Abstract B24: Development of small-molecule RAS inhibitors using Affimer reagents
Katarzyna Z. HazaHeather L. MartinChristian TiedeKevin W. TippingChi H. TrinhHolly FosterRachel TrowbridgeRichard FosterThomas A. EdwardsAlexander L. BreezeMichael J. McPhersonDarren C. Tomlinson
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Abstract Ras proteins are small GTPases that are mutationally activated in around 30% of all human cancers. Oncogenic mutations in Ras trigger uncontrolled cellular differentiation and division through uninhibited Ras-GTP signaling. Despite major efforts in developing inhibitors, lack of treatments directly targeting Ras in cancer led to the current assumption that Ras is undruggable. A new approach, which involves use of biologics, has shown great potential for development of Ras inhibitors, as demonstrated by recent increase in the number of antibody mimetic reagents targeting Ras, including single domain antibodies, monobodies and DARPins. We have developed modulators of Ras activity using novel artificial binding proteins, termed Affimers. Affimers are small 91-amino-acid scaffold proteins that constrain one or two randomized nine amino acid loop regions for molecular recognition. Affimers isolated against KRas, the most commonly mutated Ras family member, displayed low nanomolar binding affinities, were shown to be effective at inhibiting nucleotide exchange and blocked interaction between Ras and its effector Raf. When expressed in mammalian cells, Affimers bound with endogenous Ras and inhibited Ras-mediated signaling. Site-directed mutagenesis of Affimer variable regions revealed three residues critical for binding and inhibition. X-ray crystal structure of Affimer in complex with KRas demonstrated that these residues bind into a hydrophobic pocket on Ras, previously described with small molecules. Currently, we are determining whether this interaction gives insight into new modes of therapeutic development using molecular docking in attempt to mimic the Affimer residues with small molecules. This project provides a unique opportunity to further our understanding of Ras biology through the development of reagents that modulate Ras activity. In addition, structural insights into mode of inhibition allow the modelling and design of small-molecule compounds, providing novel therapeutic strategies to slow Ras-addicted tumor growth. Citation Format: Katarzyna Haza, Heather Martin, Christian Tiede, Kevin Tipping, Chi Trinh, Holly Foster, Rachel Trowbridge, Richard Foster, Thomas Edwards, Alexander Breeze, Michael McPherson, Darren Tomlinson. Development of small-molecule RAS inhibitors using Affimer reagents [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B24.Keywords:
HRAS
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Small GTPase
Live cell imaging
Conformational change
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[Objective] The aim was to summarize the research progress of small GTPase family,to provide reference for deeply analyzing the structure,function and its molecular action model of small GTPase.[Methods] The structure,mechanism of action and classification of small GTPase were introduced,moreover,the biological functions of each subfamily were summarized emphatically.[Result] The small GTPase is a large superfamily in eukaryotes,which contains more than 100 members.It is classified into Ras,Rho,Rab,Sar/Arf and Ran five distinct families,each of them participate in various physiological processes of organism such as cytoskeletal reorganization,gene expression,cell wall synthesis,vesicle trafficking,nucleocytoplasmic trafficking,microtubules formation,yeast budding process,spindle assembly and polarity of the cell growth.[Conclusion] The small GTPase has many members,its molecular action mechanism and regulatory complex network remain to be further studied.
Rab
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Ran
Ras superfamily
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Newly synthesized small GTPases in the Ras and Rho families are prenylated by cytosolic prenyltransferases and then escorted by chaperones to membranes, the nucleus, and other sites where the GTPases participate in a variety of signaling cascades. Understanding how prenylation and trafficking are regulated will help define new therapeutic strategies for cancer and other disorders involving abnormal signaling by these small GTPases. A growing body of evidence indicates that splice variants of SmgGDS (gene name RAP1GDS1) are major regulators of the prenylation, post-prenylation processing, and trafficking of Ras and Rho family members. SmgGDS-607 binds pre-prenylated small GTPases, while SmgGDS-558 binds prenylated small GTPases. This review discusses the history of SmgGDS research and explains our current understanding of how SmgGDS splice variants regulate the prenylation and trafficking of small GTPases. We discuss recent evidence that mutant forms of RabL3 and Rab22a control the release of small GTPases from SmgGDS, and review the inhibitory actions of DiRas1, which competitively blocks the binding of other small GTPases to SmgGDS. We conclude with a discussion of current strategies for therapeutic targeting of SmgGDS in cancer involving splice-switching oligonucleotides and peptide inhibitors.
Small GTPase
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Ras superfamily
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Brefeldin A
Small GTPase
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Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.
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Ras GTPases belong to a subfamily of signal transduction switches found in the Ras superfamily. Three isoforms of Ras (H‐, K‐, and N‐Ras) are expressed to regulate cellular proliferation, growth, and apoptosis. Unregulated activity of these proteins is closely related to disease, particularly the growth of tumors.1 The GTP/GDP cycle is a common regulatory feature of signaling output across GTPases. Exchange of GDP for GTP within the Ras active site results in an active signaling cascade. GAPs accelerate the hydrolysis of GTP to GDP, turning off the signal. In addition to exchange factors and activating proteins, the Ras GTPase cycle is also controlled by post‐translational modifications. An acetylation mimic (K104Q) in KRas G12V has been shown to negate oncogenic growth of the double mutant cell lines. However, KRas K104A/G12V does not reduce the oncogenicity of the cell line.2 The purpose of this study is to investigate the structural‐functional implications of K104Q and K104A mutations in HRas through X‐ray crystallography and to obtain their intrinsic hydrolysis rates through kinetic experiments with γ‐ 32 P‐GTP. These data show a novel hydrogen‐bonding network present in K104Q, but not WT or K104A HRas. Intrinsic hydrolysis of GTP to GDP is significantly impaired in HRas K104Q, but is not affected by a K104A mutation. The unique network may control helix 5 motility in HRas, resulting in a lower intrinsic hydrolysis rate. Connectivity from K104Q towards helix 5 passes through a region of amino acids that are different between the H‐, K‐, and N‐ Ras isoforms. This suggests that Ras acetylation of K104 may affect the function of the isoforms in subtly different ways. Support or Funding Information This work is funded by a grant from the NSF MCB‐1244203
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Ras GTPases are small proteins that are involved in various signal transduction pathways that regulate cellular proliferation, survival, migration, and apoptosis. Although the first crystal structure of Ras was determined more than two decades ago, biochemical and structural differences in the G‐domain of the three isoforms, H‐, K‐ and N‐Ras is only now beginning to emerge. Each isoform functions as a molecular switch where guanine nucleotide exchange factors (GEFs) facilitate the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) turning its signal on. Ras signaling is turned off via intrinsic hydrolysis or through the aid of GTPase activating proteins (GAP). Ras mutants are involved in 20% of human cancers, yet there are no inhibitors that target these proteins effectively. Most of the effort was originally focused on HRas as the classical representative of all three isoforms and more recently on KRas, the most frequently mutated in cancers. Thus, the structural biology and biochemistry of NRas have been largely overlooked. We have determined hydrolysis rate constants for NRas and determined the crystal structure of wild‐type NRas bound to GppNHp (GNP), a non‐hydrolyzable analogue of GTP, for comparison with H‐Ras and K‐Ras. Although each of the three isoforms follow a one‐step mechanism of hydrolysis, K‐ and NRas have lower rates than HRas, with structural features that promote disorder in switch II near the active site. N‐Ras mutants at G12 and Q61 are commonly found in melanoma tumors. These conserved residues are found in the phosphate‐binding loop (G12) and switch II (Q61) that help make up the active site of NRas. The structures of NRas mutants reveal distinct mechanisms through which they affect catalysis, consistent with lower hydrolysis rate constants found for G12D, G12S, Q61H, and Q61K compared to wild‐type NRas. Biochemical and structural understanding of NRas mutants will help further influence the development of novel therapeutic targets of NRas mutant melanoma. Support or Funding Information This research was funded by NSF Grant Number MCB‐1517295.
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Small GTPases cycle between an inactive GDP-bound and an active GTP-bound state to control various cellular events, such as cell proliferation, cytoskeleton organization, and membrane trafficking. Clarifying the guanine nucleotide-bound states of small GTPases is vital for understanding the regulation of small GTPase functions and the subsequent cellular responses. Although several methods have been developed to analyze small GTPase activities, our knowledge of the activities for many small GTPases is limited, partly because of the lack of versatile methods to estimate small GTPase activity without unique probes and specialized equipment. In the present study, we developed a versatile and straightforward HPLC-based assay to analyze the activation status of small GTPases by directly quantifying the amounts of guanine nucleotides bound to them. This assay was validated by analyzing the RAS-subfamily GTPases, including HRAS, which showed that the ratios of GTP-bound forms were comparable with those obtained in previous studies. Furthermore, we applied this assay to the investigation of psychiatric disorder-associated mutations of RHEB (RHEB/P37L and RHEB/S68P), revealing that both mutations cause an increase in the ratio of the GTP-bound form in cells. Mechanistically, loss of sensitivity to TSC2 (a GTPase-activating protein for RHEB) for RHEB/P37L, as well as both decreased sensitivity to TSC2 and accelerated guanine-nucleotide exchange for RHEB/S68P, is involved in the increase of their GTP-bound forms, respectively. In summary, the HPLC-based assay developed in this study provides a valuable tool for analyzing small GTPases for which the activities and regulatory mechanisms are less well understood. Small GTPases cycle between an inactive GDP-bound and an active GTP-bound state to control various cellular events, such as cell proliferation, cytoskeleton organization, and membrane trafficking. Clarifying the guanine nucleotide-bound states of small GTPases is vital for understanding the regulation of small GTPase functions and the subsequent cellular responses. Although several methods have been developed to analyze small GTPase activities, our knowledge of the activities for many small GTPases is limited, partly because of the lack of versatile methods to estimate small GTPase activity without unique probes and specialized equipment. In the present study, we developed a versatile and straightforward HPLC-based assay to analyze the activation status of small GTPases by directly quantifying the amounts of guanine nucleotides bound to them. This assay was validated by analyzing the RAS-subfamily GTPases, including HRAS, which showed that the ratios of GTP-bound forms were comparable with those obtained in previous studies. Furthermore, we applied this assay to the investigation of psychiatric disorder-associated mutations of RHEB (RHEB/P37L and RHEB/S68P), revealing that both mutations cause an increase in the ratio of the GTP-bound form in cells. Mechanistically, loss of sensitivity to TSC2 (a GTPase-activating protein for RHEB) for RHEB/P37L, as well as both decreased sensitivity to TSC2 and accelerated guanine-nucleotide exchange for RHEB/S68P, is involved in the increase of their GTP-bound forms, respectively. In summary, the HPLC-based assay developed in this study provides a valuable tool for analyzing small GTPases for which the activities and regulatory mechanisms are less well understood. Small GTPases function as molecular switches through conformational changes between an inactive GDP-bound form and an active GTP-bound form. More than 150 small GTPases that are known in humans are classified into five distinct subfamilies (i.e., RAS, RHO/RAC, RAB, ARF/ARL, and RAN) based on the similarities of their primary structure (1Colicelli J. Human RAS superfamily proteins and related GTPases.Sci. STKE. 2004; 2004: RE13Crossref PubMed Scopus (562) Google Scholar, 2Wennerberg K. Rossman K.L. Der C.J. The Ras superfamily at a glance.J. Cell Sci. 2005; 118: 843-846Crossref PubMed Scopus (980) Google Scholar). Small GTPases generally interact with effector proteins in their activated form (GTP-bound form) to regulate various biological events, such as cell proliferation and differentiation, cell motility, and intracellular transport. The balance between the activated and inactive forms of small GTPases is tightly regulated by several regulatory factors: guanine-nucleotide exchange factors promote the dissociation of GDP from small GTPases, thereby facilitating the binding of GTP (which is about 10 times more abundant than GDP in cells) to small GTPases. In turn, GTPase-activating proteins (GAPs) promote the conversion of GTP-bound forms to GDP-bound forms by stimulating the intrinsic GTPase activity of small GTPases. For the RHO and RAB proteins, a guanine-nucleotide dissociation inhibitor (GDI) protein (RHO-GDI and RAB-GDI, respectively) prevents the dissociation of GDP from small GTPases (3Cherfils J. Zeghouf M. Regulation of small GTPases by GEFs, GAPs, and GDIs.Physiol. Rev. 2013; 93: 269-309Crossref PubMed Scopus (712) Google Scholar, 4Boulter E. Garcia-Mata R. Guilluy C. Dubash A. Rossi G. Brennwald P.J. Burridge K. Regulation of Rho GTPase crosstalk, degradation and activity by RhoGDI1.Nat. Cell Biol. 2010; 12: 477-483Crossref PubMed Scopus (240) Google Scholar, 5Müller M.P. Goody R.S. Molecular control of Rab activity by GEFs, GAPs and GDI.Small GTPases. 2017; 9: 5-21Crossref PubMed Scopus (103) Google Scholar, 6Sztul E. Chen P.-W. Casanova J.E. Cherfils J. Dacks J.B. Lambright D.G. Lee F.-J.S. Randazzo P.A. Santy L.C. Schürmann A. Wilhelmi I. Yohe M.E. Kahn R.A. ARF GTPases and their GEFs and GAPs: Concepts and challenges.Mol. Biol. Cell. 2019; 30: 1249-1271Crossref PubMed Scopus (86) Google Scholar). Mutations in small GTPases that perturb the balance of their guanine-nucleotide binding states are associated with various diseases, including cancer. For example, the glycine 12 (G12) mutation in RAS, which is frequently observed in cancer, leads to RAS activation by preventing GAP-mediated GTP hydrolysis (7Hobbs G.A. Der C.J. Rossman K.L. RAS isoforms and mutations in cancer at a glance.J. Cell Sci. 2016; 129: 1287-1292Crossref PubMed Scopus (425) Google Scholar). The mutations in RAC1 (e.g., P29S and N92I) identified in melanoma increase the proportion of activated RAC1 due to the enhanced GDP/GTP-exchange reactions associated with accelerated GDP dissociation (8Krauthammer M. Kong Y. Ha B.H. Evans P. Bacchiocchi A. McCusker J.P. Cheng E. Davis M.J. Goh G. Choi M. Ariyan S. Narayan D. Dutton-Regester K. Capatana A. Holman E.C. et al.Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma.Nat. Genet. 2012; 44: 1006-1014Crossref PubMed Scopus (842) Google Scholar, 9Hodis E. Watson I.R. Kryukov G.V. Arold S.T. Imielinski M. Theurillat J.-P. Nickerson E. Auclair D. Li L. Place C. DiCara D. Ramos A.H. Lawrence M.S. Cibulskis K. Sivachenko A. et al.A landscape of driver mutations in melanoma.Cell. 2012; 150: 251-263Abstract Full Text Full Text PDF PubMed Scopus (1782) Google Scholar, 10Kawazu M. Ueno T. Kontani K. Ogita Y. Ando M. Fukumura K. Yamato A. Soda M. Takeuchi K. Miki Y. Transforming mutations of RAC guanosine triphosphatases in human cancers.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 3029-3034Crossref PubMed Scopus (86) Google Scholar, 11Davis M.J. Ha B.H. Holman E.C. Halaban R. Schlessinger J. Boggon T.J. RAC1P29S is a spontaneously activating cancer-associated GTPase.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 912-917Crossref PubMed Scopus (116) Google Scholar, 12Toyama Y. Kontani K. Katada T. Shimada I. Conformational landscape alternations promote oncogenic activities of Ras-related C3 botulinum toxin substrate 1 as revealed by NMR.Sci. Adv. 2019; 5eaav8945Crossref PubMed Scopus (11) Google Scholar, 13Toyama Y. Kontani K. Katada T. Shimada I. Decreased conformational stability in the oncogenic N92I mutant of Ras-related C3 botulinum toxin substrate 1.Sci. Adv. 2019; 5eaax1595Crossref PubMed Scopus (4) Google Scholar). Unlike these activating mutations, the ARL6/T31R mutation identified in ciliopathies causes reduced binding affinity for GTP, resulting in a constitutive GDP-bound (inactive) ARL6 (14Fan Y. Esmail M.A. Ansley S.J. Blacque O.E. Boroevich K. Ross A.J. Moore S.J. Badano J.L. May-Simera H. Compton D.S. Green J.S. Lewis R.A. Haelst M. M. van Parfrey P.S. Baillie D.L. et al.Mutations in a member of the Ras superfamily of small GTP-binding proteins causes Bardet-Biedl syndrome.Nat. Genet. 2004; 36: 989-993Crossref PubMed Scopus (268) Google Scholar, 15Kobayashi T. Hori Y. Ueda N. Kajiho H. Muraoka S. Shima F. Kataoka T. Kontani K. Katada T. Biochemical characterization of missense mutations in the Arf/Arl-family small GTPase Arl6 causing Bardet–Biedl syndrome.Biochem. Biophys. Res. Commun. 2009; 381: 439-442Crossref PubMed Scopus (22) Google Scholar, 16Wiens C. Tong Y. Esmail M. Oh E. Gerdes J.M. Wang J. Tempel W. Rattner J.B. Katsanis N. Park H.-W. Leroux M.R. Bardet-Biedl syndrome-associated small GTPase ARL6 (BBS3) functions at or near the ciliary gate and modulates Wnt signaling.J. Biol. Chem. 2010; 285: 16218-16230Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The clarification of the small GTPase activities and the impact of disease states on their activity are necessary for understanding the mechanisms of the physiological responses and drug-discovery targeting small GTPases. To date, several assays have been developed to analyze the activities of small GTPases; however, many small GTPases remain poorly understood in terms of the regulation of their guanine-nucleotide binding states in cells. Ras homolog enriched in brain (RHEB) is a member of the RAS-family GTPases and was initially identified as an immediate-early gene expressed in the brain (17Yamagata K. Sanders L.K. Kaufmann W.E. Yee W. Barnes C.A. Nathans D. Worley P.F. Rheb, a growth factor- and synaptic activity-regulated gene, encodes a novel Ras-related protein.J. Biol. Chem. 1994; 269: 16333-16339Abstract Full Text PDF PubMed Google Scholar). RHEB functions as a positive regulator of the mechanistic target of rapamycin complex 1 (mTORC1) in mammals (18Stocker H. Radimerski T. Schindelholz B. Wittwer F. Belawat P. Daram P. Breuer S. Thomas G. Hafen E. Rheb is an essential regulator of S6K in controlling cell growth in Drosophila.Nat. Cell Biol. 2003; 5: 559-566Crossref PubMed Scopus (423) Google Scholar, 19Saucedo L.J. Gao X. Chiarelli D.A. Li L. Pan D. Edgar B.A. Rheb promotes cell growth as a component of the insulin/TOR signalling network.Nat. Cell Biol. 2003; 5: 566-571Crossref PubMed Scopus (524) Google Scholar, 20Inoki K. Li Y. Xu T. Guan K.-L.L. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling.Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1378) Google Scholar, 21Long X. Lin Y. Ortiz-Vega S. Yonezawa K. Avruch J. Rheb binds and regulates the mTOR kinase.Curr. Biol. 2005; 15: 702-713Abstract Full Text Full Text PDF PubMed Scopus (726) Google Scholar). Although the regulatory mechanism of GTP loading onto RHEB remains to be determined, the GTP-bound form of RHEB activates mTORC1 on lysosomal membranes upon stimulation by insulin or growth factors (22Menon S. Dibble C.C. Talbott G. Hoxhaj G. Valvezan A.J. Takahashi H. Cantley L.C. Manning B.D. Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome.Cell. 2014; 156: 771-785Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar, 23Angarola B. Ferguson S.M. Coordination of Rheb lysosomal membrane interactions with mTORC1 activation.F1000Res. 2020; 9: 450Crossref Scopus (11) Google Scholar). The activated mTORC1 phosphorylates its substrates, such as the ribosomal protein S6 kinase (S6K), promoting protein synthesis and cell proliferation. RHEB activity is negatively regulated by the TSC complex, which acts as a GAP for RHEB (20Inoki K. Li Y. Xu T. Guan K.-L.L. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling.Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1378) Google Scholar, 24Thomas G. Hall M.N. TOR signalling and control of cell growth.Curr. Opin. Cell Biol. 1997; 9: 782-787Crossref PubMed Scopus (411) Google Scholar, 25Dibble C.C. Elis W. Menon S. Qin W. Klekota J. Asara J.M. Finan P.M. Kwiatkowski D.J. Murphy L.O. Manning B.D. TBC1D7 is a third subunit of the TSC1-TSC2 complex upstream of mTORC1.Mol. Cell. 2012; 47: 535-546Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). In the absence of insulin or growth factors, the TSC complex stimulates RHEB GTPase activity, leading to the conversion of the active RHEB-GTP to the inactive RHEB-GDP. Two RHEB mutations (RHEB/P37L and RHEB/S68P) were recently reported as being involved in psychiatric disorders, such as autism (26Reijnders M. Kousi M. Woerden G. van Klein M. Bralten J. Mancini G. Essen T. van Proietti-Onori M. Smeets E. Gastel M. van Stegmann A. Stevens S. Lelieveld S. Gilissen C. Pfundt R. et al.Variation in a range of mTOR-related genes associates with intracranial volume and intellectual disability.Nat. Commun. 2017; 8: 1052Crossref PubMed Scopus (36) Google Scholar, 27Onori M.P. Koene L.M.C. Schäfer C.B. Nellist M. Velze M. de B. van Gao Z. Elgersma Y. Woerden G. M. van RHEB/mTOR hyperactivity causes cortical malformations and epileptic seizures through increased axonal connectivity.PLoS Biol. 2021; 19e3001279Crossref PubMed Scopus (6) Google Scholar). The expression of RHEB/P37L or RHEB/S68P increased cell size, suggesting that both mutants are gain-of-function mutants. However, the effects of these mutations on the biochemical properties of RHEB remain to be clarified. The present study reported an HPLC-based assay that is broadly applicable to the analysis of the guanine-nucleotide bound status of small GTPases. Using this assay, we investigated the disease-associated RHEB/P37L and RHEB/S68P mutations, demonstrating that these mutations lead to RHEB activation in a distinct manner. We sought to analyze the activation state of small GTPases by directly quantifying the GDP and GTP bound to them. For this purpose, we performed ion-pair reversed-phase HPLC (IP-RP-HPLC) to measure GDP and GTP. Ion-pair reagents bind with counter-ions of charged compounds (such as nucleotides) to neutralize their charge, which allows the retention of the nucleotides in the reverse-phase columns, for analysis (28Werner A. Reversed-phase and ion-pair separations of nucleotides, nucleosides and nucleobases: Analysis of biological samples in health and disease.J. Chromatogr. 1993; 618: 3-14Crossref PubMed Scopus (38) Google Scholar, 29Contreras-Sanz A. Scott-Ward T.S. Gill H.S. Jacoby J.C. Birch R.E. Malone-Lee J. Taylor K.M. Peppiatt-Wildman C.M. Wildman S.S. Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC.Purinergic Signal. 2012; 8: 741-751Crossref PubMed Scopus (41) Google Scholar). Under our optimized IP-RP-HPLC condition, a mixture of four nucleotides (GDP, GTP, ADP, and ATP; 10 pmol each) was well separated (Figs. 1 and S1). We determined the retention time, CV, and limit of quantification of all standard substances (Table 1). The correlation coefficient (r2) obtained from the linear regression of the calibration concentration range was ≥0.999 for GDP and GTP (0.5–100 pmol).Table 1Analytical data for the four nucleotides standards using IP-RP-HPLCNucleotideRetention timeCVaCoefficient of variation.LoQbLimit of quantification.GDP5.4 min1.21%0.47 pmolGTP8.6 min1.21%0.50 pmolADP6.2 min1.40%0.27 pmolATP10.9 min2.64%0.34 pmola Coefficient of variation.b Limit of quantification. Open table in a new tab We tested whether the guanine nucleotides bound to small GTPases can be quantified using IP-RP-HPLC. Our previous study using metabolic labeling with [32P]orthophosphate has shown that HRAS existed predominantly in the GDP-bound form (30Kontani K. Tada M. Ogawa T. Okai T. Saito K. Araki Y. Katada T. Di-Ras: A distinct subgroup of Ras-family GTPases with unique biochemical properties.J. Biol. Chem. 2002; 277: 41070-41078Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In contrast, DIRAS1 and DIRAS2 displayed considerable GTP-bound forms, probably because of the accelerated intrinsic guanine-nucleotide exchange reaction. Thus, we thought that these small GTPases with different ratios of GTP-bound forms would be suitable for validating IP-RP-HPLC analysis. We established HeLa cell lines expressing Flag-tagged RAS-family small GTPases (HRAS, DIRAS1, and DIRAS2) in a doxycycline (dox)-dependent manner (Fig. 2A). The Flag-tagged proteins were immunopurified from the corresponding cell lysates and denatured by heat to dissociate the bound guanine nucleotides. We could reproducibly quantify the amount of GDP and GTP bound to each small GTPase using IP-RP-HPLC (Fig. 2B). The ratios of the GTP-bound form of HRAS, DIRAS1, and DIRAS2 were 4.7%, 88.9%, and 50.2%, respectively (Fig. 2C), which were comparable with those obtained using metabolic labeling with [32P]orthophosphate (30Kontani K. Tada M. Ogawa T. Okai T. Saito K. Araki Y. Katada T. Di-Ras: A distinct subgroup of Ras-family GTPases with unique biochemical properties.J. Biol. Chem. 2002; 277: 41070-41078Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). These results showed that IP-RP-HPLC can be used to analyze the guanine-nucleotide-bound status of small GTPases in cells. The small GTPase RHEB functions as a molecular switch to regulate mTORC1 activity in mammals. The GTP-bound form of RHEB stimulates mTORC1 activity on lysosomal membranes (22Menon S. Dibble C.C. Talbott G. Hoxhaj G. Valvezan A.J. Takahashi H. Cantley L.C. Manning B.D. Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome.Cell. 2014; 156: 771-785Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar, 23Angarola B. Ferguson S.M. Coordination of Rheb lysosomal membrane interactions with mTORC1 activation.F1000Res. 2020; 9: 450Crossref Scopus (11) Google Scholar). In turn, the activated mTORC1 phosphorylates its substrates, such as S6K and the eukaryotic translation initiation factor 4E-binding protein 1, thus promoting protein synthesis and cell growth. RHEB inactivation is mediated by the TSC complex (consisting of TSC1, TSC2, and TBC1D7 in mammals), which functions as a GAP for RHEB via the TSC2 GAP domain (20Inoki K. Li Y. Xu T. Guan K.-L.L. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling.Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1378) Google Scholar, 25Dibble C.C. Elis W. Menon S. Qin W. Klekota J. Asara J.M. Finan P.M. Kwiatkowski D.J. Murphy L.O. Manning B.D. TBC1D7 is a third subunit of the TSC1-TSC2 complex upstream of mTORC1.Mol. Cell. 2012; 47: 535-546Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). To elucidate the guanine-nucleotide-bound state of RHEB in cells, we established a HeLa cell line expressing Flag-RHEB in a dox-dependent manner. We performed an IP-RP-HPLC analysis of Flag-RHEB proteins expressed in HeLa cells, which showed that the ratio of the GTP-bound form of Flag-RHEB was about 16% (Fig. 3A), which was comparable with those reported in previous studies (31Long X. Lin Y. Ortiz-Vega S. Busch S. Avruch J. The Rheb switch 2 segment is critical for signaling to target of rapamycin complex 1.J. Biol. Chem. 2007; 282: 18542-18551Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Dox-induced expression of Flag-RHEB enhanced the phosphorylation levels of S6K and S6 via mTORC1 activation (Fig. 3B), as reported previously (31Long X. Lin Y. Ortiz-Vega S. Busch S. Avruch J. The Rheb switch 2 segment is critical for signaling to target of rapamycin complex 1.J. Biol. Chem. 2007; 282: 18542-18551Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). We further analyzed the effect of the loss of TSC2 on the guanine-nucleotide-bound state of Flag-RHEB. Previous studies have shown that loss of TSC2 causes RHEB activation via the suppression of RHEB-GTPase activity (20Inoki K. Li Y. Xu T. Guan K.-L.L. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling.Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1378) Google Scholar). In fact, RNAi against TSC2 (siTSC2) increased the proportion of the GTP-bound form of RHEB with a concomitant elevation of the phosphorylation levels of S6K and S6 (Fig. 3, C and D). These results indicate that IP-RP-HPLC can be used to analyze the RHEB activation status in cells. A recent study reported that the P37L and S68P mutations in RHEB were associated with neurological disorders, such as autism (26Reijnders M. Kousi M. Woerden G. van Klein M. Bralten J. Mancini G. Essen T. van Proietti-Onori M. Smeets E. Gastel M. van Stegmann A. Stevens S. Lelieveld S. Gilissen C. Pfundt R. et al.Variation in a range of mTOR-related genes associates with intracranial volume and intellectual disability.Nat. Commun. 2017; 8: 1052Crossref PubMed Scopus (36) Google Scholar). That research suggested that these mutations cause RHEB activation based on the enhanced mTORC1 activity detected in the RHEB-mutant-expressing cells. However, no detailed biochemical analysis has been performed using these RHEB mutants. Thus, we generated HeLa cell lines expressing Flag-tagged RHEB/P37L, RHEB/S68P, and RHEB/Q64L (an active RHEB mutant as a control) for their biochemical characterization. Ectopic expression of these mutants caused the activation of mTORC1 signaling (as estimated by the phosphorylation levels of S6K and S6), as reported previously (Fig. 4A) (20Inoki K. Li Y. Xu T. Guan K.-L.L. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling.Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1378) Google Scholar, 26Reijnders M. Kousi M. Woerden G. van Klein M. Bralten J. Mancini G. Essen T. van Proietti-Onori M. Smeets E. Gastel M. van Stegmann A. Stevens S. Lelieveld S. Gilissen C. Pfundt R. et al.Variation in a range of mTOR-related genes associates with intracranial volume and intellectual disability.Nat. Commun. 2017; 8: 1052Crossref PubMed Scopus (36) Google Scholar, 27Onori M.P. Koene L.M.C. Schäfer C.B. Nellist M. Velze M. de B. van Gao Z. Elgersma Y. Woerden G. M. van RHEB/mTOR hyperactivity causes cortical malformations and epileptic seizures through increased axonal connectivity.PLoS Biol. 2021; 19e3001279Crossref PubMed Scopus (6) Google Scholar). The RHEB/Q64L mutant is less sensitive to the GAP activity of TSC2 than is RHEB/WT and exists predominantly in the GTP-bound form in cells (32Li Y. Inoki K. Guan K.-L.L. Biochemical and functional characterizations of small GTPase Rheb and TSC2 GAP activity.Mol. Cell Biol. 2004; 24: 7965-7975Crossref PubMed Scopus (186) Google Scholar). In fact, the IP-RP-HPLC analysis showed a marked increase in the ratio of the GTP-bound form of RHEB/Q64L compared with RHEB/WT (Fig. 4B). Furthermore, we found that both RHEB/P37L and RHEB/S68P showed increases in their GTP-bound forms (Fig. 4B). These results suggest that the P37L and S68P disease-associated mutations in RHEB cause its activation, thus leading to enhanced mTORC1 signaling. To elucidate the mechanism underlying the increases in the ratio of the GTP-bound forms of the RHEB/P37L and RHEB/S68P mutants, we investigated the effects of the loss of TSC2 on the guanine-nucleotide-bound status of these mutants (Fig. 4C). Similar to RHEB/Q64L, the GTP-bound ratio of RHEB/P37L was unaffected by siTSC2, suggesting that the RHEB/P37L GTP hydrolysis is less sensitive to TSC2 GAP. In contrast, siTSC2 significantly increased the GTP-bound ratio of RHEB/S68P, suggesting that RHEB/S68P retained sensitivity to the GAP activity of TSC2 to some extent. To analyze the effect of TSC2 on the GTPase activity of RHEB mutants in greater detail, we performed an in vitro GAP assay using GST-RHEB and the Flag–TSC1/2 complex. To analyze RHEB-GTPase activity, GST-RHEB was incubated with or without Flag-TSC1/2, and the reaction mixture was subjected to IP-RP-HPLC analysis. The guanine-nucleotide bound form of GST-RHEB/WT showed no apparent changes in the absence of Flag-TSC1/2 (Fig. 5A, left panel), whereas the amounts of the GTP-bound form were time-dependently decreased with a concomitant increase in those of the GDP-bound form in the presence of Flag-TSC1/2 (Fig. 5A, right panel), indicating that Flag-TSC1/2 stimulated the GTPase activity of GST-RHEB/WT. We found that Flag-TSC1/2 did not affect the GTPase activity of GST-RHEB/P37L (Fig. 5B, P37L). In contrast, it weakly but significantly promoted that of GST-RHEB/S68P (Fig. 5B, S68P). We have obtained similar results using untagged RHEB proteins, indicating that the GST-tag does not interfere with the assay (Fig. S2). Together with the results of the RNAi experiments (Fig. 4C), these data indicate that the loss and reduction of the sensitivity of RHEB/P37L and RHEB/S68P to TSC2-GAP, respectively, are responsible for the increases in their GTP-bound forms. In addition to the decrease in GTP hydrolysis, the promotion of GDP/GTP exchange reactions can also cause an increase in the ratio of the GTP-bound forms of small GTPases. For example, HRAS/F28L exists mainly in a GTP-bound form because of an increased nucleotide-dissociation rate, despite its sensitivity to the GTPase activation proteins (33Reinstein J. Schlichting I. Frech M. Goody R.S. Wittinghofer A. p21 with a phenylalanine 28—leucine mutation reacts normally with the GTPase activating protein GAP but nevertheless has transforming properties.J. Biol. Chem. 1991; 266: 17700-17706Abstract Full Text PDF PubMed Google Scholar). Oncogenic RAC1 mutants, such as RAC1/P29S and RAC1/N92I, also display high GDP-dissociation rates, which leads to the existence of the mutants predominantly in their GTP-bound active states (10Kawazu M. Ueno T. Kontani K. Ogita Y. Ando M. Fukumura K. Yamato A. Soda M. Takeuchi K. Miki Y. Transforming mutations of RAC guanosine triphosphatases in human cancers.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 3029-3034Crossref PubMed Scopus (86) Google Scholar, 11Davis M.J. Ha B.H. Holman E.C. Halaban R. Schlessinger J. Boggon T.J. RAC1P29S is a spontaneously activating cancer-associated GTPase.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 912-917Crossref PubMed Scopus (116) Google Scholar, 12Toyama Y. Kontani K. Katada T. Shimada I. Conformational landscape alternations promote oncogenic activities of Ras-related C3 botulinum toxin substrate 1 as revealed by NMR.Sci. Adv. 2019; 5eaav8945Crossref PubMed Scopus (11) Google Scholar, 13Toyama Y. Kontani K. Katada T. Shimada I. Decreased conformational stability in the oncogenic N92I mutant of Ras-related C3 botulinum toxin substrate 1.Sci. Adv. 2019; 5eaax1595Crossref PubMed Scopus (4) Google Scholar). Thus, we investigated whether the acceleration of the GDP/GTP exchange rate is involved in the increases in the GTP-bound form of the RHEB/P37L and RHEB/S68P mutants. Therefore, we performed an in vitro GDP-dissociation assay using purified Escherichia coli recombinant RHEB proteins. We found that the dissociation rates of the WT and P37L proteins were comparable, whereas that of S68P was increased by comparison to them (Fig. 6A). Consistently, an in vitro [35S]GTPγS binding assay revealed that the GTPγS-binding rate of RHEB/S68P was enhanced compared with that of RHEB/WT and RHEB/P37L (Fig. 6B). These data indicate that the acceleration of the GDP/GTP exchange rate, together with a reduced sensitivity to TSC2-GAP, is involved in the increased ratio of the RHEB/S68P GTP-bound form in cells. This study established a straightforward and versatile HPLC-based method to analyze the guanine-nucleotide binding state of small GTPases. The IP-RP-HPLC analysis enabled the quantification of picomolar levels of GDP and GTP bound to small GTPases. Using this method, we found that the disease-associated RHEB mutants (RHEB/P37L and RHEB/S68P) displayed an increased proportion of GTP-bound forms in cells. Furthermore, an in vitro analysis using purified proteins indicated that loss of sensitivity to TSC2 GAP for RHEB/P37L and both reduced sensitivity to TSC2 GAP and enhanced guanine-nucleotide exchange reaction for RHEB/S68P are involved in the increase of their GTP-bound forms, respectively. Thus, IP-RP-HPLC analysis will be an effective tool in the analysis of the activation status and regulatory mechanisms of various small GTPases. Various methods have been developed to analyze the intracellular guanine-nucleotide binding states of small GTPases. The classic method consists in metabolic labeling using [32P]orthophosphate, in which small GTPases are loaded with radiolabeled GDP or GTP in cells (20Inoki K. Li Y. Xu T. Guan K.-L.L. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling.Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1378) Google Scholar, 30Kontani K. Tada M. Ogawa T. Okai T. Saito K. Araki Y. Katada T. Di-Ras: A distinct subgroup of Ras-family GTPases with unique biochemical properties.J. Biol. Chem. 2002; 277: 41070-41078Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 34Satoh T. Endo M. Nakamura S. Kaziro Y. Analysis of guanine nucleotide bound to ras protein in PC12 cells.FEBS Lett. 1988; 236: 185-189Crossref PubMed Scopus (22) Google Scholar). After immunoisolation of small GTPases from the cells, the bound GDP and GTP are separated by TLC to be analyzed by autoradiography. This method can detect GDP and GTP with high sensitivity; however, its drawbacks include the need for facilities to use radioisotopes, radiation eff
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Coordination of the activity of multiple small GTPases is required for the regulation of many physiological processes, including cell migration. There are now several examples of functional interplay between small GTPase pairs, but the mechanisms that control GTPase activity in time and space are only partially understood. Here, we build on the hypothesis that small GTPases are part of a large, integrated network and propose that key proteins within this network integrate multiple signaling events and coordinate multiple small GTPase activities. Specifically, we identify the scaffolding protein IQGAP1 as a master regulator of multiple small GTPases, including Cdc42, Rac1, Rap1, and RhoA. In addition, we demonstrate that IQGAP1 promotes Arf6 activation downstream of β1 integrin engagement. Furthermore, following literature-curated searches and recent mass spectrometric analysis of IQGAP1-binding partners, we report that IQGAP1 recruits other small GTPases, including RhoC, Rac2, M-Ras, RhoQ, Rab10, and Rab5, small GTPase regulators, including Tiam1, RacGAP1, srGAP2 and HERC1, and small GTPase effectors, including PAK6, N-WASP, several sub-units of the Arp2/3 complex and the formin mDia1. Therefore, we propose that IQGAP1 acts as a small GTPase scaffolding platform within the small GTPase network, and recruits and/or regulates small GTPases, small GTPase regulators and effectors to orchestrate cell behavior. Finally, to identify other putative key regulators of small GTPase crosstalk, we have assembled a small GTPase network using protein-protein interaction databases.
IQGAP1
Small GTPase
Crosstalk
Rap1
Formins
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