia to permit the dynamic actin rearrangement required for cilia elongation. Together, our results establish an unexpected link between Rho GTPase regulation, ciliogenesis and glomerulocystic kidney disease.
Project description:The tumor oncoproteins HRAS, KRAS, and NRAS are the founding members of a larger family of at least 35 related human proteins. Using a somewhat broader definition of sequence similarity reveals a more extended superfamily of more than 170 RAS-related proteins. The RAS superfamily of GTP (guanosine triphosphate) hydrolysis-coupled signal transduction relay proteins can be subclassified into RAS, RHO, RAB, and ARF families, as well as the closely related Galpha family. The members of each family can, in turn, be arranged into evolutionarily conserved branches. These groupings reflect structural, biochemical, and functional conservation. Recent findings have provided insights into the signaling characteristics of representative members of most RAS superfamily branches. The analysis presented here may serve as a guide for predicting the function of numerous uncharacterized superfamily members. Also described are guanosine triphosphatases (GTPases) distinct from members of the RAS superfamily. These related proteins employ GTP binding and GTPase domains in diverse structural contexts, expanding the scope of their function in humans.
Project description:P190A and p190B Rho GTPase activating proteins (GAPs) are essential genes that have distinct, but overlapping roles in the developing nervous system. Previous studies from our laboratory demonstrated that p190B is required for mammary gland morphogenesis, and we hypothesized that p190A might have a distinct role in the developing mammary gland. To test this hypothesis, we examined mammary gland development in p190A-deficient mice. P190A expression was detected by in situ hybridization in the developing E14.5day embryonic mammary bud and within the ducts, terminal end buds (TEBs), and surrounding stroma of the developing virgin mammary gland. In contrast to previous results with p190B, examination of p190A heterozygous mammary glands demonstrated that p190A deficiency disrupted TEB morphology, but did not significantly delay ductal outgrowth indicating haploinsufficiency for TEB development. To examine the effects of homozygous deletion of p190A, embryonic mammary buds were rescued by transplantation into the cleared fat pads of SCID/Beige mice. Complete loss of p190A function inhibited ductal outgrowth in comparison to wildtype transplants (51% vs. 94% fat pad filled). In addition, the transplantation take rate of p190A deficient whole gland transplants from E18.5 embryos was significantly reduced compared to wildtype transplants (31% vs. 90%, respectively). These results suggest that p190A function in both the epithelium and stroma is required for mammary gland development. Immunostaining for p63 demonstrated that the myoepithelial cell layer is disrupted in the p190A deficient glands, which may result from the defective cell adhesion between the cap and body cell layers detected in the TEBs. The number of estrogen- and progesterone receptor-positive cells, as well as the expression levels of these receptors was increased in p190A deficient outgrowths. These data suggest that p190A is required in both the epithelial and stromal compartments for ductal outgrowth and that it may play a role in mammary epithelial cell differentiation.
Project description:The negative regulator of Rho family GTPases, p190A RhoGAP, is one of six mammalian proteins harboring so-called FF motifs. To explore the function of these and other p190A segments, we identified interacting proteins by tandem mass spectrometry. Here we report that endogenous human p190A, but not its 50% identical p190B paralog, associates with all 13 eIF3 subunits and several other translational preinitiation factors. The interaction involves the first FF motif of p190A and the winged helix/PCI domain of eIF3A, is enhanced by serum stimulation and reduced by phosphatase treatment. The p190A/eIF3A interaction is unaffected by mutating phosphorylated p190A-Tyr308, but disrupted by a S296A mutation, targeting the only other known phosphorylated residue in the first FF domain. The p190A-eIF3 complex is distinct from eIF3 complexes containing S6K1 or mammalian target of rapamycin (mTOR), and appears to represent an incomplete preinitiation complex lacking several subunits. Based on these findings we propose that p190A may affect protein translation by controlling the assembly of functional preinitiation complexes. Whether such a role helps to explain why, unique among the large family of RhoGAPs, p190A exhibits a significantly increased mutation rate in cancer remains to be determined.
Project description:ARHGAP35 encoding p190A RhoGAP is a cancer-associated gene with a mutation spectrum suggestive of a tumor-suppressor function. In this study, we demonstrate that loss of heterozygosity for ARHGAP35 occurs in human tumors. We sought to identify tumor-suppressor capacities for p190A RhoGAP (p190A) and its paralog p190B in epithelial cells. We reveal an essential role for p190A and p190B to promote contact inhibition of cell proliferation (CIP), a function that relies on RhoGAP activity. Unbiased mRNA sequencing analyses establish that p190A and p190B modulate expression of genes associated with the Hippo pathway. Accordingly, we determine that p190A and p190B induce CIP by repressing YAP-TEAD-regulated gene transcription through activation of LATS kinases and inhibition of the Rho-ROCK pathway. Finally, we demonstrate that loss of a single p190 paralog is sufficient to elicit nuclear translocation of YAP and perturb CIP in epithelial cells cultured in Matrigel. Collectively, our data reveal a novel mechanism consistent with a tumor-suppressor function for ARHGAP35.