Biochemical Clustering of Monomeric GTPases of the Ras Superfamily

To date phylogeny has been used to compare entire families of proteins based on their nucleotide or amino acid sequence. Here we developed a novel analytical platform allowing a systematic comparison of protein families based on their biochemical properties. This approach was validated on the Rho subfamily of GTPases. We used two high throughput methods, referred to as AlphaScreen™ and FlashPlate®, to measure nucleotide binding capacity, exchange, and hydrolysis activities of small monomeric GTPases. These two technologies have the characteristics to be very sensitive and to allow homogenous and high throughput assays. To analyze and integrate the data obtained, we developed an algorithm that allows the classification of GTPases according to their enzymatic activities. Integration and hierarchical clustering of these results revealed unexpected features of the small Rho GTPases when compared with primary sequence-based trees. Hence we propose a novel phylobiochemical classification of the Ras superfamily of GTPases. To date phylogeny has been used to compare entire families of proteins based on their nucleotide or amino acid sequence. Here we developed a novel analytical platform allowing a systematic comparison of protein families based on their biochemical properties. This approach was validated on the Rho subfamily of GTPases. We used two high throughput methods, referred to as AlphaScreen™ and FlashPlate®, to measure nucleotide binding capacity, exchange, and hydrolysis activities of small monomeric GTPases. These two technologies have the characteristics to be very sensitive and to allow homogenous and high throughput assays. To analyze and integrate the data obtained, we developed an algorithm that allows the classification of GTPases according to their enzymatic activities. Integration and hierarchical clustering of these results revealed unexpected features of the small Rho GTPases when compared with primary sequence-based trees. Hence we propose a novel phylobiochemical classification of the Ras superfamily of GTPases. Ras GTPases are small GTP-binding proteins involved in diverse cellular processes such as apoptosis, cell proliferation/differentiation, cytoskeleton reorganization, and membrane trafficking. These proteins cycle between an inactive GDP-bound form and an active GTP-bound form. Under their GTP-bound form their active conformation allows them to interact with effector molecules and to generate specific biological responses (1Takai Y. Sasaki T. Matozaki T. Small GTP-binding proteins.Physiol. Rev. 2001; 81: 153-208Google Scholar). Two intrinsic enzymatic activities regulate the balance between GTP-bound and GDP-bound conformations: 1) GDP/GTP exchange and 2) GTP hydrolysis activities (2Colicelli J. Human RAS superfamily proteins and related GTPases.Sci. STKE. 2004; 250: RE13Google Scholar). Whereas the intrinsic enzymatic activities have been characterized for a few of these proteins, such studies were carried out in non-systematic manners and by different research groups (3Kontani 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: 41070-41078Google Scholar, 4Trahey M. Milley R.J. Cole G.E. Innis M. Paterson H. Marshall C.J. Hall A. McCormick F. Biochemical and biological properties of the human N-ras p21 protein.Mol. Cell. Biol. 1987; 7: 541-544Google Scholar, 5Shao H. Kadono-Okuda K. Finlin B.S. Andres D.A. Biochemical characterization of the Ras-related GTPases Rit and Rin.Arch. Biochem. Biophys. 1999; 371: 207-219Google Scholar, 6Haeusler L.C. Blumenstein L. Stege P. Dvorsky R. Ahmadian M.R. Comparative functional analysis of the Rac GTPases.FEBS Lett. 2003; 555: 556-560Google Scholar). The analysis of full genome sequences has allowed large scale biology experimental approaches consisting of systematic characterization of entire families of proteins instead of investigating them individually. Developing such “functional proteomic” approaches requires the design of high throughput and versatile experimental procedures (7Luan C.H. Qiu S. Finley J.B. Carson M. Gray R.J. Huang W. Johnson D. Tsao J. Reboul J. Vaglio P. Hill D.E. Vidal M. Delucas L.J. Luo M. High-throughput expression of C. elegans proteins.Genome Res. 2004; 14: 2102-2110Google Scholar, 8Brasch M.A. Hartley J.L. Vidal M. ORFeome cloning and systems biology: standardized mass production of the parts from the parts-list.Genome Res. 2004; 14: 2001-2009Google Scholar). For the study of small G proteins, conventional filtration assays, although powerful at small scale, are not appropriate for systematic studies. Indeed this methodology has a low to very low throughput, is not versatile, and requires large amounts of reagents. To overcome these issues and characterize GTP-binding proteins of the Ras superfamily in a systematic manner, we developed a robust assay platform using two well adopted high throughput technologies, AlphaScreen™ (9Beaudet L. Bedard J. Breton B. Mercuri R.J. Budarf M.L. Homogeneous assays for single-nucleotide polymorphism typing using AlphaScreen.Genome Res. 2001; 11: 600-608Google Scholar) and FlashPlate® (10Mullinax T.R. Henrich G. Kasila P. Ahern D.G. Wenske E.A. Hou C. Argentieri D. Bembenek M.E. Monitoring inositol-specific phospholipase C activity using a phospholipid FlashPlate®.J. Biomol. Screen. 1999; 4: 151-155Google Scholar). AlphaScreen is a bead-based non-radioactive and homogenous proximity assay used to measure interaction between biological binding partners. The principle of this technology relies on the use of a Donor bead and an Acceptor bead that generate a light signal when brought into proximity (<200 nm). Upon laser excitation at 680 nm, the Donor beads, containing a photosensitizer, will generate short lived singlet oxygen that can diffuse only a short distance before returning to the ground state. The Acceptor beads, containing chemiluminescers and fluorophores, will react with this singlet oxygen and will emit an amplified light signal measurable at ∼600 nm. AlphaScreen (PerkinElmer) provides highly versatile, sensitive, and homogeneous assays that allow us to perform studies at a higher throughput and at a lower cost. FlashPlates are white polystyrene microplates in which the interior of wells is coated with a thin layer of polystyrene-based scintillation reagent. The principle of this homogeneous radiometric technology is based on proximity between a radioligand and the scintillation reagent. A target protein, such as antibodies or GSH, is also labeled on the wall of the plate and will bring the protein of interest close to the scintillation reagent. The interaction between the radioisotopes and the proteins of interest will activate the scintillation reagent and emit a luminescent signal. FlashPlate (PerkinElmer) technology reduces considerably the amount of reagent to use and eliminates the time-consuming washes normally needed to separate bound from unbound radioligands. In this study we took advantage of these two technologies to characterize in a systematic manner four biochemical properties of the Ras family of small G proteins and we reclassified them according to their activities instead of their primary amino acid sequences. Due to their high level of interspecies conservation, we selected the Rho GTPases as prototype proteins to develop and validate our assays. [γ-35S]GTPγS and [γ-33P]GTP were purchased form PerkinElmer Life Sciences. ATP, GDP, GTP, and GTPγS 1The abbreviations used are: GTPγS, guanosine 5′-3-O-(thio)triphosphate; b-GDP, biotinylated GDP; SB, sequence-based; b-GTPγS, biotinylated GTPγS; PEG, polyethylene glycol; NHS, N-hydroxysuccinimide; UPGMA, unweighted pair-group method arithmetic averaged; mRAC-1, mouse RAC-1. were purchased form Sigma. Goat anti-GST antibodies were from Amersham Biosciences. Caenorhabditiselegans CDC-42 (RO7G3.1), RAC-2 (KO3D3.10b), CED-10 (C09G12.8b), MIG-2 (C35C5.4), and RHO-1 (Y51H4A.3) plus one putative uncharacterized GTPase, CRP-1 (Y32F6B.3), were obtained from the C. elegans ORFeome (11Reboul J. Vaglio P. Rual J.F. Lamesch P. Martinez M. Armstrong C.M. Li S. Jacotot L. Bertin N. Janky R. Moore T. Hudson Jr., J.R. Hartley J.L. Brasch M.A. Vandenhaute J. Boulton S. Endress G.A. Jenna S. Chevet E. Papasotiropoulos V. Tolias P.P. Ptacek J. Snyder M. Huang R. Chance M.R. Lee H. Doucette-Stamm L. Hill D.E. Vidal M. C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression.Nat. Genet. 2003; 34: 35-41Google Scholar). The mRAC-1 and mRAC-1N17 were described previously (12Liu J.F. Chevet E. Kebache S. Lemaitre G. Barritault D. Larose L. Crepin M. Functional Rac-1 and Nck signaling networks are required for FGF-2-induced DNA synthesis in MCF-7 cells.Oncogene. 1999; 18: 6425-6433Google Scholar). The above ORFs were recombined in a bacterial expression vector (pGEX-2TK) to produce N-terminally tagged GST fusion proteins using the Gateway technology (Invitrogen). GTPγS and GDP were biotinylated, respectively, using biotin-PEG-maleinate (Pierce) dissolved in MES, pH 6, and biotin-PEG-COO-NHS (Pierce) dissolved in carbonate buffer, pH 8.5, according to the manufacturer’s instructions. For both nucleotides, reaction mixtures were incubated for 2 h at 37 °C and purified using HPLC. Concentrations were measured by optical density at 260 nm. Biotinylated nucleotides were validated for their biological properties toward GTPases (data not shown). Bacterial recombinant GST-GTPases were expressed and purified as described previously (13Braun P. Hu Y. Shen B. Halleck A. Koundinya M. Harlow E. LaBaer J. Proteome-scale purification of human proteins from bacteria.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2654-2659Google Scholar). AlphaScreen assays were performed in Costar 384-well microplates in a final reaction volume of 25 μl. Streptavidin Donor beads and protein G Acceptor beads (PerkinElmer BioSignal) were used at a final concentration of 0.02 mg/ml per well. The assays were performed in AlphaScreen buffer (50 mm HEPES, pH 7.4, 100 mm NaCl, 5 mm MgCl2, 1 mm DTT, and 1 mg/ml BSA). All incubations were performed at 23 °C. Laser excitations were carried out at 680 nm, and readings were performed at 520–620 nm using an AlphaQuest reader (Packard). During purification, GST-GTPases were unloaded from their intrinsic nucleotide by adding 5 mm EDTA in lysate before purification. Different concentrations of nucleotides (ranging from 0.4 nm to 3 μm) were incubated with a 30 nm concentration of unloaded GST-GTPases and a 30 nm concentration of a biotinylated derivative of GTPγS (b-GTPγS). 25 nm goat anti-GST antibody (Amersham Biosciences) was added and incubated for 20 min. Donor and Acceptor beads were added simultaneously and incubated for 1 h before reading. Purified GST-GTPases were preloaded with a biotinylated derivative of GDP (b-GDP) as follows. 150 nm proteins were incubated with 100 nm b-GDP in loading buffer (25 mm Tris, pH 7.4, 100 mm NaCl, 0.1 mm DTT, and 5 mm EDTA) for 30 min at 30 °C. Reactions were then placed on ice, and 10 mm MgCl2 was added. 5 μl of preloaded GST-GTPases (final concentration, 30 nm) were then added in microplate wells with different concentration of unlabeled GTP (ranging from 0.4 nm to 3 μm). The plate was incubated for 20 min, and 25 nm goat anti-GST (Amersham Biosciences) was added. After 20 min of incubation, Donor and Acceptor beads were added simultaneously, and the plate was analyzed after 1 h of incubation at 23 °C. These assays were performed in glutathione-coated 96-wells FlashPlates (PerkinElmer). Bacterial lysates containing GST-GTPases were diluted with PBS (50 μl) and incubated in the FlashPlate for 45 min at 4 °C in the presence of 5 mm EDTA to allow the binding of GST-GTPases on the GSH plate and to unload the protein from its intrinsic nucleotide. The lysate was removed, and the wells were washed two times with loading buffer (20 mm Tris, pH 7.4, 100 mm NaCl, and 0.1 mm DTT). Then 50 μl of loading buffer containing different concentrations of [γ-35S]GTPγS (20–1000 nCi) were added. The signal produced was monitored on a β scintillation counter (Packard) every 5 min until the plateau was The plate was placed on ice, and 10 mm MgCl2 was added. The reaction was then from the and 50 μl of buffer (20 mm Tris, pH 7.4, 0.1 mm DTT, 5 mm MgCl2, 1 mg/ml and 100 were added. was monitored on a β scintillation counter (Packard) every 5 min for 30 min to measure nucleotide Bacterial lysate containing EDTA was by 50 μl of loading buffer (20 mm Tris, pH 7.4, 100 mm NaCl, and 0.1 mm containing of [γ-35S]GTPγS or of The interaction between GTPases and these was carried out for 30 min after which 10 mm MgCl2 was added. The reaction was then by 50 μl of hydrolysis buffer (20 mm Tris, pH 7.4, 100 mm NaCl, 5 mm MgCl2, 1 mm DTT, and 1 and the was monitored for min using a β scintillation counter (Packard). C. elegans amino acid sequences were using form. were from the using according to the The hierarchical clustering was then using the based on the method and using Biochemical obtained for were and 1) and used to generate distance based on or in These were based on and hierarchical clustering were then using the based on the method and using comparison was carried out using The ORFeome which of the cloning of ORFs from the to the from genome are the of biology The C. elegans ORFeome is one of the and in (11Reboul J. Vaglio P. Rual J.F. Lamesch P. Martinez M. Armstrong C.M. Li S. Jacotot L. Bertin N. Janky R. Moore T. Hudson Jr., J.R. Hartley J.L. Brasch M.A. Vandenhaute J. Boulton S. Endress G.A. Jenna S. Chevet E. Papasotiropoulos V. Tolias P.P. Ptacek J. Snyder M. Huang R. Chance M.R. Lee H. Doucette-Stamm L. Hill D.E. Vidal M. C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression.Nat. Genet. 2003; 34: 35-41Google Scholar). In C. have been to Rho and the amino acid sequences were and compared with the mouse protein as in The ORFs were from the C. elegans ORFeome P. S. T. J. Li N. R. S. Doucette-Stamm L. Vandenhaute J. Hill D.E. Vidal M. C. elegans ORFeome version the of ORFeome with Res. 2004; 14: Scholar, J. Vaglio P. N. N. Moore T. C. T. Y. D. J. Lee H. J. Doucette-Stamm L. Hartley J.L. Brasch M.A. Vandenhaute J. Lamesch Hill D.E. Vidal M. the of at in C. Genet. 2001; Scholar) and expressed as N-terminally tagged GST fusion proteins in The GST fusion proteins were expressed at high and purified to these small G proteins, we developed four distinct assays to measure their for nucleotides as well as their exchange and hydrolysis activities. The two assays, developed using both FlashPlates and at the of small G proteins for GTP and FlashPlates were used to perform binding assays concentrations of [γ-35S]GTPγS were incubated in the presence of a amount of GST was also used as a [γ-35S]GTPγS with the proteins with the β from activate the scintillation reagent in the of the A luminescent signal is then and in the signal to the concentration and a to the concentration of [γ-35S]GTPγS to of the signal was measured in a using CDC-42 nm). obtained with the C. elegans putative Rho GTPases and were then compared to a of GTP binding In the well characterized mRAC-1 (12Liu J.F. Chevet E. Kebache S. Lemaitre G. Barritault D. Larose L. Crepin M. Functional Rac-1 and Nck signaling networks are required for FGF-2-induced DNA synthesis in MCF-7 cells.Oncogene. 1999; 18: 6425-6433Google Scholar, L. E. R.J. R. J.R. a with high intrinsic activity and distinct biochemical J. Biochem. Scholar) and the GTP mRAC-1N17 of cell by a ras protein with for Cell. Biol. Scholar) were used as results that these proteins in two groups according to their toward on one and mRAC-1 a high [γ-35S]GTPγS binding with from to nm, and on the and mRAC-1N17 a low for [γ-35S]GTPγS with from to nm The results obtained with mRAC-1N17 were with that this low GTP of cell by a ras protein with for Cell. Biol. Scholar). was to develop assays to specific nucleotide These assays were carried out using both AlphaScreen and results for CDC-42 for GTP or GDP To compare the C. elegans Rho we used AlphaScreen of its higher throughput and its non-radioactive In this was produced and used as a binding to GST-GTPases as described In our the binding of to GST-GTPases anti-GST Acceptor beads into proximity of Donor beads allowing the of an AlphaScreen signal are by adding concentrations of unlabeled nucleotides, which binding to A signal is then with GTP were used to generate which are an to the GTP concentration of signal in GTP the binding of to CDC-42 with an of 45 nm. specific for GTPases were then measured for mRAC-1N17 for which an signal was to its low for the were compared Rho GTPases in the two groups previously with binding studies using the Rho GTPases from to nm, the Rho GTPases from to nm was used as a nucleotide was not to the binding of to the GTPases. In the assays, GST was as to measure A assay to measure the GDP/GTP exchange activity was developed using GST-GTPases were with b-GDP and incubated with concentrations of Donor and Acceptor beads were and the exchange of b-GDP to GTP was measured as described described above for the binding exchange of b-GDP to GTP to a signal The exchange activity was also characterized by an obtained when concentrations of GTP were used to the binding of b-GDP to a Rho such as CDC-42 The to the concentration of GTP for of the b-GDP To compare exchange activities for the specific activity of the GTPases was and a amount of was used in the of the exchange activities by the Rho GTPases that and mRAC-1 have high exchange activities with from to nm, MIG-2 and mRAC-1N17 lower exchange activities of and nm, with their lower for GTP exchange activity was for This is to the lower of this protein to to measure the of the GTPases for nucleotides using assay in revealed that CRP-1 a lower for the GTP GDP These results were with the data previously obtained using filtration assay and that CRP-1 a very low exchange activity and characteristics S. A. M. Li S. R. Reboul R. Vidal M. R. Chevet E. of membrane by a novel protein in C. elegans Biol. Cell. Scholar). A assay was developed with FlashPlates to measure GTP hydrolysis GTPases were with and the level of with the GTPases was measured as a of The signal is produced from a of hydrolysis of the [γ-33P]GTP and of the nucleotide. To for the GTP from the was measured after loading with a GTP The measured is the of binding to the Rho GTPases. This to the required to hydrolysis after signal to for nucleotide A using CDC-42 revealed that this protein has a high hydrolysis activity In our and mRAC-1 hydrolysis activities with from to min, RHO-1 and CRP-1 in with of and min, results were with in the for Rho GTPases using conventional filtration assays 1 and W. L. A of the ras the from and analysis of of and biochemical characterization of the Biol. Chem. Scholar). To compare and integrate our we hierarchical clustering based on the biochemical of the proteins from our the enzymatic based on GTP binding and GTPγS the lower of MIG-2 and CRP-1 for These two GTPases in a separate from the of the GTPases 6, A and the exchange that MIG-2 from the and the hydrolysis the of CRP-1 and RHO-1 from the of the GTPases these with the that CRP-1 and RHO-1 are the GTPases with the which is for GTP hydrolysis We then that different biochemical characteristics be of the amino acid sequence-based in the of families of proteins, and the sequence-based be to the of biochemical characteristics in the trees. the hierarchical clustering the four biochemical properties of Rho GTPases was compared with the amino acid sequence-based we as the distance was when the was compared with the based on biochemical characteristics In the GST was used as a for For GST out of the Rho GTPases family the of We then the distance between the and based on biochemical properties of the Rho GTPases or four the between the and biochemical were when only two biochemical characteristics were and the distance to the was obtained when the for GTP was with the hydrolysis the large of amino acid involved in two In the that the exchange activity to an in distance to the that the small of involved in this not be the Rho our results that sequence-based only a classification of the Rho GTPases. In this we developed four different high throughput enzymatic assays to characterize the biochemical properties of small G To the by ORFeome scale we used two well high throughput technologies, and we developed the allowing the analysis of the The allowed us to measure GTP and exchange activity for the small G proteins with a very high with and at low cost. In to allowing the characterization of the of GTPases for different nucleotides, this technology a powerful to characterize such as that have properties toward the AlphaScreen platform was not to measure GTP hydrolysis we for the FlashPlate technology to measure [γ-35S]GTPγS binding as well as the [γ-33P]GTP hydrolysis compared with filtration methods, FlashPlate a high throughput to in a homogenous manner the hydrolysis activity of small G We used AlphaScreen and FlashPlate in to the entire of small G protein enzymatic activities. results were obtained for C. elegans and two mouse GTPases of the Rho In the biochemical activities of CED-10 and its mouse mRAC-1 revealed that these two proteins for GTP as well as GDP/GTP exchange and GTP hydrolysis activities only in their amino acid activities obtained for mRAC-1, and CDC-42 were to described in the H. Kadono-Okuda K. Finlin B.S. Andres D.A. Biochemical characterization of the Ras-related GTPases Rit and Rin.Arch. Biochem. Biophys. 1999; 371: 207-219Google Scholar, L. E. R.J. R. J.R. a with high intrinsic activity and distinct biochemical J. Biochem. Scholar, S. A. M. Li S. R. Reboul R. Vidal M. R. Chevet E. of membrane by a novel protein in C. elegans Biol. Cell. Scholar, W. L. The from of Biol. Chem. Scholar, and characterization of A GTP-binding protein that human Biol. Chem. Scholar). These that our novel approach provides results in to the data previously from these results are between proteins, our experimental platform allows a high of the enzymatic activities of the small G protein, and our assays are sensitive and to enzymatic between of the Ras superfamily of This analytical platform have been the an analysis of our experimental We hierarchical amino acid proximity between groups of a we hierarchical clustering the distance between protein groups based on their biochemical The comparison of the amino acid sequence-based and the allowed us to that only the functional characteristics of small G In we were to that the amino acid sequence-based the of two biochemical the for GTP and the GTP hydrolysis our analytical platform allowed us to propose an classification of the Rho GTPases based on their enzymatic In this we and a platform AlphaScreen and FlashPlate technologies to the enzymatic characteristics of small GTPases of the Ras The of of these assays was by using the subfamily of Rho GTPases from both and C. elegans This platform to for to study the between phylogeny and functional characteristics by a study of different proteins from the the this of platform be to the for the of The of specific characteristics such as exchange or hydrolysis activities a specific of this We the Chevet for

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