Molecular Basis of a Novel Oncogenic Mutation in GNAO1

M Garcia-Marcos1, P Ghosh2 and MG Farquhar1

1Department of Cellular and Molecular Medicine, San Diego School of Medicine, University of California, La Jolla, CA, USA and 2Department of Medicine, University of California, San Diego, School of Medicine, La Jolla, CA, USA

Heterotrimeric G proteins are molecular switches that Introduction control signal transduction, and their dysregulation can promote oncogenesis. Somatic mutations in GNAS, Identification of oncogenic mutations in G proteins GNAI2 and GNAQ genes induce oncogenesis by rendering has provided insights into the molecular mechanisms Ga subunits constitutively activated. Recently the first that drive tumorigenesis, and has also highlighted the somatic mutation, arginine243-histidine (R243H) in the importance of maintaining the critical balance between GNAO1 (Gao) gene was identified in breast carcinomas G-protein activation and deactivation. Mutation of and shown to promote oncogenic transformation when the small G-protein Ras is very common in cancer, for introduced into cells. Here, we provide the molecular basis example, K-ras is mutated B20% of all tumors (Forbes for the oncogenic properties of the Gao R243H mutant. et al., 2008). Ga subunits of heterotrimeric G proteins, Using limited proteolysis assays, nucleotide-binding which have structural similarities to the Ras superfamily assays, and single-turnover and steady-state GTPase of G proteins, have also been reported to be mutated in assays, we demonstrate that the oncogenic R234H cancer. For example, mutations in codons 201 and 227 mutation renders Gao constitutively active by accelerating of GNAS (Gas) are found in B25% of pituitary the rate of nucleotide exchange; however, this mutation adenomas (Landis et al., 1989; Forbes et al., 2008), does not affect Gao’s ability to become deactivated by and mutation of the corresponding codons in GNAI2 GTPase-activating proteins (GAPs) or by its intrinsic (Gai2) (Lyons et al., 1990) and GNAQ (Gaq) (Lamba GTPase activity. This mechanism differs from that of et al., 2009; Van Raamsdonk et al., 2009) has also been previously reported oncogenic mutations that impair reported in human tumors. All these mutations in Ras GTPase activity and GAP sensitivity without affecting and heterotrimeric G proteins promote malignant nucleotide exchange. The constitutively active Gao R243H transformation and tumorigenesis by rendering the mutant also enhances Src-STAT3 signaling in NIH-3T3 G proteins constitutively active in the guanosine cells, a pathway previously shown to be directly triggered 50-triphosphate (GTP)-bound conformation (Landis by active Gao proteins to promote cellular transformation. et al., 1989; Iiri et al., 1998; Farfel et al., 1999; Based on structural analyses, we propose that the enhanced Downward, 2003). The molecular mechanism by which rate of nucleotide exchange in Gao R243H results from this is achieved is also highly conserved in that these loss of the highly conserved electrostatic interaction of oncogenic mutations block the deactivation step of the R243 with E43, located in the in the P-loop that represents G protein by impairing its intrinsic ability to hydrolyze the binding site for the a-andb-phosphates of the GTP (GTPase deficient) (Landis et al., 1989) and its nucleotide. We conclude that the novel R234H mutation sensitivity to the action of GTPase-activating proteins imparts oncogenic properties to Gaobyaccelerating (GAPs) (Berman et al., 1996; Downward, 2003). No nucleotide exchange and rendering it constitutively active, such mutations in other Ga subunits (Gao, Ga12, Ga13 thereby enhancing signaling pathways, for example, src- and Gaz) have been described in carcinomas, but they STAT3, responsible for neoplastic transformation. were found to be oncogenic when artificially introduced Oncogene (2011) 30, 2691–2696; doi:10.1038/onc.2010.645; into cultured cells (Kroll et al., 1992; Xu et al., 1993, published online 14 February 2011 1994; Vara Prasad et al., 1994; Voyno-Yasenetskaya et al., 1994; Wong et al., 1995; Marinissen and Gutkind, Keywords: oncoprotein; somatic mutation; hetero- 2001; Radhika and Dhanasekaran, 2001). trimeric G protein; GAIP/RGS19; neoplasia; STAT3 Very recently, the first somatic mutation for GNAO1 (Gao) has been described in breast cancer (Kan et al., 2010). This oncogenic mutation, R243H, is unique because it does not correspond to any of the previously Correspondence: Dr M Garcia-Marcos, Department Cellular and described mutations in other G proteins. However, the Molecular Medicine, San Diego School of Medicine, University of molecular basis for its ability to induce oncogenic California, George E. Palade Laboratories for Cellular and Molecular transformation was not investigated. Here, we studied Medicine, 9500 Gilman Drive, Room 218, La Jolla, CA 92093-0651, the biochemical properties of Gao R243H and its USA. E-mail: [email protected] impact on cellular signaling. We found that Gao Received 11 September 2010; revised and accepted 16 December 2010; R243H is rendered constitutively active by a molecular published online 14 February 2011 mechanism distinct from that reported for any of the Molecular insights into Gao R243H oncogenicity M Garcia-Marcos et al 2692 previously characterized oncogenic mutations in G (De Vries et al., 1995; Berman et al., 1996). The experi- proteins, and discuss the structural basis for this mental conditions of the single-turnover assay allow mechanism using insights from the available G-protein direct measurement of the rate of GTP hydrolysis crystal structures. Finally, we show that this mutant without any interference from the rate of nucleotide triggers a signaling program responsible for malignant binding. We found that Gao R243H hydrolyzed GTP transformation. These data advance our understanding virtually at the same rate (that is, same Kcat)aswtGao, of how heterotrimeric G proteins can be transformed and that GAIP/RGS19 accelerated the GTPase activity from protooncogenes to oncogenes by naturally occur- of Gao R243H or wt Gao equally (Figure 1b). These ring mutations in key residues. results demonstrate that the R243H mutation does not affect the basal or GAP-enhanced GTPase activity of Gao, indicating that the deactivation step in the G-protein cycle of Gao R243H is intact. This property Results and discussion of Gao R234H differs from the previously reported oncogenic mutations in GNAS, GNAI2 and GNAQ that Gao R234H is capable of binding nucleotides and of impair the intrinsic GTPase activity of the G protein changing conformation upon activation (Lyons et al., 1990) and its deactivation by GAPs To investigate the effect of the R234H mutation on the (Berman et al., 1996). biochemical properties of Gao and to gain insights into the molecular basis of its oncogenicity (Kan et al., 2010), we purified hexahistidine-tagged wild-type (wt) Gaoand Nucleotide exchange rate of Gao R243H is accelerated the R234H mutant from Escherichia coli (498%purity). Because G proteins are activated by exchanging GDP Because some mutations in Ga subunits affect their for GTP, we hypothesized that the R243H mutation ability to fold properly, bind nucleotides and/or change might render Gao constitutively active by increasing the et al. conformation upon activation (Kleuss , 1994; Grishina rate of nucleotide exchange. To investigate the effect of and Berlot, 1998; Warner and Weinstein, 1999; Pereira the R243H mutation on the rate of nucleotide exchange and Cerione, 2005), we determined whether Gao R234H by Gao, we used two well-established assays—steady- retains the native properties of the wt protein by using a state GTPase and GTPgS-binding assays. Steady-state well-established assay based on differential resistance to GTP hydrolysis by Ga subunits is a reaction with proteolysis (Kleuss et al., 1994). When Ga subunits are two major steps—nucleotide exchange (release of GDP in the inactive guanosine 50-diphosphate (GDP)-bound and GTP loading) and GTP hydrolysis to GDP. The conformation, they are readily digested by trypsin, nucleotide exchange step is rate limiting for the steady- whereas upon binding of either AlFÀ (complexes with 4 state GTPase reaction because it is 10–100 times slower GDP to mimic GTP in the G-protein active site) or than the GTP hydrolysis step (Mukhopadhyay and guanosine 50[g-thio]-triphosphate (GTPgS) (a non-hydro- Ross, 2002). For this reason, steady-state GTP hydro- lyzable GTP analog) and adoption of the active con- lysis normally reflects the rate of nucleotide exchange. formation, only a short sequence can be cleaved, and the We found that the steady-state rate of GTP hydrolysis remainder of the protein remains trypsin resistant. Gao (K )ofGao R243H was approximately fourfold faster R234H behaved like wt Gao in that it was hydrolyzed by ss than wt Gao (Figure 1c). The rate of nucleotide trypsinwhenpreloadedwithGDP,butgenerateda exchange (K ) for Gao R243H was calculated based trypsin-resistant form when preloaded with GDP and GTP on the approximation K ¼ 1/K À1/K (Higashijima AlFÀ or GTPgS (Figure 1a). Thus, Gao R234H can GTP ss cat 4 et al., 1987; Makita et al., 2007), assuming that K efficiently adopt the active conformation upon AlFÀ or cat 4 remains unchanged (Figure 1b) and K is increased GTPgS binding. Given that activation by AlFÀ requires ss 4 fourfold (Figure 1c) compared with the constant values the nucleotide-binding site to contain GDP and the reported for wt Gao(Linderet al., 1990). This analysis G protein to be in an appropriate conformation, these predicts that K for Gao R243H is approximately six results also indicate that Gao R234H is properly folded GTP times faster than wt Gao. To validate this result, we and is capable of binding both GDP and GTP. performed GTPgS-binding, which is a direct measure of nucleotide exchange activity, and found that the rate of Intrinsic GTPase activity of Gao R234H is intact GTPgS binding to Gao R243H is approximately six times and is efficiently enhanced by GAPs faster than to wt Gao (Figure 1d). This result demon- Previously reported oncogenic mutations in G proteins strates that the R243H mutation accelerates the rate of have been shown to be GTPase deficient and GAP nucleotide exchange by Gao. The fractional occupancy insensitive (Landis et al., 1989; Berman et al., 1996; Iiri of GTP in Gao R243H was estimated based on the et al., 1998), thereby blocking the G-protein deactiva- approximation 100(KGTP/(KGTP þ Kcat) (Higashijima tion step and rendering it constitutively active. We et al., 1987; Johnston et al., 2007), considering that Kcat investigated whether the GTPase activity and GAP remains unchanged (Figure 1b) and KGTP is increased sensitivity of Gao R243H was similarly affected. sixfold(Figure1d)comparedwithwtGao. This analysis For this, we performed single-turnover GTPase assays predicts that 460% of Gao R243H is loaded with GTP (Berman et al., 1996; Garcia-Marcos et al., 2010) with (active) at steady state, whereas the GTP occupancy in wt purified wt Gao and Gao R243H alone or in the Gao under the same conditions is predicted to be only presence of GAIP/RGS19, a GAP for Gai/o subunits 10%. These results suggest that Gao R243H would behave

Oncogene Molecular insights into Gao R243H oncogenicity M Garcia-Marcos et al 2693

Figure 1 The R243H mutation enhances the rate of nucleotide exchange by GaobuthasnoeffectontheintrinsicorGAP-enhanced GTPase activity of the G protein. (a)TheGao R243H mutant retains the ability to adopt a trypsin-resistant conformation upon activation. Rat Gao(isoform1;Gao1, hereafter referred to as Gao) cloned into pET28b (Garcia-Marcos et al.,2010)wasusedas template to generate the R243H mutant (Quikchange mutagenesis kit, Stratagene, La Jolla, CA, USA). Hexahistidine-tagged wt Gaoand the R243H mutant were purified from BL21(DE3) Escherichia coli as previously described (Garcia-Marcos et al., 2010). A gel filtration chromatographic step (Superdex 200, GE Healthcare, Boston, MA, USA) was substituted for the final steps of dialysis and buffer exchange. Limited trypsin proteolysis was performed as previously described (Garcia-Marcos et al.,2010).WtGao (lanes 1–4) and À R243H mutant (lanes 5–8) (0.5 mg/ml) were incubated at 30 1C in the presence of GDP (30 mM, lanes 2 and 6), GDP Á AlF4 (30 mM AlCl3, 10 mM NaF, lanes 3 and 7) or GTPgS(30mM, lanes 4 and 8) and treated (lanes 2–4 and 6–8) or not (lanes 1 and 4) with trypsin (12.5 mg/ ml) for 10 min as indicated. The reaction was stopped by adding SDS sample buffer and boiling. Proteins (B5 mg) were separated by SDS–polyacrylamide gel electrophoresis and stained with Coomassie blue. One representative experiment out of three is shown. The star (*) denotes the position of the non-trypsinized, full-length proteins loaded (lanes 1 and 4), and the arrowhead denotes the trypsin-resistant form of the Ga subunit in the active conformation (lanes 3, 4, 7 and 8). Both wt Gao and R234H are digested when preloaded with GDP À (lanes 2 and 5), and adopt the trypsin-resistant conformation after incubation with GDP Á AlF4 (lanes 3 and 7) or GTPgS(lanes4and8). (b). The R243H mutation does not affect either the intrinsic or the GAP-enhanced GTPase activity of Gao. Single-turnover GTPase assays were performed exactly as previously described (Garcia-Marcos et al.,2010).WtGao (blue circles) and Gao R243H mutant (red triangles) were preloaded with 4 mM [g-32P]GTP (B100 cpm/fmol) in the absence of Mg2 þ to prevent GTP hydrolysis (Berman et al., 1996). The tubes were cooled on ice, and the reaction started by diluting (10 Â ) the GTP-loaded G proteins (50 nM final concentration) into buffer containing B2mM free Mg2 þ and excess cold GTP in the presence (open symbols, dotted lines) or absence (closed symbols, solid lines) of GST–GAIP (1 mM). Aliquots were removed at the indicated time points, and reactions were stopped with ice-cold 5% 32 (w/v)-activated charcoal in 20 mM H3PO4, pH 3. The amount of [ P]Pi released was quantified by liquid scintillation counting after centrifugation. One representative experiment of three is shown. The rates of GTP hydrolysis by wt Gao (blue closed circles) and Gao R243H (red closed triangles) are virtually identical. GAIP enhances the rate of GTP hydrolysis by wt Gao (blue open circles) and Gao R243H (red open triangles) to a simlar extent. (c)Gao R234H displays increased steady-state GTPase activity. Steady-state GTPase assays were performed as previously described (Garcia-Marcos et al., 2009, 2010). Wt Gao(50nM, blue circles) and Gao R243H mutant (50 nM, red triangles) were incubated in the presence of 500 nM [g-32P]GTP (B50 cpm/fmol) at 30 1C. Duplicate aliquots were removed at the indicated time points, and reactions were stopped with ice-cold 5% (w/v)-activated charcoal in 20 mM H3PO4, pH 3. The amount of 32 [ P]Pi released was quantified by liquid scintillation counting after centrifugation. Results are expressed as mean±s.d. of one representative experiment of three. The steady-state GTPase activity of Gao R243H (red triangles) is increased approximately fourfold compared with wt Gao (blue circles) in the linear region (2–6 min) of the kinetic curve. (d)GTPgS binding to Gao R234H is accelerated. GTPgS-binding assays were performed as previously described (Sternweis and Robishaw, 1984; Garcia-Marcos et al.,2010).WtGao (50 nM, blue circles) and Gao R243H mutant (50 nM, red triangles) were incubated in the presence of 500 nM [35S]GTPgS(B50 cpm/fmol) at 30 1C. Duplicate aliquots were removed at the indicated time points, 2 ml ice-cold wash buffer was added to stop binding of radioactive nucleotide, and the quenched reactions were rapidly passed through BA-85 nitrocellulose filters. Filters were dried and counted. Radioactive counts were normalized to maximal binding and fitted to a non-linear, one phase exponential association curve (solid lines) using Prism 4.0 (GraphPad Software, La Jolla, CA, USA) to determine the rate constant. Results are expressed as mean±s.d. of one representative experiment of three. The rate of GTPgS binding by Gao R243H is approximately sixfold faster than wt Gao.

Oncogene Molecular insights into Gao R243H oncogenicity M Garcia-Marcos et al 2694 as a constitutively active protein in vivo because its acceleratedrateofnucleotideexchangealongwiththe high intracellular GTP:GDP ratio would promote sponta- neous activation of the G protein without the need for guanine nucleotide exchange factors (GEFs). Thus, Gao R243H shares in common with other G-protein oncogenic mutants that it is constitutively active, but differs in that increased activation arises by accelerated nucleotide exchange rather than decreased inactivation by impaired GTPase activity.

Gao R243H enhances signaling pathways required for Gao-mediated cellular transformation Although no direct effector for Gao has been characterized to date, previous reports have demonstrated that constitutively active Q205L mutant enhances Src- dependent activation of signal transducer and activator of transcription 3 (STAT3) to promote neoplastic transformation of NIH3T3 fibroblasts (Ram et al., 2000). Next, we investigated whether constitutively active Gao R243H similarly activates the STAT3/Src signaling pathway because it also induces oncogenic transformation (Kan et al., 2010). NIH3T3 cells were Figure 2 Gao R243H enhances STAT3 and Src activation in NIH3T3 cells. Rat Gao was cloned into pcDNA3.1( þ ) transfected with plasmids encoding for wt Gao, Gao (EcoRI/NotI), and R243H and Q205L mutants were generated R243H or Gao Q205L (as a positive control), and the using a Quikchange mutagenesis kit. NIH3T3 cells were transfected level of STAT3/Src activation was determined by with the indicated plasmids using Genejuice (Novagen, San Diego, quantifying the extent of phosphorylation at Y705 CA, USA) as previously described (Ghosh et al., 2008, 2010) and of STAT3 and Y416 of c-Src. Expression of similar maintained in Dulbecco’s modied Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). After B32 h amounts of Gao R243H or Gao Q205L enhanced the of transfection, the cells were switched to DMEM supplemented levels of pSTAT3 and pSrc approximately threefold, with 2% FBS and cultured overnight. Cells were lysed in sample whereas expression of wt Gao had no significant effect buffer, boiled and proteins separated by SDS–polyacrylamide gel and was similar to vector control (Figure 2a). Phos- electrophoresis. Samples were analyzed by immunoblotting (IB) for Y705 phospho-STAT3 (pSTAT3, rabbit pAb, Cell Signaling, phorylation of Akt and ERK1/2 was unaffected Beverly, MA, USA), total STAT3 (tSTAT3 mouse mAb, Santa (Figure 2a), indicating that the effect of Gao R243H Cruz Biotechnology, Santa Cruz, CA, USA), Y416 phospho-Src and Gao Q205L on signaling was specific for the (pSrc, rabbit pAb, Cell Signaling), total Src (tSrc, mouse mAb, Src–STAT3 pathway. Identical results were obtained in Santa Cruz Biotechnology), phospho-ERK1/2 (pERK1/2, rabbit MDA-MB 231 breast carcinoma cells (data not shown). pAb, Cell Signaling), S473 phospho-Akt (pAkt, rabbit pAb, Cell Signaling) and Gao (rabbit pAb, Santa Cruz Biotechnology). Thus, Gao R243H triggers activation of the Src–STAT3 When the Gao R243H (lane 3) and Gao Q205L (lane 4) mutants pathway and behaves like constitutively active Gao were expressed, the levels of pSTAT3 and pSrc increased in vivo. Because blocking Src-STAT3 activation aboli- approximately three times compared with vector-transfected shes neoplastic transformation by constitutively active controls (lane 1), whereas wt Gao (lane 2) had no effect. There was no effect on Akt and ERK1/2 phosphorylation when the Gao Q205L (Ram et al., 2000), we propose that the con- different Gao constructs were expressed. stitutively active Gao R243H mutant is likely to promote oncogenic transformation (Kan et al., 2010) similarly by 1994). It has been proposed (Bourne, 1997) that this activating the Src–STAT3 pathway. Further investigation residue works as an ‘arginine finger’, that is, interacts is required to unequivocally validate this point. with GTP to facilitate nucleotide hydrolysis, because it structurally and functionally resembles the ‘arginine Structural basis for the constitutive activation promoted finger’ found in GAPs for G proteins of the Ras by the R243H mutation superfamily. We propose that the heterotrimeric Structural and functional analysis of oncogenic mutants G-protein residue corresponding to R243 in Gao be- has unraveled the similarities and differences between haves as yet another ‘arginine finger’ whose primary heterotrimeric G proteins and the Ras superfamily of G function is to stabilize nucleotide binding. This R243 proteins (Sprang, 1997). The Q227L mutation in GNAS ‘finger’ points at E43, which is located in the highly (or the equivalent mutation in other heterotrimeric G conserved P-loop responsible for binding of the a- and proteins) is fully analogous to the oncogenic mutation b-phosphates of the nucleotide (Figure 3a). Based on Q61L found in Ras (Coleman et al., 1994; Kleuss et al., our finding that the R243H mutation facilitates nucleo- 1994), whereas the R201C mutation in GNAS (or the tide exchange without affecting GTP hydrolysis, we equivalent in other Ga subunits) is unique to hetero- propose that this mutation disrupts the electrostatic trimeric G proteins because this residue is not shared contact of R243 with E43 by loss of its negative charge with G proteins of the Ras superfamily (Coleman et al., when mutated to H, and that by doing so destabilizes

Oncogene Molecular insights into Gao R243H oncogenicity M Garcia-Marcos et al 2695 the interaction of the P-loop with the nucleotide which is then released more easily. Importantly, R243 and E43 are highly conserved across Ga subunits but not among members of the Ras superfamily (Figure 3b, left panel), and the electrostatic contact between them is observed in the crystal structures of all Ga subunits resolved to date (Figure 3b, right panel). Thus, like the previously described ‘arginine finger’ in Ga subunits (Bourne, 1997), the R234 ‘arginine finger’ seems to be a unique and conserved feature of heterotrimeric G proteins that is not shared with members of the Ras superfamily. This conserved ‘arginine finger’ has also been investigated in Ga subunits other than Gao. For example, its mutation to Q in the Gaq homolog of Caenorhabditis elegans is known to promote a gain-of-function pheno- type (Doi and Iwasaki, 2002). Subsequent studies have revealed that mutation of the corresponding arginine to E in two different Gat/i chimeras or Gas gives rise to G proteins with different biochemical properties—GTPase deficient (Barren et al., 2006), incompetent for nucleotide binding (Pereira and Cerione, 2005), or facilitated activation by GTPgS (Zurita and Birnbaumer, 2008). These different biochemical properties associated to the same mutation (R-E) were attributed to subtle differences in the molecular environment of the arginine in the different G proteins (Zurita and Birnbaumer, Figure 3 Structural basis for Gao R243H properties. (a) View of 2008). It is therefore not surprising that substitution of the nucleotide-binding site of Gao. The coordinates of Gaowere extracted from the Protein Data Bank ID number 3CK7 and that same critical arginine by a different amino acid visualized using Molsoft ICM Browser (Molsoft, L.L.C., San Diego, (R-H) in a different G protein (Gao) in this current CA, USA). Only key residues and their corresponding segments of study reveals a completely different set of biochemical the backbone chain are depicted for clarity. R179 and Q205 are À properties compared with those described in the above positioned toward AlF4 (small green spheres), which mimics the g-phosphate of GTP in the transition state, to catalyze the GTP to mentioned studies, that is, unaltered GTP hydrolysis GDP þ Pi reaction. R243 points toward E43, which is located in the and enhanced nucleotide exchange. Although the net P-loop (yellow) known to make contact with a-andb-phosphates of result of mutating this conserved ‘arginine finger’ varies, GDP. The negatively charged guanidino group of R243 interacts the findings highlight its importance in controlling the with the negatively charged carboxyl group of E43 located in close activation–deactivation kinetics of Ga subunits. proximity (B4A˚). Disruption of this electrostatic interaction by loss of the negative charge in the R243H mutant is predicted to affect the interaction between the P-loop and the nucleotide, thereby affecting the rate of nucleotide exchange. The large blue sphere represents Conclusions Mg2 þ and the red ribbon corresponds to the a3helix.(b)The electrostatic contact between R243 and E43 is conserved across Ga Taken together, our results provide the molecular basis subunits but not among members of the Ras superfamily. Left, sequence alignment of Ga subunits and Ras-related G proteins. for the oncogenic properties of GaoR243H.GaoR234H Sequences of the indicated proteins were obtained form the NCBI behaves like a constitutively active protein in vitro and database and aligned using CLUSTAL W (http://www.ebi.ac.uk/ in vivo because of the accelerated rate of nucleotide Tools/msa/clustalw2/). Conserved identical residues are shaded in exchange and enhances cellular signaling responsible for black; similar residues in gray. Structural elements (P-loop, oncogenic transformation. This work reveals a new SwIII ¼ switch III, a3 ¼ a-helix 3) indicated above the alignment are named according to the Gao crystal structure. The positions mechanism by which heterotrimeric G proteins can be corresponding to E43 (in red) and R243 (in green) of Gao are turned into oncoproteins by somatic mutations. highly conserved across Ga subunits (with the exception of Gaz, which has biochemical properties different from other Ga subunits (Casey et al., 1990)), but they are not present in Ras and other Conflict of interest members of the Ras superfamily. Right, structural view of the electrostatic contact between E43 and R243 in different Ga subunits. The coordinates of different Ga subunits crystallized to The authors declare no conflict of interest. date were extracted from the following ID numbers of the Protein Data Bank: Gao, 3CK7; Gai1, 1GDD; Gat, 1TAG; Gas, 1AZT; Ga13, 2BCJ; and Gaq, 1SHZ. These structures were overlapped Acknowledgements and displayed using Molsoft ICM Browser following the color code indicated in the black box on the bottom right corner. Only key residues with their corresponding segments of the backbone chain This work was funded by NIH grants CA100768 and DKI7780 are depicted for clarity. The residues corresponding to R243 (to MGF). MGM was supported by a Susan G Komen and E43 in Gao are positioned in close proximity in all the other postdoctoral fellowship KG080079 and PG by Burroughs Ga-subunit crystal structures, indicating that this electrostatic Wellcome Fund and Research Scholar Award (American interaction is highly conserved in heterotrimeric G proteins. Gastroenterology Association FDN).

Oncogene Molecular insights into Gao R243H oncogenicity M Garcia-Marcos et al 2696 References

Barren B, Natochin M, Artemyev NO. (2006). Mutation R238E in Lamba S, Felicioni L, Buttitta F, Bleeker FE, Malatesta S, Corbo V transducin-alpha yields a GTPase and effector-deficient, but not et al. (2009). Mutational profile of GNAQQ209 in human tumors. dominant-negative, G-protein alpha-subunit. Mol Vis 12: 492–498. PLoS One 4: e6833. Berman DM, Wilkie TM, Gilman AG. (1996). GAIP and RGS4 are Landis CA, Masters SB, Spada A, Pace AM, Bourne HR, Vallar L. GTPase-activating proteins for the Gi subfamily of G protein alpha (1989). GTPase inhibiting mutations activate the alpha chain of Gs subunits. Cell 86: 445–452. and stimulate adenylyl cyclase in human pituitary tumours. Nature Bourne HR. (1997). G proteins. The arginine finger strikes again. 340: 692–696. Nature 389: 673–674. Linder ME, Ewald DA, Miller RJ, Gilman AG. (1990). Purification Casey PJ, Fong HK, Simon MI, Gilman AG. (1990). Gz, a guanine and characterization of Go alpha and three types of Gi alpha after nucleotide-binding protein with unique biochemical properties. expression in Escherichia coli. J Biol Chem 265: 8243–8251. J Biol Chem 265: 2383–2390. Lyons J, Landis CA, Harsh G, Vallar L, Grunewald K, Feichtinger H Coleman DE, Berghuis AM, Lee E, Linder ME, Gilman AG, Sprang et al. (1990). Two G protein oncogenes in human endocrine tumors. SR. (1994). Structures of active conformations of Gi alpha 1 and the Science 249: 655–659. mechanism of GTP hydrolysis. Science 265: 1405–1412. Makita N, Sato J, Rondard P, Fukamachi H, Yuasa Y, Aldred MA De Vries L, Mousli M, Wurmser A, Farquhar MG. (1995). GAIP, a et al. (2007). Human G(salpha) mutant causes pseudohypopar- protein that specifically interacts with the trimeric G protein G athyroidism type Ia/neonatal diarrhea, a potential cell-specific alpha i3, is a member of a protein family with a highly conserved role of the palmitoylation cycle. Proc Natl Acad Sci USA 104: core domain. Proc Natl Acad Sci USA 92: 11916–11920. 17424–17429. Doi M, Iwasaki K. (2002). Regulation of retrograde signaling at Marinissen MJ, Gutkind JS. (2001). G-protein-coupled receptors and neuromuscular junctions by the novel C2 domain protein AEX-1. signaling networks: emerging paradigms. Trends Pharmacol Sci 22: Neuron 33: 249–259. 368–376. Downward J. (2003). Targeting RAS signalling pathways in cancer Mukhopadhyay S, Ross EM. (2002). Quench-flow kinetic measure- therapy. Nat Rev Cancer 3: 11–22. ment of individual reactions of G-protein-catalyzed GTPase cycle. Farfel Z, Bourne HR, Iiri T. (1999). The expanding spectrum of G Methods Enzymol 344: 350–369. protein diseases. N Engl J Med 340: 1012–1020. Pereira R, Cerione RA. (2005). A switch 3 point mutation in the alpha Forbes SA, Bhamra G, Bamford S, Dawson E, Kok C, Clements J subunit of transducin yields a unique dominant-negative inhibitor. et al. (2008). The Catalogue of Somatic Mutations in Cancer J Biol Chem 280: 35696–35703. (COSMIC). Curr Protoc Hum Genet Chapter 10: Unit 10.11. Radhika V, Dhanasekaran N. (2001). Transforming G proteins. Garcia-Marcos M, Ghosh P, Farquhar MG. (2009). GIV is a Oncogene 20: 1607–1614. nonreceptor GEF for G alpha i with a unique motif that regulates Ram PT, Horvath CM, Iyengar R. (2000). Stat3-mediated transforma- Akt signaling. Proc Natl Acad Sci USA 106: 3178–3183. tion of NIH-3T3 cells by the constitutively active Q205L Galphao Garcia-Marcos M, Ghosh P, Ear J, Farquhar MG. (2010). A protein. Science 287: 142–144. structural determinant that renders G alpha(i) sensitive to activation Sprang SR. (1997). G protein mechanisms: insights from structural by GIV/girdin is required to promote cell migration. J Biol Chem analysis. Annu Rev Biochem 66: 639–678. 285: 12765–12777. Sternweis PC, Robishaw JD. (1984). Isolation of two proteins with Ghosh P, Beas AO, Bornheimer SJ, Garcia-Marcos M, Forry EP, high affinity for guanine nucleotides from membranes of bovine Johannson C et al. (2010). A G{alpha}i-GIV molecular complex brain. J Biol Chem 259: 13806–13813. binds epidermal growth factor receptor and determines whether cells Van Raamsdonk CD, Bezrookove V, Green G, Bauer J, Gaugler L, migrate or proliferate. Mol Biol Cell 21: 2338–2354. O’Brien JM et al. (2009). Frequent somatic mutations of GNAQ in Ghosh P, Garcia-Marcos M, Bornheimer SJ, Farquhar MG. (2008). uveal melanoma and blue naevi. Nature 457: 599–602. Activation of Galphai3 triggers cell migration via regulation of Vara Prasad MV, Shore SK, Dhanasekaran N. (1994). Activated GIV. J Cell Biol 182: 381–393. mutant of G alpha 13 induces Egr-1, c-fos, and transformation in Grishina G, Berlot CH. (1998). Mutations at the domain interface of NIH 3T3 cells. Oncogene 9: 2425–2429. GSalpha impair receptor-mediated activation by altering receptor Voyno-Yasenetskaya TA, Pace AM, Bourne HR. (1994). Mutant and guanine nucleotide binding. J Biol Chem 273: 15053–15060. alpha subunits of G12 and G13 proteins induce neoplastic Higashijima T, Ferguson KM, Smigel MD, Gilman AG. (1987). The transformation of Rat-1 fibroblasts. Oncogene 9: 2559–2565. effect of GTP and Mg2+ on the GTPase activity and the Warner DR, Weinstein LS. (1999). A mutation in the heterotrimeric fluorescent properties of Go. J Biol Chem 262: 757–761. stimulatory guanine nucleotide binding protein alpha-subunit with Iiri T, Farfel Z, Bourne HR. (1998). G-protein diseases furnish a model impaired receptor-mediated activation because of elevated GTPase for the turn-on switch. Nature 394: 35–38. activity. Proc Natl Acad Sci USA 96: 4268–4272. Johnston CA, Taylor JP, Gao Y, Kimple AJ, Grigston JC, Chen JG Wong YH, Chan JS, Yung LY, Bourne HR. (1995). Mutant alpha et al. (2007). GTPase acceleration as the rate-limiting step in subunit of Gz transforms Swiss 3T3 cells. Oncogene 10: 1927–1933. Arabidopsis G protein-coupled sugar signaling. Proc Natl Acad Sci Xu N, Bradley L, Ambdukar I, Gutkind JS. (1993). A mutant alpha USA 104: 17317–17322. subunit of G12 potentiates the eicosanoid pathway and is highly Kan Z, Jaiswal BS, Stinson J, Janakiraman V, Bhatt D, Stern HM oncogenic in NIH 3T3 cells. Proc Natl Acad Sci USA 90: et al. (2010). Diverse somatic mutation patterns and pathway 6741–6745. alterations in human cancers. Nature 466: 869–873. Xu N, Voyno-Yasenetskaya T, Gutkind JS. (1994). Potent transform- Kleuss C, Raw AS, Lee E, Sprang SR, Gilman AG. (1994). ing activity of the G13 alpha subunit defines a novel family of Mechanism of GTP hydrolysis by G-protein alpha subunits. oncogenes. Biochem Biophys Res Commun 201: 603–609. Proc Natl Acad Sci USA 91: 9828–9831. Zurita AR, Birnbaumer L. (2008). The same mutation in Gsalpha and Kroll SD, Chen J, De Vivo M, Carty DJ, Buku A, Premont RT et al. transducin alpha reveals behavioral differences between these highly (1992). The Q205LGo-alpha subunit expressed in NIH-3T3 cells homologous G protein alpha-subunits. Proc Natl Acad Sci USA induces transformation. J Biol Chem 267: 23183–23188. 105: 2363–2368.

Oncogene