Ras-related GTP-binding Proteins Research Articles

Tissue-specific expression differences in Ras-related GTP-binding proteins in male rats.

The protein kinase Mechanistic Target of Rapamycin (mTOR) in Complex 1 (mTORC1) is regulated in part by the Ras-related GTP-binding proteins (Rag GTPases). Rag GTPases form a heterodimeric complex consisting of either RagA or RagB associated with either RagC or RagD and act to localize mTORC1 to the lysosomal membrane. Until recently, RagA and RagB were thought to be functionally redundant, as were RagC and RagD. However, recent research suggests that the various isoforms differentially activate mTORC1. Here, the mRNA expression and protein abundance of the Rag GTPases was compared across male rat skeletal muscle, heart, liver, kidney, and brain. Whereas mRNA expression of RagA was higher than RagB in nearly all tissues studied, RagB protein abundance was higher than RagA in all tissues besides skeletal muscle. RagC mRNA expression was more abundant or equal to RagD mRNA, and RagD protein was more abundant than RagC protein in all tissues. Moreover, the proportion of RagB in the short isoform was greater than the long in liver, whereas the opposite was true in brain. These results serve to further elucidate Rag GTPase expression and offer potential explanations for the differential responses to amino acids that are observed in different tissues.

Insights into the complex interactions between Rab22a and extracellular vesicles in cancers.

Oncogenic Ras-related GTP-binding proteins, referred to as Rabs, are characterized by their intricate interactions with upstream, downstream molecules, and notably, extracellular vesicles (EVs). While the expansive family of Rabs and their associated signaling pathways have been exhaustively dissected, Rab22a emerges as an entity of outstanding interest, owing to its potent influence in many biological processes and its conspicuous correlation with cancer metastasis and migration. A burgeoning interest in the interactions between Rab22a and EVs in the field of oncology underscores the necessity for more in-depth reviews and scholarly discourses. We performed a review based on published original and review articles related to Rab22a, tumor, microRNA, exosome, microvesicles, EVs, CD147, lysosome, degradation, endosomal recycling, etc. from PubMed, Web of Science and Google Scholar databases. We summarize the regulatory processes governing the expression of Rab22a and the mutants of Rab22a. Notably, the present understanding of complex interactions between Rab22a and EVs are highlighted, encompassing both the impact of Rab22a on the genesis of EVs and the role of EVs that are affected by Rab22a mutants in propelling tumor advancement. The dynamic interaction between Rab22a and EVs plays a significant role in the progression of tumors, and it can provide novel insights into the pathogenesis of cancers and the development of new therapeutic targets.

Crucial roles of Rab22a in endosomal cargo recycling.

Endosomal cargo recycling lies at the heart of subcellular trafficking processes under the management of several Ras-related GTP-binding proteins (Rabs) which are coordinated by their upstream regulators and require their downstream effectors to display their functions. In this regard, several Rabs have been well-reviewed except Rab22a. Rab22a is a crucial regulator of vesicle trafficking, early endosome and recycling endosome formation. Notably, recent studies demonstrated the immunological roles of Rab22a, which are closely associated with cancers, infection and autoimmune disorders. This review provides an overview of the regulators and effectors of Rab22a. Also, we highlight the current knowledge of the role of Rab22a in endosomal cargo recycling, including the biogenesis of recycling tubules with the help of a complex with Rab22a at its core, and how different internalized cargo chooses different recycling routes thanks to the cooperation of Rab22a, its effectors and its regulators. Of note, contradictions and speculation related to endosomal cargo recycling that Rab22a brings impacts on are also discussed. Finally, this review endeavors to briefly introduce the various events impacted by Rab22a, particularly focusing on the commandeered Rab22a-associated endosomal maturation and endosomal cargo recycling, in addition to the extensively investigated oncogenic role of Rab22a.

Specific microRNA–mRNA Regulatory Network of Colon Cancer Invasion Mediated by Tissue Kallikrein–Related Peptidase 6

Metastatic colon cancer is a major cause of deaths among colorectal cancer (CRC) patients. Elevated expression of kallikrein 6 (KLK6), a member of a kallikrein subfamily of peptidase S1 family serine proteases, has been reported in CRC and is associated with low patient survival rates and poor disease prognosis. We knocked down KLK6 expression in HCT116 colon cancer cells to determine the significance of KLK6 expression for metastatic dissemination and to identify the KLK6-associated microRNAs (miRNAs) signaling networks in metastatic colon cancer. KLK6 suppression resulted in decreased cells invasion in vitro with a minimal effect on the cell growth and viability. In vivo, animals with orthotopic colon tumors deficient in KLK6 expression had the statistically significant increase in survival rates (P=.005) and decrease in incidence of distant metastases. We further performed the integrated miRNA and messenger RNA (mRNA) expression profiling to identify functional miRNA-mRNA interactions associated with KLK6-mediated invasiveness of colon cancer.Through bioinformatics analysis we identified and functionally validated the top two up-regulated miRNAs, miR-182 and miR-203, and one down-regulated miRNA, miRNA-181d, and their seven mRNA effectors. The established miRNA-mRNA interactions modulate cellular proliferation, differentiation and epithelial–mesenchymal transition (EMT) in KLK6-expressing colon cancer cells via the TGF-β signaling pathway and RAS-related GTP-binding proteins. We confirmed the potential tumor suppressive properties of miR-181d and miR-203 in KLK6-expressing HCT116 cells using Matrigel invasion assay. Our data provide experimental evidence that KLK6 controls metastasis formation in colon cancer via specific downstream network of miRNA-mRNA effectors.

Increased expression of ARF GTPases in prostate cancer tissue.

PurposeARFs are a family of Ras-related GTP binding proteins, ARF6, in particular, is implicated in cancer invasion and metastasis. However, the role of ARF proteins in prostate cancer have yet to be investigated.MethodsImmunohistochemical staining for ARF6 was performed on a prostate cancer tissue microarray with patient matched normal specimens.ResultsAntibody staining was significantly over-expressed in prostate cancer patient samples compared to normal patient tissue and a trend towards increased staining intensity in cancer samples with Gleason scores of 8 and above (metastatic disease).ConclusionDue to high homology between members of the ARF family we could not determine if ARF 6 was the only ARF over-expressed in the prostate cancer samples. However, we are the first to show that ARF-GTPases are over expressed in prostate cancer which provides further insight into the molecular biology of prostate cancer.

The GTPase TcRjl of the human pathogen Trypanosoma cruzi is involved in the cell growth and differentiation

The protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas Disease, undergoes through a complex life cycle where rounds of cell division and differentiation occur initially in the gut of triatominae vectors and, after transmission, inside of infected cells in vertebrate hosts. Members of the Ras superfamily of GTPases are molecular switches which play pivotal regulatory functions in cell growth and differentiation. We have previously described a novel GTPase in T. cruzi, TcRjl, which belongs to the RJL family of Ras-related GTP binding proteins. Here we show that most of TcRjl protein is found bound to GTP nucleotides and may be locked in this stage. In addition, we show that TcRjl is located close to the kinetoplast, in a region corresponding possibly to flagellar pocket of the parasite and the expression of a dominant-negative TcRjl construct (TcRjlS37N) displays a significative growth phenotype in reduced serum medium. Remarkably, overexpression of TcRjl inhibits differentiation of epimastigotes to trypomastigote forms and promotes the accumulation of intermediate differentiation stages. Our data suggest that TcRjl might play a role in the control of the parasite growth and differentiation.

Functional analysis of Rig, a novel Ras family small GTPase protein, in neurons

Starting with mRNA from field bean (Vicia faba) cotyledons, PCR fragments were obtained by using degenerate primers from conserved domains of ras-related GTP-binding proteins. These were used to isolate a number of clones from a field bean cotyledon cDNA-library. Homologs of rab1 (ypt1), rab2, rab11 (ypt3) and rab5 (ypt5) were identified. DNA sequencing yielded complete sequences of five clones named Vfa-ypt1, Vfa-ypt3a, Vfa-ypt3b, Vfa-ypt3x, and Vfa-ypt5. The deduced peptide sequences showed clear homology to the family of small GTP-binding proteins, including the effector loop region, the four conserved domains involved in GTP-binding, and the C-terminal cysteine-motif. Vfa-ypt3a and -3b are 99% and 100% identical to pea Pra6 and Pra5, respectively. Vfa-ypt3x is 60% identical to Ara1 and Ara4 from A. thaliana, Vfa-ypt1 is 93 % identical to Ara5 and 100 % identical to Pra9c from pea, and Vfa-ypt5 is 90% identical to Rhn1 from tobacco. RNA blotting showed a relatively low expression level of these clones, the highest level was observed with clone Vfa-ypt3 b. The five clones were expressed in bacteria and the products were shown to bind GTP. Antibodies were raised against synthetic peptides from variable regions and used to detect the protein expressed in bacteria as well as proteins in membrane preparations from different organs of field bean.

An evolutionarily conserved Rit GTPase–p38 MAPK signaling pathway mediates oxidative stress resistance

Ras-related small GTP-binding proteins control a wide range of cellular processes by regulating a variety of effector pathways, including prominent roles in the control of mitogen-activated protein kinase (MAPK) cascades. Although the regulatory role(s) for many Ras family GTPases are well established, the physiological function for the Rit/Rin subfamily has been lacking. Here, using both knockout mice and Drosophila models, we demonstrate an evolutionarily conserved role for Rit subfamily GTPases (mammalian Rit and Rin, and the Drosophila RIC homologue) in governing survival in response to oxidative stress. Primary embryonic fibroblasts derived from Rit knockout mice display increased apoptosis and selective disruption of MAPK signaling following reactive oxygen species (ROS) exposure but not in response to endoplasmic reticulum stress or DNA damage. These deficits include a reduction in ROS-mediated stimulation of a p38-MK2-HSP27 signaling cascade that controls Akt activation, directing Bad phosphorylation to promote cell survival. Furthermore, D-RIC null flies display increased susceptibility to environmental stresses and reduced stress-dependent p38 signaling, extending the Rit-p38 survival pathway to Drosophila. Together, our studies establish the Rit GTPases as critical regulators of an evolutionarily conserved, p38 MAPK-dependent signaling cascade that functions as an important survival mechanism for cells in response to oxidative stress.

An Early Response Transcription Factor, Egr-1, Enhances Insulin Resistance in Type 2 Diabetes with Chronic Hyperinsulinism

One of the most important characteristics of type 2 diabetes is insulin resistance, during which the patients normally experienced hyperinsulinism stress that would alter insulin signal transduction in insulin target tissues. We have previously found that early growth responsive gene-1 (Egr-1), a zinc finger transcription factor, is highly expressed in db/db mice and in the fat tissue of individuals with type 2 diabetes. In this report, we found that chronic exposure to hyperinsulinism caused persistent Erk/MAPK activity in adipocytes and enhanced insulin resistance in an Egr-1-dependent manner. An elevation in Egr-1 augmented Erk1/2 activation via geranylgeranyl diphosphate synthase (GGPPS). Egr-1-promoted GGPPS transcription increased Ras prenylation and caused Erk1/2 activation. The sustained activation of Erk1/2 resulted in the phosphorylation of insulin receptor substrate-1 at Serine 612. Phosphorylation at this site impaired insulin signaling in adipocytes and reduced glucose uptake. The loss of Egr-1 function, knockdown of GGPPS, or inhibition of Erk1/2 activity in insulin-resistant adipocytes restored insulin receptor substrate-1 tyrosine phosphorylation and increased insulin sensitivity. Our results suggest a new mechanism by which the Egr-1/GGPPS/Erk1/2 pathway is responsible for insulin resistance during hyperinsulinism. This pathway provides a new therapeutic target for increasing insulin sensitivity: inhibiting the function of Egr-1.

Differential Effect Of Zoledronic Acid On Quiescent Vs. Proliferating Vascular Smooth Muscle Cells

Introduction: Restenosis following arterial interventions is primarily due to phenotypic alteration vascular smooth-muscle cells. Zoledronic Acid (ZA), the most potent member of the nitrogen containing Bisphosphonates (BP), has proven to be effective in treating bone resorption and bone malignancies in humans. BP are known to exert their effects by inhibiting the post-translational modification of the Ras-related GTP binding proteins (e.g. Rap1A) by an isoprenoid lipid (prenylation) which are part of the mevalonate pathway. These experiments were designed to compare the relative sensitivity and the dose response of actively proliferating vs. quiescent cultured human vascular smooth cells to ZA treatment. Finally, to test whether ZA will modulate the prenylation of Rap-1A, an important mediator of multiple different cellular activities. Methods: Human aortic smooth muscle cells (HASMC) were cultured under growth condition until they reached ∼60% confluence. Cells were switched to quiescent media for 48 hours to achieve growth arrest. After which, cells were divided into two groups; proliferating cells group which was fed with growth media, or the quiescent cells group which continued to be fed with quiescent media. Both groups were supplemented with 0, 1, 10, 100μM ZA. Relative metabolic activity and cytotoxicity were measured by WST-1 and Cyto-tox fluor assays respectively at 48 hours. The effect of ZA on proliferating vs. quiescent HASMC migration was assessed by modified Boyden chamber. The relative prenylation of Rap-1A protein (index of mevolanate pathway activity) was evaluated by western blotting. Data was expressed as percent of the untreated control cells (0 μM ZA) from the same group. Results: Proliferating HASMC treated with 1μMZA did not differ in their metabolic activity or cytotoxicity index compared to untreated cells (Metabolic activity: p=0.99, Cytotoxicity: p=0.19). In contrast at 10 and 100μMZA treatment remarkably decreased metabolic activity (10μM p=0.03, 100μM p=0.03) and increased cytotoxicity in proliferating cells compared to quiescent cells (10μM p=0.007, 100μM p=0.03). The decreased metabolic activity and increased cytotoxicity correlated with the appearance of unprenylated Rap-1A protein. Further more ZA was more effective in inhibiting cellular migration in actively proliferating cells compared to quiescent cells at all ZA concentrations (1μM: p<0.002, 10μM: p<0.002, 100μM: p<0.002). Conclusions: These data suggest that ZA is more effective in inhibiting migration and promoting cytotoxicity in proliferating rather than quiescent HASMC. Since the expression of unprenylated RAP-1A correlated with inhibition of migration and increased cellular toxicity and reduced metabolic activity, this suggest that mevolante pathway may play a role in the inhibition of these crucial cellular functions. ZA might have a favorable therapeutic profile as a candidate drug to prevent intimal hyperplasia.

Rem GTPase interacts with the proximal CaV1.2 C-terminus and modulates calcium-dependent channel inactivation

The Rem, Rem2, Rad, and Gem/Kir (RGK) GTPases, comprise a subfamily of small Ras-related GTP-binding proteins, and have been shown to potently inhibit high voltage-activated Ca2+ channel current following overexpression. Although the molecular mechanisms underlying RGK-mediated Ca2+ channel regulation remains controversial, recent studies suggest that RGK proteins inhibit Ca2+ channel currents at the plasma membrane in part by interactions with accessory channel β subunits. In this paper, we extend our understanding of the molecular determinants required for RGK-mediated channel regulation by demonstrating a direct interaction between Rem and the proximal C-terminus of CaV1.2 (PCT), including the CB/IQ domain known to contribute to Ca2+/calmodulin (CaM)-mediated channel regulation. The Rem2 and Rad GTPases display similar patterns of PCT binding, suggesting that the CaV1.2 C-terminus represents a common binding partner for all RGK proteins. In vitro Rem:PCT binding is disrupted by Ca2+/CaM, and this effect is not due to Ca2+/CaM binding to the Rem C-terminus. In addition, co-overexpression of CaM partially relieves Rem-mediated L-type Ca2+ channel inhibition and slows the kinetics of Ca2+-dependent channel inactivation. Taken together, these results suggest that the association of Rem with the PCT represents a crucial molecular determinant in RGK-mediated Ca2+ channel regulation and that the physiological function of the RGK GTPases must be re-evaluated. Rather than serving as endogenous inhibitors of Ca2+ channel activity, these studies indicate that RGK proteins may play a more nuanced role, regulating Ca2+ currents via modulation of Ca2+/CaM-mediated channel inactivation kinetics.

P190RhoGAP and Rap-dependent RhoGAP (ARAP3) inactivate RhoA in response to nerve growth factor leading to neurite outgrowth from PC12 cells

Rat pheochromocytoma (PC12) cells have been used to investigate neurite outgrowth. Nerve growth factor (NGF) has been well known to induce neurite outgrowth from PC12 cells. RhoA belongs to Ras-related small GTP-binding proteins, which regulate a variety of cellular processes, including cell morphology alteration, actin dynamics, and cell migration. NGF suppressed GTP-RhoA levels after 12 h in PC12 cells and was consistently required for a long time to induce neurite outgrowth. Constitutively active (CA)-RhoA suppressed neurite outgrowth from PC12 cells in response to NGF, whereas dominant-negative (DN)-RhoA stimulated it, suggesting that RhoA inactivation is essential for neurite outgrowth. Here, we investigated the mechanism of RhoA inactivation. DN-p190RhoGAP abrogated neurite outgrowth, whereas wild-type (WT)-p190RhoGAP and WT-Src synergistically stimulated it along with accelerating RhoA inactivation, suggesting that p190RhoGAP, which can be activated by Src, is a major component in inhibiting RhoA in response to NGF in PC12 cells. Contrary to RhoA, Rap1 was activated by NGF, and DN-Rap1 suppressed neurite outgrowth, suggesting that Rap1 is also essential for neurite outgrowth. RhoA was co-immunoprecipitated with Rap1, suggesting that Rap1 interacts with RhoA. Furthermore, a DN-Rap-dependent RhoGAP (ARAP3) prevented RhoA inactivation, abolishing neurite formation from PC12 cells in response to NGF. These results suggest that NGF activates Rap1, which, in turn, up-regulates ARAP3 leading to RhoA inactivation and neurite outgrowth from PC12 cells. Taken together, p190RhoGAP and ARAP3 seem to be two main factors inhibiting RhoA activity during neurite outgrowth in PC12 cells in response to NGF.

A New Paradigm for Gem Regulation of Voltage-Gated Ca2+ Channels

The RGK (Rem, Rem2, Rad, Gem/Kir) family of Ras-related monomeric small GTP-binding proteins has emerged as potent inhibitors of high-voltage activated (HVA) Ca2+ channels. All RGK proteins bind all four subfamilies of HVA Ca2+ channel β subunits (Cavβs), and Cavβ is required for RGK-induced inhibition. Two modes of RGK action have been reported: (1) RGKs interfere with channel trafficking to the plasma membrane and hence reduce the number of surface channels; (2) RGKs inhibit channels already on the plasma membrane. It is generally believed that both forms of inhibition absolutely rely on the RGK/Cavβ interaction. However, this central hypothesis has not been tested directly. We investigated the molecular mechanism of Gem inhibition of P/Q-type Ca2+ channels expressed in Xenopus oocytes and HEK 293T cells. Gem inhibited P/Q channels without affecting their surface expression. Application of a purified Gem protein domain in inside-out membrane patches acutely inhibited P/Q channels. This acute inhibition was completely abolished when Cavβ was removed from surface P/Q channels, but it was fully restored after the channels regain Cavβ. These results unequivocally demonstrate that Cavβ is indispensable for Gem inhibition of surface P/Q channels. Surprisingly, however, complete disruption of the Gem/Cavβ interaction, as shown biochemically, did not affect Gem inhibition. On the other hand, we discovered that Gem associated with Cav2.1 in a Cavβ-independent manner. Finally, we identified a 12-amino acid region in the C-terminus of Gem that was sufficient to produce inhibition in inside-out patches and another site in the core region of Gem that was also involved in Gem inhibition. Based on these findings, we propose that Gem directly binds and inhibits CaVβ-primed HVA Ca2+ channels on the plasma membrane.

The RJL family of small GTPases is an ancient eukaryotic invention probably functionally associated with the flagellar apparatus

A patchily distributed gene family is often taken as evidence for horizontal gene transfer (HGT) events, but it may also result solely from multiple gene losses. The RJL family of uncharacterised Ras-like GTPases was previously suggested to have undergone HGT events between protists and deuterostome metazoans, owing to the apparent absence of RJL in intermediate groups (Nepomuceno-Silva, J.L., de Melo, L.D., Mendonca, S.M., Paixao, J.C., Lopes, U.G., 2004. RJLs: a new family of Ras-related GTP-binding proteins. Gene 327, 221–232). We have reanalysed the phylogenetic distribution and phylogeny of the RJL family, taking advantage of the recent expansion of sequence data available from diverse eukaryotes. We found that RJL orthologs are much more widely distributed than previously assumed. At least one representative encoding an RJL protein could be identified for each of the six major eukaryotic “supergroups” (Opisthokonta, Amoebozoa, Excavata, Archaeplastida, Chromalveolata, and Rhizaria) as well as for a species of Apusomonadida, a deep lineage that may not be specifically related to any of the recognized supergroups. Phylogenetic analyses do not support HGT of RJL genes between the major eukaryotic lineages, indicating that the RJL family was present in the last eukaryotic common ancestor and was lost several times over the course of eukaryotic evolution. Interestingly, RJL was lost from all taxa lacking flagellated cells and from a few lineages that build structurally unusual or reduced flagella, raising the intriguing possibility that RJL proteins are functionally associated with the flagellar apparatus. The RJL GTPase domain has been fused with the DnaJ domain on two separate occasions: in the Holozoa (before the split of Metazoa and choanoflagellates), giving rise to the previously known Rbj type of RJL with the DnaJ domain at the C-terminus, and independently in Alveolata resulting in novel proteins with the DnaJ domain at the N-terminus. These independent fusions suggest that RJL proteins may generally function via regulating the DnaJ–Hsp70 module.

Rem Inhibits Skeletal Muscle EC Coupling by Reducing the Number of Functional L-Type Ca2+ Channels

In skeletal muscle, the L-type voltage-gated Ca2+ channel (1,4-dihydropyridine receptor) serves as the voltage sensor for excitation-contraction (EC) coupling. In this study, we examined the effects of Rem, a member of the RGK (R_em,R_em2,R_ad,R_em/R_ir) family of Ras-related monomeric GTP-binding proteins, on the function of the skeletal muscle L-type Ca2+ channel. EC coupling was found to be weakened in myotubes expressing Rem tagged with enhanced yellow fluorescent protein (YFP-Rem), as assayed by electrically evoked contractions and myoplasmic Ca2+ transients. This impaired EC coupling was not a consequence of altered function of the type 1 ryanodine receptor, or of reduced Ca2+ stores, since the application of 4-chloro-m-cresol, a direct type 1 ryanodine receptor activator, elicited myoplasmic Ca2+ release in YFP-Rem-expressing myotubes that was not distinguishable from that in control myotubes. However, YFP-Rem reduced the magnitude of L-type Ca2+ current by ∼75% and produced a concomitant reduction in membrane-bound charge movements. Thus, our results indicate that Rem negatively regulates skeletal muscle EC coupling by reducing the number of functional L-type Ca2+ channels in the plasma membrane.

New Insights into the Role of Conserved, Essential Residues in the GTP Binding/GTP Hydrolytic Cycle of Large G Proteins

The GTP hydrolytic (GTPase) reaction terminates signaling by both large (heterotrimeric) and small (Ras-related) GTP-binding proteins (G proteins). Two residues that are necessary for GTPase activity are an arginine (often called the "arginine finger") found either in the Switch I domains of the alpha subunits of large G proteins or contributed by the GTPase-activating proteins of small G proteins, and a glutamine that is highly conserved in the Switch II domains of Galpha subunits and small G proteins. However, questions still exist regarding the mechanism of the GTPase reaction and the exact role played by the Switch II glutamine. Here, we have characterized the GTP binding and GTPase activities of mutants in which the essential arginine or glutamine residue has been changed within the background of a Galpha chimera (designated alpha(T)*), comprised mainly of the alpha subunit of retinal transducin (alpha(T)) and the Switch III region from the alpha subunit of G(i1). As expected, both the alpha(T)*(R174C) and alpha(T)*(Q200L) mutants exhibited severely compromised GTPase activity. Neither mutant was capable of responding to aluminum fluoride when monitoring changes in the fluorescence of Trp-207 in Switch II, although both stimulated effector activity in the absence of rhodopsin and Gbetagamma. Surprisingly, each mutant also showed some capability for being activated by rhodopsin and Gbetagamma to undergo GDP-[(35)S]GTPgammaS exchange. The ability of the mutants to couple to rhodopsin was not consistent with the assumption that they contained only bound GTP, prompting us to examine their nucleotide-bound states following their expression and purification from Escherichia coli. Indeed, both mutants contained bound GDP as well as GTP, with 35-45% of each mutant being isolated as GDP-P(i) complexes. Overall, these findings suggest that the R174C and Q200L mutations reveal Galpha subunit states that occur subsequent to GTP hydrolysis but are still capable of fully stimulating effector activity.

The regulation of mDia1 by autoinhibition and its release by Rho•GTP

Formins induce the nucleation and polymerisation of unbranched actin filaments via the formin-homology domains 1 and 2. Diaphanous-related formins (Drfs) are regulated by a RhoGTPase-binding domain situated in the amino-terminal (N-terminal) region and a carboxy-terminal Diaphanous-autoregulatory domain (DAD), whose interaction stabilises an autoinhibited inactive conformation. Binding of active Rho releases DAD and activates the catalytic activity of mDia. Here, we report on the interaction of DAD with the regulatory N-terminus of mDia1 (mDia(N)) and its release by Rho*GTP. We have defined the elements required for tight binding and solved the three-dimensional structure of a complex between an mDia(N) construct and DAD by X-ray crystallography. The core DAD region is an alpha-helical peptide, which binds in the most highly conserved region of mDia(N) using mainly hydrophobic interactions. The structure suggests a two-step mechanism for release of autoinhibition whereby Rho*GTP, although having a partially nonoverlapping binding site, displaces DAD by ionic repulsion and steric clashes. We show that Rho*GTP accelerates the dissociation of DAD from the mDia(N)*DAD complex.

Rin GTPase Couples Nerve Growth Factor Signaling to p38 and b-Raf/ERK Pathways to Promote Neuronal Differentiation

In neuronal precursor cells, the magnitude and longevity of mitogen-activated protein (MAP) kinase cascade activation contribute to the nature of the cellular response, differentiation, or proliferation. However, the mechanisms by which neurotrophins promote prolonged MAP kinase signaling are not well understood. Here we defined the Rin GTPase as a novel component of the regulatory machinery contributing to the selective integration of MAP kinase signaling and neuronal development. Rin is expressed exclusively in neurons and is activated by neurotrophin signaling, and loss-of-function analysis demonstrates that Rin makes an essential contribution to nerve growth factor (NGF)-mediated neuronal differentiation. Most surprisingly, although Rin was unable to stimulate MAP kinase activity in NIH 3T3 cells, it potently activated isoform-specific p38alpha MAP kinase signaling and weakly stimulated ERK signaling in pheochromocytoma (PC6) cells. This cell-type specificity is explained in part by the finding that Rin binds and stimulates b-Raf but does not activate c-Raf. Accordingly, selective down-regulation of Rin in PC6 cells suppressed neurotrophin-elicited activation of b-Raf and p38, without obvious effects on NGF-induced ERK activation. Moreover, the ability of NGF to promote neurite outgrowth was inhibited by Rin knockdown. Together, these observations establish Rin as a neuronal specific regulator of neurotrophin signaling, required to couple NGF stimulation to sustain activation of p38 MAP kinase and b-Raf signaling cascades required for neuronal development.

The RGK family: a regulatory tail of small GTP-binding proteins

RGK proteins are small Ras-related GTP-binding proteins that function as potent inhibitors of voltage-dependent calcium channels, and two members of the family, Gem and Rad, modulate Rho-dependent remodeling of the cytoskeleton. Within the Ras superfamily, RGK proteins have distinct structural and regulatory characteristics. It is an open question as to whether RGK proteins catalyze GTP hydrolysis in vivo. Binding of calmodulin and the 14-3-3 protein to RGK proteins controls downstream pathways. Here, we discuss the structural and functional properties of RGK proteins and highlight recent work by Beguin and colleagues addressing the mechanism of Gem regulation by calmodulin and 14-3-3.

Localization and function of Arf family GTPases

Arfs are a family of Ras-related GTP-binding proteins that function in the regulation of membrane trafficking and structure. The six mammalian Arf proteins are expressed ubiquitously and so it is anticipated that each will have a distinct localization and function within the cell. It has been assumed that much of this specificity will be defined by determining which regulators of Arfs, the GEFs (guanine nucleotide-exchange factors) and GAPs (GTPase-activating proteins) function with which Arf proteins. Although in vitro assays may indicate Arf preferences for the numerous Arf GEFs and GAPs that have been identified, in the cell the different Arfs, GEFs and GAPs are targeted to specific compartments where they carry out their functions. We have embarked on studies to define regions of the Arf1 and Arf6 proteins that determine their sites of action and specific activities at the Golgi and plasma membrane respectively. Chimaeras were made between Arf1 and Arf6 in order to identify regions of the protein that contributed to targeting and function. Whereas Arf6 is targeted to the plasma membrane through multiple regions along the protein, we have found a Golgi-targeting region in Arf1 that is sufficient to target Arf6 to the Golgi complex.