p190-RhoGAP as an integral component of the Tiam1/Rac1-induced downregulation of Rho

The Rho family of small GTPases plays a central role in intracellular signal transduction, particularly in reorganization of the actin cytoskeleton. Rho activity induces cell contractility, whereas Rac promotes cellular protrusion, which counteracts Rho signaling. In this regard, the reciprocal balance between these GTPases determines cell morphology and migratory behavior. Here we demonstrate that Tiam1/Rac1 signaling is able to antagonize Rho activity directly at the GTPase level in COS-7 cells. p190-RhoGAP plays a central regulatory role in this signaling pathway. Interfering with its activation by Src-kinase-dependent tyrosine phosphorylation or its recruitment to the membrane through interaction with the SH2 domains of p120-RasGAP blocks the Tiam1-mediated rapid downregulation of Rho. This process is mediated by Rac1, but not by Rac2 or Rac3 isoforms. Our data provide evidence for a biochemical pathway of the reciprocal regulation of two related small GTPases, which are key elements in cell migration.     

Rho1 interacts with p120ctn and alpha-catenin,and regulates cadherin-based adherens junction components in Drosophila

Rho GTPases are important regulators of cellular behavior through their effects on processes such as cytoskeletal organization. Here we show interactions between Drosophila Rho1 and the adherens junction components alpha-catenin and p120(ctn). We find that while Rho1 protein is present throughout the cell, it accumulates apically, particularly at sites of cadherin-based adherens junctions. Cadherin and catenin localization is disrupted in Rho1 mutants, implicating Rho1 in their regulation. p120(ctn) has recently been suggested to inhibit Rho activity through an unknown mechanism. We find that Rho1 accumulates in response to lowered p120(ctn) activity. Significantly, we find that Rho1 binds directly to alpha-catenin and p120(ctn) in vitro, and these interactions map to distinct surface-exposed regions of the protein not previously assigned functions. In addition, we find that both alpha-catenin and p120(ctn) co-immunoprecipitate with Rho1-containing complexes from embryo lysates. Our observations suggest that alpha-catenin and p120(ctn) are key players in a mechanism of recruiting Rho1 to its sites of action.     

The PRK2 kinase is a potential effector target of both Rho and Rac GTPases and regulates actin cytoskeletal organization.

The Ras-related Rho family GTPases mediate signal transduction pathways that regulate a variety of cellular processes. Like Ras, the Rho proteins (which include Rho, Rac, and CDC42) interact directly with protein kinases, which are likely to serve as downstream effector targets of the activated GTPase. Activated RhoA has recently been reported to interact directly with several protein kinases, p120 PKN, p150 ROK alpha and -beta, p160 ROCK, and p164 Rho kinase. Here, we describe the purification of a novel Rho-associated kinase, p140, which appears to be the major Rho-associated kinase activity in most tissues. Peptide microsequencing revealed that p140 is probably identical to the previously reported PRK2 kinase, a close relative of PKN. However, unlike the previously described Rho-binding kinases, which are Rho specific, p140 associates with Rac as well as Rho. Moreover, the interaction of p140 with Rho in vitro is nucleotide independent, whereas the interaction with Rac is completely GTP dependent. The association of p140 with either GTPase promotes kinase activity substantially, and expression of a kinase-deficient form of p140 in microinjected fibroblasts disrupts actin stress fibers. These results indicate that p140 may be a shared kinase target of both Rho and Rac GTPases that mediates their effects on rearrangements of the actin cytoskeleton.     

VEGF isoforms

The Rho-family of p21 small GTPases are directly linked to the regulation of actin-based motile machinery and play a key role in the control of cell migration. Aside from the original and most well-characterized canonical Rho GTPases RhoA, Rac1, and Cdc42, numerous isoforms of these key proteins have been identified and shown to have specific roles in regulating various cellular motility processes. The major difficulty in addressing these isoform-specific effects is that isoforms typically contain highly similar primary amino acid sequences and thus are able to interact with the same upstream regulators and the downstream effector targets. Here, we will introduce the major members of each GTPase subfamily and discuss recent advances in the design and application of fluorescent resonance energy transfer-based probes, which are at the forefront of the technologies available to directly probe the differential, spatiotemporal activation dynamics of these proteins in live single cells. Currently, it is possible to specifically detect the activation status of RhoA vs. RhoC isoforms, as well as Cdc42 vs. TC-10 isoforms in living cells. Clearly, additional efforts are still required to produce biosensor systems capable of detecting other isoforms of Rho GTPases including RhoB, Rac2/3, RhoG, etc. Through such efforts, we will uncover the isoform-specific roles of these near-identical proteins in living cells, clearly an important area of the Rho GTPase biology that is not yet fully appreciated.     

Rho GTPase isoforms in cell motility: Don't fret,we have FRET

The Rho-family of p21 small GTPases are directly linked to the regulation of actin-based motile machinery and play a key role in the control of cell migration. Aside from the original and most well-characterized canonical Rho GTPases RhoA, Rac1, and Cdc42, numerous isoforms of these key proteins have been identified and shown to have specific roles in regulating various cellular motility processes. The major difficulty in addressing these isoform-specific effects is that isoforms typically contain highly similar primary amino acid sequences and thus are able to interact with the same upstream regulators and the downstream effector targets. Here, we will introduce the major members of each GTPase subfamily and discuss recent advances in the design and application of fluorescent resonance energy transfer-based probes, which are at the forefront of the technologies available to directly probe the differential, spatiotemporal activation dynamics of these proteins in live single cells. Currently, it is possible to specifically detect the activation status of RhoA vs. RhoC isoforms, as well as Cdc42 vs. TC-10 isoforms in living cells. Clearly, additional efforts are still required to produce biosensor systems capable of detecting other isoforms of Rho GTPases including RhoB, Rac2/3, RhoG, etc. Through such efforts, we will uncover the isoform-specific roles of these near-identical proteins in living cells, clearly an important area of the Rho GTPase biology that is not yet fully appreciated.     

Rho GTPase expression in tumourigenesis: evidence for a significant link

Rho proteins belong to the small GTPases superfamily. They function as molecular switches that, in response to diverse stimuli, control key signaling and structural aspects of the cell. Although early studies proposed a role for Rho GTPases in cellular transformation, this effect was underestimated due to the fact that no genetic mutations affecting Rho-encoding genes were found in tumors. Recently, it has become evident that Rho GTPases participate in the carcinogenic process by either overexpression of some of the members of the family with oncogenic activity, downmodulation of other members with suggested tumor suppressor activity, or by alteration of upstream modulators or downstream effectors. Thus, alteration of the levels of expression of different members of the family of Rho GTPases has been detected in many types of human tumors leading to a great interest in the cellular effects elicited by these oncoproteins. This essay reviews the current evidence of dysregulation of Rho signaling by overexpression in human tumors.     

Tumor progression: Small GTPases and loss of cell-cell adhesion

Tumor progression involves the transition from normal to malignant cells, through a series of cumulative alterations. During this process, invasive and migratory properties are acquired, enabling cells to metastasize (reach and grow in tissues far from their origin). Numerous cellular changes take place during epithelial malignancy, and disruption of E-cadherin based cell-cell adhesion is a major event. The small Rho GTPases (Rho, Rac and Cdc42) have been implicated in multiple steps during cellular transformation, including alterations on the adhesion status of the tumor cells. This review focuses on recent in vivo evidence that implicates RhoGTPases in epithelial tumor progression. In addition, we discuss different hypotheses to explain disruption of cadherin-mediated cell-cell adhesion, directly or indirectly, through activation of Rho GTPases. Understanding the molecular mechanism of how cadherin adhesion and RhoGTPases interplay in normal cells and how this balance is altered during cellular transformation will provide clues as to how to interfere with tumor progression.     

p120‐catenin is a binding partner and substrate for Group B Pak kinases

Pak5 is a member of the Group B p21‐activated kinases, which are effectors of the Rho family GTPases Cdc42 and Rac. Pak5 has been shown to promote cytoskeletal reorganization, inducing filopodia formation and neurite outgrowth in neuroblastoma cells. In this study, we used affinity chromatography followed by SDS–PAGE and mass spectrometry to identify potential downstream effectors of Pak5. Using this approach, we isolated p120‐catenin (p120), a known regulator of cytoskeletal reorganization and Rho GTPases. Using co‐immunoprecipitation assays we found that p120 preferentially interacts with Pak5 among the Group B Paks. Results from immunofluorescence studies revealed that Pak5 and p120 co‐localize in cells. Both Pak5 and constitutively active Pak4, the founding member of the Group B Paks, directly phosphorylate p120 in vitro. The phosphorylation was shown by Western blot and immunofluorescence to take place specifically on serine 288. This study is the first report of an upstream serine/threonine kinase that phosphorylates p120. J. Cell. Biochem. 110: 1244–1254, 2010. Published 2010 Wiley‐Liss, Inc.     

Rho GTPases and spermatogenesis

Rho GTPases, such as Rho, Rac and Cdc42, are known to regulate many cellular processes including cell movement and cell adhesion. While the cellular events of germ cell movement are crucial to spermatogenesis since developing germ cells must migrate progressively from the basal to the adluminal compartment but remain attached to the seminiferous epithelium, the physiological significance of Rho GTPases in spermatogenesis remains largely unexplored. This paper reviews some recent findings on Rho GTPases in the field with emphasis on the studies in the testis, upon which future studies can be designed to delineate the role of Rho GTPases in spermatogenesis.     

How important are Rho GTPases in neurosecretion?

Rho GTPases are small GTP binding proteins belonging to the Ras superfamily which act as molecular switches that regulate many cellular function including cell morphology, cell to cell interaction, cell migration and adhesion. In neuronal cells, Rho GTPases have been proposed to regulate neuronal development and synaptic plasticity. However, the role of Rho GTPases in neurosecretion is poorly documented. In this review, we discuss data that highlight the importance of Rho GTPases and their regulators into the control of neurotransmitter and hormone release in neurons and neuroendocrine cells, respectively.     

p120 catenin regulates the actin cytoskeleton via Rho family GTPases

Cadherins are calcium-dependent adhesion molecules responsible for the establishment of tight cell-cell contacts. p120 catenin (p120ctn) binds to the cytoplasmic domain of cadherins in the juxtamembrane region, which has been implicated in regulating cell motility. It has previously been shown that overexpression of p120ctn induces a dendritic morphology in fibroblasts (Reynolds, A.B. , J. Daniel, Y. Mo, J. Wu, and Z. Zhang. 1996. Exp. Cell Res. 225:328-337.). We show here that this phenotype is suppressed by coexpression of cadherin constructs that contain the juxtamembrane region, but not by constructs lacking this domain. Overexpression of p120ctn disrupts stress fibers and focal adhesions and results in a decrease in RhoA activity. The p120ctn-induced phenotype is blocked by dominant negative Cdc42 and Rac1 and by constitutively active Rho-kinase, but is enhanced by dominant negative RhoA. p120ctn overexpression increased the activity of endogenous Cdc42 and Rac1. Exploring how p120ctn may regulate Rho family GTPases, we find that p120ctn binds the Rho family exchange factor Vav2. The behavior of p120ctn suggests that it is a vehicle for cross-talk between cell-cell junctions and the motile machinery of cells. We propose a model in which p120ctn can shuttle between a cadherin-bound state and a cytoplasmic pool in which it can interact with regulators of Rho family GTPases. Factors that perturb cell-cell junctions, such that the cytoplasmic pool of p120ctn is increased, are predicted to decrease RhoA activity but to elevate active Rac1 and Cdc42, thereby promoting cell migration.     

Inhibition of RhoGAP activity is sufficient for the induction of Rho-mediated actin reorganization.

It is generally believed that the induction of actin cytoskeleton rearrangements by extracellular stimuli results from the activation of guanine nucleotide exchange factors for the Rho GTPases. Here, we present evidence that the inactivation of RhoGAP (GTPase activating protein) activity is an equally effective means of promoting Rho-mediated cellular processes. We observed that exposure of cultured fibroblasts to sodium fluoride (NaF) results in a rapid and potent stimulation of actin stress fiber formation. This effect is mediated by the Rho GTPase and is associated with the inactivation of cellular RhoGAP activity. Specifically, NaF promotes formation of a high-affinity complex between Rho and the two cellular p190 RhoGAPs in vivo, apparently sequestering limiting amounts of RhoGAP activity, thereby resulting in Rho activation. p190 RhoGAP activity was found to account for approximately 60% of the total RhoGAP activity detected in whole cell extracts, indicating that relatively small changes in cellular RhoGAP activity can have potent effects on Rho activation. We also found that sub-effective concentrations of NaF combined with sub-effective concentrations of the Rho pathway activator, lysophosphatidic acid, which stimulates guanine nucleotide exchange activity on the Rho GTPase, results in the rapid induction of actin stress fibers. Together, these results suggest that the Rho GTPase is regulated by a fine balance of nucleotide exchange and RhoGAP activities, and that inactivation of RhoGAP activity may be a physiologically important regulatory mechanism for activating the Rho GTPase.     

p120 catenin associates with microtubules: inverse relationship between microtubule binding and Rho GTPase regulation

p120 catenin (p120ctn), an armadillo protein and component of the cadherin adhesion complex, has been found recently to induce a dendritic morphology by regulating Rho family GTPases. We have identified specific serines within the Arm repeat domain that, when mutated to alanine, promote p120ctn association with interphase microtubules, leading to microtubule reorganization and stabilization. The mutant p120ctn also localized to the mitotic spindle and centrosomes. In contrast to wild-type p120ctn, the microtubule-associated p120ctn mutant did not activate Rac1 and did not induce a dendritic morphology. In addition, we show that a basic motif within the p120ctn Arm repeat domain known to be required for the inhibition of RhoA is also required for binding to microtubules. We therefore propose that binding of p120ctn to microtubules is inversely related to its ability to regulate Rho GTPases.     

The function of p120 catenin in filopodial growth and synaptic vesicle clustering in neurons

At the developing neuromuscular junction (NMJ), physical contact between motor axons and muscle cells initiates presynaptic and postsynaptic differentiation. Using Xenopus nerve-muscle cocultures, we previously showed that innervating axons induced muscle filopodia (myopodia), which facilitated interactions between the synaptic partners and promoted NMJ formation. The myopodia were generated by nerve-released signals through muscle p120 catenin (p120ctn), a protein of the cadherin complex that modulates the activity of Rho GTPases. Because axons also extend filopodia that mediate early nerve-muscle interactions, here we test p120ctn's function in the assembly of these presynaptic processes. Overexpression of wild-type p120ctn in Xenopus spinal neurons leads to an increase in filopodial growth and synaptic vesicle (SV) clustering along axons, whereas the development of these specializations is inhibited following the expression of a p120ctn mutant lacking sequences important for regulating Rho GTPases. The p120ctn mutant also inhibits the induction of axonal filopodia and SV clusters by basic fibroblast growth factor, a muscle-derived molecule that triggers presynaptic differentiation. Of importance, introduction of the p120ctn mutant into neurons hinders NMJ formation, which is observed as a reduction in the accumulation of acetylcholine receptors at innervation sites in muscle. Our results suggest that p120ctn signaling in motor neurons promotes nerve-muscle interaction and NMJ assembly.     

Rho family GTPases as key regulators for neuronal network formation

Rho family GTPases act as transducers of signals from extracellular stimuli to the cytoskeleton and gene expression. Their actions are temporal and spatial determinants for cellular functions. The cellular functions of Rho family GTPases have been studied in fibroblasts and endothelial cells, and recent advances have revealed their roles in the regulation of neuronal network formation, including migration, neurite outgrowth, polarity, axon guidance, dendrite maturation and synapse formation. In addition, a significant number of X-linked mental retardation genes have been shown to encode components directly involved in signal transduction pathways of Rho family GTPases, underscoring the view that Rho family GTPases essentially participate in the neuronal network formation. In this review, we will overview current understanding of the functions of Rho family GTPases in neuronal network formation.     

p190A RhoGAP is a glycogen synthase kinase-3-beta substrate required for polarized cell migration

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.     

Serum-activated assembly and membrane translocation of an endogenous Rac1:effector complex

Rho family GTPases (Cdc42, Rac1, and RhoA) function downstream of Ras [1], and in a variety of cellular processes [2]. Studies to examine these functions have not directly linked endogenous protein interactions with specific in vivo functions of Rho GTPases. Here, we show that endogenous Rac1 and two known binding partners, Rho GDP dissociation inhibitor (RhoGDI) and p21-activated kinase (PAK), fractionate as distinct cytosolic complexes. A Rac1:PAK complex is translocated from the cytosol to ruffling membranes upon cell activation by serum. Overexpression of dominant-negative (T17N) Rac1 does not affect the assembly or distribution of this Rac1:PAK complex. This is the first direct evidence of how a specific function of Rac1 is selected by the assembly and membrane translocation of a distinct Rac1:effector complex.     

Cell type-specific functions of Rho GTPases revealed by gene targeting in mice

Mammalian Rho family GTPases are intracellular signal transducers known to regulate multiple signaling pathways involved in actin organization and cell proliferation. However, previous knowledge of their cellular functions came mostly from studies using a dominant-negative or constitutively active mutant expression approach in various clonal cell lines. Such an approach has increasingly been recognized to impose experimental limitations related to specificity, dosage and/or clonal variation. Recent progress in mammalian Rho GTPase cell biology by gene targeting individual Rho GTPases in mice has provided more convincing evidence of their physiological roles and signaling pathways in diverse primary cells. Although adaptive compensation by related Rho GTPase members remains a potential concern in the gene targeting approach, in many cases these studies enable an elucidation of the unique functions of individual Rho GTPases in different cell types in vivo.     

Bead-based protein-protein interaction assays for the analysis of Rho GTPase signaling

Bead-based interaction assays are excellently suited to study protein-protein interactions, as they require only minimal amounts of sample material. Miniaturized protein-protein interaction assays were designed to analyze Rho GTPase activation based on its interaction with Rho GDI or p21-activated kinase (PAK).Rho GDI plays a key role in the regulation of a variety of cellular functions through its interaction with Rho GTPases. Rho GDI is frequently overexpressed in many human cancers. Therefore, there is a growing and as yet unfulfilled demand for screening assays to identify biologically active compounds that may inhibit the Rho GTPase-Rho GDI interaction. Bead-based interaction assays provide an interesting alternative that facilitate such assays to be performed faster with only small amounts of material compared to routinely used co-immunoprecipitation followed by Western Blot analysis.Bead-based protein interaction assays for overexpressed HA-tagged Rho GTPases were established to study the GTPγS-dependent interaction of five different Rho GTPases with the regulatory protein Rho GDIα and the downstream effector PAK1. In addition, it was demonstrated that the ability of Rho GTPases to interact with Rho GDI in this experimental system was markedly, but differentially sensitive to post-translational modification of their carboxyl terminus. Importantly, this modification also notably affected the ability of Rac1 and Rac2, but not of Cdc42, to interact with PAK1.