Pragmin, a novel effector of Rnd2 GTPase, stimulates RhoA activity

The Rho family of small GTPases has been implicated in the reorganization of actin cytoskeleton and subsequent morphological changes in various cells. Rnd2 is a member of the Rnd subfamily, comprising Rnd1, Rnd2, and Rnd3. In contrast to Rnd1 and Rnd3, displaying an antagonistic action for RhoA signaling, signaling pathways of Rnd2 are not well known. Here we have performed a yeast two-hybrid screen using Rnd2 as bait and identified a novel Rnd2 effector protein, predominantly expressed in neurons, including cortical and hippocampal neurons. We named it Pragmin (pragma of Rnd2). In in vivo and in vitro binding assays, Pragmin specifically binds to Rnd2 among the Rho family GTPases in a GTP-dependent manner. Rnd2-bound Pragmin significantly stimulates RhoA activity and induces cell contraction through RhoA and the Rho-kinase pathway in HeLa cells. In PC12 cells, expressing Pragmin inhibits nerve growth factor-induced neurite outgrowth in response to Rnd2, and knock-down of Pragmin by Pragmin-specific small interfering RNA enhances neurite elongation. Therefore, Rnd2 regulates neurite outgrowth by functioning as the RhoA activator through Pragmin, in contrast to Rnd1 and Rnd3 inhibiting RhoA signaling.     

Identification of splicing variants of Rapostlin, a novel RND2 effector that interacts with neural Wiskott-Aldrich syndrome protein and induces neurite branching

Rho family GTPases regulate neuronal morphology. Rnd subfamily is a new branch of Rho family GTPases. Of these GTPases, Rnd2 is specifically expressed in brain. We recently identified Rapostlin as a novel effector of Rnd2. Rapostlin induces neurite branching in response to Rnd2 in PC12 cells. During the cloning of Rapostlin, we have found two mainly expressed splicing variants of Rapostlin (renamed as RapostlinL), RapostlinM and RapostlinS, lacking 29 residues and 61 residues within the unique insert region at the center, respectively, and three minor variants, RapostlinLd, RapostlinMd, and RapostlinSd, each with the identical five-amino acid deletion from RapostlinL, RapostlinM, and RapostlinS, respectively. RapostlinL is predominantly expressed in brain, whereas RapostlinS is expressed ubiquitously. In a dot-blot assay, all splicing variants bind to Rnd2 in a GTP-dependent manner. However, RapostlinM and RapostlinS induce less neurite branching when coexpressed with Rnd2 in PC12 cells, indicating that the insert region is important for the branching activity of RapostlinL. All splicing variants bind to N-WASP in vitro and in vivo through the SH3 domain at the carboxyl terminus, and the SH3 domain is essential for branching activity of RapostlinL. In immunoprecipitation experiments, Rnd2 reduces RapostlinL-N-WASP interaction, whereas it has little effect on the interaction of RapostlinM or RapostlinS with N-WASP. Therefore, we found that functionally different splicing variants of Rapostlin have different responses to Rnd2 in association with N-WASP.     

STI1 antagonizes cytoskeleton collapse mediated by small GTPase Rnd1 and regulates neurite growth

Rnd proteins comprise a branch of the Rho family of small GTP-binding proteins, which have been implicated in rearrangements of the actin cytoskeleton and microtubule dynamics. Particularly in the nervous system, Rnd family proteins regulate neurite formation, dendrite development and axonal branching. A secreted form of the co-chaperone Stress-Inducible Protein 1 (STI1) has been described as a prion protein partner that is involved in several processes of the nervous system, such as neurite outgrowth, neuroprotection, astrocyte development, and the self-renewal of neural progenitor cells. We show that cytoplasmic STI1 directly interacts with the GTPase Rnd1. This interaction is specific for the Rnd1 member of the Rnd family. In the COS collapse assay, overexpression of STI1 prevents Rnd1–plexin-A1-mediated cytoskeleton retraction. In PC-12 cells, overexpression of STI1 enhances neurite outgrowth in cellular processes initially established by Rnd1. Therefore, we propose that STI1 participates in Rnd1-induced signal transduction pathways that are involved in the dynamics of the actin cytoskeleton.     

Binding of Rac1, Rnd1, and RhoD to a novel Rho GTPase interaction motif destabilizes dimerization of the plexin-B1 effector domain

Plexins are the first known transmembrane receptors that interact directly with small GTPases. On binding to certain Rho family GTPases, the receptor regulates the remodeling of the actin cytoskeleton and alters cell movement in response to semaphorin guidance cues. In a joint solution NMR spectroscopy and x-ray crystallographic study, we characterize a 120-residue cytoplasmic independent folding domain of plexin-B1 that directly binds three Rho family GTPases, Rac1, Rnd1, and RhoD. The NMR data show that, surprisingly, the Cdc42/Rac interactive binding-like motif of plexin-B1 is not involved in this interaction. Instead, all three GTPases interact with the same region, beta-strands 3 and 4 and a short alpha-helical segment of the plexin domain. The 2.0 A resolution x-ray structure shows that these segments are brought together by the tertiary structure of the ubiquitin-like fold. In the crystal, the protein is dimerized with C2 symmetry through a four-stranded antiparallel beta-sheet that is formed outside the fold by a long loop between the monomers. This region is adjacent to the GTPase binding motifs identified by NMR. Destabilization of the dimer in solution by binding of any one of the three GTPases suggests a model for receptor regulation that involves bidirectional signaling. The model implies a multifunctional role for the GTPase-plexin interaction that includes conformational change and a localization of active receptors in the signaling mechanism.     

Pak6 protein kinase is a novel effector of an atypical Rho family GTPase Chp/RhoV

Chp/RhoV is an atypical Rho GTPase whose functions are far from being fully understood. To date several effector proteins of Chp have been identified, including p21-activated kinases Pak1, Pak2, and Pak4. Using a yeast two-hybrid system and co-immunoprecipitation, here we show that another p21-activated kinase, Pak6, is a novel Chp-binding protein. Interaction between Chp and Pak6 depends on the activation state of the GTPase, suggesting that Pak6 is an effector protein for Chp. Point mutations in the effector domain of Chp or in the CRIB motif of Pak6 significantly impair the interaction between Chp and Pak6 upon co-immunoprecipitation, suggesting that the binding interface involves the effector domain of Chp and the CRIB motif in Pak6. We found that Chp does not affect the phosphorylation status of the S560 residue in the catalytic domain of Pak6 when Chp and Pak6 are co-expressed in HEK293 cells. Therefore, similarly to Cdc42, Chp is not likely to activate Pak6. In NCI-H1299 cells, Chp co-localizes with Pak6 on vesicular structures in activation state-dependent manner. Taking the data together, we report here the identification of p21-activated kinase Pak6 as a novel effector of the atypical Rho GTPase Chp. Our data suggest further directions in elucidating biological functions of these proteins.     

RASSF2 is a novel K-Ras-specific effector and potential tumor suppressor

Ras proteins regulate a wide range of biological processes by interacting with a broad assortment of effector proteins. Although activated forms of Ras are frequently associated with oncogenesis, they may also provoke growth-antagonistic effects. These include senescence, cell cycle arrest, differentiation, and apoptosis. The mechanisms that underlie these growth-inhibitory activities are relatively poorly understood. Recently, two related novel Ras effectors, NORE1 and RASSF1, have been identified as mediators of apoptosis and cell cycle arrest. Both of these proteins exhibit many of the properties normally associated with tumor suppressors. We now identify a novel third member of this family, designated RASSF2. RASSF2 binds directly to K-Ras in a GTP-dependent manner via the Ras effector domain. However, RASSF2 only weakly interacts with H-Ras. Moreover, RASSF2 promotes apoptosis and cell cycle arrest and is frequently down-regulated in lung tumor cell lines. Thus, we identify RASSF2 as a new member of the RASSF1 family of Ras effectors/tumor suppressors that exhibits a specificity for interacting with K-Ras.     

Rnd1, a novel rho family GTPase, induces the formation of neuritic processes in PC12 cells

Rho family GTPases have been shown to be involved in the regulation of neuronal cell morphology, including neurite extension and retraction. Rho activation leads to neurite retraction and cell rounding, whereas Rac and Cdc42 are implicated in the promotion of filopodia and lamellipodia formation in growth cones and, therefore, in neurite extension. In this study, we examined the morphological role of Rnd1, a new member of Rho family GTPases, in PC12 cells, and found that expression of Rnd1 by itself caused the formation of many neuritic processes from the cell body with disruption of the cortical actin filaments, the processes having microtubules but few filamentous actin and neurofilaments. Treatment with cytochalasin D, an inhibitor of actin polymerization, could mimic the effects of expression of Rnd1, in that this inhibitor disrupted the cortical actin filaments and induced the formation of many thin processes containing microtubules. The process formation induced by Rnd1 was inhibited by dominant negative Rac1. These results suggest that Rnd1 induces the Rac-dependent neuritic process formation in part by disruption of the cortical actin filaments.     

NleB2 from enteropathogenic Escherichia coli is a novel arginine-glucose transferase effector

During infection, enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) directly manipulate various aspects of host cell function through the translocation of type III secretion system (T3SS) effector proteins directly into the host cell. Many T3SS effector proteins are enzymes that mediate post-translational modifications of host proteins, such as the glycosyltransferase NleB1, which transfers a single N-acetylglucosamine (GlcNAc) to arginine residues, creating an Arg-GlcNAc linkage. NleB1 glycosylates death-domain containing proteins including FADD, TRADD and RIPK1 to block host cell death. The NleB1 paralogue, NleB2, is found in many EPEC and EHEC strains but to date its enzymatic activity has not been described. Using in vitro glycosylation assays combined with mass spectrometry, we found that NleB2 can utilize multiple sugar donors including UDP-glucose, UDP-GlcNAc and UDP-galactose during glycosylation of the death domain protein, RIPK1. Sugar donor competition assays demonstrated that UDP-glucose was the preferred substrate of NleB2 and peptide sequencing identified the glycosylation site within RIPK1 as Arg603, indicating that NleB2 catalyses arginine glucosylation. We also confirmed that NleB2 catalysed arginine-hexose modification of Flag-RIPK1 during infection of HEK293T cells with EPEC E2348/69. Using site-directed mutagenesis and in vitro glycosylation assays, we identified that residue Ser252 in NleB2 contributes to the specificity of this distinct catalytic activity. Substitution of Ser252 in NleB2 to Gly, or substitution of the corresponding Gly255 in NleB1 to Ser switches sugar donor preference between UDP-GlcNAc and UDP-glucose. However, this switch did not affect the ability of the NleB variants to inhibit inflammatory or cell death signalling during HeLa cell transfection or EPEC infection. NleB2 is thus the first identified bacterial Arg-glucose transferase that, similar to the NleB1 Arg-GlcNAc transferase, inhibits host protein function by arginine glycosylation.     

Small GTPase RhoG is a key regulator for neurite outgrowth in PC12 cells

The Rho family of small GTPases has been implicated in cytoskeletal reorganization and subsequent morphological changes in various cell types. Among them, Rac and Cdc42 have been shown to be involved in neurite outgrowth in neuronal cells. In this study, we examined the role of RhoG, another member of Rho family GTPases, in nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Expression of wild-type RhoG in PC12 cells induced neurite outgrowth in the absence of NGF, and the morphology of wild-type RhoG-expressing cells was similar to that of NGF-differentiated cells. Constitutively active RhoG-transfected cells extended short neurites but developed large lamellipodial or filopodial structures at the tips of neurites. RhoG-induced neurite outgrowth was inhibited by coexpression with dominant-negative Rac1 or Cdc42. In addition, expression of constitutively active RhoG elevated endogenous Rac1 and Cdc42 activities. We also found that the NGF-induced neurite outgrowth was enhanced by expression of wild-type RhoG whereas expression of dominant-negative RhoG suppressed the neurite outgrowth. Furthermore, constitutively active Ras-induced neurite outgrowth was also suppressed by dominant-negative RhoG. Taken together, these results suggest that RhoG is a key regulator in NGF-induced neurite outgrowth, acting downstream of Ras and upstream of Rac1 and Cdc42 in PC12 cells.     

Direct interaction of Rnd1 with FRS2 beta regulates Rnd1-induced down-regulation of RhoA activity and is involved in fibroblast growth factor-induced neurite outgrowth in PC12 cells

The Rho family of small GTPases has been implicated in the reorganization of the actin cytoskeleton and subsequent morphological changes in various cells. Rnd1, a member of this family, has a low intrinsic GTPase activity and exerts antagonistic effects on RhoA signaling. However, how the activity of Rnd1 is regulated has not yet been elucidated. Here we have demonstrated that Rnd1 directly associates with FRS2alpha and FRS2beta, which are docking proteins of fibroblast growth factor (FGF) receptors and play important roles in the intracellular signals induced by FGFs. The interaction of FRS2beta with Rnd1 suppresses the inhibitory effect of Rnd1 on RhoA. Rnd1 binds to the COOH-terminal region of FRS2beta including tyrosine residues essential for the interaction with Shp2. When FGF receptor 1 is activated, it phosphorylates FRS2beta, recruits Shp2, and releases Rnd1 from FRS2beta. The liberated Rnd1 then inhibits RhoA activity. Furthermore, knockdown of Rnd1 by Rnd1-specific short interfering RNAs suppress the FGF-induced neurite outgrowth in PC12 cells. These results suggest that the activity of Rnd1 is regulated by FGF receptor through FRS2beta and that Rnd1 plays an important role in the FGF signaling during neurite outgrowth.     

Socius is a novel Rnd GTPase-interacting protein involved in disassembly of actin stress fibers

Rho family small GTPases are key regulators of the actin cytoskeleton in various cell types. The Rnd proteins, Rnd1, Rnd2, and Rnd3/RhoE, have been recently identified as new members of the Rho family of GTPases, and expression of Rnd1 or Rnd3 in fibroblasts causes the disassembly of actin stress fibers and the retraction of the cell body to produce extensively branching cellular processes. Here we have performed a yeast two-hybrid screening by using Rnd1 as bait and identified a novel protein that specifically binds to Rnd GTPases. We named this protein Socius. Socius directly binds to Rnd GTPases through its COOH-terminal region. When transfected into COS-7 cells, Socius is translocated to the cell periphery in response to Rnd1 and Rnd3 and colocalized with the GTPases. While expression of wild-type Socius in Swiss 3T3 fibroblasts has little effect on the actin cytoskeleton, the expression of a membrane-targeted form of Socius, containing a COOH-terminal farnesylation motif (Socius-CAAX), induces a dramatic loss of stress fibers. The inhibitory effect of Socius-CAAX on stress fiber formation is enhanced by truncation of its NH(2) terminus. On the other hand, the expression of Socius-CAAX or its NH(2) terminus-truncated form suppresses the Rnd-induced retraction of the cell body and the production of extensively branching cellular processes, although the disassembly of stress fibers is observed. We propose that Socius participates in the Rnd GTPase-induced signal transduction pathways, leading to reorganization of the actin cytoskeleton.     

ICA69 is a novel Rab2 effector regulating ER-Golgi trafficking in insulinoma cells

Islet cell autoantigen of 69kDa (ICA69) is a small GTPase-binding protein of unknown function. ICA69 is enriched in the Golgi complex and its N-terminal half contains a BAR domain, a module that can bind/bend membranes and interacts with phospholipids. Here we show that in insulinoma INS-1 cells ICA69 binds to the small GTPase Rab2, which regulates the transport of COPI vesicles between the endoplasmic reticulum and the Golgi complex. Rab2 binds to ICA69 in a GTP-dependent fashion and recruits it to membranes. Over-expression of either Rab2 or ICA69 in INS-1 cells results in a phenotype characterized by: (i) impaired anterograde transport of the secretory granule protein precursors pro-ICA512 and chromogranin A; (ii) reduction of stimulated insulin secretion. Taken together, these data identify ICA69 as a novel Rab2 effector and point to its role in regulating the early transport of insulin secretory granule proteins.     

The scaffold protein JIP3 functions as a downstream effector of the small GTPase ARF6 to regulate neurite morphogenesis of cortical neurons

The small GTPase ADP-ribosylation factor 6 (ARF6) plays crucial roles in a wide variety of cell functions. To better understand the molecular mechanisms of ARF6-mediated signaling and cellular functions, we sought new ARF6-binding proteins in the mouse brain. We identified the signaling scaffold protein JNK-interacting protein 3 (JIP3), which is exclusively expressed in neurons, as a downstream effector of ARF6. Overexpression of a unique dominant negative mutant of ARF6, which was unable to interact with JIP3, and knockdown of JIP3 in mouse cortical neurons stimulated the elongation and branching of neurites. These results provide evidence that ARF6/JIP3 signaling regulates neurite morphogenesis.

Structured summary

MINT-7892698: PIP5K gamma 661 (uniprotkb:O70161) physically interacts (MI:0915) with Arf6 (uniprotkb:P62331) by anti tag coimmunoprecipitation (MI:0007)MINT-7892333, MINT-7892573, MINT-7892594, MINT-7892629, MINT-7892644, MINT-7892522, MINT-7892716: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JLP (uniprotkb:Q58A65) by anti tag coimmunoprecipitation (MI:0007)MINT-7892509: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JIP3 (uniprotkb:Q9ESN9) by pull down (MI:0096)MINT-7892770: Arf6 (uniprotkb:P62331) binds (MI:0407) to JIP3 (uniprotkb:Q9ESN9) by pull down (MI:0096)MINT-7892755: Arf6 (uniprotkb:P62331) binds (MI:0407) to JLP (uniprotkb:Q58A65) by pull down (MI:0096)MINT-7892289, MINT-7892314: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JLP (uniprotkb:Q58A65) by pull down (MI:0096)MINT-7892353, MINT-7892615, MINT-7892657, MINT-7892672, MINT-7892549, MINT-7892738: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JIP3 (uniprotkb:Q9ESN9) by anti tag coimmunoprecipitation (MI:0007)     

Src tyrosine kinase is a novel direct effector of G proteins

Heterotrimeric G proteins transduce signals from cell surface receptors to modulate the activity of cellular effectors. Src, the product of the first characterized proto-oncogene and the first identified protein tyrosine kinase, plays a critical role in the signal transduction of G protein-coupled receptors. However, the mechanism of biochemical regulation of Src by G proteins is not known. Here we demonstrate that Galphas and Galphai, but neither Galphaq, Galpha12 nor Gbetay, directly stimulate the kinase activity of downregulated c-Src. Galphas and Galphai similarly modulate Hck, another member of Src-family tyrosine kinases. Galphas and Galphai bind to the catalytic domain and change the conformation of Src, leading to increased accessibility of the active site to substrates. These data demonstrate that the Src family tyrosine kinases are direct effectors of G proteins.     

Arabidopsis SHORT INTEGUMENTS 2 is a mitochondrial DAR GTPase

The Arabidopsis short integuments 2-1 (sin2-1) mutant produces ovules with short integuments due to early cessation of cell division in these structures. SIN2 was isolated and encodes a putative GTPase sharing features found in the novel DAR GTPase family. DAR proteins share a signature DAR motif and a unique arrangement of the four conserved GTPase G motifs. We found that DAR GTPases are present in all examined prokaryotes and eukaryotes and that they have diversified into four paralogous lineages in higher eukaryotes. Eukaryotic members of the SIN2 clade of DAR GTPases have been found to localize to mitochondria and are related to eubacterial proteins that facilitate essential steps in biogenesis of the large ribosomal subunit. We propose a similar role for SIN2 in mitochondria. A sin2 insertional allele has ovule effects similar to sin2-1, but more pronounced pleiotropic effects on vegetative and floral development. The diverse developmental effects of the mitochondrial SIN2 GTPase support a mitochondrial role in the regulation of multiple developmental pathways.     

Sec15 is an effector for the Rab11 GTPase in mammalian cells

Rab/Ypt GTPases play key roles in the regulation of vesicular trafficking. They perform most of their functions in a GTP-bound form by interacting with specific downstream effectors. The exocyst is a complex of eight polypeptides involved in constitutive secretion and functions as an effector for multiple Ras-related small GTPases, including the Rab protein Sec4p in yeast. In this study, we have examined the localization and function of the Sec15 exocyst subunit in mammalian cells. Overexpressed Sec15 associated with clusters of tubular/vesicular elements that were concentrated in the perinuclear region. The tubular/vesicular clusters were dispersed throughout the cytoplasm upon treatment with the microtubule-depolymerizing agent nocodazole and were accessible to endocytosed transferrin, but not exocytic cargo (vesicular stomatitis virus glycoprotein). Consistent with these observations, Sec15 colocalized selectively with the recycling endosome marker Rab11 and exhibited a GTP-dependent interaction with the Rab11 GTPase, but not with Rab4, Rab6, or Rab7. These findings provide the first evidence that the exocyst functions as a Rab effector complex in mammalian cells.     

A novel ROP/RAC GTPase effector integrates plant cell form and pattern formation

ROPs/RACs are the only known signaling Ras superfamily small GTPases in plants. As such they have been suggested to function as central regulators of diverse signaling cascades. The ROP/RAC signaling networks are largely unknown, however, because only few of their effector proteins have been identified. In a paper that was published in the June 5, 2007 issue of Current Biology we described the identification of a novel ROP/RAC effector designated ICR1 (Interactor of Constitutive active ROPs 1). We demonstrated that ICR1 functions as a scaffold that interacts with diverse but specific group of proteins including SEC3 subunit of the exocyst vesicle tethering complex. ICR1-SEC3 complexes can interact with ROPs in vivo and are thereby recruited to the plasma membrane. ICR1 knockdown or silencing leads to cell deformation and loss of the root stem cells population, and ectopic expression of ICR1 phenocopies activated ROPs/RACs. ICR1 presents a new paradigm in ROP/RAC signaling and integrates mechanisms regulating cell form and pattern formation at the whole plant level.Key words: Rho, auxin, root development, vesicle trafficking, RAC, ROP, polarity, Arabidopsis, exocyst     

Collapsin response mediator protein-2 regulates neurite formation by modulating tubulin GTPase activity

Collapsin response mediator protein-2 (CRMP-2) plays a key role in axonal development by regulating microtubule dynamics. However, the molecular mechanisms underlying this function have not been clearly elucidated. In this study, we demonstrated that hCRMP-2, specifically amino acid residues 480–509, is essential for stimulating tubulin GTPase activity. We also found that the GTPase-activating protein (GAP) activity of hCRMP-2 was important for microtubule assembly and neurite formation in differentiated PC12 pheochromocytoma cell lines. Mutant hCRMP-2, lacking arginine residues responsible for GAP activity, inhibited microtubule assembly and neurite formation. Interestingly, we found that the N-terminal region (amino acids150–299) of hCRMP-2 had an inhibitory role on GAP activity via a direct interaction with the C-terminal region (amino acids 480–509). Our results suggest that CRMP-2 as a tubulin direct binder may be a GAP of tubulin in neurite formation and that its GAP activity may be regulated by an intramolecular interaction with an N-terminal inhibitory region.