Detection of Rho GEF and GAP activity through a sensitive split luciferase assay system

Rho GTPases regulate the assembly of cellular actin structures and are activated by GEFs (guanine-nucleotide-exchange factors) and rendered inactive by GAPs (GTPase-activating proteins). Using the Rho GTPases Cdc42, Rac1 and RhoA, and the GTPase-binding portions of the effector proteins p21-activated kinase and Rhophilin1, we have developed split luciferase assays for detecting both GEF and GAP regulation of these GTPases. The system relies on purifying split luciferase fusion proteins of the GTPases and effectors from bacteria, and our results show that the assays replicate GEF and GAP specificities at nanomolar concentrations for several previously characterized Rho family GEFs (Dbl, Vav2, Trio and Asef) and GAPs [p190, Cdc42 GAP and PTPL1-associated RhoGAP]. The assay detected activities associated with purified recombinant GEFs and GAPs, cell lysates expressing exogenous proteins, and immunoprecipitates of endogenous Vav1 and p190. The results demonstrate that the split luciferase system provides an effective sensitive alternative to radioactivity-based assays for detecting GTPase regulatory protein activities and is adaptable to a variety of assay conditions.     

Regulation of Rho-GEF Rgf3 by the arrestin Art1 in fission yeast cytokinesis

Rho GTPases, activated by guanine nucleotide exchange factors (GEFs), are essential regulators of polarized cell growth, cytokinesis, and many other cellular processes. However, the regulation of Rho-GEFs themselves is not well understood. Rgf3 is an essential GEF for Rho1 GTPase in fission yeast. We show that Rgf3 protein levels and localization are regulated by arrestin-related protein Art1. art1∆ cells lyse during cell separation with a thinner and defective septum. As does Rgf3, Art1 concentrates to the contractile ring starting at early anaphase and spreads to the septum during and after ring constriction. Art1 localization depends on its C-terminus, and Art1 is important for maintaining Rgf3 protein levels. Biochemical experiments reveal that the Rgf3 C-terminus binds to Art1. Using an Rgf3 conditional mutant and mislocalization experiments, we found that Art1 and Rgf3 are interdependent for localization to the division site. As expected, active Rho1 levels at the division site are reduced in art1∆ and rgf3 mutant cells. Taken together, these data reveal that the arrestin family protein Art1 regulates the protein levels and localization of the Rho-GEF Rgf3, which in turn modulates active Rho1 levels during fission yeast cytokinesis.     

Rho-specific Guanine nucleotide exchange factors (Rho-GEFs) inhibition affects macrophage phenotype and disrupts Golgi complex

Macrophages play crucial role in tissue homeostasis and the innate and adaptive immune response. Depending on the state of activation macrophages acquire distinct phenotypes that depend on actin, which is regulated by small GTPase RhoA. The naive M0 macrophages are slightly elongated, pro-inflammatory M1 are round and M2 anti-inflammatory macrophages are elongated. We showed previously that interference with RhoA pathway (RhoA deletion or RhoA/ROCK kinase inhibition) disrupted actin, produced extremely elongated (hummingbird) macrophage phenotype and inhibited macrophage movement toward transplanted hearts. The RhoA function depends on the family of guanine-nucleotide exchange factors (GEFs), which catalyze the exchange of GDP for GTP and activate RhoA that reorganizes actin cytoskeleton. Using actin staining, immunostaining, Western blotting, flow cytometry and transmission electron microscopy we studied how a direct inhibition of Rho-GEFs with Rhosin (Rho GEF-binding domain blocker) and Y16 (Rho GEF DH-PH domain blocker) affects M0, M1 and M2 macrophage phenotypes. We also studied how Rho-GEFs inhibition and RhoA deletion affects organization of Golgi complex that is crucial for normal macrophage functions such as phagocytosis, antigen presentation and receptor recycling. We found that GEFs inhibition differently affected M0, M1 and M2 macrophages phenotype and that GEFs inhibition and RhoA deletion both caused changes in the ultrastructure of the Golgi complex. These results suggest that actin/RhoA- dependent shaping of macrophage phenotype has different requirements for activity of RhoA/GEFs pathway in M0, M1 and M2 macrophages, and that RhoA and Rho-GEFs functions are necessary for the maintenance of actin-dependent organization of Golgi complex.     

A Highlights from MBoC Selection: The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis

Rho GTPases, activated by Rho guanine nucleotide exchange factors (GEFs), are conserved molecular switches for signal transductions that regulate diverse cellular processes, including cell polarization and cytokinesis. The fission yeast Schizosaccharomyces pombe has six Rho GTPases (Cdc42 and Rho1–Rho5) and seven Rho GEFs (Scd1, Rgf1–Rgf3, and Gef1–Gef3). The GEFs for Rho2–Rho5 have not been unequivocally assigned. In particular, Gef3, the smallest Rho GEF, was barely studied. Here we show that Gef3 colocalizes with septins at the cell equator. Gef3 physically interacts with septins and anillin Mid2 and depends on them to localize. Gef3 coprecipitates with GDP-bound Rho4 in vitro and accelerates nucleotide exchange of Rho4, suggesting that Gef3 is a GEF for Rho4. Consistently, Gef3 and Rho4 are in the same genetic pathways to regulate septum formation and/or cell separation. In gef3∆ cells, the localizations of two potential Rho4 effectors—glucanases Eng1 and Agn1—are abnormal, and active Rho4 level is reduced, indicating that Gef3 is involved in Rho4 activation in vivo. Moreover, overexpression of active Rho4 or Eng1 rescues the septation defects of mutants containing gef3∆. Together our data support that Gef3 interacts with the septin complex and activates Rho4 GTPase as a Rho GEF for septation in fission yeast.     

Function of the N-terminus of zizimin1: autoinhibition and membrane targeting

Rho family small GTPases are critical regulators of multiple cellular functions. Dbl-homology-domain-containing proteins are the classical GEFs (guanine nucleotide exchange factors) responsible for activation of Rho proteins. Zizimin1 is a Cdc42-specific GEF that belongs to a second family of mammalian Rho-GEFs, CZH [CDM (Ced-5/DOCK180/Myoblast city)-zizimin homology] proteins, which possess a novel type of GEF domain. CZH proteins can be divided into a subfamily related to DOCK 180 and a subfamily related to zizimin1. The two groups share two conserved regions named the CZH1 (or DHR1) domain and the CZH2 (DHR2 or DOCKER) domains, the latter exhibiting GEF activity. We now show that limited proteolysis of zizimin1 suggests the existence of structural domains that do not correspond to those identified on the basis of homologies. We demonstrate that the N-terminal half binds to the GEF domain through three distinct areas, including the CZH1, to inhibit the interaction with Cdc42. The N-terminal PH (pleckstrin homology) domain binds phosphoinositides and mediates zizimin1 membrane targeting. These results define two novel functions for the N-terminal region of zizimin1.     

Activation of the guanine nucleotide exchange factor Dbl following ACK1-dependent tyrosine phosphorylation

Signals triggered by diverse receptors modulate the activity of Rho family proteins, although the regulatory mechanism remains largely unknown. On the basis of their biochemical activity as guanine nucleotide exchange factors (GEFs), Dbl family proteins are believed to be implicated in the regulation of Rho family GTP-binding proteins in response to a variety of extracellular stimuli. Here we show that GEF activity of full-length proto-Dbl is enhanced upon tyrosine phosphorylation. When transiently coexpressed with the activated form of the non-receptor tyrosine kinase ACK1, a downstream target of Cdc42, Dbl became tyrosine-phosphorylated. In vitro GEF activity of Dbl toward Rho and Cdc42 was augmented following tyrosine phosphorylation. Moreover, accumulation of the GTP-bound form of Rho and Rac within the cell paralleled ACK-1-dependent tyrosine phosphorylation of Dbl. Consistently, activation of c-Jun N-terminal kinase downstream of Rho family GTP-binding proteins was also enhanced when Dbl was tyrosine-phosphorylated. Collectively, these findings suggest that the tyrosine kinase ACK1 may act as a regulator of Dbl, which in turn activates Rho family proteins.     

鸟苷酸交换因子P92GEF通过靶向调控RhoA抑制肿瘤细胞增殖侵袭（英文稿）

摘要：Dbl家族鸟苷交换因子（GEFs）是Rho家族蛋白发生恶性转化的主要调控单位,它通过使Rho蛋白从无活性的GDP形式转换为GTP形式的Rho蛋白而发挥作用,参与细胞骨架重排,细胞的生长和活力。P92GEF是一GEFs家族分子。本研究通过Real time PCR对P92GEF在人体48种正常组织中的表达情况进行了测定;GST-pulldown技术对P92GEF的体内GEF活性进行了检测;双荧光素酶报告基因检测技术对下游小分子进行转录因子活性检测;应用免疫荧光双染标记法完成了高表达P92GEF对正常细胞骨架形态的影响;在细胞表型实验中分别使用CCK8法、Transwell法及软琼脂克隆形成实验检测了高表达P92GEF对细胞增殖侵袭迁移及体外成瘤能力的影响。研究结果显示P92GEF有841个氨基酸,具有典型的Dbl家族分子结构域,在肺组织中表达量最高,能够促使正常成纤维细胞中的应力纤维（stress fiber）增多,P92GEF转染的NIH3T3细胞可以独立生长和形成继发性病灶,同时促使细胞增殖,侵袭及克隆形成能力增强,体外转录因子活性检测发现该基因可能与JAK/STAT通路有关。因此,P92GEF是一个典型的鸟苷交换因子家族分子,能激活Rho家族分子RhoA,具有明显的癌基因特征。     

Catching a GEF by its tail

The activation of Rho GTPases is mediated by guanine-nucleotide exchange factors (GEFs), which catalyze the exchange of GDP for GTP. Rho-GEFs are a very diverse family, with >70 members in humans. Bioinformatics analysis of the human Rho-GEFs shows that approximately 40% contain a putative PDZ-binding motif at the C-terminus. PDZ domains are protein-protein interaction domains that act as scaffolds to concentrate signaling molecules at specialized regions in the cell. We propose that the interaction between Rho-GEFs and PDZ-domain proteins is a general mechanism that controls Rho-GEF targeting and activation, helping to restrict and concentrate the exchange activity to appropriate subcellular destinations. Here, we summarize recent data that highlight the importance of these interactions in Rho-GEF regulation.     

Zizimin1, a novel Cdc42 activator,reveals a new GEF domain for Rho proteins

The Rho family GTPases Rac, Rho and Cdc42 are critical in regulating the actin-based cytoskeleton, cell migration, growth, survival and gene expression. These GTPases are activated by guanine nucleotide-exchange factors (GEFs). A biochemical search for Cdc42 activators led to the cloning of zizimin1, a new protein whose overexpression induces Cdc42 activation. Sequence comparison combined with mutational analysis identified a new domain, which we named CZH2, that mediates direct interaction with Cdc42. CZH2-containing proteins constitute a new superfamily that includes the so-called 'CDM' proteins that bind to and activate Rac. Together, the results suggest that CZH2 is a new GEF domain for the Rho family of proteins.     

Role of guanine nucleotide exchange factors for Rho family GTPases in the regulation of cell morphology and actin cytoskeleton in fission yeast

Rho GTPases regulate fundamental processes including cell morphology and migration in various organisms. Guanine nucleotide exchange factor (GEF) has a crucial role in activating small GTPase by exchange GDP for GTP. In fission yeast Schizosaccharomyces pombe, six members of the Rho small GTPase family were identified and reported to be involved in cell morphology and polarized cell growth. We identified seven genes encoding Rho GEF domain from genome sequence and analyzed. Overexpressions of identified genes in cell lead to change of morphology, suggesting that all of them are involved in the regulation of cell morphology. Although all of null mutants were viable, two of seven null cells had morphology defects and five of seven displayed altered actin cytoskeleton arrangements. Most of the double mutants were viable and biochemical analysis revealed that each of GEFs bound to several small G proteins. These data suggest that identified Rho GEFs are involved in the regulation of cell morphology and share signals via small GTPase Rho family.     

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

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

Paxillin kinase linker (PKL) regulates Vav2 signaling during cell spreading and migration

The Rho family of GTPases plays an important role in coordinating dynamic changes in the cell migration machinery after integrin engagement with the extracellular matrix. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) and negatively regulated by GTPase-activating proteins (GAPs). However, the mechanisms by which GEFs and GAPs are spatially and temporally regulated are poorly understood. Here the activity of the proto-oncogene Vav2, a GEF for Rac1, RhoA, and Cdc42, is shown to be regulated by a phosphorylation-dependent interaction with the ArfGAP PKL (GIT2). PKL is required for Vav2 activation downstream of integrin engagement and epidermal growth factor (EGF) stimulation. In turn, Vav2 regulates the subsequent redistribution of PKL and the Rac1 GEF β-PIX to focal adhesions after EGF stimulation, suggesting a feedforward signaling loop that coordinates PKL-dependent Vav2 activation and PKL localization. Of interest, Vav2 is required for the efficient localization of PKL and β-PIX to the leading edge of migrating cells, and knockdown of Vav2 results in a decrease in directional persistence and polarization in migrating cells, suggesting a coordination between PKL/Vav2 signaling and PKL/β-PIX signaling during cell migration.     

Guanine nucleotide exchange factors: Activators of the Ras superfamily of proteins

Ras proteins function as critical relay switches that regulate diverse signaling pathways between cell surface receptors and the nucleus. Over the past 2-3 years researchers have identified many components of these pathways that mediate Ras activation and effector function. Among these proteins are several guanine nucleotide exchange factors (GEFs), which are responsible for directly interacting with and activating Ras in response to extracellular stimuli. Analogous GEFs regulate Ras-related proteins that serve other diverse cellular functions. In particular, a growing family of proteins (Dbl homology proteins) has recently been identified, which may function as GEFs for the Rho family of Ras-related proteins. This review summarizes our current knowledge of the structure, biochemistry and biology of Ras and Rho family GEFs. Additionally, we describe mechanisms of GEF activation of Ras in signal transduction and address the potential that deregulated GEFs might contribute to malignant transformation through chronic Ras protein activation.     

Vav2 is an activator of Cdc42, Rac1, and RhoA

Vav and Vav2 are members of the Dbl family of proteins that act as guanine nucleotide exchange factors (GEFs) for Rho family proteins. Whereas Vav expression is restricted to cells of hematopoietic origin, Vav2 is widely expressed. Although Vav and Vav2 share highly related structural similarities and high sequence identity in their Dbl homology domains, it has been reported that they are active GEFs with distinct substrate specificities toward Rho family members. Whereas Vav displayed GEF activity for Rac1, Cdc42, RhoA, and RhoG, Vav2 was reported to exhibit GEF activity for RhoA, RhoB, and RhoG but not for Rac1 or Cdc42. Consistent with their distinct substrate targets, it was found that constitutively activated versions of Vav and Vav2 caused distinct transformed phenotypes when expressed in NIH 3T3 cells. In contrast to the previous findings, we found that Vav2 can act as a potent GEF for Cdc42, Rac1, and RhoA in vitro. Furthermore, we found that NH(2)-terminally truncated and activated Vav and Vav2 caused indistinguishable transforming actions in NIH 3T3 cells that required Cdc42, Rac1, and RhoA function. In addition, like Vav and Rac1, we found that Vav2 activated the Jun NH(2)-terminal kinase cascade and also caused the formation of lamellipodia and membrane ruffles in NIH 3T3 cells. Finally, Vav2-transformed NIH 3T3 cells showed up-regulated levels of Rac-GTP. We conclude that Vav2 and Vav share overlapping downstream targets and are activators of multiple Rho family proteins. Therefore, Vav2 may mediate the same cellular consequences in nonhematopoietic cells as Vav does in hematopoietic cells.     

Molecular basis for Rac1 recognition by guanine nucleotide exchange factors.

Rho GTPases are activated by a family of guanine nucleotide exchange factors (GEFs) known as Dbl family proteins. The structural basis for how GEFs recognize and activate Rho GTPases is presently ill defined. Here, we utilized the crystal structure of the DH/PH domains of the Rac-specific GEF Tiam1 in complex with Rac1 to determine the structural elements of Rac1 that regulate the specificity of this interaction. We show that residues in the Rac1 beta2-beta3 region are critical for Tiam1 recognition. Additionally, we determined that a single Rac1-to-Cdc42 mutation (W56F) was sufficient to abolish Rac1 sensitivity to Tiam1 and allow recognition by the Cdc42-specific DH/PH domains of Intersectin while not impairing Rac1 downstream activities. Our findings identified unique GEF specificity determinants in Rac1 and provide important insights into the mechanism of DH/PH selection of GTPase targets.     

The many faces of the guanine-nucleotide exchange factor trio

Small Rho-GTPases are enzymes that are bound to GDP or GTP, which determines their inactive or active state, respectively. The exchange of GDP for GTP is catalyzed by so-called Rho-guanine nucleotide exchange factors (GEFs). Rho-GEFs are characterized by a Dbl-homology (DH) and adjacent Pleckstrin-homology (PH) domain that serves as enzymatic unit for the GDP/GTP exchange. Rho-GEFs show different GTPase specificities, meaning that a particular GEF can activate either multiple GTPases or only one specific GTPase. We recently reported that the Rho-GEF Trio, known to be able to exchange GTP on Rac1, RhoG and RhoA, regulates lamellipodia formation to mediate cell spreading and migration in a Rac1-dependent manner. In this commentary, we review the current knowledge of Trio in several aspects of cell biology.     

The many faces of the guanine-nucleotide exchange factor trio

Small Rho-GTPases are enzymes that are bound to GDP or GTP, which determines their inactive or active state, respectively. The exchange of GDP for GTP is catalyzed by so-called Rho-guanine nucleotide exchange factors (GEFs). Rho-GEFs are characterized by a Dbl-homology (DH) and adjacent Pleckstrin-homology (PH) domain that serves as enzymatic unit for the GDP/GTP exchange. Rho-GEFs show different GTPase specificities, meaning that a particular GEF can activate either multiple GTPases or only one specific GTPase. We recently reported that the Rho-GEF Trio, known to be able to exchange GTP on Rac1, RhoG and RhoA, regulates lamellipodia formation to mediate cell spreading and migration in a Rac1-dependent manner. In this commentary, we review the current knowledge of Trio in several aspects of cell biology.