Cdk1 phosphorylation negatively regulates the activity of Net1 towards RhoA during mitosis

The Neuroepithelial transforming gene 1 (Net1) is a RhoA subfamily guanine nucleotide exchange factor that is overexpressed in a number of cancers and contributes to cancer cell motility and proliferation. Net1 also plays a Rho GTPase independent role in mitotic progression, where it promotes centrosomal activation of Aurora A and Pak2, and aids in chromosome alignment during prometaphase. To understand regulatory mechanisms controlling the mitotic function of Net1, we examined whether it was phosphorylated by the mitotic kinase Cdk1. We observed that Cdk1 phosphorylated Net1 on multiple sites in its N-terminal regulatory domain and C-terminus in vitro. By raising phospho-specific antibodies to two of these sites, we also demonstrated that both endogenous and transfected Net1 were phosphorylated by Cdk1 in cells. Substitution of the major Cdk1 phosphorylation sites with aliphatic or acidic residues inhibited the interaction of Net1 with RhoA, and treatment of metaphase cells with a Cdk1 inhibitor increased Net1 activity. Cdk1 inhibition also increased Net1 localization to the plasma membrane and stimulated cortical F-actin accumulation. Moreover, Net1 overexpression caused spindle polarity defects that were reduced in frequency by acidic substitution of the major Cdk1 phosphorylation sites. These data indicate that Cdk1 phosphorylates Net1 during mitosis and suggest that this negatively regulates its ability to signal to RhoA and alter actin cytoskeletal organization.     

Direct Activation of RhoA by Reactive Oxygen Species Requires a Redox-Sensitive Motif

Background

Rho family GTPases are critical regulators of the cytoskeleton and affect cell migration, cell-cell adhesion, and cell-matrix adhesion. As with all GTPases, their activity is determined by their guanine nucleotide-bound state. Understanding how Rho proteins are activated and inactivated has largely focused on regulatory proteins such as guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). However, recent in vitro studies have indicated that GTPases may also be directly regulated by redox agents. We hypothesized that this redox-based mechanism occurs in cells and affects cytoskeletal dynamics, and in this report we conclude this is indeed a novel mechanism of regulating the GTPase RhoA.

Methodology/Principal Findings

In this report, we show that RhoA can be directly activated by reactive oxygen species (ROS) in cells, and that this requires two critical cysteine residues located in a unique redox-sensitive motif within the phosphoryl binding loop. First, we show that ROS can reversibly activate RhoA and induce stress fiber formation, a well characterized readout of RhoA activity. To determine the role of cysteine residues in this mechanism of regulation, we generated cysteine to alanine RhoA mutants. Mutation of these cysteines abolishes ROS-mediated activation and stress fiber formation, indicating that these residues are critical for redox-regulation of RhoA. Importantly, these mutants maintain the ability to be activated by GEFs.

Conclusions/Significance

Our findings identify a novel mechanism for the regulation of RhoA in cells by ROS, which is independent of classical regulatory proteins. This mechanism of regulation may be particularly relevant in pathological conditions where ROS are generated and the cellular redox-balance altered, such as in asthma and ischemia-reperfusion injury.     

PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility

Integrin binding to matrix proteins such as fibronectin (FN) leads to formation of focal adhesion (FA) cellular contact sites that regulate migration. RhoA GTPases facilitate FA formation, yet FA-associated RhoA-specific guanine nucleotide exchange factors (GEFs) remain unknown. Here, we show that proline-rich kinase-2 (Pyk2) levels increase upon loss of focal adhesion kinase (FAK) in mouse embryonic fibroblasts (MEFs). Additionally, we demonstrate that Pyk2 facilitates deregulated RhoA activation, elevated FA formation, and enhanced cell proliferation by promoting p190RhoGEF expression. In normal MEFs, p190RhoGEF knockdown inhibits FN-associated RhoA activation, FA formation, and cell migration. Knockdown of p190RhoGEF-related GEFH1 does not affect FA formation in FAK^−/− or normal MEFs. p190RhoGEF overexpression enhances RhoA activation and FA formation in MEFs dependent on FAK binding and associated with p190RhoGEF FA recruitment and tyrosine phosphorylation. These studies elucidate a compensatory function for Pyk2 upon FAK loss and identify the FAK–p190RhoGEF complex as an important integrin-proximal regulator of FA formation during FN-stimulated cell motility.     

Intramolecular Interactions between the Dbl Homology (DH) Domain and the Carboxyl-terminal region of Myosin II-interacting Guanine Nucleotide Exchange Factor (MyoGEF) Act as an Autoinhibitory Mechanism for the Regulation of MyoGEF Functions

We have reported previously that nonmuscle myosin II-interacting guanine nucleotide exchange factor (MyoGEF) plays an important role in the regulation of cell migration and cytokinesis. Like many other guanine nucleotide exchange factors (GEFs), MyoGEF contains a Dbl homology (DH) domain and a pleckstrin homology domain. In this study, we provide evidence demonstrating that intramolecular interactions between the DH domain (residues 162–351) and the carboxyl-terminal region (501–790) of MyoGEF can inhibit MyoGEF functions. In vitro and in vivo pulldown assays showed that the carboxyl-terminal region (residues 501–790) of MyoGEF could interact with the DH domain but not with the pleckstrin homology domain. Expression of a MyoGEF carboxyl-terminal fragment (residues 501–790) decreased RhoA activation and suppressed actin filament formation in MDA-MB-231 breast cancer cells. Additionally, Matrigel invasion assays showed that exogenous expression of the MyoGEF carboxyl-terminal region decreased the invasion activity of MDA-MB-231 cells. Moreover, coimmunoprecipitation assays showed that phosphorylation of the MyoGEF carboxyl-terminal region by aurora B kinase interfered with the intramolecular interactions of MyoGEF. Furthermore, expression of the MyoGEF carboxyl-terminal region interfered with RhoA localization during cytokinesis and led to an increase in multinucleation. Together, our findings suggest that binding of the carboxyl-terminal region of MyoGEF to its DH domain acts as an autoinhibitory mechanism for the regulation of MyoGEF activation.     

Regulation of Focal Adhesion Kinase Activation,Breast Cancer Cell Motility,and Amoeboid Invasion by the RhoA Guanine Nucleotide Exchange Factor Net1

Net1 is a RhoA guanine nucleotide exchange factor (GEF) that is overexpressed in a subset of human cancers and contributes to cancer cell motility and invasion in vitro. However, the molecular mechanism accounting for its role in cell motility and invasion has not been described. In the present work, we show that expression of both Net1 isoforms in breast cancer cells is required for efficient cell motility. Although loss of Net1 isoform expression only partially blocks RhoA activation, it inhibits lysophosphatidic acid (LPA)-stimulated migration as efficiently as knockdown of RhoA itself. However, we demonstrate that the Net1A isoform predominantly controls myosin light-chain phosphorylation and is required for trailing edge retraction during migration. Net1A interacts with focal adhesion kinase (FAK), localizes to focal adhesions, and is necessary for FAK activation and focal adhesion maturation during cell spreading. Net1A expression is also required for efficient invasion through a Matrigel matrix. Analysis of invading cells demonstrates that Net1A is required for amoeboid invasion, and loss of Net1A expression causes cells to shift to a mesenchymal phenotype characterized by high β₁-integrin activity and membrane type 1 matrix metalloproteinase (MT1-MMP) expression. These results demonstrate a previously unrecognized role for the Net1A isoform in controlling FAK activation during planar cell movement and amoeboid motility during extracellular matrix (ECM) invasion.     

Phospholipase D2 induces stress fiber formation through mediating nucleotide exchange for RhoA

Phospholipase D (PLD) is involved in diverse cellular processes including cell movement, adhesion, and vesicle trafficking through cytoskeletal rearrangements. However, the mechanism by which PLD induces cytoskeletal reorganization is still not fully understood. Here, we describe a new link to cytoskeletal changes that is mediated by PLD2 through direct nucleotide exchange on RhoA. We found that PLD2 induces RhoA activation independent of its lipase activity. PLD2 directly interacted with RhoA, and the PX domain of PLD2 specifically recognized nucleotide-free RhoA. Finally, we found that the PX domain of PLD2 has guanine nucleotide-exchange factor (GEF) activity for RhoA in vitro. In addition, we verified that overexpression of the PLD2-PX domain induces RhoA activation, thereby provoking stress fiber formation. Together, our findings suggest that PLD2 functions as an upstream regulator of RhoA, which enables us to understand how PLD2 regulates cytoskeletal reorganization in a lipase activity-independent manner.     

Rac activation by lysophosphatidic acid LPA1 receptors through the guanine nucleotide exchange factor Tiam1

Lysophosphatidic acid (LPA) is a serum-borne phospholipid that activates its own G protein-coupled receptors present in numerous cell types. In addition to stimulating cell proliferation, LPA also induces cytoskeletal changes and promotes cell migration in a RhoA- and Rac-dependent manner. Whereas RhoA is activated via Galpha(12/13)-linked Rho-specific guanine nucleotide exchange factors, it is unknown how LPA receptors may signal to Rac. Here we report that the prototypic LPA(1) receptor (previously named Edg2), when expressed in B103 neuroblastoma cells, mediates transient activation of RhoA and robust, prolonged activation of Rac leading to cell spreading, lamellipodia formation, and stimulation of cell migration. LPA-induced Rac activation is inhibited by pertussis toxin and requires phosphoinositide 3-kinase activity. Strikingly, LPA fails to activate Rac in cell types that lack the Rac-specific exchange factor Tiam1; however, enforced expression of Tiam1 restores LPA-induced Rac activation in those cells. Tiam1-deficient cells show enhanced RhoA activation, stress fiber formation, and cell rounding in response to LPA, consistent with Tiam1/Rac counteracting RhoA. We conclude that LPA(1) receptors couple to a G(i)-phosphoinositide 3-kinase-Tiam1 pathway to activate Rac, with consequent suppression of RhoA activity, and thereby stimulate cell spreading and motility.     

Guanine Nucleotide Exchange Factor-H1 Regulates Cell Migration via Localized Activation of RhoA at the Leading Edge

Cell migration involves the cooperative reorganization of the actin and microtubule cytoskeletons, as well as the turnover of cell–substrate adhesions, under the control of Rho family GTPases. RhoA is activated at the leading edge of motile cells by unknown mechanisms to control actin stress fiber assembly, contractility, and focal adhesion dynamics. The microtubule-associated guanine nucleotide exchange factor (GEF)-H1 activates RhoA when released from microtubules to initiate a RhoA/Rho kinase/myosin light chain signaling pathway that regulates cellular contractility. However, the contributions of activated GEF-H1 to coordination of cytoskeletal dynamics during cell migration are unknown. We show that small interfering RNA-induced GEF-H1 depletion leads to decreased HeLa cell directional migration due to the loss of the Rho exchange activity of GEF-H1. Analysis of RhoA activity by using a live cell biosensor revealed that GEF-H1 controls localized activation of RhoA at the leading edge. The loss of GEF-H1 is associated with altered leading edge actin dynamics, as well as increased focal adhesion lifetimes. Tyrosine phosphorylation of focal adhesion kinase and paxillin at residues critical for the regulation of focal adhesion dynamics was diminished in the absence of GEF-H1/RhoA signaling. This study establishes GEF-H1 as a critical organizer of key structural and signaling components of cell migration through the localized regulation of RhoA activity at the cell leading edge.     

Phosphorylation of Myosin II-interacting Guanine Nucleotide Exchange Factor (MyoGEF) at Threonine 544 by Aurora B Kinase Promotes the Binding of Polo-like Kinase 1 to MyoGEF

We previously reported that phosphorylation of myosin II-interacting guanine nucleotide exchange factor (MyoGEF) by polo-like kinase 1 (Plk1) promotes the localization of MyoGEF to the central spindle and increases MyoGEF activity toward RhoA during mitosis. In this study we report that aurora B-mediated phosphorylation of MyoGEF at Thr-544 creates a docking site for Plk1, leading to the localization and activation of MyoGEF at the central spindle. In vitro kinase assays show that aurora B can phosphorylate MyoGEF. T544A mutation drastically decreases aurora B-mediated phosphorylation of MyoGEF in vitro and in transfected HeLa cells. Coimmunoprecipitation and in vitro pulldown assays reveal that phosphorylation of MyoGEF at Thr-544 enhances the binding of Plk1 to MyoGEF. Immunofluorescence analysis shows that aurora B colocalizes with MyoGEF at the central spindle and midbody during cytokinesis. Suppression of aurora B activity by an aurora B inhibitor disrupts the localization of MyoGEF to the central spindle. In addition, T544A mutation interferes with the localization of MyoGEF to the cleavage furrow and decreases MyoGEF activity toward RhoA during mitosis. Taken together, our results suggest that aurora B coordinates with Plk1 to regulate MyoGEF activation and localization, thus contributing to the regulation of cytokinesis.     

Real-time NMR Study of Three Small GTPases Reveals That Fluorescent 2��(3��)-O-(N-Methylanthraniloyl)-tagged Nucleotides Alter Hydrolysis and Exchange Kinetics

The Ras family of small GTPases control diverse signaling pathways through a conserved “switch” mechanism, which is turned on by binding of GTP and turned off by GTP hydrolysis to GDP. Full understanding of GTPase switch functions requires reliable, quantitative assays for nucleotide binding and hydrolysis. Fluorescently labeled guanine nucleotides, such as 2′(3′)-O-(N-methylanthraniloyl) (mant)-substituted GTP and GDP analogs, have been widely used to investigate the molecular properties of small GTPases, including Ras and Rho. Using a recently developed NMR method, we show that the kinetics of nucleotide hydrolysis and exchange by three small GTPases, alone and in the presence of their cognate GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors, are affected by the presence of the fluorescent mant moiety. Intrinsic hydrolysis of mantGTP by Ras homolog enriched in brain (Rheb) is ∼10 times faster than that of GTP, whereas it is 3.4 times slower with RhoA. On the other hand, the mant tag inhibits TSC2GAP-catalyzed GTP hydrolysis by Rheb but promotes p120 RasGAP-catalyzed GTP hydrolysis by H-Ras. Guanine nucleotide exchange factor-catalyzed nucleotide exchange for both H-Ras and RhoA was inhibited by mant-substituted nucleotides, and the degree of inhibition depends highly on the GTPase and whether the assay measures association of mantGTP with, or dissociation of mantGDP from the GTPase. These results indicate that the mant moiety has significant and unpredictable effects on GTPase reaction kinetics and underscore the importance of validating its use in each assay.     

EspM2 is a RhoA guanine nucleotide exchange factor

We investigated how the type III secretion system WxxxE effectors EspM2 of enterohaemorrhagic Escherichia coli, which triggers stress fibre formation, and SifA of Salmonella enterica serovar Typhimurium, which is involved in intracellular survival, modulate Rho GTPases. We identified a direct interaction between EspM2 or SifA and nucleotide‐free RhoA. Nuclear Magnetic Resonance Spectroscopy revealed that EspM2 has a similar fold to SifA and the guanine nucleotide exchange factor (GEF) effector SopE. EspM2 induced nucleotide exchange in RhoA but not in Rac1 or H‐Ras, while SifA induced nucleotide exchange in none of them. Mutating W70 of the WxxxE motif or L118 and I127 residues, which surround the catalytic loop, affected the stability of EspM2. Substitution of Q124, located within the catalytic loop of EspM2, with alanine, greatly attenuated the RhoA GEF activity in vitro and the ability of EspM2 to induce stress fibres upon ectopic expression. These results suggest that binding of SifA to RhoA does not trigger nucleotide exchange while EspM2 is a unique Rho GTPase GEF.     

The Src Homology 3 Domain-containing Guanine Nucleotide Exchange Factor Is Overexpressed in High-grade Gliomas and Promotes Tumor Necrosis Factor-like Weak Inducer of Apoptosis-Fibroblast Growth Factor-inducible 14-induced Cell Migration and Invasion via Tumor Necrosis Factor Receptor-associated Factor 2

Glioblastoma (GB) is the highest grade of primary adult brain tumors, characterized by a poorly defined and highly invasive cell population. Importantly, these invading cells are attributed with having a decreased sensitivity to radiation and chemotherapy. TNF-like weak inducer of apoptosis (TWEAK)-Fn14 ligand-receptor signaling is one mechanism in GB that promotes cell invasiveness and survival and is dependent upon the activity of multiple Rho GTPases, including Rac1. Here we report that Src homology 3 domain-containing guanine nucleotide exchange factor (SGEF), a RhoG-specific guanine nucleotide exchange factor, is overexpressed in GB tumors and promotes TWEAK-Fn14-mediated glioma invasion. Importantly, levels of SGEF expression in GB tumors inversely correlate with patient survival. SGEF mRNA expression is increased in GB cells at the invasive rim relative to those in the tumor core, and knockdown of SGEF expression by shRNA decreases glioma cell migration in vitro and invasion ex vivo. Furthermore, we showed that, upon TWEAK stimulation, SGEF is recruited to the Fn14 cytoplasmic tail via TRAF2. Mutation of the Fn14-TRAF domain site or depletion of TNF receptor-associated factor 2 (TRAF2) expression by siRNA oligonucleotides blocked SGEF recruitment to Fn14 and inhibited SGEF activity and subsequent GB cell migration. We also showed that knockdown of either SGEF or RhoG diminished TWEAK activation of Rac1 and subsequent lamellipodia formation. Together, these results indicate that SGEF-RhoG is an important downstream regulator of TWEAK-Fn14-driven GB cell migration and invasion.     

GIV/Girdin is a rheostat that fine-tunes growth factor signals during tumor progression

GIV/Girdin is a multidomain signaling molecule that enhances PI3K-Akt signals downstream of both G protein-coupled and growth factor receptors. We previously reported that GIV triggers cell migration via its C-terminal guanine nucleotide exchange factor (GEF) motif that activates Gαi. Recently we discovered that GIV''s C-terminus directly interacts with the epidermal growth factor receptor (EGFR) and when its GEF function is intact, a Gαi-GIV-EGFR signaling complex assembles. By coupling G proteins to growth factor receptors, GIV is uniquely poised to intercept the incoming receptor-initiated signals and modulate them via G protein intermediates. Subsequent work has revealed that expression of the highly specialized C-terminus of GIV undergoes a bipartite dysregulation during oncogenesis—full-length GIV with an intact C-terminus is expressed at levels ∼20–50-fold above normal in highly invasive cancer cells and metastatic tumors, but its C-terminus is truncated by alternative splicing in poorly invasive cancer cells and non-invasive tumors. The consequences of such dysregulation on graded signal transduction and cellular phenotypes in the normal epithelium and its implication during tumor progression are discussed herein. Based on the fact that GIV grades incoming signals initiated by ligand-activated receptors by linking them to cyclical activation of G proteins, we propose that GIV is a molecular rheostat for signal transduction.Key words: G proteins, girdin, guanine nucleotide exchange factor, epidermal growth factor-receptor, G protein coupled receptors, metastasis, migration-proliferation dichotomy, growth factors, alternative splicing, PI3-kinase, Akt, rheostat, actin cytoskeleton     

Structure of Shigella IpgB2 in Complex with Human RhoA: IMPLICATIONS FOR THE MECHANISM OF BACTERIAL GUANINE NUCLEOTIDE EXCHANGE FACTOR MIMICRY*

A common theme in bacterial pathogenesis is the manipulation of eukaryotic cells by targeting the cytoskeleton. This is in most cases achieved either by modifying actin, or indirectly via activation of key regulators controlling actin dynamics such as Rho-GTPases. A novel group of bacterial virulence factors termed the WXXXE family has emerged as guanine nucleotide exchange factors (GEFs) for these GTPases. The precise mechanism of nucleotide exchange, however, has remained unclear. Here we report the structure of the WXXXE-protein IpgB2 from Shigella flexneri and its complex with human RhoA. We unambiguously identify IpgB2 as a bacterial RhoA-GEF and dissect the molecular mechanism of GDP release, an essential prerequisite for GTP binding. Our observations uncover that IpgB2 induces conformational changes on RhoA mimicking DbI- but not DOCK family GEFs. We also show that dissociation of the GDP·Mg²⁺ complex is preceded by the displacement of the metal ion to the α-phosphate of the nucleotide, diminishing its affinity to the GTPase. These data refine our understanding of the mode of action not only of WXXXE GEFs but also of mammalian GEFs of the DH/PH family.     

A novel function for Cyclin A2: control of cell invasion via RhoA signaling

Cyclin A2 plays a key role in cell cycle regulation. It is essential in embryonic cells and in the hematopoietic lineage yet dispensable in fibroblasts. In this paper, we demonstrate that Cyclin A2-depleted cells display a cortical distribution of actin filaments and increased migration. These defects are rescued by restoration of wild-type Cyclin A2, which directly interacts with RhoA, or by a Cyclin A2 mutant unable to associate with Cdk. In vitro, Cyclin A2 potentiates the exchange activity of a RhoA-specific guanine nucleotide exchange factor. Consistent with this, Cyclin A2 depletion enhances migration of fibroblasts and invasiveness of transformed cells via down-regulation of RhoA activity. Moreover, Cyclin A2 expression is lower in metastases relative to primary colon adenocarcinoma in matched human tumors. All together, these data show that Cyclin A2 negatively controls cell motility by promoting RhoA activation, thus demonstrating a novel Cyclin A2 function in cytoskeletal rearrangements and cell migration.     

The trio guanine nucleotide exchange factor is a RhoA target. Binding of RhoA to the trio immunoglobulin-like domain

Trio is a complex protein containing two guanine nucleotide exchange factor domains each with associated pleckstrin homology domains, a serine/threonine kinase domain, two SH3 domains, an immunoglobulin-like domain, and spectrin-like repeats. Trio was originally identified as a LAR tyrosine phosphatase-binding protein and is involved in actin remodeling, cell migration, and cell growth. Herein we provide evidence that Trio not only activates RhoA but is also a RhoA target. The RhoA-binding site was mapped to the Trio immunoglobulin-like domain. RhoA isoprenylation is necessary for the RhoA-Trio interaction, because mutation of the RhoA carboxyl-terminal cysteine residue blocked binding. The existence of an intramolecular functional link between RhoA activation and RhoA binding is suggested by the finding that Trio exchange activity enhanced RhoA binding to Trio. Furthermore, immunofluorescence studies of HeLa cells showed that although ectopically expressed Trio was evenly distributed within the cell, co-expression of Trio with RhoA resulted in relocalization of Trio into punctate structures. Relocalization was not observed with Trio constructs lacking the immunoglobulin-like domain, indicating that RhoA acts to regulate Trio localization via binding to the immunoglobulin-like domain. We propose that Trio-mediated RhoA activation and subsequent RhoA-mediated relocalization of Trio functions to modulate and coordinate Trio signaling.     

RhoA activation during polarization and cytokinesis of the early Caenorhabditis elegans embryo is differentially dependent on NOP-1 and CYK-4

The GTPase RhoA is a central regulator of cellular contractility in a wide variety of biological processes. During these events, RhoA is activated by guanine nucleotide exchange factors (GEFs). These molecules are highly regulated to ensure that RhoA activation occurs at the proper time and place. During cytokinesis, RhoA is activated by the RhoGEF ECT-2. In human cells, ECT-2 activity requires its association with CYK-4, which is a component of the centralspindlin complex. In contrast, in early Caenorhabditis elegans embryos, not all ECT-2–dependent functions require CYK-4. In this study, we identify a novel protein, NOP-1, that functions in parallel with CYK-4 to promote RhoA activation. We use mutations in nop-1 and cyk-4 to dissect cytokinesis and cell polarization. NOP-1 makes a significant, albeit largely redundant, contribution to cytokinesis. In contrast, NOP-1 is required for the preponderance of RhoA activation during the establishment phase of polarization.