Ras-like GTPase Research Articles

Rag GTPases are cardioprotective by regulating lysosomal function.

The Rag family proteins are Ras-like small GTPases that play a critical role in amino acid-stimulated mTORC1 activation by recruiting mTORC1 to lysosome. Despite progress in the mechanistic understanding of Rag GTPases in mTORC1 activation, little is known about the physiological function of Rag GTPases in vivo. Here, we show that loss of RagA and RagB (RagA/B) in cardiomyocytes results in hypertrophic cardiomyopathy and phenocopies lysosomal storage diseases although mTORC1 activity is not substantially impaired in vivo. We demonstrate that despite upregulation of lysosomal protein expression by constitutive activation of the transcription factor EB (TFEB) in RagA/B knockout mouse embryonic fibroblasts, lysosomal acidification is compromised due to decreased v-ATPase level in the lysosome fraction. Our study uncovers RagA/B GTPases as key regulators of lysosomal function and cardiac protection.

Amino acid residues required for Gtr1p‐Gtr2p complex formation and its interactions with the Ego1p‐Ego3p complex and TORC1 components in yeast

The yeast Ras-like GTPases Gtr1p and Gtr2p form a heterodimer, are implicated in the regulation of TOR complex 1 (TORC1) and play pivotal roles in cell growth. Gtr1p and Gtr2p bind Ego1p and Ego3p, which are tethered to the endosomal and vacuolar membranes where TORC1 functions are regulated through a relay of amino acid signaling interactions. The mechanisms by which Gtr1p and Gtr2p activate TORC1 remain obscure. We probed the interactions of the Gtr1p-Gtr2p complex with the Ego1p-Ego3p complex and TORC1 subunits. Mutations in the region (179-220 a.a.) following the nucleotide-binding region of Gtr1p and Gtr2p abrogated their mutual interaction and resulted in a loss in function, suggesting that complex formation between Gtr1p and Gtr2p was indispensable for TORC1 function. A modified yeast two-hybrid assay showed that Gtr1p-Gtr2p complex formation is important for its interaction with the Ego1p-Ego3p complex. GTP-bound Gtr1p interacted with the region containing the HEAT repeats of Kog1p and the C-terminal region of Tco89p. The GTP-bound Gtr2p suppressed a Kog1p mutation. Our findings indicate that the interactions of the Gtr1p-Gtr2p complex with the Ego1p-Ego3p complex and TORC1 components Kog1p and Tco89p play a role in TORC1 function.

Regulation of Bacterial Cell Polarity by Small GTPases

Bacteria are polarized with many proteins localizing dynamically to specific subcellular sites. Two GTPase families have important functions in the regulation of bacterial cell polarity, FlhF homologues and small GTPases of the Ras superfamily. The latter consist of only a G domain and are widespread in bacteria. The rod-shaped Myxococcus xanthus cells have two motility systems, one for gliding and one that depends on type IV pili. The function of both systems hinges on proteins that localize asymmetrically to the cell poles. During cellular reversals, these asymmetrically localized proteins are released from their respective poles and then bind to the opposite pole, resulting in an inversion of cell polarity. Here, we review genetic, cell biological, and biochemical analyses that identified two modules containing small Ras-like GTPases that regulate the dynamic polarity of motility proteins. The GTPase SofG interacts directly with the bactofilin cytoskeletal protein BacP to ensure polar localization of type IV pili proteins. In the second module, the small GTPase MglA, its cognate GTPase activating protein (GAP) MglB, and the response regulator RomR localize asymmetrically to the poles and sort dynamically localized motility proteins to the poles. During reversals, MglA, MglB, and RomR switch poles, in that way inducing the relocation of dynamically localized motility proteins. Structural analyses have demonstrated that MglB has a Roadblock/LC7 fold, the central β2 strand in MglA undergoes an unusual screw-type movement upon GTP binding, MglA contains an intrinsic Arg finger required for GTP hydrolysis, and MglA and MglB form an unusual G protein/GAP complex with a 1:2 stoichiometry.

Prediction of the Repeat Domain Structures and Impact of Parkinsonism-Associated Variations on Structure and Function of all Functional Domains of Leucine-Rich Repeat Kinase 2 (LRRK2)

Genetic variations of leucine-rich repeat kinase 2 (LRRK2) are the major cause of dominantly inherited Parkinson disease (PD). LRRK2 protein contains seven predicted domains: a tandem Ras-like GTPase (ROC) domain and C-terminal of Roc (COR) domain, a protein kinase domain, and four repeat domains. PD-causative variations arise in all domains, suggesting that aberrant functioning of any domain can contribute to neurotoxic mechanisms of LRRK2. Determination of the three-dimensional structure of LRRK2 is one of the best avenues to decipher its neurotoxic mechanism. However, with the exception of the Roc domain, the three-dimensional structures of the functional domains of LRRK2 have yet to be determined. Based on the known three-dimensional structures of repeat domains of other proteins, the tandem Roc-COR domains of the Chlorobium tepidum Rab family protein, and the kinase domain of the Dictyostelium discoideum Roco4 protein, we predicted (1) the motifs essential for protein-protein interactions in all domains, (2) the motifs critical for catalysis and substrate recognition in the tandem Roc-COR and kinase domains, and (3) the effects of some PD-associated missense variations on the neurotoxic action of LRRK2. Results of our analysis provide a conceptual framework for future investigation into the regulation and the neurotoxic mechanism of LRRK2.

Regulation of autophagy by the Rab GTPase network

Autophagy (macroautophagy) is a highly conserved intracellular and lysosome-dependent degradation process in which autophagic substrates are enclosed and degraded by a double-membrane vesicular structure in a continuous and dynamic vesicle transport process. The Rab protein is a small GTPase that belongs to the Ras-like GTPase superfamily and regulates the vesicle traffic process. Numerous Rab proteins have been shown to be involved in various stages of autophagy. Rab1, Rab5, Rab7, Rab9A, Rab11, Rab23, Rab32, and Rab33B participate in autophagosome formation, whereas Rab9 is required in non-canonical autophagy. Rab7, Rab8B, and Rab24 have a key role in autophagosome maturation. Rab8A and Rab25 are also involved in autophagy, but their role is unknown. Here, we summarize new findings regarding the involvement of Rabs in autophagy and provide insights regarding future research on the mechanisms of autophagy regulation.

RalGTPase Controls Cell Polarity Organization during Epithelial Tissue Remodeling

Ral, a Ras-like GTPase, is an essential component of Ras-driven oncogenesis that supports tumor initiation and progression. We explored the role of Ral in the polarity organization of epithelial tissue in Drosophila. We found that Ral is dispensable for the polarization of proliferative epithelial tissue, and that Ral is required for the maintenance of apical-basal polarity of post-mitotic epithelial cells during tissue remodeling. More precisely, lack of Ral activity results in loss of cortical apical aPKC and lateral Lgl, and in apical spreading and accumulation of Armadillo/betacatenin. Our analysis demonstrates that Ral regulates polarity organization by acting as a negative regulator of JNK and p38 MAPK signaling. Ral controls aPKC apical localization by down-regulating JNK and controls Armadillo localization at adherens junction by down-regulating p38 MAPK signaling, whereas Lgl membrane localization depends on both pathways. Finally, in the absence of Ral function aPKC becomes dispensable for Lgl basolateral localization and Armadillo is recruited to the cell membrane independently of DE-Cadherin and Bazooka localization. This suggests that additional mechanisms control Lgl and Arm distribution in polarized epithelia.

Activation Cycle of GαS Protein Elucidated by Elastic Network Based Simulation

Gαs, a subunit of the heterotrimeric G protein, has a crucial role in mediating the signal transduction from G protein coupled-receptor (GPCR) into intracellular proteins. This cascade process is accompanied by conformational change of Gαs protein such as binding to β2AR as well as contributing to GDP/GTP exchange. We investigated dynamic features of Gαs based on the X-ray crystal structure of β2AR-Gαs complex and elastic network based simulation was performed to reveal the whole conformational change pathway of β2AR-Gαs complex. The normal mode results strongly propose that N terminus of Gαs initially binds to the activated β2AR and then C terminus binding is followed. These two binding events would break the densely connected GDP pocket site located between two Gαs subdomains (Ras-like GTPase and α-helical domain), leading to the increase of the GDP solvent-accessible surface area. We also identified a flexible hinge point of switch 1 linker region from the proposed large swing motion of Gαs between closed and open conformation. B-factor comparison between GDP and GTP binding to Gαs indicates that GTP binding much more suppresses the mobility of both switch 1 and switch 2 regions, while both N and C termini vibrate more actively. It facilitates reorientation of Gαs to its initial closed form in order to complete the entire cycle of conformational change. This atomistic simulation not only confirms various experimental observations regarding β2AR-Gαs complex but also newly reveals the binding affinity of Gαs termini for β2AR.

Cardiomyocyte Endpoints Distinguish Non-Canonical Regulation of the RGK Gtpases Rad and Rem

Rad/Rem/Rem2/Gem/Kir (RGK) proteins are Ras-like GTPases with diverse (and expanding) functions including: regulating cytoskeleton dynamics, cell proliferation, synaptogenesis, and inhibiting voltage-dependent calcium (CaV) channels. Whether RGKs are canonical GTP-regulated ON-OFF switches similar to Ras is ambiguous. Here, we used a range of functional measurements in cardiomyocytes (CaV1.2 currents, Ca2+ transients, CaVβ binding) as biosensors to probe nucleotide regulation of Rad and Rem. We utilized as tools RadS105N and RemT94N, two mutants analogous to RasS17N which is a Ras variant that displays a strongly decreased GTP binding affinity. As a reference for expected canonical behavior, RasS17N loses effector binding, but acts as a dominant negative in situ due to increased avidity for guanine nucleotide exchange factor. Adenovirus mediated expression of either wild type (wt) Rad or RadS105N in cardiomyocytes dramatically blocked endogenous ICa,L, contradicting reports that RadS105N acts as a dominant negative in heart. Both Rad and RadS105N diminished cardiac EC coupling as reported by the amplitude of rhod-2-reported Ca2+ transients. However, RadS105N increased the propensity for cardiac arrhythmias compared to wt Rad, hinting at a functional difference. By contrast with Rad/RadS105N, RemT94N was significantly less effective than wt Rem in inhibiting either ICa,L or Ca2+ transients, revealing a functional dichotomy between the two RGKs. FRET analyses of binding interactions between auxiliary CaVβs and RGKs revealed a reduced binding affinity for RadS105N and RemT94N relative to their wt counterparts. Finally, proteomic analyses of Rem and RemT94N interactomes in cardiomyocytes displayed subtle differences in binding partners. The results indicate that while Rad and Rem are non-canonical GTPases, their GTP-binding status can be sensed to yield a spectrum of functions in an isoform-dependent manner.

Mammalian Target of Rapamycin Complex I (mTORC1) Activity in Ras Homologue Enriched in Brain (Rheb)-Deficient Mouse Embryonic Fibroblasts

The Ras-like GTPase Rheb has been identified as a crucial activator of mTORC1. Activation most likely requires a direct interaction between Rheb and mTOR, but the exact mechanism remains unclear. Using a panel of Rheb-deficient mouse embryonic fibroblasts (MEFs), we show that Rheb is indeed essential for the rapid increase of mTORC1 activity following stimulation with insulin or amino acids. However, mTORC1 activity is less severely reduced in Rheb-deficient MEFs in the continuous presence of serum or upon stimulation with serum. This remaining mTORC1 activity is blocked by depleting the cells for amino acids or imposing energy stress. In addition, MEK inhibitors and the RSK-inhibitor BI-D1870 interfere in mTORC1 activity, suggesting that RSK acts as a bypass for Rheb in activating mTORC1. Finally, we show that this rapamycin-sensitive, Rheb-independent mTORC1 activity is important for cell cycle progression. In conclusion, whereas rapid adaptation in mTORC1 activity requires Rheb, a second Rheb-independent activation mechanism exists that contributes to cell cycle progression.

The Small Gtpase Rap1b Negatively Regulates Neutrophil Migration During Inflammation By Limiting Trans-Cellular Diapedesis

Neutrophils are the first line of cellular defense against infecting microorganisms by moving rapidly toward sites of infection. Impaired neutrophil recruitment and functions can cause life-threatening infections, while excessive neutrophil tissue infiltration contributes to inflammatory disorders and tissue injury. A number of key positive regulators of neutrophil tissue infiltration have been identified. However, the mechanisms that protect from unwanted inflammation by negative regulation of neutrophil recruitment and functions are still unrecognized.Rap1b is an evolutionary conserved protein of the Ras-like GTPase superfamily. The mammalian genome encodes two Rap1 genes, Rap1a and Rap1b, which are highly homologous. Rap1 is historically known to control functional activation of integrins to positively regulate a number of cellular processes, including cell adhesion, cell polarity, cell migration, platelet aggregation. Rap1b is the main Rap1 isoform expressed in neutrophils; yet, its functions in neutrophils are poorly understood.Here, we found that, quite unexpectedly, Rap1b is a key suppressor of neutrophil migration, and inflammation. Rap1b loss enhanced neutrophil emigration into lungs, associated with increased susceptibility to endotoxin shock. To further understand the role of Rap1b in neutrophil migration, we used a 3-D migration assay in which neutrophils are plated onto activated endothelial cells. This assay enables examination of critical steps of the extravasation cascade, ie neutrophil adhesion onto the endothelium, lateral crawling to the nearest endothelial cell junction and permissive sites for transmigration. This assay confirmed increased transendothelial migration of Rap1b-/- neutrophils compared to WT cells. However, Rap1b-/- neutrophils were unable to reach endothelial junction and shifted their mode of transmigration to a trans-cellular (through endothelial cells) diapedesis instead of the canonical paracellular route (between two endothelial cells). Indeed, using immunostaining with VE-Cadherin, ICAM-1 and CD11b to identify endothelial junctions and neutrophils, respectively, we found only 5-10% WT neutrophils used the transcellular route of diapedesis. Up to 30-35% Rap1b–/– neutrophils transmigrated via the transcellular route. Transcellular diapedesis requires the formation of invadopodia-like actin protrusions, an extracellular matrix-degrading structure enabling penetration into tissue. Transmission electron microscopy indicated increased invadopodia-like structures in Rap1b–/– neutrophils that penetrated deeper into endothelial surfaces than WT cells. Likewise, Rap1b–/– neutrophils had increased ability to degrade gelatin matrix in vitro. Mechanistically, the Rap1b-null phenotype was mediated by enhanced Pi3K-Akt activation. Rap1b-/- neutrophils manifested increased phosphorylation of Akt, in response to chemokine and integrin stimuli. Treatment of a small molecule Akt inhibitor, MK-2206 reversed elevated transmigration, matrix degradation and rescued crawling of Rap1b–/– neutrophils to endothelial junctions. Importantly, Akt inhibition in vivo suppressed excessive Rap1b–/– neutrophil migration into lungs and associated endotoxin shock. The inhibitory action of Rap1b on PI3K signaling may be mediated by the phosphatase SHP-1, as its activation and localization was Rap1b-dependent. These findings uncover a novel mechanism of neutrophil migration, and reveal an unexpected role for Rap1b as a key suppressor of neutrophilic lung inflammation. This work has far reaching importance for inflammatory processes. It may represent new avenues for the treatment of pathological inflammation conditions. Disclosures:No relevant conflicts of interest to declare.

The deubiquitylase USP33 discriminates between RALB functions in autophagy and innate immune response

The RAS-like GTPase RALB mediates cellular responses to nutrient availability or viral infection by respectively engaging two components of the exocyst complex, EXO84 and SEC5. RALB employs SEC5 to trigger innate immunity signalling, whereas RALB-EXO84 interaction induces autophagocytosis. How this differential interaction is achieved molecularly by the RAL GTPase remains unknown. We found that whereas GTP binding turns on RALB activity, ubiquitylation of RALB at Lys47 tunes its activity towards a particular effector. Specifically, ubiquitylation at Lys47 sterically inhibits RALB binding to EXO84, while facilitating its interaction with SEC5. Double-stranded RNA promotes RALB ubiquitylation and SEC5-TBK1 complex formation. In contrast, nutrient starvation induces RALB deubiquitylation by accumulation and relocalization of the deubiquitylase USP33 to RALB-positive vesicles. Deubiquitylated RALB promotes the assembly of the RALB-EXO84-beclin-1 complexes driving autophagosome formation. Thus, ubiquitylation within the effector-binding domain provides the switch for the dual functions of RALB in autophagy and innate immune responses.

A Loss-Of-Function Analysis Reveals That Endogenous Rem2 Promotes Functional Glutamatergic Synapse Formation and Restricts Dendritic Complexity

Rem2 is a member of the RGK family of small Ras-like GTPases whose expression and function is regulated by neuronal activity in the brain. A number of questions still remain as to the endogenous functions of Rem2 in neurons. RNAi-mediated Rem2 knockdown leads to an increase in dendritic complexity and a decrease in functional excitatory synapses, though a recent report challenged the specificity of Rem2-targeted RNAi reagents. In addition, overexpression in a number of cell types has shown that Rem2 can inhibit voltage-gated calcium channel (VGCC) function, while studies employing RNAi-mediated knockdown of Rem2 have failed to observe a corresponding enhancement of VGCC function. To further investigate these discrepancies and determine the endogenous function of Rem2, we took a comprehensive, loss-of-function approach utilizing two independent, validated Rem2-targeted shRNAs to analyze Rem2 function. We sought to investigate the consequence of endogenous Rem2 knockdown by focusing on the three reported functions of Rem2 in neurons: regulation of synapse formation, dendritic morphology, and voltage-gated calcium channels. We conclude that endogenous Rem2 is a positive regulator of functional, excitatory synapse development and a negative regulator of dendritic complexity. In addition, while we are unable to reach a definitive conclusion as to whether the regulation of VGCCs is an endogenous function of Rem2, our study reports important data regarding RNAi reagents for use in future investigation of this issue.

MiR-21 coordinates tumor growth and modulates KRIT1 levels

miR-21 is overexpressed in tumors and it displays oncogenic activity. Here, we show that expression of miR-21 in primary tumors anticorrelates with KRIT1/CCM1, an interacting partner of the Ras-like GTPase Rap1, involved in Cerebral Cavernous Malformations (CCM). We present evidences that miR-21 silences KRIT1 by targeting its mRNA 3′UTR and that this interaction is involved in tumor growth control. In fact, miR-21 over-expression or KRIT1 knock-down promote anchorage independent tumor cell growth compared to controls, whereas the opposite is observed when anti-miR-21 or KRIT1 overexpression are employed. Our findings suggest that miR-21 promotes tumor cell growth, at least in part, by down-modulating the potential tumor suppressor KRIT1.

The Small GTPase RALA Controls c-Jun N-terminal Kinase-mediated FOXO Activation by Regulation of a JIP1 Scaffold Complex

FOXO (forkhead box O) transcription factors are tumor suppressors and increase the life spans of model organisms. Cellular stress, in particular oxidative stress caused by an increase in levels of reactive oxygen species (ROS), activates FOXOs through JNK-mediated phosphorylation. Importantly, JNK regulation of FOXO is evolutionarily conserved. Here we identified the pathway that mediates ROS-induced JNK-dependent FOXO regulation. Following increased ROS, RALA is activated by the exchange factor RLF (RalGDS-like factor), which is in complex with JIP1 (C-Jun-amino-terminal-interacting protein 1) and JNK. Active RALA consequently regulates assembly and activation of MLK3, MKK4, and JNK onto the JIP1 scaffold. Furthermore, regulation of FOXO by RALA and JIP1 is conserved in C. elegans, where both ral-1 and jip-1 depletion impairs heat shock-induced nuclear translocation of the FOXO orthologue DAF16.

The tRNA-modifying function of MnmE is controlled by post-hydrolysis steps of its GTPase cycle

MnmE is a homodimeric multi-domain GTPase involved in tRNA modification. This protein differs from Ras-like GTPases in its low affinity for guanine nucleotides and mechanism of activation, which occurs by a cis, nucleotide- and potassium-dependent dimerization of its G-domains. Moreover, MnmE requires GTP hydrolysis to be functionally active. However, how GTP hydrolysis drives tRNA modification and how the MnmE GTPase cycle is regulated remains unresolved. Here, the kinetics of the MnmE GTPase cycle was studied under single-turnover conditions using stopped- and quench-flow techniques. We found that the G-domain dissociation is the rate-limiting step of the overall reaction. Mutational analysis and fast kinetics assays revealed that GTP hydrolysis, G-domain dissociation and Pi release can be uncoupled and that G-domain dissociation is directly responsible for the ‘ON’ state of MnmE. Thus, MnmE provides a new paradigm of how the ON/OFF cycling of GTPases may regulate a cellular process. We also demonstrate that the MnmE GTPase cycle is negatively controlled by the reaction products GDP and Pi. This feedback mechanism may prevent inefficacious GTP hydrolysis in vivo. We propose a biological model whereby a conformational change triggered by tRNA binding is required to remove product inhibition and initiate a new GTPase/tRNA-modification cycle.

Downregulation of the Novel Tumor Suppressor DIRAS1 Predicts Poor Prognosis in Esophageal Squamous Cell Carcinoma

Loss of chromosome 19p is one of the most frequent allelic imbalances in esophageal squamous cell carcinoma (ESCC), suggesting the existence of one or more tumor suppressor genes within this region. In this study, we investigated a role in ESCCs for a candidate tumor suppressor gene located at 19p13.3, the Ras-like small GTPase DIRAS1. Downregulation of DIRAS1 occurred in approximately 50% of primary ESCCs where it was associated significantly with advanced clinical stage, lymph node metastasis, and poor overall survival. LOH and promoter methylation analyses suggested that loss of DIRAS1 expression was mediated by epigenetic mechanisms. Functional studies established that ectopic re-expression of DIRAS1 in ESCC cells inhibited cell proliferation, clonogenicity, cell motility, and tumor formation. Mechanistic investigations suggested that DIRAS1 acted through extracellular signal-regulated kinase (ERK1/2; MAPK3/1) and p38 mitogen-activated protein kinase (MAPK; MAPK14) signaling to trigger BAD Ser112 dephosphorylation and matrix metalloproteinase (MMP)2/9 transcriptional inactivation to promote apoptosis and inhibit metastasis, respectively. Taken together, our results revealed that DIRAS1 has a pivotal function in ESCC pathogenesis, with possible use as a biomarker and intervention point for new therapeutic strategies.

Role of guanine-nucleotide exchange factor Epac in renal physiology and pathophysiology.

Exchange proteins directly activated by cAMP [Epac(s)] were discovered more than a decade ago as new sensors for the second messenger cAMP. The Epac family members, including Epac1 and Epac2, are guanine nucleotide exchange factors for the Ras-like small GTPases Rap1 and Rap2, and they function independently of protein kinase A. Given the importance of cAMP in kidney homeostasis, several molecular and cellular studies using specific Epac agonists have analyzed the role and regulation of Epac proteins in renal physiology and pathophysiology. The specificity of the functions of Epac proteins may depend upon their expression and localization in the kidney as well as their abundance in the microcellular environment. This review discusses recent literature data concerning the involvement of Epac in renal tubular transport physiology and renal glomerular cells where various signaling pathways are known to be operative. In addition, the potential role of Epac in kidney disorders, such as diabetic kidney disease and ischemic kidney injury, is discussed.

C. elegans Rassf homolog, rasf‐1, is functionally associated with rab‐39 Rab GTPase in oxidative stress response

The Ras association domain family (Rassf) is one of the Ras effectors, which can bind to several GTP-charged Ras-like GTPases. The Rassf proteins are widely conserved beyond species from nematode to human. To explore the novel functions of Rassf proteins, we took advantage of nematode C. elegans as a model animal with only one Rassf homolog, T24F1.3 (rasf-1). The rasf-1-mutant as well as rasf-1-knockdown animals were found to be more sensitive to oxidative stress of arsenite than in wild type, indicating that rasf-1 is involved in oxidative stress response. We next screened for proteins that interact with RASF-1 by the yeast two-hybrid system and identified RAB-39 Rab GTPase as an interacting partner of RASF-1. We not only confirmed specific binding between these molecules but also demonstrated that RASF-1 binds to GTP-bound form but not GDP-bound form of RAB-39. Importantly, rab-39 mutant animals were also sensitive to oxidative stress, which was dependent on rasf-1 according to the epistasis analysis. Moreover, Rassf1 and Rab39, mammalian homologs of rasf-1 and rab-39, respectively, were shown to interact with each other in vitro. These results indicate that the RASF-1 functionally interacts with RAB-39 and that the interaction between their homologs is conserved in mammals.

RAC1P29S is a spontaneously activating cancer-associated GTPase.

RAC1 is a small, Ras-related GTPase that was recently reported to harbor a recurrent UV-induced signature mutation in melanoma, resulting in substitution of P29 to serine (RAC1(P29S)), ranking this the third most frequently occurring gain-of-function mutation in melanoma. Although the Ras family GTPases are mutated in about 30% of all cancers, mutations in the Rho family GTPases have rarely been observed. In this study, we demonstrate that unlike oncogenic Ras proteins, which are primarily activated by mutations that eliminate GTPase activity, the activated melanoma RAC1(P29S) protein maintains intrinsic GTP hydrolysis and is spontaneously activated by substantially increased inherent GDP/GTP nucleotide exchange. Determination and comparison of crystal structures for activated RAC1 GTPases suggest that RAC1(F28L)--a known spontaneously activated RAC1 mutant--and RAC1(P29S) are self-activated in distinct fashions. Moreover, the mechanism of RAC1(P29S) and RAC1(F28L) activation differs from the common oncogenic mutations found in Ras-like GTPases that abrogate GTP hydrolysis. The melanoma RAC1(P29S) gain-of-function point mutation therefore represents a previously undescribed class of cancer-related GTPase activity.

Negative regulation of p53 by Ras superfamily protein RBEL1A

We had previously reported that RBEL1A, a novel Ras-like GTPase, was overexpressed in multiple human malignancies and that its depletion suppressed cell growth. However, the underlying molecular mechanism remained to be elucidated. Here we report that depletion of endogenous RBEL1A results in p53 accumulation due to increased p53 half-life whereas increased expression of RBEL1A reduces p53 levels under unstressed and genotoxic stress conditions. RBEL1A directly interacts with p53 and MDM2, and strongly enhances MDM2-dependent p53 ubiquitylation and degradation. We also found that RBEL1A modulation of p53 ubiquitylation by MDM2 does not depend on its GTPase activity. We have also defined the p53 oligomeric domain and RBEL1A GTPase domain to be the crucial regions for p53-RBEL1A interactions. Importantly, we have found that RBEL1A strongly interferes with p53 transactivation function; thus our results indicate that RBEL1A appears to function as a novel p53 negative regulator that facilitates MDM2-dependent p53 ubiquitylation and degradation.