Regulation of epidermal growth factor receptor traffic by the small GTPase rhoB.

Members of the Rho family of small GTPases control cell adhesion and motility through dynamic regulation of the actin cytoskeleton. Although twelve family members have been identified, only three of these - RhoA, Rac and Cdc42 - have been studied in detail. RhoA regulates the formation of focal adhesions and the bundling of actin filaments into stress fibres. It is also involved in other cell signalling pathways including the regulation of gene expression and the generation of lipid second messengers [1] [2]. RhoA is very closely related to two other small GTPases about which much less is known: RhoB and RhoC (which are approximately 83% identical). Perhaps the most intriguing of these is RhoB. RhoA is largely cytosolic but translocates to the plasma membrane on activation. RhoB, however, is entirely localised to the cytosolic face of endocytic vesicles [3] [4]. This suggests a potential role for RhoB in regulating endocytic traffic; however, no evidence has been presented to support this. RhoA has been shown to act at the plasma membrane to regulate the clathrin-mediated internalisation of transferrin receptor [5] and of the muscarinic acetylcholine receptor [6]. We have recently demonstrated that RhoB binds the RhoA effector, PRK1 and targets it to the endosomal compartment [7]. We show here that RhoB acts through PRK1 to regulate the kinetics of epidermal growth factor receptor traffic.     

Farnesylated RhoB Prevents Cell Cycle Arrest and Actin Cytoskeleton Disruption Caused by the Geranylgeranyltransferase I Inhibitor GGTI-298

Here we demonstrate that the geranylgeranyltransferase-I inhibitor GGTI-298 inhibits the RhoB pathway and disrupts stress fiber and focal adhesion formation in NIH-3T3 cells. Farnesylated V14RhoB-CAIM (resistant to GGTI-298), but not geranylgeranylated V14RhoB (-CLLL), prevented inhibition of actin stress fiber and focal adhesion formation, underlining the critical role of RhoB. In contrast, farnesylated, V14RhoA (-CVLS) was unable to prevent effects of GGTI 298 on cytoskeleton organization. Furthermore, the ability of GGTI-298 to induce p21WAF and to block cells in the G0/G1 phase of the cell cycle was also prevented by farnesylated V14RhoB but not by farnesylated V14RhoA. Moreover, treatment with GGTI-298 of cells expressing farnesylated RhoB results in accumulation of these cells in the G2/M phase. Therefore, the RhoB pathway is a critical target of GGTI-298.     

The Abl-related gene tyrosine kinase acts through p190RhoGAP to inhibit actomyosin contractility and regulate focal adhesion dynamics upon adhesion to fibronectin

In migrating cells, actin polymerization promotes protrusion of the leading edge, whereas actomyosin contractility powers net cell body translocation. Although they promote F-actin-dependent protrusions of the cell periphery upon adhesion to fibronectin (FN), Abl family kinases inhibit cell migration on FN. We provide evidence here that the Abl-related gene (Arg/Abl2) kinase inhibits fibroblast migration by attenuating actomyosin contractility and regulating focal adhesion dynamics. arg-/- fibroblasts migrate at faster average speeds than wild-type (wt) cells, whereas Arg re-expression in these cells slows migration. Surprisingly, the faster migrating arg-/- fibroblasts have more prominent F-actin stress fibers and focal adhesions and exhibit increased actomyosin contractility relative to wt cells. Interestingly, Arg requires distinct functional domains to inhibit focal adhesions and actomyosin contractility. The kinase domain-containing Arg N-terminal half can act through the RhoA inhibitor p190RhoGAP to attenuate stress fiber formation and cell contractility. However, Arg requires both its kinase activity and its cytoskeleton-binding C-terminal half to fully inhibit focal adhesions. Although focal adhesions do not turn over efficiently in the trailing edge of arg-/- cells, the increased contractility of arg-/- cells tears the adhesions from the substrate, allowing for the faster migration observed in these cells. Together, our data strongly suggest that Arg inhibits cell migration by restricting actomyosin contractility and regulating its coupling to the substrate through focal adhesions.     

A novel strategy for specifically down-regulating individual Rho GTPase activity in tumor cells

The Rho family GTPases RhoA, RhoB, and RhoC regulate the actin cytoskeleton, cell movement, and cell growth. Unlike Ras, up-regulation or overexpression of these GDP/GTP binding molecular switches, but not activating point mutations, has been associated with human cancer. Although they share over 85% sequence identity, RhoA, RhoB, and RhoC appear to play distinct roles in cell transformation and metastasis. In NIH 3T3 cells, RhoA or RhoB overexpression causes transformation whereas RhoC increases the cell migration rate. To specifically target RhoA, RhoB, or RhoC function, we have generated a set of chimeric molecules by fusing the RhoGAP domain of p190, a GTPase-activating protein that accelerates the intrinsic GTPase activity of all three Rho GTPases, with the C-terminal hypervariable sequences of RhoA, RhoB, or RhoC. The p190-Rho chimeras were active as GTPase-activating proteins toward RhoA in vitro, co-localized with the respective active Rho proteins, and specifically down-regulated Rho protein activities in cells depending on which Rho GTPase sequences were included in the chimeras. In particular, the p190-RhoA-C chimera specifically inhibited RhoA-induced transformation whereas p190-RhoC-C specifically reversed the migration phenotype induced by the active RhoC. In human mammary epithelial-RhoC breast cancer cells, p190-RhoC-C, but not p190-RhoA-C or p190-RhoB-C, reversed the anchorage-independent growth and invasion phenotypes caused by RhoC overexpression. In the highly metastatic A375-M human melanoma cells, p190-RhoC-C specifically reversed migration, and invasion phenotypes attributed to RhoC up-regulation. Thus, we have developed a novel strategy utilizing RhoGAP-Rho chimeras to specifically down-regulate individual Rho activity and demonstrate that this approach may be applied to multiple human tumor cells to reverse the growth and/or invasion phenotypes associated with disregulation of a distinct subtype of Rho GTPase.     

Farnesylated RhoB prevents cell cycle arrest and actin cytoskeleton disruption caused by the geranylgeranyltransferase I inhibitor GGTI-298

Here we demonstrate that the geranylgeranyltransferase-I inhibitor GGTI-298 inhibits the RhoB pathway and disrupts stress fiber and focal adhesion formation in NIH-3T3 cells. Farnesylated (V14)RhoB-CAIM (resistant to GGTI-298), but not geranylgeranylated (V14)RhoB (-CLLL), prevented inhibition of actin stress fiber and focal adhesion formation, underlining the critical role of RhoB. In contrast, farnesylated, (V14)RhoA (-CVLS) was unable to prevent effects of GGTI 298 on cytoskeleton organization. Furthermore, the ability of GGTI-298 to induce p21(WAF) and to block cells in the G(0)/G(1) phase of the cell cycle was also prevented by farnesylated (V14)RhoB but not by farnesylated (V14)RhoA. Moreover, treatment with GGTI-298 of cells expressing farnesylated RhoB results in accumulation of these cells in the G(2)/M phase. Therefore, the RhoB pathway is a critical target of GGTI-298.     

The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton.

The GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, motility, and cytokinesis. We recently reported on a p150 serine/threonine kinase (termed ROK alpha) binding RhoA only in its active GTP-bound state and on its cDNA; introduction of RhoA into HeLa cells resulted in translocation of the cytoplasmic kinase to plasma membranes, consistent with ROK alpha being a target for RhoA (T. Leung, E. Manser, L. Tan, and L. Lim, J. Biol. Chem. 256:29051-29054, 1995). Reanalysis of the cDNA revealed that ROK alpha contains an additional N-terminal region. We also isolated another cDNA which encoded a protein (ROK beta) with 90% identity to ROK alpha in the kinase domain. Both ROK alpha and ROK beta, which had a molecular mass of 160 kDa, contained a highly conserved cysteine/histidine-rich domain located within a putative pleckstrin homology domain. The kinases bound RhoA, RhoB, and RhoC but not Rac1 and Cdc42. The Rho-binding domain comprises about 30 amino acids. Mutations within this domain caused partial or complete loss of Rho binding. The morphological effects of ROK alpha were investigated by microinjecting HeLa cells with DNA constructs encoding various forms of ROK alpha. Full-length ROK alpha promoted formation of stress fibers and focal adhesion complexes, consistent with its being an effector of RhoA. ROK alpha truncated at the C terminus promoted this formation and also extensive condensation of actin microfilaments and nuclear disruption. The proteins exhibited protein kinase activity which was required for stress fiber formation; the kinase-dead ROK alpha K112A and N-terminally truncated mutants showed no such promotion. The latter mutant instead induced disassembly of stress fibers and focal adhesion complexes, accompanied by cell spreading. These effects were mediated by the C-terminal region containing Rho-binding, cysteine/histidine-rich, and pleckstrin homology domains. Thus, the multidomained ROK alpha appears to be involved in reorganization of the cytoskeleton, with the N and C termini acting as positive and negative regulators, respectively, of the kinase domain whose activity is crucial for formation of stress fibers and focal adhesion complexes.     

RhoA-GDP regulates RhoB protein stability. Potential involvement of RhoGDIalpha

RhoA plays a significant role in actin stress fibers formation. However, silencing RhoA alone or RhoA and RhoC did not completely suppress the stress fibers suggesting a residual "Rho-like" activity. RhoB, the third member of the Rho subclass, is a shortlived protein barely detectable in basal conditions. In various cell types, the silencing of RhoA induced a strong up-regulation of both total and active RhoB protein levels that were rescued by re-expressing RhoA and related to an enhanced half-life of the protein. The RhoA-dependent regulation of RhoB does not depend on the activity of RhoA but is mediated by its GDP-bound form. The stabilization of RhoB was not dependent on isoprenoid biosynthesis, Rho kinase, extracellular signal-regulated kinase, p38 mitogen-activated kinase, or phosphatidylinositol 3'-OH kinase pathways but required RhoGDIalpha. The forced expression of RhoGDIalpha increased RhoB half-life, whereas its knock-down antagonized the induction of RhoB following RhoA silencing. Moreover, a RhoA mutant (RhoAR68E) unable to bind RhoGDIalpha was significantly less efficient as compared with wild-type RhoA in reversing RhoB up-regulation upon RhoA silencing. These results suggest that, in basal conditions, RhoGDIalpha is rate-limiting and the suppression of RhoA makes it available to stabilize RhoB. Our results highlight RhoGDIalpha-dependent cross-talks that regulate the stability of RhoGTPases.     

Why three Rho proteins? RhoA, RhoB, RhoC, and cell motility

Higher vertebrates have 3 Rho GTPases, RhoA, RhoB, and RhoC, which share 85% amino acid sequence identity. Here, we compare and contrast the roles of RhoA, B, and C in the regulation of the cytoskeleton and cell motility. Despite their similarity, some regulators and effectors show preferential interaction with RhoA, B, or C, and the three proteins show differences in function in cells. RhoA plays a key role in the regulation of actomyosin contractility. RhoB, which is localized primarily on endosomes, has been shown to regulate cytokine trafficking and cell survival, while RhoC may be more important in cell locomotion. In cancer cells, the expression and activity of RhoA, B, and C is altered in different ways. Together, this evidence suggests that although the 3 isoforms of Rho are structurally highly homologous, they have different cellular functions.     

Genetic deletion of Rac1 GTPase reveals its critical role in actin stress fiber formation and focal adhesion complex assembly

Rac1 is an intracellular signal transducer regulating a variety of cell functions. Previous studies by overexpression of dominant-negative or constitutively active mutants of Rac1 in clonal cell lines have established that Rac1 plays a key role in actin lamellipodia induction, cell-matrix adhesion, and cell anoikis. In the present studies, we have examined the cellular behaviors of Rac1 gene-targeted primary mouse embryonic fibroblasts (MEFs) after Cre recombinase-mediated deletion of Rac1 gene. Rac1-null MEFs became contracted and elongated in morphology and were defective in lamellipodia formation, cell spreading, cell-fibronectin adhesion, and focal contact formation in response to platelet-derived growth factor or serum. Unexpectedly, deletion of Rac1 also abolished actin stress fibers in the cells without detectable alteration of endogenous RhoA activity. Although the expression and/or activation status of focal adhesion complex components such as Src, FAK, and vinculin were not affected by Rac1 deletion, the number and size of adhesion plaques were significantly reduced, and the molecular complex between Src, FAK, and vinculin was dissembled in Rac1-null cells. Overexpression of an active RhoA mutant or ROK failed to rescue the stress fiber and adhesion plaque defects of the Rac1-null cells. Although Rac1 deletion caused a significant reduction in phospho-PAK1, -AKT, and -ERK under serum stimulation, reconstitution of active PAK1, but not AKT or MEK1, was able to rescue the actin cytoskeleton and adhesion phenotypes of the Rac1-deficient cells. Furthermore, Rac1 deletion led to a marked increase in spontaneous apoptosis that could be rescued by active PAK1, AKT, or MEK1 expression. Our results obtained from gene-targeted primary MEFs indicate that Rac1 is essential not only for lamellipodia induction but also for the RhoA-regulated actin stress fiber and focal adhesion complex formation and that Rac1 is involved in cell survival regulation through anoikis-dependent as well as -independent mechanisms.     

XPLN,a guanine nucleotide exchange factor for RhoA and RhoB,but not RhoC

Rho proteins cycle between an inactive, GDP-bound state and an active, GTP-bound state. Activation of these GTPases is mediated by guanine nucleotide exchange factors (GEFs), which promote GDP to GTP exchange. In this study we have characterized XPLN, a Rho family GEF. Like other Rho GEFs, XPLN contains a tandem Dbl homology and pleckstrin homology domain topography, but lacks homology with other known functional domains or motifs. XPLN protein is expressed in the brain, skeletal muscle, heart, kidney, platelets, and macrophage and neuronal cell lines. In vitro, XPLN stimulates guanine nucleotide exchange on RhoA and RhoB, but not RhoC, RhoG, Rac1, or Cdc42. Consistent with these data, XPLN preferentially associates with RhoA and RhoB. The specificity of XPLN for RhoA and RhoB, but not RhoC, is surprising given that they share over 85% sequence identity. We determined that the inability of XPLN to exchange RhoC is mediated by isoleucine 43 in RhoC, a position occupied by valine in RhoA and RhoB. When expressed in cells, XPLN activates RhoA and RhoB, but not RhoC, and stimulates the assembly of stress fibers and focal adhesions in a Rho kinase-dependent manner. We also found that XPLN possesses transforming activity, as determined by focus formation assays. In conclusion, here we describe a Rho family GEF that can discriminate between the closely related RhoA, RhoB, and RhoC, possibly giving insight to the divergent functions of these three proteins.     

RhoE Regulates Actin Cytoskeleton Organization and Cell Migration

The actin cytoskeleton is regulated by Rho family proteins: in fibroblasts, Rho mediates the formation of actin stress fibers, whereas Rac regulates lamellipodium formation and Cdc42 controls filopodium formation. We have cloned the mouse RhoE gene, whose product is a member of the Rho family that shares (except in one amino acid) the conserved effector domain of RhoA, RhoB, and RhoC. RhoE is able to bind GTP but does not detectably bind GDP and has low intrinsic GTPase activity compared with Rac. The role of RhoE in regulating actin organization was investigated by microinjection in Bac1.2F5 macrophages and MDCK cells. In macrophages, RhoE induced actin reorganization, leading to the formation of extensions resembling filopodia and pseudopodia. In MDCK cells, RhoE induced the complete disappearance of stress fibers, together with cell spreading. However, RhoE did not detectably affect the actin bundles that run parallel to the outer membranes of cells at the periphery of colonies, which are known to be dependent on RhoA. In addition, RhoE induced an increase in the speed of migration of hepatocyte growth factor/scatter factor-stimulated MDCK cells, in contrast to the previously reported inhibition produced by activated RhoA. The subcellular localization of RhoE at the lateral membranes of MDCK cells suggests a role in cell-cell adhesion, as has been shown for RhoA. These results suggest that RhoE may act to inhibit signalling downstream of RhoA, altering some RhoA-regulated responses, such as stress fiber formation, but not affecting others, such as peripheral actin bundle formation.     

Genetic ablation of zyxin causes Mena/VASP mislocalization, increased motility, and deficits in actin remodeling

Focal adhesions are specialized regions of the cell surface where integrin receptors and associated proteins link the extracellular matrix to the actin cytoskeleton. To define the cellular role of the focal adhesion protein zyxin, we characterized the phenotype of fibroblasts in which the zyxin gene was deleted by homologous recombination. Zyxin-null fibroblasts display enhanced integrin-dependent adhesion and are more migratory than wild-type fibroblasts, displaying reduced dependence on extracellular matrix cues. We identified differences in the profiles of 75- and 80-kD tyrosine-phosphorylated proteins in the zyxin-null cells. Tandem array mass spectrometry identified both modified proteins as isoforms of the actomyosin regulator caldesmon, a protein known to influence contractility, stress fiber formation, and motility. Zyxin-null fibroblasts also show deficits in actin stress fiber remodeling and exhibit changes in the molecular composition of focal adhesions, most notably by severely reduced accumulation of Ena/VASP proteins. We postulate that zyxin cooperates with Ena/VASP proteins and caldesmon to influence integrin-dependent cell motility and actin stress fiber remodeling.     

LIM-kinase 2 and cofilin phosphorylation mediate actin cytoskeleton reorganization induced by transforming growth factor-beta

Reorganization of the actin cytoskeleton in response to growth factor signaling, such as transforming growth factor beta (TGF-beta), controls cell adhesion, motility, and growth of diverse cell types. In Swiss3T3 fibroblasts, a widely used model for studies of actin reorganization, TGF-beta1 induced rapid actin polymerization into stress fibers and concomitantly activated RhoA and RhoB small GTPases. Consequently, dominant-negative RhoA and RhoB mutants blocked TGF-beta1-induced actin reorganization. Because Rho GTPases are known to regulate the activity of LIM-kinases (LIMK), we found that TGF-beta1 induced LIMK2 phosphorylation with similar kinetics to Rho activation. Cofilin and LIMK2 co-precipitated and cofilin became phosphorylated in response to TGF-beta1, whereas RNA interference against LIMK2 blocked formation of new stress fibers by TGF-beta1. Because the kinase ROCK1 links Rho GTPases to LIMK2, we found that inhibiting ROCK1 activity blocked completely TGF-beta1-induced LIMK2/cofilin phosphorylation and downstream stress fiber formation. We then tested whether the canonical TGF-beta receptor/Smad pathway mediates regulation of the above effectors and actin reorganization. Adenoviruses expressing constitutively activated TGF-beta type I receptor led to robust actin reorganization and Rho activation, whereas the constitutively activated TGF-beta type I receptor with mutated Smad docking sites (L45 loop) did not affect either actin organization or Rho activity. In line with this, ectopic expression of the inhibitory Smad7 inhibited TGF-beta1-induced Rho activation and cytoskeletal reorganization. Our data define a novel pathway emanating from the TGF-beta type I receptor and leading to regulation of actin assembly, via the kinase LIMK2.     

The headpiece domain of dematin regulates cell shape, motility, and wound healing by modulating RhoA activation

RhoA is known to participate in cytoskeletal remodeling events through several signaling pathways, yet the precise mechanism of its activation remains unknown. Here, we provide the first evidence that dematin functions upstream of RhoA and regulates its activation. Primary mouse embryonic fibroblasts were generated from a dematin headpiece domain null (HPKO) mouse, and the visualization of the actin morphology revealed a time-dependent defect in stress fiber formation, membrane protrusions, cell motility, and cell adhesion. Rescue experiments using RNA interference and transfection assays revealed that the observed phenotypes are due to a null effect and not a gain of function in the mutant fibroblasts. In vivo wounding of adult HPKO mouse skin showed a decrease in wound healing (reepithelialization and granulation) compared to the wild-type control. Biochemical analysis of the HPKO fibroblasts revealed a sustained hyperphosphorylation of focal adhesion kinase (FAK) at tyrosine 397 as well as a twofold increase in RhoA activation. Inhibition of both RhoA and FAK signaling using C3 toxin and FRNK (focal adhesion kinase nonrelated kinase), respectively, revealed that dematin acts upstream of RhoA. Together, these results unveil a new function of dematin as a negative regulator of the RhoA activation pathway with physiological implications for normal and pathogenic signaling pathways.     

Generation of a Single Chain Antibody Variable Fragment (scFv) to Sense Selectively RhoB Activation

Determining the cellular level of activated form of RhoGTPases is of key importance to understand their regulatory functions in cell physiopathology. We previously reported scFvC1, that selectively bind to the GTP-bound form of RhoA, RhoB and RhoC. In this present study we generate, by molecular evolution, a new phage library to isolate scFvs displaying high affinity and selectivity to RhoA and RhoB. Using phage display affinity maturation against the GTP-locked mutant RhoAL63, we isolated scFvs against RhoA active conformation that display K_d values at the nanomolar range, which corresponded to an increase of affinity of three orders of magnitude compared to scFvC1. Although a majority of these evolved scFvs remained selective towards the active conformation of RhoA, RhoB and RhoC, we identified some scFvs that bind to RhoA and RhoC but not to RhoB activated form. Alternatively, we performed a substractive panning towards RhoB, and isolated the scFvE3 exhibiting a 10 times higher affinity for RhoB than RhoA activated forms. We showed the peculiar ability of scFvE3 to detect RhoB but not RhoA GTP-bound form in cell extracts overexpressing Guanine nucleotide Exchange Factor XPLN as well as in EGF stimulated HeLa cells. Our results demonstrated the ability of scFvs to distinguish RhoB from RhoA GTP-bound form and provide new selective tools to analyze the cell biology of RhoB GTPase regulation.     

p160ROCK mediates RhoA activation of Na-H exchange.

The ubiquitously expressed Na-H exchanger, NHE1, acts downstream of RhoA in a pathway regulating focal adhesion and actin stress fiber formation. p160ROCK, a serine/threonine protein kinase, is a direct RhoA target mediating RhoA-induced assembly of focal adhesions and stress fibers. Here, stress fiber formation induced by p160ROCK was inhibited by the addition of a specific NHE1 inhibitor, ethylisopropylamiloride, in CCL39 fibroblasts, and was absent in PS120 mutant fibroblasts lacking NHE1. In CCL39 cells, NHE1 activity was stimulated by expression of mutationally active p160ROCK, but not by mutationally active protein kinase N, another RhoA target kinase. Expression of a dominant interfering p160ROCK inhibited RhoA-, but not Cdc42- or Rac-activation of NEH1. In addition, the p160ROCK-specific inhibitor Y-27632 inhibited increases in NHE1 activity in response to RhoA, and to lysophosphatidic acid (LPA), which stimulates RhoA, and it also inhibited LPA-increased phosphorylation of NHE1. A C-terminal truncation of NHE1 abolished both LPA-induced phosphorylation and activation of the exchanger. Furthermore, mutationally active p160ROCK phosphorylated an NHE1 C-terminal fusion protein in vitro, and this was inhibited in the presence of Y-27632. Phosphopeptide maps indicated that identical residues in NHE1 were phosphorylated by p160ROCK in vivo and in vitro. These findings identify p160ROCK as an upstream, possibly direct, activator of NHE1, and suggest that NHE1 activity and phosphorylation are necessary for actin stress fiber assembly induced by p160ROCK.     

Phosphorylation of RhoB by CK1 impedes actin stress fiber organization and epidermal growth factor receptor stabilization

RhoB is a small GTPase implicated in cytoskeletal organization, EGF receptor trafficking and cell transformation. It is an immediate-early gene, regulated at many levels of its biosynthetic pathway. Herein we show that the serine/threonine protein kinase CK1 phosphorylates RhoB in vitro but not RhoA or RhoC. With the use of specific CK1 inhibitors, IC261 and D4476, we show that the kinase phosphorylates also RhoB in HeLa cells. Mass spectrometry analysis demonstrates that RhoB is monophosphorylated by CK1, in its C-terminal end, on serine 185. The substitution of Ser185 by Ala dramatically inhibited the phosphorylation of RhoB in cultured cells. Lastly we show that the inhibition of CK1 activates RhoB and promotes RhoB dependent actin fiber formation and EGF-R level. Our data provide the first demonstration of RhoB phosphorylation and indicate that this post-translational maturation would be a novel critical mechanism to control the RhoB functions.