Functional interaction between the cytoplasmic leucine-zipper domain of HIV-1 gp41 and p115-RhoGEF.

The long cytoplasmic tail of the human immunodeficiency virus (HIV)-1 transmembrane protein gp41 (gp41C) is implicated in the replication and cytopathicity of HIV-1 [1]. Little is known about the specific functions of gp41C, however. HIV-1 or simian immunodeficiency virus (SIV) mutants with defective gp41C have cell-type- or species-dependent phenotypes [2] [3] [4] [5] [6]. Thus, host factors are implicated in mediating the functions of gp41C. We report here that gp41C interacted with the carboxy-terminal regulatory domain of p115-RhoGEF [7], a specific guanine nucleotide exchange factor (GEF) and activator of the RhoA GTPase, which regulates actin stress fiber formation, activation of serum response factor (SRF) and cell proliferation [8] [9]. We demonstrate that gp41C inhibited p115-mediated actin stress fiber formation and activation of SRF. An amphipathic helix region with a leucine-zipper motif in gp41C is involved in its interaction with p115. Mutations in gp41C leading to loss of interaction with p115 impaired HIV-1 replication in human T cells. These findings suggest that an important function of gp41C is to modulate the activity of p115-RhoGEF and they thus reveal a new potential anti-HIV-1 target.     

A Highlights from MBoC Selection: Involvement of p114-RhoGEF and Lfc in Wnt-3a– and Dishevelled-Induced RhoA Activation and Neurite Retraction in N1E-115 Mouse Neuroblastoma Cells

The Wnt-induced planar cell polarity (PCP) signaling pathway is essential for polarized cell migration and morphogenesis. Dishevelled (Dvl) and its binding protein Daam1 mediate RhoA activation in this pathway. WGEF, a member of the Rho-guanine nucleotide exchange factor (Rho-GEF) family, was shown to play a role in Wnt-induced RhoA activation in Xenopus embryos. However, it has remained unknown which member(s) of a Rho-GEF family are involved in Wnt/Dvl-induced RhoA activation in mammalian cells. Here we identified p114-RhoGEF and Lfc (also called GEF-H1) as the Rho-GEFs responsible for Wnt-3a–induced RhoA activation in N1E-115 mouse neuroblastoma cells. We screened for Rho-GEF–silencing short-hairpin RNAs (shRNAs) that are capable of suppressing Dvl-induced neurite retraction in N1E-115 cells and found that p114-RhoGEF and Lfc shRNAs, but not WGEF shRNA, suppressed Dvl- and Wnt-3a–induced neurite retraction. p114-RhoGEF and Lfc shRNAs also inhibited Dvl- and Wnt-3a–induced RhoA activation, and p114-RhoGEF and Lfc proteins were capable of binding to Dvl and Daam1. Additionally, the Dvl-binding domains of p114-RhoGEF and Lfc inhibited Dvl-induced neurite retraction. Our results suggest that p114-RhoGEF and Lfc are critically involved in Wnt-3a– and Dvl-induced RhoA activation and neurite retraction in N1E-115 cells.     

Differences in Gα12- and Gα13-mediated plasma membrane recruitment of p115-RhoGEF

Regulator of G protein signaling domain-containing Rho guanine-nucleotide exchange factors (RGS-RhoGEFs) directly links activated forms of the G12 family of heterotrimeric G protein α subunits to the small GTPase Rho. Stimulation of G_12/13-coupled GPCRs or expression of constitutively activated forms of α₁₂ and α₁₃ has been shown to induce the translocation of the RGS-RhoGEF, p115-RhoGEF, from the cytoplasm to the plasma membrane (PM). However, little is known regarding the functional importance and mechanisms of this regulated PM recruitment, and thus PM recruitment of p115-RhoGEF is the focus of this report. A constitutively PM-localized mutant of p115-RhoGEF shows a much greater activity compared to wild type p115-RhoGEF in promoting Rho-dependent neurite retraction of NGF-differentiated PC12 cells, providing the first evidence that PM localization can activate p115-RhoGEF signaling. Next, we uncovered the unexpected finding that Rho is required for α₁₃-induced PM translocation of p115-RhoGEF. However, inhibition of Rho did not prevent α₁₂-induced PM translocation of p115-RhoGEF. Additional differences between α₁₃ and α₁₂ in promoting PM recruitment of p115-RhoGEF were revealed by analyzing RGS domain mutants of p115-RhoGEF. Activated α₁₂ effectively recruits the isolated RGS domain of p115-RhoGEF to the PM, whereas α₁₃ only weakly does. On the other hand, α₁₃ strongly recruits to the PM a p115-RhoGEF mutant containing amino acid substitutions in an acidic region at the N-terminus of the RGS domain; however, α₁₂ is unable to recruit this p115-RhoGEF mutant to the PM. These studies provide new insight into the function and mechanisms of α_12/13-mediated PM recruitment of p115-RhoGEF.     

Mutation of an N-terminal acidic-rich region of p115-RhoGEF dissociates alpha13 binding and alpha13-promoted plasma membrane recruitment

The Ras homology (Rho) guanine nucleotide exchange factor p115-RhoGEF couples the alpha(13) heterotrimeric guanine nucleotide binding protein (G protein) subunit to Rho GTPase. Alpha(13) binds to a regulator of G protein signaling (RGS) domain in p115-RhoGEF, but the mechanism of alpha(13) activation of p115-RhoGEF is poorly understood. In this report, we demonstrate in cell-based assays that the acidic-rich N-terminus, adjacent to the RGS domain, is required for binding to activated alpha(13), and refine the importance of this region by showing that mutation of glutamic acids 27 and 29 in full-length p115-RhoGEF is sufficient to prevent interaction with activated alpha(13). However, alpha(13)-interacting deficient N-terminal mutants of p115-RhoGEF retain alpha(13)-dependent plasma membrane recruitment. Overall, these findings demonstrate a critical role for the N-terminal extension of p115-RhoGEF in mediating binding to alpha(13) and dissociate two activities of p115-RhoGEF: binding to activated alpha(13) and translocation to the PM in response to activated alpha(13).     

Leukemia-associated Rho guanine nucleotide exchange factor promotes G alpha q-coupled activation of RhoA

Leukemia-associated Rho guanine-nucleotide exchange factor (LARG) belongs to the subfamily of Dbl homology RhoGEF proteins (including p115 RhoGEF and PDZ-RhoGEF) that possess amino-terminal regulator of G protein signaling (RGS) boxes also found within GTPase-accelerating proteins (GAPs) for heterotrimeric G protein alpha subunits. p115 RhoGEF stimulates the intrinsic GTP hydrolysis activity of G alpha 12/13 subunits and acts as an effector for G13-coupled receptors by linking receptor activation to RhoA activation. The presence of RGS box and Dbl homology domains within LARG suggests this protein may also function as a GAP toward specific G alpha subunits and couple G alpha activation to RhoA-mediating signaling pathways. Unlike the RGS box of p115 RhoGEF, the RGS box of LARG interacts not only with G alpha 12 and G alpha 13 but also with G alpha q. In cellular coimmunoprecipitation studies, the LARG RGS box formed stable complexes with the transition state mimetic forms of G alpha q, G alpha 12, and G alpha 13. Expression of the LARG RGS box diminished the transforming activity of oncogenic G protein-coupled receptors (Mas, G2A, and m1-muscarinic cholinergic) coupled to G alpha q and G alpha 13. Activated G alpha q, as well as G alpha 12 and G alpha 13, cooperated with LARG and caused synergistic activation of RhoA, suggesting that all three G alpha subunits stimulate LARG-mediated activation of RhoA. Our findings suggest that the RhoA exchange factor LARG, unlike the related p115 RhoGEF and PDZ-RhoGEF proteins, can serve as an effector for Gq-coupled receptors, mediating their functional linkage to RhoA-dependent signaling pathways.     

A p27(kip1)-binding protein, p27RF-Rho, promotes cancer metastasis via activation of RhoA and RhoC

Rho family proteins regulate multiple cellular functions including motility and invasion through regulation of the actin cytoskeleton and gene expression. Activation of Rho proteins is controlled precisely by multiple regulators in a spatiotemporal manner. RhoA and/or RhoC are key players that regulate the metastatic activity of malignant tumor cells, and it is therefore of particular interest to understand how activation of these Rho proteins is controlled. We recently identified an upstream regulator of RhoA activation, p27RF-Rho (p27(kip1) releasing factor from RhoA) that acts by freeing RhoA from inhibition by p27(kip1). p27(kip1) is a cell cycle regulator when it is localized to the nucleus, but it binds RhoA and inhibits activation of the latter when it is localized to the cytoplasm. Here, we show that a metastatic variant of mouse melanoma B16 cells (F10) exhibits greater expression of p27RF-Rho, RhoA, and RhoC than the nonmetastatic parental cells (F0). Injection of F10 cells into mouse tail vein resulted in the formation of metastatic lung colonies, whereas prior knockdown of expression of either one of the three proteins using specific shRNA sequences decreased metastasis markedly. p27RF-Rho regulated the activation of RhoA and RhoC and thereby modulated cellular adhesion and motility, in addition to pericellular proteolysis. The Rho activities enhanced by p27RF-Rho had a marked effect upon efficiency of lodging of F10 cells in the lung, which represents an early step of metastasis. p27RF-Rho also regulated metastasis of human melanoma and fibrosarcoma cells. Thus, p27RF-Rho is a key upstream regulator of RhoA and RhoC that controls spreading of tumor cells.     

The RhoA effector mDia is induced during T cell activation and regulates actin polymerization and cell migration in T lymphocytes

Regulation of actin polymerization is critical for many different functions of T lymphocytes, including cell migration. Here we show that the RhoA effector mDia is induced in vitro in activated PBL and is highly expressed in vivo in diseased tissue-infiltrating activated lymphocytes. mDia localizes at the leading edge of polarized T lymphoblasts in an area immediately posterior to the leading lamella, in which its effector protein profilin is also concentrated. Overexpression of an activated mutant of mDia results in an inhibition of both spontaneous and chemokine-directed T cell motility. mDia does not regulate the shape of the cell, which involves another RhoA effector, p160 Rho-coiled coil kinase, and is not involved in integrin-mediated cell adhesion. However, mDia activation blocked CD3- and PMA-mediated cell spreading. mDia activation increased polymerized actin levels, which resulted in the blockade of chemokine-induced actin polymerization by depletion of monomeric actin. Moreover, mDia was shown to regulate the function of the small GTPase Rac1 through the control of actin availability. Together, our data demonstrate that RhoA is involved in the control of the filamentous actin/monomeric actin balance through mDia, and that this balance is critical for T cell responses.     

Reversible translocation of p115-RhoGEF by G(12/13)-coupled receptors

G protein-coupled receptors (GPCRs) are important targets for medicinal agents. Four different G protein families, G(s), G(i), G(q), and G(12), engage in their linkage to activation of receptor-specific signal transduction pathways. G(12) proteins were more recently studied, and upon activation by GPCRs they mediate activation of RhoGTPase guanine nucleotide exchange factors (RhoGEFs), which in turn activate the small GTPase RhoA. RhoA is involved in many cellular and physiological aspects, and a dysfunction of the G(12/13)-Rho pathway can lead to hypertension, cardiovascular diseases, stroke, impaired wound healing and immune cell functions, cancer progression and metastasis, or asthma. In this study, regulator of G protein signaling (RGS) domain-containing RhoGEFs were tagged with enhanced green fluorescent protein (EGFP) to detect their subcellular localization and translocation upon receptor activation. Constitutively active Galpha(12) and Galpha(13) mutants induced redistribution of these RhoGEFs from the cytosol to the plasma membrane. Furthermore, a pronounced and rapid translocation of p115-RhoGEF from the cytosol to the plasma membrane was observed upon activation of several G(12/13)-coupled GPCRs in a cell type-independent fashion. Plasma membrane translocation of p115-RhoGEF stimulated by a GPCR agonist could be completely and rapidly reversed by subsequent application of an antagonist for the respective GPCR, that is, p115-RhoGEF relocated back to the cytosol. The translocation of RhoGEF by G(12/13)-linked GPCRs can be quantified and therefore used for pharmacological studies of the pathway, and to discover active compounds in a G(12/13)-related disease context.     

LIM kinase and Diaphanous cooperate to regulate serum response factor and actin dynamics

The small GTPase RhoA controls activity of serum response factor (SRF) by inducing changes in actin dynamics. We show that in PC12 cells, activation of SRF after serum stimulation is RhoA dependent, requiring both actin polymerization and the Rho kinase (ROCK)-LIM kinase (LIMK)-cofilin signaling pathway, previously shown to control F-actin turnover. Activation of SRF by overexpression of wild-type LIMK or ROCK-insensitive LIMK mutants also requires functional RhoA, indicating that a second RhoA-dependent signal is involved. This is provided by the RhoA effector mDia: dominant interfering mDia1 derivatives inhibit both serum- and LIMK-induced SRF activation and reduce the ability of LIMK to induce F-actin accumulation. These results demonstrate a role for LIMK in SRF activation, and functional cooperation between RhoA-controlled LIMK and mDia effector pathways.     

Integrin engagement suppresses RhoA activity via a c-Src-dependent mechanism

The Rho family GTPases Cdc42, Rac1 and RhoA control many of the changes in the actin cytoskeleton that are triggered when growth factor receptors and integrins bind their ligands [1] [2]. Rac1 and Cdc42 stimulate the formation of protrusive structures such as membrane ruffles, lamellipodia and filopodia. RhoA regulates contractility and assembly of actin stress fibers and focal adhesions. Although prolonged integrin engagement can stimulate RhoA [3] [4] [5], regulation of this GTPase by early integrin-mediated signals is poorly understood. Here we show that integrin engagement initially inactivates RhoA, in a c-Src-dependent manner, but has no effect on Cdc42 or Rac1 activity. Additionally, early integrin signaling induces activation and tyrosine phosphorylation of p190RhoGAP via a mechanism that requires c-Src. Dynamic modulation of RhoA activity appears to have a role in motility, as both inhibition and activation of RhoA hinder migration [6] [7] [8]. Transient suppression of RhoA by integrins may alleviate contractile forces that would otherwise impede protrusion at the leading edge of migrating cells.     

Cdc42 and RhoA have opposing roles in regulating membrane type 1-matrix metalloproteinase localization and matrix metalloproteinase-2 activation

Proteolysis of the basement membrane and interstitial matrix occurs early in the angiogenic process and requires matrix metalloproteinase (MMP) activity. Skeletal muscle microvascular endothelial cells exhibit robust actin stress fibers, low levels of membrane type 1 (MT1)-MMP expression, and minimal MMP-2 activation. Depolymerization of the actin cytoskeleton increases MT1-MMP expression and MMP-2 activation. Rho family GTPases are regulators of actin cytoskeleton dynamics, and their activity can be modulated in response to angiogenic stimuli such as vascular endothelial growth factor (VEGF). Therefore, we investigated their roles in MMP-2 and MT1-MMP production. Endothelial cells treated with H1152 [an inhibitor of Rho kinase (ROCK)] induced stress fiber depolymerization and an increase in cortical actin. Both MMP-2 and MT1-MMP mRNA increased, which translated into greater MMP-2 protein production and activation. ROCK inhibition rapidly increased cell surface localization of MT1-MMP and increased PI3K activity, which was required for MMP-2 activation. Constitutively active Cdc42 increased cortical actin polymerization, phosphatidylinositol 3-kinase activity, MT1-MMP cell surface localization, and MMP-2 activation similarly to inhibition of ROCK. Activation of Cdc42 was sufficient to decrease RhoA activity. Capillary sprout formation in a three-dimensional collagen matrix was increased in cultures treated with RhoAN19 or Cdc42QL and, conversely, decreased in cultures treated with dominant negative Cdc42N17. VEGF stimulation also induced activation of Cdc42 while inhibiting RhoA activity. Furthermore, VEGF-dependent activation of MMP-2 was reduced by inhibition of Cdc42. These results suggest that Cdc42 and RhoA have opposing roles in regulating cell surface localization of MT1-MMP and MMP-2 activation.     

Rnd proteins function as RhoA antagonists by activating p190 RhoGAP

BACKGROUND: The Rnd proteins Rnd1, Rnd2, and Rnd3 (RhoE) comprise a unique branch of Rho-family G-proteins that lack intrinsic GTPase activity and consequently remain constitutively "active." Prior studies have suggested that Rnd proteins play pivotal roles in cell regulation by counteracting the biological functions of the RhoA GTPase, but the molecular basis for this antagonism is unknown. Possible mechanisms by which Rnd proteins could function as RhoA antagonists include sequestration of RhoA effector molecules, inhibition of guanine nucleotide exchange factors, and activation of GTPase-activating proteins (GAPs) for RhoA. However, effector molecules of Rnd proteins with such properties have not been identified. RESULTS: Here we identify p190 RhoGAP (p190), the most abundant GAP for RhoA in cells, as an interactor with Rnd proteins and show that this interaction is mediated by a p190 region that is distinct from the GAP domain. Using Rnd3-RhoA chimeras and Rnd3 mutants defective in p190 binding, as well as p190-deficient cells, we demonstrate that the cellular effects of Rnd expression are mediated by p190. We moreover show that Rnd proteins increase the GAP activity of p190 toward GTP bound RhoA and, finally, demonstrate that expression of Rnd3 leads to reduced cellular levels of RhoA-GTP by a p190-dependent mechanism. CONCLUSIONS: Our results identify p190 RhoGAPs as effectors of Rnd proteins and demonstrate a novel mechanism by which Rnd proteins function as antagonists of RhoA.     

HIV-1 Nef disrupts the podocyte actin cytoskeleton by interacting with diaphanous interacting protein

The ability of the human immunodeficiency virus, type 1 (HIV-1) protein Nef to induce cytoskeleton changes in infected host cells is a key event in viral replication. In renal podocytes, we found that Nef induced loss of stress fibers and increased lamellipodia, pathological changes leading to proteinuria in HIV-associated nephropathy. These morphological changes were mediated by Nef-induced Rac1 activation and RhoA inhibition. We identified a new interaction between Nef and diaphanous interacting protein (DIP), a recently described regulator of Rho and Rac signaling. We found that the Src homology 3 binding domain of DIP and the Nef PXXP motif were required for this interaction. Nef also interacts with Vav2 in podocytes. DIP and Vav2 both interact directly with Nef in a competitive manner. DIP interacts with p190RhoGAP, and intact DIP was required for Nef-induced phosphorylation of p190RhoGAP. DIP also interacts with Vav2, and although DIP enhanced baseline phosphorylation of Vav2, it was not required for Nef-induced Vav2 activation. In Nef-infected podocytes, Src kinase induces phosphorylation of DIP, p190RhoGAP, and Vav2, leading to RhoA inhibition and Rac1 activation. Inhibition of the Nef-induced signaling pathway by using a dominant negative of either Src or DIP or siRNA for DIP or p190RhoAGAP restored RhoA activity and stress fiber formation in Nef-infected podocytes, whereas siRNA for Vav2 reduced Rac1 activity and formation of lamellipodia. We conclude that in HIV-infected podocytes, Nef, through the recruitment of DIP and p190RhoAGAP to Nef-Src complex, activates p190RhoAGAP and down-regulates RhoA activity.