Identification and molecular characterization of the G alpha12-Rho guanine nucleotide exchange factor pathway in Caenorhabditis elegans

G alpha 12/13-mediated pathways have been shown to be involved in various fundamental cellular functions in mammalian cells such as axonal guidance, apoptosis, and chemotaxis. This study identified a homologue of Rho-guanine nucleotide exchange factor (GEF) in Caenorhabditis elegans (CeRhoGEF), which functions downstream of gpa-12, the C. elegans homologue of G alpha 12/13. CeRhoGEF contains a PSD-95/Dlg/ZO-1 domain and a regulator of G protein signaling (RGS) domain upstream of the Dbl homology-pleckstrin homology region similar to mammalian RhoGEFs with RGS domains, PSD-95/Dlg/ZO-1-RhoGEF and leukemia-associated RhoGEF. It has been shown in mammalian cells that these RhoGEFs interact with activated forms of G alpha 12 or G alpha 13 through their RGS domains. This study demonstrates by coimmunoprecipitation that the RGS domain of CeRhoGEF interacts with GPA-12 in an AIF4- activation-dependent manner and confirms that the Dbl homology-pleckstrin homology domain of CeRhoGEF was capable of Rho-dependent signaling. These results proved conservation of the G alpha 12-RhoGEF pathway in C. elegans. Expression of DsRed or GFP under the control of the promoter of CeRhoGEF or gpa-12 revealed an overlap of their expression patterns in ventral cord motor neurons and several neurons in the head. RNA-mediated gene interference for CeRhoGEF and gpa-12 results in similar phenotypes such as embryonic lethality and sensory and locomotive defects in adults. Thus, the G alpha 12/13-RhoGEF pathway is likely to be involved in embryonic development and neuronal function in C. elegans (Yau, 2003; full text of article)

The Galpha12-RGS RhoGEF-RhoA signalling pathway regulates neurotransmitter release in C. elegans

In C. elegans adults, the single Rho GTPase orthologue, RHO-1, stimulates neurotransmitter release at synapses. One of the pathways acting upstream of RHO-1 in acetylcholine (ACh)-releasing motor neurons depends on Galpha12 (GPA-12), which acts via the single C. elegans RGS RhoGEF (RHGF-1). Activated GPA-12 has the same effect as activated RHO-1, inducing the accumulation of diacylglycerol and the neuromodulator UNC-13 at release sites, and increased ACh release. RHO-1 stimulates ACh release by two separate pathways-one that requires UNC-13 and a second that does not. A non-DAG-binding-UNC-13 mutant that partially blocks increased ACh release by activated RHO-1 completely blocks increased ACh release by activated GPA-12. Thus, the upstream GPA-12/RHGF-1 pathway stimulates only a subset of RHO-1 downstream effectors, suggesting that either the RHO-1 effectors require different levels of activated RHO-1 for activation or there are two distinct pools of RHO-1 within C. elegans neurons (Hiley, 2006).

PDZ-RhoGEF links heterotrimeric G proteins to Rho

Small GTP-binding proteins of the Rho family play a critical role in signal transduction. However, there is still very limited information on how they are activated by cell surface receptors. This study used a consensus sequence for Dbl domains of Rho guanine nucleotide exchange factors (GEFs) to search DNA data bases, and identified a novel human GEF for Rho-related GTPases harboring structural features indicative of its possible regulatory mechanism(s). This protein contained a tandem DH/PH domain closely related to those of Rho-specific GEFs, a PDZ domain, a proline-rich domain, and an area of homology to Lsc, p115-RhoGEF, and a Drosophila RhoGEF that was termed Lsc-homology (LH) domain. This novel molecule, designated PDZ-RhoGEF, activated biological and biochemical pathways specific for Rho, and activation of these pathways required an intact DH and PH domain. However, the PDZ domain was dispensable for these functions, and mutants lacking the LH domain were more active, suggesting a negative regulatory role for the LH domain. A search for additional molecules exhibiting an LH domain revealed a limited homology with the catalytic region of a newly identified GTPase-activating protein for heterotrimeric G proteins, RGS14. This prompted an investigation of whether PDZ-RhoGEF could interact with representative members of each G protein family. It was found that PDZ-RhoGEF is able to form, in vivo, stable complexes with two members of the Galpha12 family, Galpha12 and Galpha13, and that this interaction is mediated by the LH domain. Furthermore, evidence was obtained to suggest that PDZ-RhoGEF mediates the activation of Rho by Galpha12 and Galpha13. Together, these findings suggest the existence of a novel mechanism whereby the large family of cell surface receptors that transmit signals through heterotrimeric G proteins activate Rho-dependent pathways: by stimulating the activity of members of the Galpha12 family which, in turn, activate an exchange factor acting on Rho (Fukuhara, 1999).

Identification of a novel sequence in PDZ-RhoGEF that mediates interaction with the actin cytoskeleton

Small GTPases of the Rho family are crucial regulators of actin cytoskeleton rearrangements. Rho is activated by members of the Rho guanine-nucleotide exchange factor (GEF) family; however, mechanisms that regulate RhoGEFs are not well understood. This report demonstrates that PDZ-RhoGEF, a member of a subfamily of RhoGEFs that contain regulator of G protein signaling domains, is partially localized at or near the plasma membranes in 293T, COS-7, and Neuro2a cells, and this localization is coincident with cortical actin. Disruption of the cortical actin cytoskeleton in cells by using latrunculin B prevents the peri-plasma membrane localization of PDZ-RhoGEF. Coimmunoprecipitation and F-actin cosedimentation assays demonstrate that PDZ-RhoGEF binds to actin. Extensive deletion mutagenesis revealed the presence of a novel 25-amino acid sequence in PDZ-RhoGEF, located at amino acids 561-585, that is necessary and sufficient for localization to the actin cytoskeleton and interaction with actin. Last, PDZ-RhoGEF mutants that fail to interact with the actin cytoskeleton display enhanced Rho-dependent signaling compared with wild-type PDZ-RhoGEF. These results identify interaction with the actin cytoskeleton as a novel function for PDZ-RhoGEF, thus implicating actin interaction in organizing PDZ-RhoGEF signaling (Banerjee, 2004).

Macroscopic stiffening of embryonic tissues via microtubules, RhoGEF and the assembly of contractile bundles of actomyosin

During morphogenesis, forces generated by cells are coordinated and channeled by the viscoelastic properties of the embryo. Microtubules and F-actin are considered to be two of the most important structural elements within living cells accounting for both force production and mechanical stiffness. This paper investigates the contribution of microtubules to the stiffness of converging and extending dorsal tissues in Xenopus laevis embryos using cell biological, biophysical and embryological techniques. Surprisingly, it was discovered that depolymerizing microtubules stiffens embryonic tissues by three- to fourfold. This tissue stiffening is attributed to Xlfc, a previously identified RhoGEF, which binds microtubules and regulates the actomyosin cytoskeleton. Combining drug treatments and Xlfc activation and knockdown lead to the conclusion that mechanical properties of tissues such as viscoelasticity can be regulated through RhoGTPase pathways and embryo. Nocodazole-induced stiffening can be rescued with drugs that reduce actomyosin contractility and can partially rescue morphogenetic defects that affect stiffened embryos. These conclusions are supported with a multi-scale analysis of cytoskeletal dynamics, tissue-scale traction and measurements of tissue stiffness to separate the role of microtubules from RhoGEF activation. These findings suggest a re-evaluation of the effects of nocodazole and increased focus on the role of Rho family GTPases as regulators of the mechanical properties of cells and their mechanical interactions with surrounding tissues (Zhou, 2010).

The crystal structure of RhoA in complex with the DH/PH fragment of PDZRhoGEF, an activator of the Ca(2+) sensitization pathway in smooth muscle

Calcium sensitization in smooth muscle is mediated by the RhoA GTPase, activated by hitherto unspecified nucleotide exchange factors (GEFs) acting downstream of Galphaq/Galpha(12/13) trimeric G proteins. At least one potential GEF, the PDZRhoGEF, is present in smooth muscle, and its isolated DH/PH fragment induces calcium sensitization in the absence of agonist-mediated signaling. In vitro, the fragment shows high selectivity for the RhoA GTPase. Full-length fragment is required for the nucleotide exchange, as the isolated DH domain enhances it only marginally. The DH/PH fragment of PDZRhoGEF was crystallized in complex with nonprenylated human RhoA and the structure was determined at 2.5 Å resolution. The refined molecular model reveals that the mutual disposition of the DH and PH domains is significantly different from other previously described complexes involving DH/PH tandems, and that the PH domain interacts with RhoA in a unique mode. The DH domain makes several specific interactions with RhoA residues not conserved among other Rho family members, suggesting the molecular basis for the observed specificity (Derewenda, 2004).

Chimeric G alpha i2/G alpha 13 proteins reveal the structural requirements for the binding and activation of the RGS-like (RGL)-containing Rho guanine nucleotide exchange factors (GEFs) by G alpha 13

The alpha-subunit of G proteins of the G(12/13) family stimulate Rho by their direct binding to the RGS-like (RGL) domain of a family of Rho guanine nucleotide exchange factors (RGL-RhoGEFs) that includes PDZ-RhoGEF (PRG), p115RhoGEF, and LARG, thereby regulating cellular functions as diverse as shape and movement, gene expression, and normal and aberrant cell growth. The structural features determining the ability of G alpha(12/13) to bind RGL domains and the mechanism by which this association results in the activation of RGL-RhoGEFs are still poorly understood. This study explored the structural requirements for the functional interaction between G alpha(13) and RGL-RhoGEFs based on the structure of RGL domains and their similarity with the area by which RGS4 binds the switch region of G alpha(i) proteins. Using G alpha(i2), which does not bind RGL domains, as the backbone in which G alpha(13) sequences were swapped or mutated, it was observed that the switch region of G alpha(13) is strictly necessary to bind PRG, and specific residues were identified that are critical for this association, likely by contributing to the binding surface. Surprisingly, the switch region of G alpha(13) is not sufficient to bind RGL domains, but instead most of its GTPase domain is required. Furthermore, membrane localization of G alpha(13) and chimeric G alpha(i2) proteins is also necessary for Rho activation. These findings revealed the structural features by which G alpha(13) interacts with RGL domains and suggest that molecular interactions occurring at the level of the plasma membrane are required for the functional activation of the RGL-containing family of RhoGEFs (Vazquez-Prado, 2004).

The molecular basis of RhoA specificity in the guanine nucleotide exchange factor PDZ-RhoGEF

The Dbl homology nucleotide exchange factors (GEFs) activate Rho family cytosolic GTPases in a variety of physiological and pathophysiological events. These signaling molecules typically act downstream of tyrosine kinase receptors and often facilitate nucleotide exchange on more than one member of the Rho GTPase superfamily. Three unique GEFs, i.e., p115, PDZ-RhoGEF, and LARG, are activated by the G-protein coupled receptors via the Galpha(12/13), and exhibit very selective activation of RhoA, although the mechanism by which this is accomplished is not fully understood. Based on the recently solved crystal structure of the DH-PH tandem of PDZ-RhoGEF in complex with RhoA, extensive mutational and functional studies of the molecular basis of the RhoA selectivity in PDZ-RhoGEF was conducted. While Trp(58) of RhoA is intimately involved in the interaction with the DH domain, it is not a selectivity determinant, and its interaction with PDZ-RhoGEF is unfavorable. The key selectivity determinants are dominated by polar contacts involving residues unique to RhoA. Selectivity for RhoA versus Cdc42 is defined by a small number of interactions (Oleksy, 2006).

Interaction of PDZRhoGEF with microtubule-associated protein 1 light chains: link between microtubules, actin cytoskeleton, and neuronal polarity

Rat PDZRhoGEF, initially identified as a glutamate transporter EAAT4-associated protein, is a member of a novel RhoGEF subfamily. The N terminus of the protein contains a PDZ and a proline-rich domain, two motifs known to be involved in protein-protein interactions. By using the yeast two-hybrid approach, a screen was performed for proteins that interact with the N terminus of rat PDZRhoGEF. The light chain 2 of microtubule-associated protein 1 (LC2) was the only protein identified from the screen that does not contain a type I PDZ-binding motif at its extreme C terminus (-(S/T)Xphi-COOH, where phi is a hydrophobic amino acid). However, the C terminus does conform to a type II-binding motif (-phiXphi). Rat PDZRhoGEF interacts with LC2 via the PDZ domain, and the interaction is abolished by mutations in the carboxylate-binding loop. The specificity of the interaction was confirmed using GST fusion protein pull-down assays and coimmunoprecipitations. Expression of rat PDZRhoGEF mutants that are unable to interact with proteins via the carboxylate-binding loop induced changes in cell morphology and actin organization. These mutants alter the activation of RhoGTPases, and coexpression of dominant-negative RhoGTPases prevent the morphological changes. Furthermore, in cells expressing wild type rat PDZRhoGEF, drug-induced microtubule depolymerization produces changes in cell morphology that are similar to those induced by PDZRhoGEF mutants. These results indicate that modulation of the guanine nucleotide exchange activity of PDZRhoGEF through interaction with microtubule-associated protein light chains may coordinate microtubule integrity and the reorganization of actin cytoskeleton. This coordinated action of the actin and microtubular cytoskeletons is essential for the development and maintenance of neuronal polarity (Longhurst, 2006).

Plexin B regulates Rho through the guanine nucleotide exchange factors leukemia-associated Rho GEF (LARG) and PDZ-RhoGEF

Plexins represent a novel family of transmembrane receptors that transduce attractive and repulsive signals mediated by the axon-guiding molecules semaphorins. Emerging evidence implicates Rho GTPases in these biological events. However, Plexins lack any known catalytic activity in their conserved cytoplasmic tails, and how they transduce signals from semaphorins to Rho is still unknown. This study shows that Plexin B2 associates directly with two members of a recently identified family of Dbl homology/pleckstrin homology containing guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and Leukemia-associated Rho GEF (LARG). This physical interaction is mediated by their PDZ domains and a PDZ-binding motif found only in Plexins of the B family. In addition, ligand-induced dimerization of Plexin B is sufficient to stimulate endogenous RhoA potently and to induce the reorganization of the cytoskeleton. Moreover, overexpression of the PDZ domain of PDZ-RhoGEF but not its regulator of G protein signaling domain prevents cell rounding and neurite retraction of differentiated PC12 cells induced by activation of endogenous Plexin B1 by semaphorin 4D. The association of Plexins with LARG and PDZ-RhoGEF thus provides a direct molecular mechanism by which semaphorins acting on Plexin B can control Rho, thereby regulating the actin-cytoskeleton during axonal guidance and cell migration (Perrot, 2002).

The semaphorin receptor plexin-B1 signals through a direct interaction with the Rho-specific nucleotide exchange factor

Semaphorins are axon guidance molecules that signal through the plexin family of receptors. Semaphorins also play a role in other processes such as immune regulation and tumorigenesis. However, the molecular signaling mechanisms downstream of plexin receptors have not been elucidated. Semaphorin 4D is the ligand for the plexin-B1 receptor and stimulation of the plexin-B1 receptor activates the small GTPase RhoA. Using the intracellular domain of plexin-B1 as an affinity ligand, two Rho-specific guanine nucleotide exchange factors, leukemia-associated Rho GEF (LARG; GEF, guanine nucleotide exchange factors) and PSD-95/Dlg/ZO-1 homology (PDZ)-RhoGEF, were isolated from mouse brain as plexin-B1-specific interacting proteins. LARG and PDZ-RhoGEF contain several functional domains, including a PDZ domain. Biochemical characterizations showed that the PDZ domain of LARG is directly involved in the interaction with the carboxy-terminal sequence of plexin-B1. Mutation of either the PDZ domain in LARG or the PDZ binding site in plexin-B1 eliminates the interaction. The interaction between plexin-B1 and LARG is specific for the PDZ domain of LARG and LARG does not interact with plexin-A1. A LARG-interaction defective mutant of the plexin-B1 receptor was created and was unable to stimulate RhoA activation. The data in this report suggest that LARG plays a critical role in plexin-B1 signaling to stimulate Rho activation and cytoskeletal reorganization (Aurandt, 2002).

Plexin-B1 directly interacts with PDZ-RhoGEF/LARG to regulate RhoA and growth cone morphology

Plexins are widely expressed transmembrane proteins that, in the nervous system, mediate repulsive signals of semaphorins. However, the molecular nature of plexin-mediated signal transduction remains poorly understood. This study demonstrates that plexin-B family members associate through their C termini with the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG. Activation of plexin-B1 by semaphorin 4D regulates PDZ-RhoGEF/LARG activity leading to RhoA activation. In addition, a dominant-negative form of PDZ-RhoGEF blocks semaphorin 4D-induced growth cone collapse in primary hippocampal neurons. This study indicates that the interaction of mammalian plexin-B family members with the multidomain proteins PDZ-RhoGEF and LARG represents an essential molecular link between plexin-B and localized, Rho-mediated downstream signaling events which underly various plexin-mediated cellular phenomena including axonal growth cone collapse (Swiercz, 2002).

Direct interaction of Rnd1 with Plexin-B1 regulates PDZ-RhoGEF-mediated Rho activation by Plexin-B1 and induces cell contraction in COS-7 cells

Plexins are receptors for the axon guidance molecule semaphorins, and several lines of evidence suggest that Rho family small GTPases are implicated in the downstream signaling of Plexins. Recent studies have demonstrated that Plexin-B1 activates RhoA and induces growth cone collapse through Rho-specific guanine nucleotide exchange factor PDZ-RhoGEF. Rnd1, a member of Rho family GTPases, directly interacts with the cytoplasmic domain of Plexin-B1. In COS-7 cells, coexpression of Rnd1 and Plexin-B1 induce cell contraction in response to semaphorin 4D (Sema4D), a ligand for Plexin-B1, whereas expression of Plexin-B1 alone or coexpression of Rnd1 and a Rnd1 interaction-defective mutant of Plexin-B1 does not. The Sema4D-induced contraction in Plexin-B1/Rnd1-expressing COS-7 cells is suppressed by dominant negative RhoA, a Rho-associated kinase inhibitor, a dominant negative form of PDZ-RhoGEF, or deletion of the carboxyl-terminal PDZ-RhoGEF-binding region of Plexin-B1, indicating that the PDZ-RhoGEF/RhoA/Rho-associated kinase pathway is involved in this morphological effect. Rnd1 promotes the interaction between Plexin-B1 and PDZ-RhoGEF and thereby dramatically potentiates the Plexin-B1-mediated RhoA activation. It is proposed that Rnd1 plays an important role in the regulation of Plexin-B1 signaling, leading to Rho activation during axon guidance and cell migration (Oinuma, 2003).

Plexin-B1/RhoGEF-mediated RhoA activation involves the receptor tyrosine kinase ErbB-2

Plexins are widely expressed transmembrane proteins that mediate the effects of semaphorins. The molecular mechanisms of plexin-mediated signal transduction are still rather unclear. Plexin-B1 has recently been shown to mediate activation of RhoA through a stable interaction with the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG. However, it is unclear how the activity of plexin-B1 and its downstream effectors is regulated by its ligand Sema4D. This study shows that plexin-B family members stably associate with the receptor tyrosine kinase ErbB-2. Binding of Sema4D to plexin-B1 stimulates the intrinsic tyrosine kinase activity of ErbB-2, resulting in the phosphorylation of both plexin-B1 and ErbB-2. A dominant-negative form of ErbB-2 blocks Sema4D-induced RhoA activation as well as axonal growth cone collapse in primary hippocampal neurons. These data indicate that ErbB-2 is an important component of the plexin-B receptor system and that ErbB-2-mediated phosphorylation of plexin-B1 is critically involved in Sema4D-induced RhoA activation, which underlies cellular phenomena downstream of plexin-B1, including axonal growth cone collapse (Swiercz, 2004).

Direct interaction of p21-activated kinase 4 with PDZ-RhoGEF

Rho GTPases regulate a wide variety of cellular processes, ranging from actin cytoskeleton remodeling to cell cycle progression and gene expression. Cell surface receptors act through a complex regulatory molecular network that includes guanine exchange factors (GEFs), GTPase activating proteins, and guanine dissociation inhibitors to achieve the coordinated activation and deactivation of Rho proteins, thereby controlling cell motility and ultimately cell fate. A member of the RGL-containing family of Rho guanine exchange factors, PDZ RhoGEF, which, together with LARG and p115RhoGEF, links the G(12/13) family of heterotrimeric G proteins to Rho activation, binds through its C-terminal region to the serine-threonine kinase p21-activated kinase 4 (PAK4), an effector for Cdc42. This interaction results in the phosphorylation of PDZ RhoGEF and abolishes its ability to mediate the accumulation of Rho-GTP by Galpha13. Moreover, when overexpressed, active PAK4 dramatically decreases Rho-GTP loading in vivo and the formation of actin stress fibers in response to serum or LPA stimulation. Together, these results provide evidence that PAK4 can negatively regulate the activation of Rho through a direct protein-protein interaction with G protein-linked Rho GEFs, thus providing a novel potential mechanism for cross-talk among Rho GTPases (Barac, 2004).

Regulation of G protein-linked guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and LARG by tyrosine phosphorylation: evidence of a role for focal adhesion kinase

A recently identified family of guanine nucleotide exchange factors for Rho that includes PDZ-RhoGEF, LARG, and p115RhoGEF exhibits a unique structural feature consisting in the presence of area of similarity to regulators of G protein signaling (RGS). This RGS-like (RGL) domain provides a structural motif by which heterotrimeric G protein alpha subunits of the Galpha(12) family can bind and regulate the activity of RhoGEFs. Hence, these newly discovered RGL domain-containing RhoGEFs provide a direct link from Galpha(12) and Galpha(13) to Rho. Recently available data suggest, however, that tyrosine kinases can regulate the ability of G protein-coupled receptors (GPCRs) to stimulate Rho, although the underlying molecular mechanisms are still unknown. This study found that the activation of thrombin receptors endogenously expressed in HEK-293T cells leads to a remarkable increase in the levels of GTP-bound Rho within 1 min (11-fold) and a more limited but sustained activation (4-fold) thereafter, which lasts even for several hours. Interestingly, tyrosine kinase inhibitors did not affect the early phase of Rho activation, immediately after thrombin addition, but diminished the levels of GTP-bound Rho during the delayed phase. As thrombin receptors stimulate focal adhesion kinase (FAK) potently, whether this non-receptor tyrosine kinase participates in the activation of Rho by GPCRs was explored. Evidence was obtained that FAK can be activated by thrombin, Galpha(12), Galpha(13), and Galpha(q) through both Rho-dependent and Rho-independent mechanisms and that PDZ-RhoGEF and LARG can in turn be tyrosine-phosphorylated through FAK in response to thrombin, thereby enhancing the activation of Rho in vivo. These data indicate that FAK may act as a component of a positive feedback loop that results in the sustained activation of Rho by GPCRs, thus providing evidence of the existence of a novel biochemical route by which tyrosine kinases may regulate the activity of Rho through the tyrosine phosphorylation of RGL-containing RhoGEFs (Chikumi, 2002).

Physical and functional interactions of the lysophosphatidic acid receptors with PDZ domain-containing Rho guanine nucleotide exchange factors (RhoGEFs)

Lysophosphatidic acid (LPA) is a serum-derived phospholipid that induces a variety of biological responses in various cells via heterotrimeric G protein-coupled receptors (GPCRs) including LPA1, LPA2, and LPA3. LPA-induced cytoskeletal changes are mediated by Rho family small GTPases, such as RhoA, Rac1, and Cdc42. One of these small GTPases, RhoA, may be activated via Galpha(12/13)-linked Rho-specific guanine nucleotide exchange factors (RhoGEFs) under LPA stimulation although the detailed mechanisms are poorly understood. This study shows that the C terminus of LPA1 and LPA2 but not LPA3 interact with the PDZ domains of PDZ domain-containing RhoGEFs, PDZ-RhoGEF, and LARG, which are comprised of PDZ, RGS, Dbl homology (DH), and pleckstrin homology (PH) domains. In LPA1- and LPA2-transfected HEK293 cells, LPA-induced RhoA activation was observed although the C terminus of LPA1 and LPA2 mutants, which failed to interact with the PDZ domains, did not cause LPA-induced RhoA activation. Furthermore, overexpression of the PDZ domains of PDZ domain-containing RhoGEFs served as dominant negative mutants for LPA-induced RhoA activation. Taken together, these results indicate that formation of the LPA receptor/PDZ domain-containing RhoGEF complex plays a pivotal role in LPA-induced RhoA activation (Yamada, 2005).

The tandem BRCT domains of Ect2 are required for both negative and positive regulation of Ect2 in cytokinesis

Epithelial cell transforming protein 2 (Ect2) is a guanine nucleotide exchange factor (GEF) for Rho GTPases, molecular switches essential for the control of cytokinesis in mammalian cells. Aside from the canonical Dbl homology/pleckstrin homology cassette found in virtually all Dbl family members, Ect2 contains N-terminal tandem BRCT domains. In this study, the role was addressed of the Ect2 BRCT domains in the regulation of Ect2 activity and cytokinesis. First, it was shown that the depletion of endogenous Ect2 by small interfering RNA induces multinucleation, suggesting that Ect2 is required for cytokinesis. In addition, evidence is provided that Ect2 normally exists in an inactive conformation, which is at least partially due to an intramolecular interaction between the BRCT domains and the C-terminal domain of Ect2. This intramolecular interaction masks the catalytic domain responsible for guanine nucleotide exchange toward RhoA. Consistent with a role in regulating Ect2 GEF activity, overexpression of an N-terminal Ect2 containing the tandem BRCT domains, but not single BRCT domain or BRCT domain mutant, leads to a failure in cytokinesis. Surprisingly, although ectopically expressed wild-type Ect2 rescues the multinucleation resulting from the depletion of endogenous Ect2, expression of a BRCT mutant of Ect2 failed to restore proper cytokinesis in these cells. Taken together, the results of this study indicate that the tandem BRCT domains of Ect2 play dual roles in the regulation of Ect2. Whereas these domains negatively regulate Ect2 GEF activity in interphase cells, they are also required for the proper function of Ect2 during cytokinesis (Kim, 2005).

Cytokinesis regulator ECT2 changes its conformation through phosphorylation at Thr-341 in G2/M phase

The Rho activator ECT2 functions as a key regulator in cytokinesis. ECT2 is phosphorylated during G2/M phase, but the physiological significance of this event is not well known. This study shows that phosphorylation of ECT2 at threonine-341 (T341) affects the autoregulatory mechanism of ECT2. In G2/M phase, ECT2 was phosphorylated at T341 most likely by Cyclin B/Cyclin-dependent kinase 1 (Cdk1), and then dephosphorylated before cytokinesis. Depletion of ECT2 by RNA interference (RNAi) efficiently induced multinucleate cells. Expression of the phospho-deficient mutant of ECT2 at T341 suppressed the multinucleation induced by RNAi to ECT2, indicating that ECT2 is biologically active even when it is not phosphorylated at T341. However, the phospho-mimic mutation at T341 weakly stimulates the catalytic activity of ECT2 as detected by serum response element reporter gene assays. Since T341 is located at the hinge region of the N-terminal regulatory domain and C-terminal catalytic domain, phosphorylation of T341 may help accessing downstream signaling molecules to further activate ECT2. The phospho-mimic mutation T341D increases binding with itself or the N-terminal half of ECT2. These results suggest a conformational change of ECT2 upon phosphorylation at T341. Therefore, ECT2 activity might be regulated by the phosphorylation status of T341. It is proposed that T341 phosphorylation by Cyclin B/Cdk1 could be a trigger for further activation of ECT2 (Hara, 2006).

Centralspindlin regulates ECT2 and RhoA accumulation at the equatorial cortex during cytokinesis

During determination of the cell division plane, an actomyosin contractile ring is induced at the equatorial cell cortex by signals from the mitotic apparatus and contracts to cause cleavage furrow progression. Although the small GTPase RhoA is known to regulate the progression, probably by controlling actin filament assembly and enhancing actomyosin interaction, any involvement of RhoA in division plane determination is unknown. In this study, using a trichloroacetic acid (TCA) fixation protocol, it was shown that RhoA accumulates at the equatorial cortex before furrow initiation and continues to concentrate at the cleavage furrow during cytokinesis. Both Rho activity and microtubule organization are required for RhoA localization and proper furrowing. Selective disruption of microtubule organization revealed that both astral and central spindle microtubules can recruit RhoA at the equatorial cortex. Centralspindlin and ECT2 are required for RhoA localization and furrowing. Centralspindlin is localized both to central spindle microtubules and at the tips of astral microtubules near the equatorial cortex and recruits ECT2. Positional information for division plane determination from microtubules is transmitted to the cell cortex to organize actin cytoskeleton through a mechanism involving these proteins (Nishimura, 2006).

Phosphorylation of the cytokinesis regulator ECT2 at G2/M phase stimulates association of the mitotic kinase Plk1 and accumulation of GTP-bound RhoA

The epithelial cell transforming gene 2 (ECT2) protooncogene encodes a Rho exchange factor, and regulates cytokinesis. ECT2 is phosphorylated in G2/M phases, but its role in the biological function is not known. This study shows that two mitotic kinases, Cdk1 and polo-like kinase 1 (Plk1), phosphorylate ECT2 in vitro. An in vitro Cdk1 phosphorylation site (T412) has been identified in ECT2, which comprises a consensus phosphospecific-binding module for the Plk1 polo-box domain (PBD). Endogenous ECT2 in mitotic cells strongly associated with Plk1 PBD, and this binding is inhibited by phosphatase treatment. A phosphorylation-deficient mutant form of ECT2, T412A, does not exhibit strong association with Plk1 PBD compared with wild-type (WT) ECT2. Moreover, ECT2 T412A, but not phosphomimic T412D, displays a diminished accumulation of GTP-bound RhoA compared with WT ECT2, suggesting that phosphorylation of Thr-412 is critical for the catalytic activity of ECT2. Moreover, while overexpression of WT ECT2 or the T412D mutant causes cortical hyperactivity in U2OS cells during cell division, this activity is not observed in cells expressing ECT2 T412A. These results suggest that ECT2 is regulated by Cdk1 and Plk1 in concert (Niiya, 2006).

New functions for a vertebrate Rho guanine nucleotide exchange factor in ciliated epithelia

Human ARHGEF11, a PDZ-domain-containing Rho guanine nucleotide exchange factor (RhoGEF), has been studied primarily in tissue culture, where it exhibits transforming ability, associates with and modulates the actin cytoskeleton, regulates neurite outgrowth, and mediates activation of Rho in response to stimulation by activated Galpha12/13 or Plexin B1. The fruit fly homolog, RhoGEF2, interacts with heterotrimeric G protein subunits to activate Rho, associates with microtubules, and is required during gastrulation for cell shape changes that mediate epithelial folding. This study reports functional characterization of a zebrafish homolog of ARHGEF11 that is expressed ubiquitously at blastula and gastrula stages and is enriched in neural tissues and the pronephros during later embryogenesis. Similar to its human homolog, zebrafish Arhgef11 stimulates actin stress fiber formation in cultured cells, whereas overexpression in the embryo of either the zebrafish or human protein impairs gastrulation movements. Loss-of-function experiments utilizing a chromosomal deletion that encompasses the arhgef11 locus, and antisense morpholino oligonucleotides designed to block either translation or splicing, produced embryos with ventrally-curved axes and a number of other phenotypes associated with ciliated epithelia. Arhgef11-deficient embryos often exhibited altered expression of laterality markers, enlarged brain ventricles, kidney cysts, and an excess number of otoliths in the otic vesicles. Although cilia formed and were motile in these embryos, polarized distribution of F-actin and Na(+)/K(+)-ATPase in the pronephric ducts was disturbed. These studies in zebrafish embryos have identified new, essential roles for this RhoGEF in ciliated epithelia during vertebrate development (Panizzi, 2007).

Polo-like kinase 1 triggers the initiation of cytokinesis in human cells by promoting recruitment of the RhoGEF Ect2 to the central spindle

Cytokinesis of animal cells requires ingression of the actomyosin-based contractile ring between segregated sister genomes. Localization of the RhoGEF Ect2 to the central spindle at anaphase promotes local activation of the RhoA GTPase, which induces assembly and ingression of the contractile ring. This study used BI 2536, an inhibitor of the mitotic kinase Plk1, to analyze the functions of this enzyme during late mitosis in human cells. It is shown that Plk1 acts after Cdk1 inactivation and independently from Aurora B to promote RhoA accumulation at the equator, contractile ring formation, and cleavage furrow ingression. Inhibition of Plk1 abolishes the interaction of Ect2 with its activator and midzone anchor, HsCyk-4 (also known as Rac GTPase activating protein 1), thereby preventing localization of Ect2 to the central spindle. It is proposed that late mitotic Plk1 activity promotes recruitment of Ect2 to the central spindle, triggering the initiation of cytokinesis and contributing to cleavage plane specification in human cells (Petronczki, 2007).

Pharmacological analysis of Plk1 during late mitosis has revealed a previously unknown function for this key mitotic kinase: triggering the initiation of cytokinesis in human cells. This work was facilitated by the use of a specific and highly potent small-molecule inhibitor that provided sufficient temporal control over Plk1 activity to pinpoint essential later mitotic roles of this kinase, which have hitherto been obscured by its earlier functions. In addition, this work emphasizes the advantages and growing importance of small-molecule inhibitors for cell biological research (Petronczki, 2007).

Although it cannot be ruled out that inhibition of other kinases contributes to some of the phenotypes observed, two key points strongly support the hypothesis that inhibition of Plk1 by BI 2536 is responsible for the cytokinesis defect. First and foremost, depletion of Plk1 using a gene-specific method reproduced one crucial hallmark of the inhibitor phenotype. Second, the conclusions are substantiated by two independent studies, which came to similar conclusions using a structurally distinct small-molecule inhibitor of Plk1 and a genetically engineered ATP analog-sensitive allele of Plk1 (Petronczki, 2007 and references therein).

The results identify Plk1 as a key regulator of the Ect2/HsCyk-4 complex that lies at the heart of cleavage furrow induction in animal cells. It is proposed that Plk1's key role in triggering the initiation of cytokinesis in human cells is to induce complex formation between Ect2 and HsCyk-4. Formation of this complex is required for RhoA activation and serves to localize the RhoGEF protein Ect2 to the central spindle. Chemical epistasis experiments have shown that this function of Plk1 is required after inactivation of Cdk1. Once bound to HsCyk-4, Ect2 could then locally activate the GTPase RhoA, which would culminate in contractile ring formation and cleavage furrow ingression at the equatorial cortex. Thus, Plk1 likely plays a vital role in specifying the cleavage plane and instructing human cells where and when to divide (Petronczki, 2007).

Consistent with the above model, Plk1 inhibition and depletion of either Ect2 or HsCyk-4 result in a very similar spectrum of early cytokinesis defects: absence of contractile ring formation, failure of equatorial RhoA accumulation and cleavage furrow ingression, and, finally, failure in ectopic furrowing during forced mitotic exit. The Plk1 inhibition phenotype closely resembles the one obtained by depletion of Ect2 or HsCyk-4 and is more severe than that observed upon mere delocalization of the Ect2/HsCyk-4 complex. Hence, in addition to controlling GEF localization, Plk1-mediated complex formation presumably contributes to the activation of Ect2's GEF function. The finding that BI 2536 blocks Ect2 binding to HsCyk-4, and Ect2 but not centralspindlin recruitment to the midzone, indicates that Plk1 kinase activity might directly regulate this interaction. Recruitment of the kinase to the midzone at anaphase might increase the local concentration of the enzyme and thereby direct Ect2/HsCyk-4 complex formation to this subcellular location (Petronczki, 2007).

Ect2 activity itself and the Ect2/HsCyk-4 complex are subject to phosphoregulation. Phosphorylation of Ect2 by Cdk1 inhibits complex formation and likely contributes to the inhibition of cytokinesis before anaphase onset. In contrast, phosphorylation of HsCyk-4 at unknown residues by an uncharacterized kinase might be crucial for complex formation. These HsCyk-4 modifications might provide a docking site for the amino-terminal BRCT domains of Ect2, which mediate HsCyk-4 binding and can act as phosphopeptide binding. Thus, it is tempting to speculate that Plk1 is responsible for these phosphorylation events on HsCyk-4. However, there is also evidence supporting an alternative model. Ect2, similar to other GEFs, can be regulated by intramolecular inhibition during which the amino-terminal BRCT region binds to and inhibits the carboxy-terminal DH/GEF domain. Phosphorylation of Ect2 by Plk1 during anaphase might alleviate this intramolecular inhibition by dissociating the Ect2 amino from the carboxyl terminus. This would set free the HsCyk-4 interaction domain and the GEF domain of Ect2 and could lead to both targeting of Ect2 to the midzone and activation of its GEF function. Consistent with this model, recent data have suggested that phosphorylation of Ect2 might induce a conformational change. Furthermore, Plk1 has the ability to bind to Ect2 via the PBD and to phosphorylate the GEF protein. Future work will be required to clarify whether Plk1 directly regulates Ect2/HsCyk-4 complex formation, identify Plk1-dependent phosphorylation sites on these proteins in vivo, and analyze the molecular mechanism by which these modifications might promote complex formation (Petronczki, 2007).

Sequential Cyk-4 binding to ECT2 and FIP3 regulates cleavage furrow ingression and abscission during cytokinesis

Cytokinesis is a highly regulated and dynamic event that involves the reorganization of the cytoskeleton and membrane compartments. Recently, FIP3 has been implicated in targeting of recycling endosomes to the mid-body of dividing cells and is found required for abscission. This study demonstrates that the centralspindlin component Cyk-4 is a FIP3-binding protein. Furthermore, FIP3 is shown to bind to Cyk-4 at late telophase, and centralspindlin may be required for FIP3 recruitment to the mid-body. The FIP3-binding region on Cyk-4 was mapped; it overlaps with the ECT2-binding domain. Finally, it was demonstrated that FIP3 and ECT2 form mutually exclusive complexes with Cyk-4 and that dissociation of ECT2 from the mid-body at late telophase may be required for the recruitment of FIP3 and recycling endosomes to the cleavage furrow. Thus, it is proposed that centralspindlin complex not only regulates acto-myosin ring contraction but also endocytic vesicle transport to the cleavage furrow and it does so through sequential interactions with ECT2 and FIP3 (Simon, 2008).

Epidermal wound repair is regulated by the planar cell polarity signaling pathway

The mammalian PCP pathway regulates diverse developmental processes requiring coordinated cellular movement, including neural tube closure and cochlear stereociliary orientation. This study shows that epidermal wound repair is regulated by PCP signaling. Mice carrying mutant alleles of PCP genes Vangl2, the flamingo homolog Celsr1, off-track homolog PTK7, and Scrb1, and the Grainyhead transcription factor Grhl3, interact genetically, exhibiting failed wound healing, neural tube defects, and disordered cochlear polarity. Using phylogenetic analysis, ChIP, and gene expression in Grhl3-/- mice, RhoGEF19, a homolog of a RhoA activator involved in PCP signaling in Xenopus, was identified as a direct target of GRHL3. Knockdown of Grhl3 or RhoGEF19 in keratinocytes induced defects in actin polymerization, cellular polarity, and wound healing, and re-expression of RhoGEF19 rescued these defects in Grhl3-kd cells. These results define a role for Grhl3 in PCP signaling and broadly implicate this pathway in epidermal repair (Caddy, 2010).

RhoGef2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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