Alternative Splicing of Rac1 Generates Rac1b, a Self-activating GTPase
2004; Elsevier BV; Volume: 279; Issue: 6 Linguagem: Inglês
10.1074/jbc.m310281200
ISSN1083-351X
AutoresDennis Fiegen, Lars-Christian Haeusler, Lars Blumenstein, Ulrike Herbrand, Radovan Dvorský, Ingrid R. Vetter, Mohammad Reza Ahmadian,
Tópico(s)Cell Adhesion Molecules Research
ResumoRac1b was recently identified in malignant colorectal tumors as an alternative splice variant of Rac1 containing a 19-amino acid insertion next to the switch II region. The structures of Rac1b in the GDP- and the GppNHp-bound forms, determined at a resolution of 1.75 Å, reveal that the insertion induces an open switch I conformation and a highly mobile switch II. As a consequence, Rac1b has an accelerated GEF-independent GDP/GTP exchange and an impaired GTP hydrolysis, which is restored partially by GTPase-activating proteins. Interestingly, Rac1b is able to bind the GTPase-binding domain of PAK but not full-length PAK in a GTP-dependent manner, suggesting that the insertion does not completely abolish effector interaction. The presented study provides insights into the structural and biochemical mechanism of a self-activating GTPase. Rac1b was recently identified in malignant colorectal tumors as an alternative splice variant of Rac1 containing a 19-amino acid insertion next to the switch II region. The structures of Rac1b in the GDP- and the GppNHp-bound forms, determined at a resolution of 1.75 Å, reveal that the insertion induces an open switch I conformation and a highly mobile switch II. As a consequence, Rac1b has an accelerated GEF-independent GDP/GTP exchange and an impaired GTP hydrolysis, which is restored partially by GTPase-activating proteins. Interestingly, Rac1b is able to bind the GTPase-binding domain of PAK but not full-length PAK in a GTP-dependent manner, suggesting that the insertion does not completely abolish effector interaction. The presented study provides insights into the structural and biochemical mechanism of a self-activating GTPase. The small GTPase Rac acts as a binary molecular switch that cycles between an inactive GDP-bound state and an active GTP-bound state in response to a variety of extracellular stimuli. The interconversion between both states is controlled by nucleotide exchange and GTP hydrolysis. The structures of several GTPases in either state revealed that the switching mechanism depends on the conformational change of two regions, termed switch I and switch II (1Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1395) Google Scholar, 2Corbett K.D. Alber T. Trends Biochem. Sci. 2001; 26: 706-710Abstract Full Text Full Text PDF Scopus (79) Google Scholar). The switch regions consequently provide a surface that is in the GTP-bound state specifically recognized by downstream effectors (1Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1395) Google Scholar, 2Corbett K.D. Alber T. Trends Biochem. Sci. 2001; 26: 706-710Abstract Full Text Full Text PDF Scopus (79) Google Scholar, 3Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1682) Google Scholar) and GTPase-activating proteins (GAPs), 1The abbreviations used are: GAPGTPase-activating proteinGEFguanine nucleotide exchange factorGDIguanine nucleotide dissociation inhibitorDH-PHdouble homology-pleckstrin homologyGSTglutathione S-transferaseHPLChigh pressure liquid chromatographyGppNHpquanosine 5′-β, γ-imidotriphosphateGBDGTPase-binding domain. accelerating the slow intrinsic GTP hydrolysis reaction (1Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1395) Google Scholar, 4Gamblin S.J. Smerdon S.J. Curr. Opin. Struct. Biol. 1998; 8: 195-201Crossref PubMed Scopus (65) Google Scholar, 5Scheffzek K. Ahmadian M.R. Wittinghofer A. Trends Biochem. Sci. 1998; 23: 257-262Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 6Moon S.Y. Zheng Y. Trends Cell Biol. 2003; 13: 13-22Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar). After GTP hydrolysis, release of the cleaved γ-phosphate allows the switch regions to relax into the GDP conformation. Guanine nucleotide exchange factors (GEFs), stimulating the GDP/GTP exchange, bind independently of the nucleotide-bound state (1Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1395) Google Scholar, 7Hoffman G.R. Cerione R.A. FEBS Lett. 2002; 513: 85-91Crossref PubMed Scopus (117) Google Scholar), whereas guanine nucleotide dissociation inhibitors (GDIs), which sequester the GTPase from the membrane into the cytoplasm, interact only with the GDP-bound state (8Olofsson B. Cell Signal. 1999; 11: 545-554Crossref PubMed Scopus (412) Google Scholar). GTPase-activating protein guanine nucleotide exchange factor guanine nucleotide dissociation inhibitor double homology-pleckstrin homology glutathione S-transferase high pressure liquid chromatography quanosine 5′-β, γ-imidotriphosphate GTPase-binding domain. Rac1b was discovered in human tumors as an alternative splice variant of Rac1 containing a 19-amino acid insertion (between codons 75 and 76) at the end of the switch II region (9Jordan P. Brazao R. Boavida M.G. Gespach C. Chastre E. Oncogene. 1999; 18: 6835-6839Crossref PubMed Scopus (206) Google Scholar, 10Schnelzer A. Prechtel D. Knaus U. Dehne K. Gerhard M. Graeff H. Harbeck N. Schmitt M. Lengyel E. Oncogene. 2000; 19: 3013-3020Crossref PubMed Scopus (341) Google Scholar). It has been suggested that the insertion may create a novel effector-binding site in Rac1b and thus participate in signaling pathways related to the neoplastic growth of the intestinal mucosa (9Jordan P. Brazao R. Boavida M.G. Gespach C. Chastre E. Oncogene. 1999; 18: 6835-6839Crossref PubMed Scopus (206) Google Scholar). Most recently, it has been shown that Rac1b does not interact with Rho-GDI and PAK1 and is not involved in lamellipodia formation but able to activate the transcription factor NF-κB (11Matos P. Collard J.G. Jordan P. J. Biol. Chem. 2003; 278: 50442-50448Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). We tried to address what influence this insertion might have on the structure and the biochemical properties of Rac1b in comparison with Rac1. Therefore, we solved the crystal structures of Rac1b in the GDP- and GppNHp-bound conformations at 1.75 Å resolution. Furthermore we investigated nucleotide binding and hydrolysis of Rac1b and studied its regulation by the RacGEF Tiam1 and p50GAP and its interaction with the downstream effector PAK. The Rac1b structures explain the drastic changes of the biochemical properties of Rac1b, namely a dramatic decrease in nucleotide affinity and GTP hydrolysis. The presented data identify Rac1b as a predominantly GTP-bound form of Rac1. Plasmids—The pcDNA3-FLAG constructs of human Rac1b, Rac1, and the respective constitutive active Rac1(G12V) mutant were generated by PCR and cloned via BamHI and EcoRI restriction sites. Rac1, Rac1ΔC (1-184), Rac1b, and Rac1bΔC (1-201) were cloned in pGEX4T1, using BamHI and EcoRI restriction sites. The DH-PH domain of Tiam1 (1033-1404) was cloned into pGEX4T1 using BamHI and XhoI restriction sites. The coding region of Tiam1 contains an internal BamHI site that was removed for the cloning procedure. pGEX-PAK1-GBD was kindly provided by J. Collard (12Reid T. Bathoorn A. Ahmadian M.R. Collard J.G. J. Biol. Chem. 1999; 274: 33587-33593Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Full-length pGEX-PAK was kindly provided by A. Wittinghofer (13Buchwald G. Hostinova E. Rudolph M.G. Kraemer A. Sickmann A. Meyer H.E. Scheffzek K. Wittinghofer A. Mol. Cell. Biol. 2001; 21: 5179-5189Crossref PubMed Scopus (90) Google Scholar). Preparation of Recombinant Proteins—Rac1, Rac1ΔC, Rac1b, and Rac1bΔC, the catalytic domain of p50GAP (amino acids 198-439), the Cdc42/Rac-interacting binding domain of PAK (amino acids 57-141), full-length PAK, and the DH-PH domain of Tiam1 were produced as glutathione S-transferase (GST) fusion proteins in Escherichia coli. All of the proteins were purified as described previously for Rnd3 (14Fiegen D. Blumenstein L. Stege P. Vetter I.R. Ahmadian M.R. FEBS Lett. 2002; 525: 100-104Crossref PubMed Scopus (33) Google Scholar). Nucleotide-free GTPases as well as fluorescent nucleotide-bound GTPases were prepared, and concentration and quality were determined as described (15Ahmadian M.R. Wittinghofer A. Herrmann C. Methods Mol. Biol. 2002; 189: 45-63PubMed Google Scholar). Crystallization and Data Collection—Crystals of truncated Rac1bΔC (1-184) in complex with GDP and GppNHp (nonhydrolyzable GTP analog) were grown at 20 °C using the hanging drop method by mixing 2 μl of a 0.5 mm solution of the Rac1b G domain in 20 mm Tris/HCl, pH 7.5, 2 mm MgCl2, 2 mm dithioerythritol, 100 μm GDP or GppNHp with 2 μl of reservoir solution consisting of 100 mm Hepes buffer, pH 7.5, 18-30% polyethylene glycol 3350, and 2-6% isopropanol. The crystals of both complexes belonged to space group P212121 (a = 51.55 Å, b = 78.67 Å, c = 96.88 Å). For data collection at 100 K, the crystals were transferred to a solution containing reservoir solution and 10% glycerol. A cryo-protected crystal was then suspended in a rayon loop (Hampton Research) and flash frozen in liquid nitrogen. X-ray diffraction data were collected on an ADSC Q4 CCD detector at the beam line ID14-1 at the European Synchrotron Radiation Facility and were processed using XDS (16Kabsch W. J. Appl. Cryst. 1993; 26: 795-800Crossref Scopus (3243) Google Scholar). Details on data collection, structure determination, and crystallographic refinement are summarized in Table I.Table IData collection and refinement statistics of Rac1bGDPGppNHpIntensity data processingResolution (Å)24.92 − 1.75 Å26.26 − 1.75 ÅNumber of reflections130,220138,306Number of independent reflections38,56038,875Rsym(%)aRsym=100·∑|I-〈I〉|/∑I.5.4 (40.9)bBrackets are quantities calculated in the highest resolution bin at 1.85-1.75 Å.5.1 (40.6)bBrackets are quantities calculated in the highest resolution bin at 1.85-1.75 Å.Completeness of data (%)95.1 (88.3)bBrackets are quantities calculated in the highest resolution bin at 1.85-1.75 Å.96.0 (90.1)bBrackets are quantities calculated in the highest resolution bin at 1.85-1.75 Å.Mean 〈I/σ(I)〉15.21 (3.37)bBrackets are quantities calculated in the highest resolution bin at 1.85-1.75 Å.16.62 (3.75)bBrackets are quantities calculated in the highest resolution bin at 1.85-1.75 Å.Molecular replacement statisticsResolution range rotation translation10.0-4.0 A/8.0-4.0 Å10.0-4.0 A/8.0-4.0 ÅRotation (°)cEulerian angles (α, β, γ) are as defined in AMoRe, and translation is given in the orthogonal system.152.02, 70.81, 163.02 (151.49, 70.92, 162.12)dBrackets show the solution for the second molecule in the asymmetric unit.152.21, 70.87, 163.31 (151.46, 70.96, 161.47)dBrackets show the solution for the second molecule in the asymmetric unit.Translation (Å)cEulerian angles (α, β, γ) are as defined in AMoRe, and translation is given in the orthogonal system.− 4.99, − 7.34, − 8.63 (− 1.80, 32.33, − 9.06)dBrackets show the solution for the second molecule in the asymmetric unit.− 4.54, − 7.50, − 8.47 (− 1.89, 32.30, − 9.40)dBrackets show the solution for the second molecule in the asymmetric unit.Correlation coefficient61.5 (22.6)eBrackets are quantities of the second best solution.62.6 (25.3)eBrackets are quantities of the second best solution.Rcryst (%)fRcryst=100·∑|Fo-Fc|/∑Fo. Rfree is Rcryst that was calculated using 5% of the data, chosen randomly, and omitted from the subsequent structure refinement.47.9 (64.1)eBrackets are quantities of the second best solution.45.3 (60.7)eBrackets are quantities of the second best solution.Refinement StatisticsRcryst (%)fRcryst=100·∑|Fo-Fc|/∑Fo. Rfree is Rcryst that was calculated using 5% of the data, chosen randomly, and omitted from the subsequent structure refinement.18.818.1Rfree (%)fRcryst=100·∑|Fo-Fc|/∑Fo. Rfree is Rcryst that was calculated using 5% of the data, chosen randomly, and omitted from the subsequent structure refinement.22.321.9Root mean square bond lenghts (Å)0.0210.021Root mean square bond angles (°)1.871.88Total number of residues614 (273)gBrackets show the number of included water molecules.638 (296)gBrackets show the number of included water molecules.Residue ranges1-59 and 93-201 (1-58 and 93-199)hBrackets show the residue range for the second molecule in the asymmetric unit.1-60 and 93-201 (1-58 and 93-199)hBrackets show the residue range for the second molecule in the asymmetric unit.a Rsym=100·∑|I-〈I〉|/∑I.b Brackets are quantities calculated in the highest resolution bin at 1.85-1.75 Å.c Eulerian angles (α, β, γ) are as defined in AMoRe, and translation is given in the orthogonal system.d Brackets show the solution for the second molecule in the asymmetric unit.e Brackets are quantities of the second best solution.f Rcryst=100·∑|Fo-Fc|/∑Fo. Rfree is Rcryst that was calculated using 5% of the data, chosen randomly, and omitted from the subsequent structure refinement.g Brackets show the number of included water molecules.h Brackets show the residue range for the second molecule in the asymmetric unit. Open table in a new tab Structure Determination and Crystallographic Refinement—The initial phases were calculated by molecular replacement, using the program AMoRe (17Navaza J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1367-1372Crossref PubMed Scopus (658) Google Scholar) and a Rac1 search model based on the Rac1·GppNHp structure lacking the bound nucleotide and the switch regions (18Hirshberg M. Stockley R.W. Dodson G. Webb M.R. Nat. Struct. Biol. 1997; 4: 147-152Crossref PubMed Scopus (191) Google Scholar) (Protein Data Bank code 1mh1). After initial rigid body refinement, 20 cycles of simulated annealing and model building were performed. For the last refinement steps REFMAC5 was used (19Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar). For the detection of crystallographic water molecules, ARP/wARP (20Perrakis A. Sixma T.K. Wilson K.S. Lamzin V.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 448-455Crossref PubMed Scopus (484) Google Scholar) and REFMAC5 were used. The residue ranges that were included in the final model as well as the corresponding R-factors are listed in Table I. For all four molecules the two additional N-terminal Gly-Ser residues caused by the thrombin cleavage site could be observed and were included in the model. Fluorescence Measurements—Long time fluorescence measurements were monitored on a LS50B PerkinElmer Life Sciences spectrofluorometer, and rapid kinetics were measured with a stopped flow apparatus (Applied Photophysics SX16MV) as described (15Ahmadian M.R. Wittinghofer A. Herrmann C. Methods Mol. Biol. 2002; 189: 45-63PubMed Google Scholar). Nucleotide association was performed with 0.1 μm fluorescent nucleotide and varying concentrations of nucleotide-free Rac proteins at 20 °C as described (15Ahmadian M.R. Wittinghofer A. Herrmann C. Methods Mol. Biol. 2002; 189: 45-63PubMed Google Scholar). The dissociation of the fluorescent nucleotide from Rac proteins (0.1 μm) was measured by the addition of 200-fold excess of nonfluorescent nucleotide in the absence and the presence of 5 μm Tiam1 DH-PH at 20 °C. The equilibrium dissociation constant (Kd) for the PAK-GBD interaction with Rac1b was determined as previously described for the Ras-Raf kinase interaction (21Herrmann C. Martin G.A. Wittinghofer A. J. Biol. Chem. 1995; 270: 2901-2905Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). The measurements were carried out using 0.2 μm mantGppNHp-bound GTPase, 40 μm GppNHp, and increasing concentrations of PAK-GBD at 25 °C for Rac1 and at 10 °C in the case of Rac1b because of its fast nucleotide dissociation rate. All of the measurements were carried out in 30 mm Tris/HCl, pH 7.5, 5 mm MgCl2, 10 mm Na2HPO4/NaH2PO4 pH 7.5, 5 mm dithioerythritol. The observed rate constants were evaluated using Grafit (Erithacus software). GTPase Assay—The intrinsic and GAP-stimulated GTP hydrolysis reactions were measured by HPLC on a C18 reversed phase column using a mixture of 80 μm nucleotide-free GTPase and 70 μm GTP in the presence and the absence of 8 μm GAP at 25 °C in 30 mm Tris/HCl, pH 7.5, 5 mm dithioerythritol, 10 mm Na2HPO4/NaH2PO4, 5 mm MgCl2 as described (15Ahmadian M.R. Wittinghofer A. Herrmann C. Methods Mol. Biol. 2002; 189: 45-63PubMed Google Scholar). The relative GTP content was calculated by the ratio of [GTP]/([GTP]+[GDP]). For exponential fitting of the data, the program Grafit (Erithacus software) was used. Transfection and GTPase Pull-down Assay—COS-7 cells were transfected using DEAE-dextran as described (12Reid T. Bathoorn A. Ahmadian M.R. Collard J.G. J. Biol. Chem. 1999; 274: 33587-33593Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Pull-down assay for the active GTP-bound Rac proteins was carried out using GST-PAK-GBD (glutathione S-transferase-fused Rac-binding domain of PAK) conjugated with glutathione beads as described (12Reid T. Bathoorn A. Ahmadian M.R. Collard J.G. J. Biol. Chem. 1999; 274: 33587-33593Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The interaction of full-length GST-PAK with the Rac proteins was examined under the same conditions using purified proteins. The beads were washed four times and subjected to SDS-PAGE (15% polyacrylamide). Bound Rac proteins were detected by Western blot using a monoclonal antibody against Rac (Upstate Biotechnologies, Inc.). Rapid GEF-independent Nucleotide Dissociation Reaction of Rac1b—To investigate the influence of the 19-amino acid insertion on the nucleotide binding affinity, we first determined kinetic constants for the association of fluorescently (methylanthraniloyl- or mant-) labeled nucleotides to nucleotide-free Rac1b protein. This allowed us to monitor nucleotide association kinetics at increasing protein concentrations. As shown in Fig. 1A, the formation of the binary complex is not affected by the 19-amino acid insertion. The association rate constants (kon) for the binding of mantGDP and mantGTP to Rac1b were obtained by linear fitting of the observed rate constants at the given protein concentrations. They are only marginally slower than those of Rac1 (Table II).Table IIBiochemical properties of Rac1 and Rac1bRac1Rac1bmantGDPkon (S−1 M−1)2.47 × 1061.12 × 106koff (S−1)7.0 × 10−51.8 × 10−3Tiam 1-catalyzed koff(S−1)a5 μM Tiam 1 was applied in the reaction of 0.1 μM GTPase.3.6 × 10−31.9 × 10−3Kd (nM)0.0281.6mantGppNHpkon (S−1 M−1)1.05 × 106koff (S−1)1.1 × 10−42.9 × 10−2Kd (nM)27.6mantGTPkon (S−1 M−1)1.49 × 1061.05 × 106koff (S−1)1.0 × 10−42.7 × 10−3Kd (nM)0.0672.6GTP hydrolysisIntrinsic (min−1)0.110.0035GAP-stimulated (min−1)bThe GAP concentration (8 μM) was 10-fold below the GTPase concentration (80 μM).2.360.194PAK bindingKd (μM)0.493.55a 5 μM Tiam 1 was applied in the reaction of 0.1 μM GTPase.b The GAP concentration (8 μM) was 10-fold below the GTPase concentration (80 μM). Open table in a new tab To determine the intrinsic and GEF-accelerated nucleotide dissociation rates, Rac1b and Rac1 were loaded with the respective fluorescently labeled guanine nucleotides. The displacement of the fluorescent nucleotides was initiated by the addition of excess amounts of nonfluorescent nucleotides in the presence and absence of the DH-PH domain of Tiam1 (a Rac-specific GEF). Drastic increases in the intrinsic dissociation of mantGDP (26-fold), mantGTP (27-fold), and mantGppNHp (250-fold) from Rac1b compared with the very slow dissociation rates of the respective nucleotides from Rac1 were observed (Fig. 1B and Table II). Accordingly, the calculated Kd for nucleotide binding revealed that the 19-residue insertion dramatically affects the overall affinity for GDP, GTP, and particularly the GTP analog GppNHp (Table II). The reason for the reduced affinity of GppNHp compared with that of GTP is the disrupted hydrogen bond of the GTP-β,γ-bridging imino group to the main chain NH group of the P-loop residue Ala13. A similar observation has been reported for Ras·mantGppNHp (22Schmidt G. Lenzen C. Simon I. Deuter R. Cool R.H. Goody R.S. Wittinghofer A. Oncogene. 1996; 12: 87-96PubMed Google Scholar). Moreover, in contrast to the slow nucleotide dissociation of Rac1, which could be 50-fold accelerated in the presence of the DH-PH domain of Tiam1, the fast intrinsic mantGDP dissociation of Rac1b could not be further increased by the DH-PH domain of Tiam1 (Fig. 1C and Table II). These in vitro results show that Rac1b does not require any GEF to get activated. It rather activates itself by a very fast nucleotide dissociation and by the subsequent binding of the cellular abundant GTP. Despite the drastically increased nucleotide dissociation, Rac1b exhibits a nucleotide binding affinity in the low nanomolar range, which is still high enough to act as a GTP-binding protein in cells. However, because the DH-PH domain of Tiam1 is a weak exchange factor in vitro (51Haeusler L.C. Blumenstein L. Stege P. Dvorsky R. Ahmadian M.R. FEBS Lett. 2003; 555: 556-560Crossref PubMed Scopus (65) Google Scholar) and displays a 10 times higher activity on prenylated Rac1 bound to liposomes than on soluble unprenylated Rac1 (23Robbe K. Otto-Bruc A. Chardin P. Antonny B. J. Biol. Chem. 2003; 278: 4756-4762Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), we cannot exclude the possibility that Rac1b can in principle interact with Tiam1 under cellular conditions as shown with constitutive active Tiam1, overexpressed in NIH3T3 cells (11Matos P. Collard J.G. Jordan P. J. Biol. Chem. 2003; 278: 50442-50448Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Impaired Intrinsic GTP Hydrolysis Reaction of Rac1b—A second crucial function of small GTPases is their slow intrinsic GTP hydrolysis reaction, which needs to be stimulated by GAPs to switch off downstream signaling. Therefore, we measured the GTP hydrolysis reaction of Rac1b in direct comparison with that of Rac1 using a HPLC-based technique. Interestingly, we found that the intrinsic GTP hydrolysis reaction of Rac1b (0.0035 min-1) was about 30-fold reduced compared with that of Rac1 (0.11 min-1) (Fig. 1D and Table II). In contrast to our results it has been previously published that Rac1 and Rac1b show the same GTPase activity (10Schnelzer A. Prechtel D. Knaus U. Dehne K. Gerhard M. Graeff H. Harbeck N. Schmitt M. Lengyel E. Oncogene. 2000; 19: 3013-3020Crossref PubMed Scopus (341) Google Scholar). This discrepancy can be explained by the method this group employed. The filter binding assay seems to be inappropriate for a protein with a fast nucleotide dissociation such as Rac1b. Unlike the constitutive active mutants of Rac1 (G12V in the P-loop and Q61L in the switch II region) that also have an impaired intrinsic GTP hydrolysis (24Xu X. Wang Y. Barry D.C. Chanock S.J. Bokoch G.M. Biochemistry. 1997; 36: 626-632Crossref PubMed Scopus (28) Google Scholar), the defective GTPase reaction of Rac1b can be restored by GAP proteins. As shown in Fig. 1D, the catalytic domain of p50GAP stimulated the GTPase reaction of Rac1b up to 55-fold (21-fold for Rac1), showing that GAP is able to stabilize the catalytic elements of Rac1b and thus accelerate the GTPase reaction. High Level of Rac1b·GTP in COS-7 Cells—Considering the increased nucleotide dissociation and the decreased GTP hydrolysis, it was tempting to assume that Rac1b is GTP-bound in cells. To prove this assumption, Rac1, its constitutive active mutant Rac1(G12V) and Rac1b were overexpressed in COS-7 cells under serum-starved conditions for 48 h. The fact that wild-type Rac1b could be pulled down with GST-PAK-GBD verifies our hypothesis that Rac1b exists in an active conformation in serum-starved cells. Thereby it resembles the constitutive active Rac1(G12V) mutant (Fig. 2A). As expected wild-type Rac1 could not be detected under these conditions and obviously needs GEF proteins to be activated. A GTP-dependent Rac1b-PAK interaction was demonstrated by performing the pull-down assay with purified GDP- and GppNHp-bound Rac1b. As shown in Fig. 2B, GST-PAK-GBD selectively binds Rac1b·GppNHp but not Rac1b·GDP, similar to the Rac1 control experiment. These results reveal that Rac1b is, independent of external stimuli, GTP-bound in cells and can selectively interact with Rac effector proteins. Rac1 involvement in transcription and growth control and its requirement for Ras-induced malignant transformation is widely known (25Qiu R.G. Chen J. Kirn D. McCormick F. Symons M. Nature. 1995; 374: 457-459Crossref PubMed Scopus (813) Google Scholar, 26Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1059) Google Scholar, 27Zohn I.E. Symons M. Chrzanowska-Wodnicka M. Westwick J.K. Der C.J. Mol. Cell. Biol. 1998; 18: 1225-1235Crossref PubMed Scopus (69) Google Scholar, 28Mira J.P. Benard V. Groffen J. Sanders L.C. Knaus U.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 185-189Crossref PubMed Scopus (197) Google Scholar, 29Malliri A. van der Kammen R.A. Clark K. van der Valk M. Michiels F. Collard J.G. Nature. 2002; 417: 867-871Crossref PubMed Scopus (309) Google Scholar). This knowledge is based on experiments using the expression of a constitutively active Rac1(G12V) mutant. A fast cycling mutant of Cdc42, Cdc42(F28L), has been shown to undergo spontaneous nucleotide exchange in the absence of a GEF while maintaining full GTPase activity (30Lin R. Bagrodia S. Cerione R. Manor D. Curr. Biol. 1997; 7: 794-797Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). This mutant has an even greater cell-transforming potential in fibroblasts compared with the constitutively active Cdc42(G12V) mutant. However, our biochemical data clearly shows that Rac1b behaves as a self-activating GTPase that is predominantly GTP-bound in cells. Low Affinity Binding of Rac1b to PAK-GBD—To characterize the effect of the insertion on effector interaction, we determined equilibrium dissociation constants (Kd) of PAK-GBD binding to GppNHp-bound Rac1b and Rac1 using the GDI assay (31Herrmann C. Horn G. Spaargaren M. Wittinghofer A. J. Biol. Chem. 1996; 271: 6794-6800Abstract Full Text PDF PubMed Scopus (301) Google Scholar). As shown in Fig. 3, increasing concentrations of PAK-GBD resulted in incremental inhibition of the mantGppNHp dissociation from Rac1b and Rac1. We obtained a Kd value of 0.49 μm for the PAK-GBD interaction with Rac1, which nicely corresponds to previous reports (32Thompson G. Owen D. Chalk P.A. Lowe P.N. Biochemistry. 1998; 37: 7885-7891Crossref PubMed Scopus (123) Google Scholar). For Rac1b we observed a 7-fold reduced binding affinity of PAK-GBD. The lower Kd of 3.55 μm can be most likely attributed to the 19-amino acid insertion. MantGDP dissociation from Rac1b was not inhibited under these conditions (data not shown), confirming the GTP-dependent interaction of Rac1b with PAK-GBD. Furthermore, we examined the interaction of Rac1 and Rac1b with full-length PAK using a GST pull-down assay. Fig. 2B shows that we could not detect binding of Rac1b to full-length PAK, and hence Rac1b stands in contrast to Rac1. Compared with the high affinity binding of the isolated PAK-GBD domains to Cdc42, it has been recently shown that full-length PAK has a much lower binding affinity for Cdc42 (13Buchwald G. Hostinova E. Rudolph M.G. Kraemer A. Sickmann A. Meyer H.E. Scheffzek K. Wittinghofer A. Mol. Cell. Biol. 2001; 21: 5179-5189Crossref PubMed Scopus (90) Google Scholar, 32Thompson G. Owen D. Chalk P.A. Lowe P.N. Biochemistry. 1998; 37: 7885-7891Crossref PubMed Scopus (123) Google Scholar, 33Owen D. Mott H.R. Laue E.D. Lowe P.N. Biochemistry. 2000; 39: 1243-1250Crossref PubMed Scopus (64) Google Scholar). Assuming that this is also true for Rac1, our biochemical data suggest an extremely low affinity of full-length PAK for Rac1b. Conserved Overall Structure of Rac1b·GDP and Rac1b· GppNHp—To gain insight into the structural impact of the 19-amino acid insertion of Rac1b, we determined the crystal structures of Rac1b in the GDP- and GppNHp-bound states. The crystals diffracted to 1.75 Å resolution (Table I). Size exclusion chromatography showed that Rac1b is monomeric in solution (data not shown), but it crystallized as a dimer with two molecules/asymmetric unit in a head to head fashion. The contact surface has a size of 1347 Å2 and is build up by β1 to β3, α1, and the switch I of both molecules. Both molecules in the asymmetric unit are very similar as can be derived from the low root mean square deviation of 0.45 Å for 165 common Cα atoms. The ribbon representation in Fig. 4 shows the secondary structure elements of Rac1b·GDP and Rac1b·GppNHp superimposed on the GppNHp-bound Rac1 (18Hirshberg M. Stockley R.W. Dodson G. Webb M.R. Nat. Struct. Biol. 1997; 4: 147-152Crossref PubMed Scopus (191) Google Scholar). The overall structures of both nucleotide-bound forms of Rac1b are remarkably similar to each other and conserved as compared with the structures of Rac1·GppNHp (18Hirshberg M. Stockley R.W. Dodson G. Webb M.R. Nat. Struct. Biol. 1997; 4: 147-152Crossref PubMed Scopus (191) Google Scholar), RhoA·GTPγS (34Ihara K. Muraguchi S. Kato M. Shimizu T. Shirakawa M. Kuroda S. Kaibuchi K. Hakoshima T. J. Biol. Chem. 1998; 273: 9656-9666Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), and Cdc42·GDP (35Rudolph M.G. Wittinghofer A. Vetter I.R. Protein Sci. 1999; 8: 778-787Crossref PubMed Scopus (31) Google Scholar) with root mean square deviations of 0.67 Ε (156 common Cα atoms), 0.80 Ε (155 common Cα atoms), and 0.81 Ε (151 common Cα atoms), respectively, except for the loop regions as described below. Nucleotide binding requires five conserved sequence elements (36Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2698) Google Scholar), of which three are well order
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