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1.  SEACing the GAP that nEGOCiates TORC1 activation 
Cell Cycle  2013;12(18):2948-2952.
The target of rapamycin complex 1 (TORC1) regulates eukaryotic cell growth in response to a variety of input signals. In S. cerevisiae, amino acids activate TORC1 through the Rag guanosine triphosphatase (GTPase) heterodimer composed of Gtr1 and Gtr2 found together with Ego1 and Ego3 in the EGO complex (EGOC). The GTPase activity of Gtr1 is regulated by the SEA complex (SEAC). Specifically, SEACIT, a SEAC subcomplex containing Iml1, Npr2, and Npr3 functions as a GTPase activator (GAP) for Gtr1 to decrease the activity of TORC1 and, consequently, growth, after amino acid deprivation. Here, we present genetic epistasis data, which show that SEACAT, the other SEAC subcomplex, containing Seh1, Sea2–4, and Sec13, antagonizes the GAP function of SEACIT. Orthologs of EGOC (Ragulator), SEACIT (GATOR1), and SEACAT (GATOR2) are present in higher eukaryotes, highlighting the remarkable conservation, from yeast to man, of Rag GTPase and TORC1 regulation.
PMCID: PMC3875668  PMID: 23974112
Rag GTPases; TOR complex 1; EGO complex; SEA complex; amino acid signaling; Iml1-Npr2-Npr3 Rag GTPase GAP complex; GATOR1; GATOR2; Seh1; Sec13
2.  Putative GTP-binding protein, Gtr1, associated with the function of the Pho84 inorganic phosphate transporter in Saccharomyces cerevisiae. 
Molecular and Cellular Biology  1992;12(7):2958-2966.
We have found an open reading frame which is 1.1 kb upstream of PHO84 (which encodes a Pi transporter) and is transcribed from the opposite strand. In Saccharomyces cerevisiae, this gene is distal to the TUB3 locus on the left arm of chromosome XIII and is named GTR1. GTR1 encodes a protein consisting of 310 amino acid residues containing, in its N-terminal region, the characteristic tripartite consensus elements for binding GTP conserved in GTP-binding proteins, except for histidine in place of a widely conserved aspargine residue in element III. Disruption of the GTR1 gene resulted in slow growth at 30 degrees C and no growth at 15 degrees C; other phenotypes resembled those of pho84 mutants and included constitutive synthesis of repressible acid phosphatase, reduced Pi transport activity, and resistance to arsenate. The latter phenotypes were shown to be due to a defect in Pi uptake, and the Gtr1 protein was found to be functionally associated with the Pho84 Pi transporter. Recombination between chromosome V (at the URA3 locus) and chromosome XIII (in the GTR1-PHO84-TUB3 region) by using a plasmid-encoded site-specific recombination system indicated that the order of these genes was telomere-TUB3-PHO84-GTR1-CENXIII.
PMCID: PMC364509  PMID: 1620108
3.  Signal recognition particle (SRP) and SRP receptor: A new paradigm for multi-state regulatory GTPases 
Biochemistry  2009;48(29):6696-6704.
The GTP-binding proteins or GTPases comprise a superfamily of proteins that provide molecular switches in numerous cellular processes. The ‘GTPase switch’ paradigm, in which a GTPase acts as a bimodal switch that is turned ‘on’ and ‘off’ by external regulatory factors, has been used to interpret the regulatory mechanism of many GTPases for over two decades. Nevertheless, recent work has unveiled an emerging class of ‘multi-state’ regulatory GTPases that do not adhere to this classical paradigm. Instead of relying on external nucleotide exchange factors or GTPase activating proteins to switch between the ‘on’ and ‘off’ states, these GTPases have the intrinsic ability to exchange nucleotides and to sense and respond to upstream and downstream factors. In contrast to the bimodal nature of the ‘GTPase switch’, these GTPases undergo multiple conformational rearrangements, allowing multiple regulatory points to be built into a complex biological process to ensure the efficiency and fidelity of the pathway. We suggest that these multi-state regulatory GTPases are uniquely suited to provide spatial and temporal control over complex cellular pathways that require multiple molecular events to occur in a highly coordinated fashion.
PMCID: PMC2883566  PMID: 19469550
4.  Molecular Modeling Study for Interaction between Bacillus subtilis Obg and Nucleotides 
PLoS ONE  2010;5(9):e12597.
The bacterial Obg proteins (Spo0B-associated GTP-binding protein) belong to the subfamily of P-loop GTPase proteins that contain two equally and highly conserved domains, a C-terminal GTP binding domain and an N-terminal glycine-rich domain which is referred as the “Obg fold” and now it is considered as one of the new targets for antibacterial drug. When the Obg protein is associated with GTP, it becomes activated, because conformation of Obg fold changes due to the structural changes of GTPase switch elements in GTP binding site. In order to investigate the effects and structural changes in GTP bound to Obg and GTPase switch elements for activation, four different molecular dynamics (MD) simulations were performed with/without the three different nucleotides (GTP, GDP, and GDP + Pi) using the Bacillus subtilis Obg (BsObg) structure. The protein structures generated from the four different systems were compared using their representative structures. The pattern of Cα-Cα distance plot and angle between the two Obg fold domains of simulated apo form and each system (GTP, GDP, and GDP+Pi) were significantly different in the GTP-bound system from the others. The switch 2 element was significantly changed in GTP-bound system. Also root-mean-square fluctuation (RMSF) analysis revealed that the flexibility of the switch 2 element region was much higher than the others. This was caused by the characteristic binding mode of the nucleotides. When GTP was bound to Obg, its γ-phosphate oxygen was found to interact with the key residue (D212) of the switch 2 element, on the contrary there was no such interaction found in other systems. Based on the results, we were able to predict the possible binding conformation of the activated form of Obg with L13, which is essential for the assembly with ribosome.
PMCID: PMC2935376  PMID: 20830302
5.  Disease mutations in Rab7 result in unregulated nucleotide exchange and inappropriate activation 
Human Molecular Genetics  2009;19(6):1033-1047.
Rab GTPases are molecular switches that orchestrate vesicular trafficking, maturation and fusion by cycling between an active, GTP-bound form, and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins. Missense mutations of the GTPase Rab7 cause a dominantly inherited axonal degeneration known as Charcot-Marie-Tooth type 2B through an unknown mechanism. We present the 2.8 Å crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. Through extensive biochemical analysis, we demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that the increased activation of mutant Rab7 is counterbalanced by unregulated, GTP hydrolysis-independent membrane cycling. Notably, disease mutations are able to rescue the membrane cycling of a GTPase-deficient mutant. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease not by a gain of novel toxic function, but by misregulation of native Rab7 activity.
PMCID: PMC2830827  PMID: 20028791
6.  An expanded Ragulator is a GEF for the Rag GTPases that signal amino acid levels to mTORC1 
Cell  2012;150(6):1196-1208.
The mTOR Complex 1 (mTORC1) pathway promotes cell growth in response to a diverse set of cues, including growth factors as well as energy and amino acid levels. Amino acids signal through the Rag GTPases to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. The four mammalian Rag proteins form obligate heterodimers consisting of RagA or RagB bound to RagC or RagD. A key upstream component of the Rag GTPases is Ragulator, a trimeric complex that tethers them to the lysosome and also interacts with the v-ATPase, which is necessary for amino acid sensing by mTORC1. Amino acids stimulate the binding of GTP to RagB, a critical step in the sensing mechanism, but the factors that regulate Rag nucleotide loading are unknown. Here, we identify the proteins encoded by the HBXIP and C7orf59 genes as novel Ragulator components that are required for mTORC1 activation by amino acids. The pentameric Ragulator has nucleotide exchange activity towards RagA and RagB and interacts with the Rag heterodimers in an amino acid- and v-ATPase-dependent fashion. Thus, we provide mechanistic insight into how mTORC1 senses amino acids by revealing Ragulator to be a scaffold with guanine nucleotide exchange factor (GEF) activity for the Rag GTPases.
PMCID: PMC3517996  PMID: 22980980
7.  A P-loop Mutation in Gα Subunits Prevents Transition to the Active State: Implications for G-protein Signaling in Fungal Pathogenesis 
PLoS Pathogens  2012;8(2):e1002553.
Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active Gαβγ heterotrimer relies on nucleotide cycling by the Gα subunit: exchange of GTP for GDP activates Gα, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting Gα to its inactive state. In several genetic studies of filamentous fungi, such as the rice blast fungus Magnaporthe oryzae, a G42R mutation in the phosphate-binding loop of Gα subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that Gα(G42R) mutants are not GTPase deficient, but rather incapable of achieving the activated conformation. Two crystal structure models suggest that Arg-42 prevents a typical switch region conformational change upon Gαi1(G42R) binding to GDP·AlF4− or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. Gα(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with Gβγ and GoLoco motifs in any nucleotide state. The corresponding Gαq(G48R) mutant is not constitutively active in cells and responds poorly to aluminum tetrafluoride activation. Comparative analyses of M. oryzae strains harboring either G42R or GTPase-deficient Q/L mutations in the Gα subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two Gα mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants.
Author Summary
Heterotrimeric G-proteins function as molecular switches to convey cellular signals. When a G-protein coupled receptor encounters its ligand at the cellular membrane, it catalyzes guanine nucleotide exchange on the Gα subunit, resulting in a shift from an inactive to an active conformation. G-protein signaling pathways are conserved from mammals to plants and fungi, including the rice blast fungus Magnaporthe oryzae. A mutation in the Gα subunit (G42R), previously thought to eliminate its GTPase activity, leading to constitutive activation, has been utilized to investigate roles of heterotrimeric G-protein signaling pathways in multiple species of filamentous fungi. Here, we demonstrate through structural, biochemical, and cellular approaches that G42R mutants are neither GTPase deficient nor constitutively active, but rather are unable to transition to the activated conformation. A direct comparison of M. oryzae fungal strains harboring either G42R or truly constitutively activating mutations in two Gα subunits, MagA and MagB, revealed markedly different phenotypes. Our results suggest that activation of MagB is critical for pathogenic development of M. oryzae in response to hydrophobic surfaces, such as plant leaves. Furthermore, the lack of constitutive activity by Gα(G42R) mutants prompts a re-evaluation of its use in previous genetic experiments in multiple fungal species.
PMCID: PMC3285607  PMID: 22383884
8.  Intragenic Suppressor Mutations Restore GTPase and Translation Functions of a Eukaryotic Initiation Factor 5B Switch II Mutant▿  
Molecular and Cellular Biology  2006;27(5):1677-1685.
Structural studies of GTP-binding proteins identified the Switch I and Switch II elements as contacting the γ-phosphate of GTP and undergoing marked conformational changes upon GTP versus GDP binding. Movement of a universally conserved Gly at the N terminus of Switch II is thought to trigger the structural rearrangement of this element. Consistently, we found that mutation of this Gly in the Switch II element of the eukaryotic translation initiation factor 5B (eIF5B) from Saccharomyces cerevisiae impaired cell growth and the guanine nucleotide-binding, GTPase, and ribosomal subunit joining activities of eIF5B. In a screen for mutations that bypassed the critical requirement for this Switch II Gly in eIF5B, intragenic suppressors were identified in the Switch I element and at a residue in domain II of eIF5B that interacts with Switch II. The intragenic suppressors restored yeast cell growth and eIF5B nucleotide-binding, GTP hydrolysis, and subunit joining activities. We propose that the Switch II mutation distorts the geometry of the GTP-binding active site, impairing nucleotide binding and the eIF5B domain movements associated with GTP binding. Accordingly, the Switch I and domain II suppressor mutations induce Switch II to adopt a conformation favorable for nucleotide binding and hydrolysis and thereby reestablish coupling between GTP binding and eIF5B domain movements.
PMCID: PMC1820465  PMID: 17189426
9.  Cell motility 
The Ras proto-oncogenic proteins, prototypes of the small GTPases, work as molecular switches: they are active when bound to GTP and inactive when bound to GDP. A variety of evidence suggested that the Ras paradigm is not fully valid for the Rho-family of small GTPases. Indeed, permanent activation is not sufficient but it is rather the continuous oscillation between the GDP-bound and GTP-bound conformations (namely the GDP/GTP cycling or GTPase flux), that is required for Rho-GTPases to perform their biological functions and properly coordinate actin cytoskeleton reorganization. In our recent study, we show that Rac1 needs to cycle between the GDP and GTP states in order to efficiently control cell motility. Similarly, it was previously reported that GDP/GTP cycling is required by RhoA for cytokinesis and by Cdc42 for cell polarization. The future challenge is to understand why the GTPase flux is so important for the biological actions of Rho GTPases.
PMCID: PMC3306356  PMID: 22446552
10.  Topological characterisation and identification of critical domains within glucosyltransferase IV (GtrIV) of Shigella flexneri 
BMC Biochemistry  2011;12:67.
The three bacteriophage genes gtrA, gtrB and gtr(type) are responsible for O-antigen glucosylation in Shigella flexneri. Both gtrA and gtrB have been demonstrated to be highly conserved and interchangeable among serotypes while gtr(type) was found to be specific to each serotype, leading to the hypothesis that the Gtr(type) proteins are responsible for attaching glucosyl groups to the O-antigen in a site- and serotype- specific manner. Based on the confirmed topologies of GtrI, GtrII and GtrV, such interaction and attachment of the glucosyl groups to the O-antigen has been postulated to occur in the periplasm.
In this study, the topology of GtrIV was experimentally determined by creating different fusions between GtrIV and a dual-reporter protein, PhoA/LacZ. This study shows that GtrIV consists of 8 transmembrane helices, 2 large periplasmic loops, 2 small cytoplasmic N- and C- terminal ends and a re-entrant loop that occurs between transmembrane helices III and IV. Though this topology differs from that of GtrI, GtrII, GtrV and GtrX, it is very similar to that of GtrIc. Furthermore, both the N-terminal periplasmic and the C-terminal periplasmic loops are important for GtrIV function as shown via a series of loop deletion experiments and the creation of chimeric proteins between GtrIV and its closest structural homologue, GtrIc.
The current study provides the basis for elucidating the structure and mechanism of action of this important O-antigen modifying glucosyltransferase.
PMCID: PMC3259042  PMID: 22188643
11.  Establishing a Role for the GTPase Ypt1p at the Late Golgi 
Traffic (Copenhagen, Denmark)  2010;11(4):520-532.
GTPases of the Rab family cycle between an inactive (GDP-bound) and active (GTP-bound) conformation. The active form of the Rab regulates a variety of cellular functions via multiple effectors. Guanine nucleotide exchange factors (GEFs) activate Rabs by accelerating the exchange of GDP for GTP, while GTPase activating proteins (GAPs) inactivate Rabs by stimulating the hydrolysis of GTP. The GTPase Ypt1p is required for ER-Golgi and intra-Golgi traffic in the yeast Saccharomyces cerevisiae. Recent findings, however, have shown that a Ypt1p GEF, GAP and effector are all required for traffic from the early endosome to the Golgi. Here we describe a screen for ypt1 mutants that block traffic from the early endosome to the late Golgi, but not general secretion. This screen has led to the identification of a collection of recessive and dominant mutants that block traffic from the early endosome. While it has long been known that Ypt1p regulates the flow of biosynthetic traffic into the cis side of the Golgi, these findings have established a role for Ypt1p in the regulation of early endosome-Golgi traffic. We propose that Ypt1p regulates the flow of traffic into the cis and trans side of the Golgi via multiple effectors.
PMCID: PMC2861988  PMID: 20059749
Rab; early endosome; Golgi; membrane traffic
12.  Characterization of GTPase Activity of TrmE, a Member of a Novel GTPase Superfamily, from Thermotoga maritima 
Journal of Bacteriology  2000;182(24):7078-7082.
A gene encoding a putative GTP-binding protein, a TrmE homologue that is highly conserved in both prokaryotes and eukaryotes, was cloned from Thermotoga maritima, a hyperthermophilic bacterium. T. maritima TrmE was overexpressed in Escherichia coli and purified. TrmE has a GTPase activity but no ATPase activity. The GTPase activity can be competed with GTP, GDP, and dGTP but not with GMP, ATP, CTP, or UTP. Km and kcat at 70°C were 833 μM and 9.3 min−1, respectively. Our results indicate that TrmE is a GTP-binding protein with a very high intrinsic GTP hydrolysis rate. We also propose that TrmE homologues constitute a novel subfamily of the GTPase superfamily.
PMCID: PMC94838  PMID: 11092873
13.  Identification and Characterisation of the RalA-ERp57 Interaction: Evidence for GDI Activity of ERp57 
PLoS ONE  2012;7(11):e50879.
RalA is a membrane-associated small GTPase that regulates vesicle trafficking. Here we identify a specific interaction between RalA and ERp57, an oxidoreductase and signalling protein. ERp57 bound specifically to the GDP-bound form of RalA, but not the GTP-bound form, and inhibited the dissociation of GDP from RalA in vitro. These activities were inhibited by reducing agents, but no disulphide bonds were detected between RalA and ERp57. Mutation of all four of ERp57’s active site cysteine residues blocked sensitivity to reducing agents, suggesting that redox-dependent conformational changes in ERp57 affect binding to RalA. Mutations in the switch II region of the GTPase domain of RalA specifically reduced or abolished binding to ERp57, but did not block GTP-specific binding to known RalA effectors, the exocyst and RalBP1. Oxidative treatment of A431 cells with H2O2 inhibited cellular RalA activity, and the effect was exacerbated by expression of recombinant ERp57. The oxidative treatment significantly increased the amount of RalA localised to the cytosol. These findings suggest that ERp57 regulates RalA signalling by acting as a redox-sensitive guanine-nucleotide dissociation inhibitor (RalGDI).
PMCID: PMC3511393  PMID: 23226417
14.  Phase variation controls expression of Salmonella lipopolysaccharide modification genes by a DNA methylation-dependent mechanism 
Molecular Microbiology  2010;77(2):337-353.
The O-antigen of Salmonella lipopolysaccharide is a major antigenic determinant and its chemical composition forms the basis for Salmonella serotyping. Modifications of the O-antigen that can affect the serotype include those carried out by the products of glycosyltransferase operons (gtr), which are present on specific Salmonella and phage genomes. Here we show that expression of the gtr genes encoded by phage P22 that confers the O1 serotype is under the control of phase variation. This phase variation occurs by a novel epigenetic mechanism requiring OxyR in conjunction with the DNA methyltransferase Dam. OxyR is an activator or a repressor of the system depending on which of its two binding sites in the gtr regulatory region is occupied. Binding is decreased by methylation at Dam target sequences in either site, and this confers heritability of the expression state to the system. Most Salmonella gtr operons share the key regulatory elements that are identified here as essential for this epigenetic phase variation.
PMCID: PMC2909390  PMID: 20487280
15.  Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy? 
Nature reviews. Cancer  2010;10(12):842-857.
There is now considerable and increasing evidence for a causal role of aberrant activity of the Ras superfamily of small GTPases in human cancers. These GTPases act as GDP-GTP-regulated binary switches that control many fundamental cellular processes. A common mechanism of GTPase deregulation in cancer is the deregulated expression and/or activity of their regulatory proteins, guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound state and GTPase activating proteins (GAPs) that return the GTPase to its GDP-bound inactive state. We assess the association of GEFs and GAPs with cancer and their druggability for cancer therapeutics.
PMCID: PMC3124093  PMID: 21102635
16.  GGAPs, a New Family of Bifunctional GTP-Binding and GTPase-Activating Proteins 
Molecular and Cellular Biology  2003;23(7):2476-2488.
G proteins are molecular switches that control a wide variety of physiological functions, including neurotransmission, transcriptional activation, cell migration, cell growth. and proliferation. The ability of GTPases to participate in signaling events is determined by the ratio of GTP-bound to GDP-bound forms in the cell. All known GTPases exist in an inactive (GDP-bound) and an active (GTP-bound) conformation, which are catalyzed by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs), respectively. In this study, we identified and characterized a new family of bifunctional GTP-binding and GTPase-activating proteins, named GGAP. GGAPs contain an N-terminal Ras homology domain, called the G domain, followed by a pleckstrin homology (PH) domain, a C-terminal GAP domain, and a tandem ankyrin (ANK) repeat domain. Expression analysis indicates that this new family of proteins has distinct cell localization, tissue distribution, and even message sizes. GTPase assays demonstrate that GGAPs have high GTPase activity through direct intramolecular interaction of the N-terminal G domain and the C-terminal GAP domain. In the absence of the GAP domain, the N-terminal G domain has very low activity, suggesting a new model of GGAP protein regulation via intramolecular interaction like the multidomain protein kinases. Overexpression of GGAPs leads to changes in cell morphology and activation of gene transcription.
PMCID: PMC150724  PMID: 12640130
17.  Evolutionary expansion of the Ras switch regulatory module in eukaryotes 
Nucleic Acids Research  2011;39(13):5526-5537.
Ras proteins control many aspects of eukaryotic cell homeostasis by switching between active (GTP-bound) and inactive (GDP-bound) conformations, a reaction catalyzed by GTPase exchange factors (GEF) and GTPase activating proteins (GAP) regulators, respectively. Here, we show that the complexity, measured as number of genes, of the canonical Ras switch genetic system (including Ras, RasGEF, RasGAP and RapGAP families) from 24 eukaryotic organisms is correlated with their genome size and is inversely correlated to their evolutionary distances from humans. Moreover, different gene subfamilies within the Ras switch have contributed unevenly to the module’s expansion and speciation processes during eukaryote evolution. The Ras system remarkably reduced its genetic expansion after the split of the Euteleostomi clade and presently looks practically crystallized in mammals. Supporting evidence points to gene duplication as the predominant mechanism generating functional diversity in the Ras system, stressing the leading role of gene duplication in the Ras family expansion. Domain fusion and alternative splicing are significant sources of functional diversity in the GAP and GEF families but their contribution is limited in the Ras family. An evolutionary model of the Ras system expansion is proposed suggesting an inherent ‘decision making’ topology with the GEF input signal integrated by a homologous molecular mechanism and bifurcation in GAP signaling propagation.
PMCID: PMC3141262  PMID: 21447561
18.  The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1 
Science (New York, N.Y.)  2008;320(5882):1496-1501.
The multiprotein mTORC1 protein kinase complex is the central component of a pathway that promotes growth in response to insulin, energy levels, and amino acids, and is deregulated in common cancers. We find that the Rag proteins—a family of four related small guanosine triphosphatases (GTPases)—interact with mTORC1 in an amino acid sensitive manner and are necessary for the activation of the mTORC1 pathway by amino acids. A Rag mutant that is constitutively bound to GTP interacted strongly with mTORC1 and its expression within cells made the mTORC1 pathway resistant to amino acid deprivation. Conversely, expression of a GDP-bound Rag mutant prevented stimulation of mTORC1 by amino acids. The Rag proteins do not directly stimulate the kinase activity of mTORC1, but, like amino acids, promote the intracellular localization of mTOR to a compartment that also contains its activator Rheb.
PMCID: PMC2475333  PMID: 18497260
19.  Rag GTPase-mediated regulation of mTORC1 by nutrients is necessary for neonatal autophagy and survival 
Nature  2012;493(7434):679-683.
The mTOR Complex 1 (mTORC1) pathway regulates organismal growth in response to many environmental cues, including nutrients and growth factors1. Cell-based studies showed that mTORC1 senses amino acids through the Rag family of GTPases 2,3, but their importance in mammalian physiology is unknown. Here, we generated knock-in mice that express a constitutively active form of RagA (RagAGTP) from its endogenous promoter. RagAGTP/GTP mice develop normally, but fail to survive postnatal day 1. When delivered by Caesarian-section, fasted RagAGTP/GTP neonates die almost twice as rapidly as wild-type littermates. Within an hour of birth, wild-type neonates strongly inhibit mTORC1, which coincides with profound hypoglycaemia and a drop in plasma amino acid levels. In contrast, mTORC1 inhibition does not occur in RagAGTP/GTP neonates, despite identical reductions in blood nutrient levels. With prolonged fasting, wild-type neonates recover their plasma glucose levels, but RagAGTP/GTP mice remain hypoglycaemic until death, despite using glycogen at a faster rate. The glucose homeostasis defect correlates with the inability of fasted RagAGTP/GTP neonates to trigger autophagy and produce amino acids for de novo glucose production. Because profound hypoglycaemia does not inhibit mTORC1 in RagAGTP/GTP neonates, we hypothesized that the Rag pathway signals glucose as well as amino acid sufficiency to mTORC1. Indeed, mTORC1 is resistant to glucose deprivation in RagAGTP/GTP fibroblasts, and glucose, like amino acids, controls its recruitment to the lysosomal surface, the site of mTORC1 activation. Thus, the Rag GTPases signal glucose and amino acid levels to mTORC1, and play an unexpectedly key role in neonates in autophagy induction and thus nutrient homeostasis and viability.
PMCID: PMC4000705  PMID: 23263183
20.  RhoE is a pro-survival p53 target gene that inhibits ROCK I-mediated apoptosis in response to genotoxic stress 
Current biology : CB  2006;16(24):2466-2472.
The Rho family of GTPases regulates many aspects of cellular behavior through alterations to the actin cytoskeleton [1-6]. The majority of the Rho family proteins function as molecular switches cycling between the active, GTP-bound, and the inactive, GDP-bound, conformations [1-6]. Unlike typical Rho family proteins, the Rnd subfamily members, including Rnd1, Rnd2, RhoE/Rnd3, and RhoH, are GTPase deficient, and thus expected to be constitutively active [7-10]. Here, we identify an unexpected role for RhoE/Rnd3 in the regulation of the p53-mediated stress response. We show that RhoE is a transcriptional p53 target gene, and that genotoxic stress triggers actin depolymerization, resulting in actin-stress fiber disassembly through p53-dependent RhoE induction. Silencing of RhoE induction in response to genotoxic stress maintains stress fiber formation and strikingly increases apoptosis, implying an antagonistic role for RhoE in p53-dependent apoptosis. We found that RhoE inhibits ROCK I (Rho associated kinase I) activity during genotoxic stress thereby suppressing apoptosis. We demonstrate that the p53-mediated induction of RhoE in response to DNA damage favors cell survival partly through inhibition of ROCK I-mediated apoptosis. Thus, RhoE is anticipated to function by regulating ROCK I signaling to control the balance between cell survival and cell death in response to genotoxic stress.
PMCID: PMC2779528  PMID: 17174923
21.  Advantages and limitations of cell-based assays for GTPase activation and regulation 
Cellular Logistics  2012;2(3):147-150.
Small GTPases of the Ras superfamily are important regulators of many cellular functions, including signal transduction, cytoskeleton assembly, metabolic regulation, organelle biogenesis and intracellular transport. Most GTPases act as binary switches, being “on” in the active, GTP-bound state and “off” in the inactive, GDP-bound state, and cycle between the two states with the aid of accessory proteins, referred to as guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). This review will focus on the ADP-ribosylation factors (Arfs), a family of G-proteins that are essential regulators of carrier vesicle formation during vesicular transport. As for most other GTPases, the Arfs themselves are vastly outnumbered by the proteins that regulate them, and a major focus in the field has been to define the functional relationships between individual GEFs and GAPs and their substrates at the cellular level. Over the years, a variety of methods have been developed to measure GTPase activation in vitro and in vivo. In vitro analysis will be discussed in the accompanying article by Randazzo and colleagues. Here we will focus on cell- and tissue-based assays and their advantages/disadvantages relative to cell-free systems.
PMCID: PMC3498073  PMID: 23181197
GTPase; Arf; GTP; GDP; GEF; GAP; effector; pulldown; FRET
22.  Rho5p Is Involved in Mediating the Osmotic Stress Response in Saccharomyces cerevisiae, and Its Activity Is Regulated via Msi1p and Npr1p by Phosphorylation and Ubiquitination ▿  
Eukaryotic Cell  2008;7(9):1441-1449.
Small GTPases of the Rho family act as molecular switches, and modulation of the GTP-bound state of Rho proteins is a well-characterized means of regulating their signaling activity in vivo. In contrast, the regulation of Rho-type GTPases by posttranslational modifications is poorly understood. Here, we present evidence of the control of the Saccharomyces cerevisiae Rho-type GTPase Rho5p by phosphorylation and ubiquitination. Rho5p binds to Ste50p, and the expression of the activated RHO5(Q91H) allele in an Δste50 strain is lethal under conditions of osmotic stress. An overexpression screen identified RGD2 and MSI1 as being high-copy suppressors of the osmotic sensitivity of this lethality. Rgd2p had been identified as being a possible Rho5p GTPase-activating protein based on an in vitro assay; this result supports its function as a regulator of Rho5p activity in vivo. MSI1 was previously identified as being a suppressor of hyperactive Ras/cyclic AMP signaling, where it antagonizes Npr1p kinase activity and promotes ubiquitination. Here, we show that Msi1p also acts via Npr1p to suppress activated Rho5p signaling. Rho5p is ubiquitinated, and its expression is lethal in a strain that is compromised for proteasome activity. These data identify Rho5p as being a target of Msi1p/Npr1p regulation and describe a regulatory circuit involving phosphorylation and ubiquitination.
PMCID: PMC2547074  PMID: 18621925
23.  Novel Protein and Mg2+ Configurations in the Mg2+ GDP Complex of the SRP GTPase Ffh 
Proteins  2004;54(2):222-230.
Ffh is the signal sequence recognition and targeting subunit of the prokaryotic signal recognition particle (SRP). Previous structural studies of the NG GTPase domain of Ffh demonstrated magnesium-dependent and magnesium-independent binding conformations for GDP and GMPPNP that are believed to reflect novel mechanisms for exchange and activation in this member of the GTPase superfamily. The current study of the NG GTPase bound to Mg2+ GDP reveals two new binding conformations—in the first the magnesium interactions are similar to those seen previously, however, the protein undergoes a conformational change that brings a conserved aspartate into its second coordination sphere. In the second, the protein conformation is similar to that seen previously, but the magnesium coordination sphere is disrupted so that only five oxygen ligands are present. The loss of the coordinating water molecule, at the position that would be occupied by the oxygen of the γ-phosphate of GTP, is consistent with that position being privileged for exchange during phosphate release. The available structures of the GDP-bound protein provide a series of structural snapshots that illuminate steps along the pathway of GDP release following GTP hydrolysis.
PMCID: PMC3540803  PMID: 14696184
signal recognition particle (SRP); SRP54; Ffh; FtsY; GTPase; crystallography; magnesium coordination
24.  Mapping the nucleotide and isoform dependent structural and dynamical features of Ras proteins 
Ras GTPases are conformational switches controlling cell proliferation, differentiation and development. Despite their prominent role in many forms of cancer, the mechanism of conformational transition between inactive GDP- and active GTP-bound states remains unclear. Here we describe a detailed analysis of available experimental structures and molecular dynamics simulations to quantitatively assess the structural and dynamical features of active and inactive states and their interconversion. We demonstrate that GTP-bound and nucleotide-free G12V H-ras sample a wide region of conformational space, and show that the inactive to active transition is a multiphase process defined by the relative rearrangement of the two switches and the orientation of Tyr32. We also modeled and simulated N- and K-ras proteins and found that K-ras is more flexible than N- and H-ras. We identified a number of isoform-specific long-range side chain interactions that define unique pathways of communication between the nucleotide binding site and the C-terminus.
PMCID: PMC2519881  PMID: 18547521
Ras; structure analysis; structure-function relationships; principal component analysis; molecular dynamics
25.  LRRK2 Kinase Activity Is Dependent on LRRK2 GTP Binding Capacity but Independent of LRRK2 GTP Binding 
PLoS ONE  2011;6(8):e23207.
Leucine rich repeat kinase 2 (LRRK2) is a Parkinson's disease (PD) gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound “on” state and a GDP bound “off” state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC) GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.
PMCID: PMC3155532  PMID: 21858031

Results 1-25 (613892)