Hyperactivation of mammalian target of rapamycin (mTOR) signaling by a gain-of-function mutant of the Rheb GTPase

Gain-of-function mutants of Ras and Rho family small GTPases have proven to be important tools in analyzing signaling downstream of these small GTPases. The Ras-related GTPase Rheb has emerged as a key player downstream of TSC1-2 in activating signaling to mammalian target of rapamycin (mTOR) effectors of cell growth such as S6K and 4E-BP1. The TSC1-2 tumor suppressor complex has been shown to act as a RhebGAP, converting Rheb from a GTP-bound to a GDP-bound form. Here we report the identification of a mutant Rheb (S16HRheb) that exhibits gain-of-function properties. At endogenous levels of expression S16HRheb exhibits increased GTP loading in vivo and is resistant to TSC1-2 GAP in vitro. Compared with wild-type Rheb, S16HRheb is more active at promoting the phosphorylation of the mTOR effectors S6K1 and 4E-BP1. Thus S16HRheb will help to identify proximal signaling events downstream of Rheb and allow potential Rheb-independent functions downstream of TSC1-2 to be investigated.     

Di-Ras2 Protein Forms a Complex with SmgGDS Protein in Brain Cytosol in Order to Be in a Low Affinity State for Guanine Nucleotides

The Ras family of small GTPases function in a wide variety of biological processes as “molecular switches” by cycling between inactive GDP-bound and active GTP-bound forms. Di-Ras1 and Di-Ras2 were originally identified as small GTPases forming a distinct subgroup of the Ras family. Di-Ras1/Di-Ras2 mRNAs are detected predominantly in brain and heart tissues. Biochemical analysis of Di-Ras1/Di-Ras2 has revealed that they have little GTPase activity and that their intrinsic guanine-nucleotide exchange rates are much faster than that of H-Ras. Yet little is known about the biological role(s) of Di-Ras1/Di-Ras2 or of how their activities are regulated. In the present study we found that endogenous Di-Ras2 co-purifies with SmgGDS from rat brain cytosol. Size-exclusion chromatography of purified recombinant proteins showed that Di-Ras2 forms a high affinity complex with SmgGDS. SmgGDS is a guanine nucleotide exchange factor with multiple armadillo repeats and has recently been shown to specifically activate RhoA and RhoC. In contrast to the effect on RhoA, SmgGDS does not act as a guanine nucleotide exchange factor for Di-Ras2 but instead tightly associates with Di-Ras2 to reduce its binding affinity for guanine nucleotides. Finally, pulse-chase analysis revealed that Di-Ras2 binds, in a C-terminal CAAX motif-dependent manner, to SmgGDS immediately after its synthesis. This leads to increased Di-Ras2 stability. We thus propose that isoprenylated Di-Ras2 forms a tight complex with SmgGDS in cytosol immediately after its synthesis, which lowers its affinity for guanine nucleotides.     

Real-time NMR Study of Three Small GTPases Reveals That Fluorescent 2��(3��)-O-(N-Methylanthraniloyl)-tagged Nucleotides Alter Hydrolysis and Exchange Kinetics

The Ras family of small GTPases control diverse signaling pathways through a conserved “switch” mechanism, which is turned on by binding of GTP and turned off by GTP hydrolysis to GDP. Full understanding of GTPase switch functions requires reliable, quantitative assays for nucleotide binding and hydrolysis. Fluorescently labeled guanine nucleotides, such as 2′(3′)-O-(N-methylanthraniloyl) (mant)-substituted GTP and GDP analogs, have been widely used to investigate the molecular properties of small GTPases, including Ras and Rho. Using a recently developed NMR method, we show that the kinetics of nucleotide hydrolysis and exchange by three small GTPases, alone and in the presence of their cognate GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors, are affected by the presence of the fluorescent mant moiety. Intrinsic hydrolysis of mantGTP by Ras homolog enriched in brain (Rheb) is ∼10 times faster than that of GTP, whereas it is 3.4 times slower with RhoA. On the other hand, the mant tag inhibits TSC2GAP-catalyzed GTP hydrolysis by Rheb but promotes p120 RasGAP-catalyzed GTP hydrolysis by H-Ras. Guanine nucleotide exchange factor-catalyzed nucleotide exchange for both H-Ras and RhoA was inhibited by mant-substituted nucleotides, and the degree of inhibition depends highly on the GTPase and whether the assay measures association of mantGTP with, or dissociation of mantGDP from the GTPase. These results indicate that the mant moiety has significant and unpredictable effects on GTPase reaction kinetics and underscore the importance of validating its use in each assay.     

To Cease or To Proliferate

Tor (Target of Rapamycin) pathway underlies a major signaling mechanism for controlling cell growth and proliferation1. Rheb (Ras homolog enriched in brain) is a small GTPase in the Tor pathway2-4. Similar to other small GTPases, Rheb cycles between a GTP-bound active state and a GDP-bound inactive state. TSC2 (Tuberous sclerosis complex 2), a gene mutated in an autosomal dominant disease Tuberous sclerosis, was shown to be the Rheb-GAP (GTPase activating protein)5, 6. However, a guanine nucleotide exchange factor (GEF) for Rheb had been missing. Human TCTP (Translationally controlled tumor protein) has been implicated in cancer, but its function in vivo has not been clearly elucidated. Recently we reported a molecular genetic characterization of TCTP function in Drosophila7. Drosophila TCTP (dTCTP) displays GEF activity to Rheb and is essential for Rheb activation in organ growth. Thus, our study provides a tight linkage of dTCTP to the Rheb-TOR pathway. In this addendum, we will briefly overview our findings and discuss our perspectives for future research on TCTP.     

Rapid parallel flow cytometry assays of active GTPases using effector beads

We describe a rapid assay for measuring the cellular activity of small guanine triphosphatases (GTPases) in response to a specific stimulus. Effector-functionalized beads are used to quantify in parallel multiple GTP-bound GTPases in the same cell lysate by flow cytometry. In a biologically relevant example, five different Ras family GTPases are shown for the first time to be involved in a concerted signaling cascade downstream of receptor ligation by Sin Nombre hantavirus.     

Hematopoietic-specific Rho GTPases Rac2 and RhoH and human blood disorders

The small guanosine triphosphotases (GTPases) Rho proteins are members of the Ras-like superfamily. Similar to Ras, most Rho GTPases cycle between active GTP-bound, and inactive GDP-bound conformations and act as molecular switches that control multiple cellular functions. While most Rho GTPases are expressed widely, the expression of Rac2 and RhoH are restricted to hematopoietic cells. RhoH is an atypical GTPase that lacks GTPase activity and remains in the active conformation. The generation of mouse knock-out lines has led to new understanding of the functions of both of these proteins in blood cells. The phenotype of these mice also led to the identification of mutations in human RAC2 and RHOH genes and the role of these proteins in immunodeficiency diseases. This review outlines the basic biology of Rho GTPases, focusing on Rac and RhoH and summarizes human diseases associated with mutations of these genes.     

Probing the GTPase cycle with real-time NMR: GAP and GEF activities in cell extracts

The Ras superfamily of small GTPases is a large family of switch-like proteins that control diverse cellular functions, and their deregulation is associated with multiple disease processes. When bound to GTP they adopt a conformation that interacts with effector proteins, whereas the GDP-bound state is generally biologically inactive. GTPase activating proteins (GAPs) promote hydrolysis of GTP, thus impeding the biological activity of GTPases, whereas guanine nucleotide exchange factors (GEFs) promote exchange of GDP for GTP and activate GTPase proteins. A number of methods have been developed to assay GTPase nucleotide hydrolysis and exchange, as well as the activity of GAPs and GEFs. The kinetics of these reactions are often studied with purified proteins and fluorescent nucleotide analogs, which have been shown to non-specifically impact hydrolysis and exchange. Most GAPs and GEFs are large multidomain proteins subject to complex regulation that is challenging to reconstitute in vitro. In cells, the activities of full-length GAPs or GEFs are typically assayed indirectly on the basis of nucleotide loading of the cognate GTPase, or by exploiting their interaction with effector proteins. Here, we describe a recently developed real-time NMR method to assay kinetics of nucleotide exchange and hydrolysis reactions by direct monitoring of nucleotide-dependent structural changes in an isotopically labeled GTPase. The unambiguous readout of this method makes it possible to precisely measure GAP and GEF activities from extracts of mammalian cells, enabling studies of their catalytic and regulatory mechanisms. We present examples of NMR-based assays of full-length GAPs and GEFs overexpressed in mammalian cells.     

All in the family? New insights and questions regarding interconnectivity of Ras,Rap1 and Ral.

Ras, Rap1 and Ral are related small GTPases. While the function of Ras in signal transduction is well established, it has been recognized only recently that Rap1 and Ral also are activated rapidly in response to a large variety of extracellular signals. Between the three GTPase an intriguing interconnectivity exists, in that guanine nucleotide exchange factors for Ral associate with the GTP-bound form of both Ras and Rap1. Furthermore, Rap1 is considered to function as an antagonist of Ras signalling by trapping Ras effectors in an inactive complex. Here, I summarize the recent developments in understanding the functional relationship between these three GTPase and argue that Rap1 functions in a signalling pathway distinct from Ras, while using similar or identical effectors.     

Analysis of Ras:RasGEF interactions by phage display and static multi-angle light scattering

Molecular switches such as small GTPases of the Ras family cycle between inactive GDP-bound and active GTP-bound states. Their essential role in controlling development and cell homeostasis requires mechanisms which determine amplitude and timing of activation. This is achieved in part by the action of guanine nucleotide exchange factors, which function as highly controlled enzymes whose activity relies on spatial segregation and intra- and intermolecular regulation. Here, we describe two experimental methodologies that permit the identification and characterization of GTPase binding sites on activators by assaying complex formation within a broad range of affinities. In the first assay system, proteins presented on the surface of filamentous phage are used to probe affinity determinants of protein-protein interactions. In this application, a protein-displayed phage library is generated by random mutagenesis and a plate-based selection is performed to identify mutations that confer higher binding affinity to an immobilized target. The second method uses light scattering as a tool for measuring the molecular weight, stoichiometry, and polydispersity of protein complexes in solution. In this application, conventional gel filtration chromatography provides initial fractionation, and in-line light scattering measurements allow accurate determination of molar masses of the eluent. This technique also provides information about conformational homogeneity which can be used as a quality     

High resolution crystal structures of human Rab5a and five mutants with substitutions in the catalytically important phosphate-binding loop

GTPase domain crystal structures of Rab5a wild type and five variants with mutations in the phosphate-binding loop are reported here at resolutions up to 1.5 A. Of particular interest, the A30P mutant was crystallized in complexes with GDP, GDP+AlF(3), and authentic GTP, respectively. The other variant crystals were obtained in complexes with a non-hydrolyzable GTP analog, GppNHp. All structures were solved in the same crystal form, providing an unusual opportunity to compare structures of small GTPases with different catalytic rates. The A30P mutant exhibits dramatically reduced GTPase activity and forms a GTP-bound complex stable enough for crystallographic analysis. Importantly, the A30P structure with bound GDP plus AlF(3) has been solved in the absence of a GTPase-activating protein, and it may resemble that of a transition state intermediate. Conformational changes are observed between the GTP-bound form and the transition state intermediate, mainly in the switch II region containing the catalytic Gln(79) residue and independent of A30P mutation-induced local alterations in the P-loop. The structures suggest an important catalytic role for a P-loop backbone amide group, which is eliminated in the A30P mutant, and support the notion that the transition state of GTPase-mediated GTP hydrolysis is of considerable dissociative character.     

Angiomyolipoma have common mutations in TSC2 but no other common genetic events

Renal angiomyolipoma are part of the PEComa family of neoplasms, and occur both in association with Tuberous Sclerosis Complex (TSC) and independent of that disorder. Previous studies on the molecular genetic alterations that occur in angiomyolipoma are very limited. We evaluated 9 angiomyolipoma for which frozen tissue was available from a consecutive surgical series. Seven of 8 samples subjected to RT-PCR-cDNA sequencing showed mutations in TSC2; none showed mutations in TSC1 or RHEB. Six of the seven mutations were deletions. We searched for 983 activating and inactivating mutations in 115 genes, and found none in these tumors. Similarly analysis for genomic regions of loss or gain, assessed by Affymetrix SNP6.0 analysis, showed no abnormalities. Loss of heterozygosity in the TSC2 region was commonly seen, except in patients with low frequency TSC2 mutations. We conclude that sporadic renal angiomyolipoma usually have mutations in TSC2, but not TSC1 or RHEB, and have no other common genomic events, among those we searched for. However, chromosomal translocations and gene fusion events were not assessed here. TSC2 inactivation by mutation is a consistent and likely necessary genetic event in the pathogenesis of most angiomyolipoma.     

Characterization of a guanine nucleotide dissociation stimulator for a ras-related GTPase.

ras-related GTPases participate in signaling for a variety of cellular processes. The GTPases cycle between a GTP-bound active state and a GDP-bound inactive state. This cycling is partially controlled by guanine nucleotide dissociation stimulators (GDS, also known as exchange factors). We report on the molecular cloning of cDNAs encoding a new mammalian GDS protein, using sequences derived from the yeast ras GDS proteins as probes. The encoded protein stimulates the dissociation of guanine nucleotides from the ras-related ralA and ralB GTPases at a rate at least 30-fold faster than the intrinsic nucleotide dissociation rate. This new GDS, ralGDS, is at least 20-fold more active on the ralA and ralB GTPases than on any other GTPase tested, including other members of the ras family (H-ras, N-ras, K-ras, R-ras, rap1a and rap2), members of the rho family (rhoA, rhoB and CDC42-Hs) and members of the rab family (rab3a and ypt1). While the ralGDS protein is phosphorylated on serine residues, we find no evidence that phosphorylation affects the activity of insect cell-expressed ralGDS towards the ralA or ralB GTPase. The 3600 nucleotide ralGDS mRNA and the 115 kDa protein were found in all tissues and cell lines examined.     

GAP control: regulating the regulators of small GTPases

The small GTPases of the Ras superfamily mediate numerous biological processes through their ability to cycle between an inactive GDP-bound and an active GTP-bound form. Among the key regulators of GTPase cycling are the GTPase-activating proteins (GAPs), which stimulate the weak intrinsic GTP-hydrolysis activity of the GTPases, thereby inactivating them. Despite the abundance of GAPs and the fact that mutations in GAP-encoding genes underlie several human diseases, these proteins have received relatively little attention. Recent studies have addressed the regulatory mechanisms that influence GAP activity. So far, findings suggest that GAP activity is regulated by several mechanisms, including protein-protein interactions, phospholipid interactions, phosphorylation, subcellular translocation and proteolytic degradation.     

Active and Inactive Cdc42 Differ in Their Insert Region Conformational Dynamics

Cell division control protein 42 homolog (Cdc42) protein, a Ras superfamily GTPase, regulates cellular activities, including cancer progression. Using all-atom molecular dynamics (MD) simulations and essential dynamic analysis, we investigated the structure and dynamics of the catalytic domains of GDP-bound (inactive) and GTP-bound (active) Cdc42 in solution. We discovered substantial differences in the dynamics of the inactive and active forms, particularly in the “insert region” (residues 122–135), which plays a role in Cdc42 activation and binding to effectors. The insert region has larger conformational flexibility in the GDP-bound Cdc42 than in the GTP-bound Cdc42. The G2 loop and switch I at the effector lobe of the catalytic domain exhibit large conformational changes in both the GDP- and the GTP-bound systems, but in the GTP-bound Cdc42, the switch I interactions with GTP are retained. Oncogenic mutations were identified in the Ras superfamily. In Cdc42, the G12V and Q61L mutations decrease the GTPase activity. We simulated these mutations in both GDP- and GTP-bound Cdc42. Although the overall structural organization is quite similar between the wild type and the mutants, there are small differences in the conformational dynamics, especially in the two switch regions. Taken together, the G12V and Q61L mutations may play a role similar to their K-Ras counterparts in nucleotide binding and activation. The conformational differences, which are mainly in the insert region and, to a lesser extent, in the switch regions flanking the nucleotide binding site, can shed light on binding and activation. We propose that the differences are due to a network of hydrogen bonds that gets disrupted when Cdc42 is bound to GDP, a disruption that does not exist in other Rho GTPases. The differences in the dynamics between the two Cdc42 states suggest that the inactive conformation has reduced ability to bind to effectors.     

Real-time in vitro measurement of GTP hydrolysis

Small GTPases require an active GTPase activity to function correctly in their cellular environment. Mutation of key residues involved in this activity renders the GTPase defective and the small G-protein constitutively active (GTP-locked). The GTPase activity is also a target for GTPase-activating proteins (GAPs) which act to attenuate GTPase signalling by accelerating the conversion of bound GTP to bound GDP. The measurement of GTP hydrolysis in vitro can therefore provide information on the intrinsic activity of the small GTPase (e.g., mutated GTPase activity) as well as help define GAP specificity. Current methods to measure GTP hydrolysis in vitro utilise either radioactivity-based filter-binding assays or measurements of GDP:GTP:P(i) ratios by high-performance liquid chromatography (HPLC). Both provide timed snapshots of the current GTP-bound state, can be prone to experimental errors, and do not provide a real-time observation of GTP hydrolysis. The method we describe here utilises a fluorescently labelled, phosphate-binding protein (PBP), which scavenges for free inorganic phosphate (P(i)). On binding of a single P(i), a change of protein conformation is coupled to a 7-fold increase in fluorescence of the fluorophore. This method therefore permits real-time monitoring of GTPase activity, through measurement of P(i) production. This review describes the process of preparing and labelling the PBP with the MDCC fluorophore, as well as an example of its use in measuring the GTPase activity of small GTPases. We also discuss the pros and cons, and implications of the technique in comparison to the radioactive and HPLC method of measuring the GTPase activity.     

Elevated small GTPase activation influences the cell proliferation signaling control in Niemann-Pick type C fibroblasts

Niemann-Pick type C (NPC) disease is characterized at the cellular level by the intracellular accumulation of free cholesterol. We have previously identified a similar phenotype in cystic fibrosis (CF) cell models that results in the activation of the small GTPase RhoA. The hypothesis of this study was that NPC cells would also exhibit an increase in small GTPase activation. An examination of the active, GTP-bound form of GTPases revealed a basal increase in the content of the active-form Ras and RhoA small GTPases in NPC fibroblasts compared to wt controls. To assess whether this increase in GTP-bound Ras and RhoA manifests a functional outcome, the expression of the proliferation control proteins p21/waf1 and cyclin D were examined. Consistent with increased GTPase signaling, p21/waf1 expression is reduced and cyclin D expression is elevated in NPC fibroblasts. Interestingly, cell growth rate is not altered in NPC fibroblasts compared to wt cells. However, NPC sensitivity to statin treatment is reversed by addition of the isoprenoid geranylgeranyl pyrophosphate (GGPP), a modifier of RhoA. It is concluded that Ras and RhoA basal activation is elevated in NPC fibroblasts and has an impact on cell survival pathways.     

Elevated small GTPase activation influences the cell proliferation signaling control in Niemann–Pick type C fibroblasts

Niemann–Pick type C (NPC) disease is characterized at the cellular level by the intracellular accumulation of free cholesterol. We have previously identified a similar phenotype in cystic fibrosis (CF) cell models that results in the activation of the small GTPase RhoA. The hypothesis of this study was that NPC cells would also exhibit an increase in small GTPase activation. An examination of the active, GTP-bound form of GTPases revealed a basal increase in the content of the active-form Ras and RhoA small GTPases in NPC fibroblasts compared to wt controls. To assess whether this increase in GTP-bound Ras and RhoA manifests a functional outcome, the expression of the proliferation control proteins p21/waf1 and cyclin D were examined. Consistent with increased GTPase signaling, p21/waf1 expression is reduced and cyclin D expression is elevated in NPC fibroblasts. Interestingly, cell growth rate is not altered in NPC fibroblasts compared to wt cells. However, NPC sensitivity to statin treatment is reversed by addition of the isoprenoid geranylgeranyl pyrophosphate (GGPP), a modifier of RhoA. It is concluded that Ras and RhoA basal activation is elevated in NPC fibroblasts and has an impact on cell survival pathways.     

Critical role of the pleckstrin homology domain in Dbs signaling and growth regulation

Dbl family proteins act as guanine nucleotide exchange factors and positive regulators of Rho GTPase function by stimulating formation of the active, GTP-bound state. All Dbl family Rho guanine nucleotide exchange factors possess an invariant tandem domain structure consisting of a Dbl homology (DH) catalytic domain followed by a pleckstrin homology (PH) regulatory domain. We determined previously that the PH domain of Dbs was critical for the intrinsic catalytic activity of the DH domain in vitro and for Dbs transformation in vivo. In this study, we evaluated the role of phosphoinositide binding to the PH domain in regulating the DH domain function of Dbs in vitro and in vivo. We determined that mutation of basic amino acids located within the beta1-beta2 and beta3-beta4 loops of the PH domain resulted in impaired phospholipid binding in vitro, yet full guanine nucleotide exchange activity in vitro was retained for RhoA and Cdc42. Surprisingly, these mutants were compromised in their ability to activate Rho GTPases in vivo and to cause transformation of NIH 3T3 cells. However, Dbs subcellular localization was impaired by these PH domain mutations, supporting a role for phospholipid interactions in facilitating membrane association. Despite the importance of phospholipid binding for Dbs function in vivo, we found that Dbs signaling and transforming activity was not stimulated by phosphatidylinositol 3-kinase activation. We suggest that the PH domain of Dbs facilitates two distinct roles in the regulation of DH domain function, one critical for GTPase association and activation in vitro and one critical for phosphoinositide binding and GTPase interaction in vivo, that together promote Dbs association with membranes.