Critical Role of Lysine 204 in Switch I Region of G 13 for Regulation of p115RhoGEF and Leukemia-Associated RhoGEF

Heterotrimeric G proteins of the G12 family regulate the Rho GTPase through RhoGEFs that contain an amino-terminal regulator of G protein signaling (RGS) domain (RGS-RhoGEFs). Direct regulation of the activity of RGS-RhoGEFs p115 or leukemia-associated RhoGEF (LARG) by G 13 has previously been demonstrated. However, the precise biochemical mechanism by which G 13 stimulates the RhoGEF activity of these proteins has not yet been well understood. Based on the crystal structure of G i1 in complex with RGS4, we mutated the G 13 residue lysine 204 to alanine (G 13K204A) and characterized the effect of this mutation in its regulation of RGS-RhoGEFs p115 or LARG. Compared with wild-type G 13, G 13K204A induced much less serum-response factor activation when expressed in HeLa cells. Recombinant G 13K204A exhibits normal function in terms of nucleotide binding, basal GTP hydrolysis, and formation of heterotrimer with . We found that lysine 204 of G 13 is important for interaction with the RGS domain of p115 or LARG and for the GTPase-activating protein activity of these proteins. In addition, the K204A mutation of G 13 impaired its regulation of the RhoGEF activity of p115 or LARG. We conclude that lysine 204 of G 13 is important for interaction with RGS-RhoGEFs and is critically involved in the regulation of their activity. Heterotrimeric G proteins are composed of , , and subunits and are activated by members of the seven-transmembrane helix family of receptors (G protein-coupled receptors) (Kaziro et al., 1991; Hepler and Gilman, 1992). Upon agonist binding, activated receptor catalyzes GDP-GTP exchange on the subunit. Nucleotide exchange induces conformational changes at three switch regions of the G subunit and facilitates the dissociation of GTP-bound G from subunits. Both GTP-bound G subunit and free subunits have the capacity to regulate various downstream effectors. The G subunit hydrolyzes bound GTP to GDP by its intrinsic GTPase activity, and this rate is accelerated by the presence of GTPase-activating proteins (GAPs), such as regulators of G protein signaling (RGS) proteins (Hollinger and Hepler, 2002). The signaling event is terminated when the GDP-bound G subunit reassociates with the subunit to form the inactive heterotrimer. In this signaling system, the strength and duration of the signal is determined by the precise control of the amount of GTP-bound G subunit. Subunits of G12 and G13 have been shown to transduce signals from G protein-coupled receptors to Rho activation (Aragay et al., 1995; Gohla et al., 1998; Kranenburg et al., 1999). It is well established that Rho family monomeric GTPases are involved in various cellular functions through regulation of the actin cytoskeleton and gene expression (Hall, 1998; Schmidt and Hall, 2002). We have identified that RhoGEFs that contain an RGS domain within their amino-terminal region (RGS-RhoGEFs) constitute direct links between heterotrimeric G12/13 and the Rho GTPase (Hart et al., 1998; Kozasa et al., 1998; Suzuki et al., 2003). Currently, three mammalian RhoGEFs, p115RhoGEF, PDZ-RhoGEF/ GTRAP48, and LARG, have been isolated in this RGS-RhoGEF subfamily. The RGS domain of each of these RhoGEFs specifically interacts with G 12 and G 13 (Kozasa et al., 1998; Fukuhara et al., 1999; Booden et al., 2002). GAP activThis work was supported by National Institutes of Health grants GM61454 and NS41441 and a grant from the American Heart Association (to T.K.). T.K. is an Established Investigator of the American Heart Association. B.K. has been supported by the National Institutes of Health training grant HL-07829 and a predoctoral fellowship from the Midwest Affiliate of the American Heart Association. S.N. and B.K. contributed equally to this study. 1 Current address: Department of Second Internal Medicine, Chiba University, Chiba Japan. 2 Current address: Department of Bioregulation, Institute of Gerontology, Nippon Medical University, Kawasaki, Japan. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.104.002287. ABBREVIATIONS: GAP, GTPase-activating protein; RGS, regulator of G protein signaling; GEF, guanine nucleotide exchange factor; PDZ, PSD-95/Dlg/ZO-1 homology; PDE, phosphodiesterase; LARG, leukemia-associated RhoGEF; SRE, serum-response element; DMEM, Dulbecco’s modified Eagle’s medium; IP, immunoprecipitation; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; NTA, nitrilotriacetic acid; GTP S, guanosine 5 -3-O-(thio)triphosphate; SRF, serum-response factor; DH, Dbl homology; PH, pleckstrin homology. 0026-895X/04/6604-1029–1034$20.00 MOLECULAR PHARMACOLOGY Vol. 66, No. 4 Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 2287/1175969 Mol Pharmacol 66:1029–1034, 2004 Printed in U.S.A. 1029 at A PE T Jornals on A uust 6, 2017 m oharm .aspeurnals.org D ow nladed from ity of the RGS domain of p115RhoGEF or LARG for G 12 and G 13 has been demonstrated (Kozasa et al., 1998; Suzuki et al., 2003). In addition, these RhoGEFs serve as direct effectors of these G subunits. In vitro reconstitution experiments using purified components demonstrated that the active form of G 13 stimulates Rho activation through p115RhoGEF or LARG (Hart et al., 1998; Suzuki et al., 2003). Members of the RGS family share a homologous domain (RGS domain) of about 120 residues. It has been demonstrated biochemically that the RGS domain of several family members possesses GAP activity for G subunits, most of them for subunits of the Gi/o or Gq subfamilies (Hollinger and Hepler, 2002). The crystal structure of the complex of G i1 with the RGS domain of RGS4 suggested that the RGS domain functions as a GAP by stabilizing the transition state of GTP hydrolysis of the G subunit (Tesmer et al., 1997). In this structure, the RGS domain makes extensive direct contacts with the switch regions of G i1. In particular, threonine 182 of G i1 forms critical contacts with several amino acid residues of the RGS domain of RGS4. This threonine residue is conserved at the corresponding region of all G subunits except for G s and G 12/13, with G 12 and G 13 each containing a lysine residue at this position. In the G i1-RGS4 complex, the side chain of a lysine residue cannot be accomodated in the position of threonine 182, supporting biochemical evidence that RGS4 does not act as a GAP for G 12 (Berman et al., 1996). Although the amino acid sequence homology of the RGS domains of RGS-RhoGEFs with the RGS domain of RGS4 is low, the recently solved crystal structures of the RGS domain of p115RhoGEF or PDZ-RhoGEF demonstrated that the overall three-dimensional structure of the RGS domain is well conserved in the RGS domains of these RhoGEFs (Chen et al., 2001; Longenecker et al., 2001). We thus tested the hypothesis that lysine 204 of G 13, which corresponds to threonine 182 of G i1, is important for interaction with the RGS domain of RGS-RhoGEFs. In the present study, we have investigated this possibility using biochemical reconstitution assays. Although lysine 204 of G 13 is not required for nucleotide binding or heterotrimer formation, we found that it is important for interaction with p115RhoGEF or LARG and is essential for the regulation of their activity. Materials and Methods Expression Constructs. The G 13K204A point-mutant was created by QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA), according to manufacturer’s instructions. Primers used to generate the K204A mutation were the following (mutated bases underlined): 5 -GCTTGCCAGAAGGCCC ACTGCAGGCATCCATGAGTACG-3 and 5 -CGTACTCATGGATGCCTGCAGTGGGC CTTCTGGCAAGC-3 . pCMV5-G 13 (wild-type), pCMV5-G 13K204A, pCMV5-G 13Q226L, pcDNAmyc-RGSp115 (amino acids 1–252), and pcDNAmycPDZ-LARG (amino acids 307-1543; Suzuki et al., 2003) constructs were used for the expression of respective proteins in serum-response element (SRE)-luciferase or coimmunoprecipitation studies. The pGL3-SRE.L reporter construct used for SRE-luciferase assays was kindly provided by Dr. Paul Sternweis (University of Texas Southwestern Medical Center, Dallas, TX). The glutathione S-transferase-rhotekin RBD construct was kindly provided by Dr. Gary Bokoch (Scripps Research Institute, La Jolla, CA). Generation of the baculovirus transfer vector for expression of His6-LARG has been described previously (Suzuki et al., 2003). G 13K204A was subcloned into pFastBac1 for preparation of its baculovirus. Each construct was confirmed by DNA sequencing. SRE-Luciferase and Rho GTP Pulldown Assays. HeLa cells were maintained in DMEM/10% fetal bovine serum and were passaged to 24-well plates at a density of 7 10 cells per well, 1 day before transfection. Transfections were performed using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA). For all conditions, cells were transfected with pGL3-SRE.L reporter (0.1 g) and pCMV5galactosidase (0.1 g). To indicated wells, cells were additionally transfected with pCMV5-G 13 wild-type, pCMV5-G 13K204A, or pCMV5-G 13Q226L (0.01 g). After 6 h, media were changed to fresh serum-free DMEM, and cells were harvested 24 h post-transfection. Luciferase activity of cell extracts was quantified according to manufacturer’s instructions (Promega, Madison, WI). Total amount of plasmids transfected per well was balanced by addition of empty vector. -Galactosidase activity in cell extracts was used to normalize for transfection efficiency. Rho GTP pulldown assays using GSTrhotekin RBD were performed as described previously (Ren and Schwartz, 2000). HeLa cells (6 10/condition) were cultured in 100-mm plates and transfected with 10 g of indicated expression plasmids. After 24 h, media were changed to fresh serum-free DMEM, and cells were harvested 48 h post-transfection. Immunoblot analyses were performed using either anti-RhoA (26C4; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or anti-G 13 (A-20; Santa Cruz Biotechnology, Inc.) antibodies. Coimmunoprecipitation Studies. COS-1 cells were cultured in 100-mm plates to a density of 6 10 cells per plate. Cells were transfected with either 5 g of pcDNA3.1-mycPDZ-LARG or pCMV5-myc-RGSp115 (residues 1–252), in combination with either 0.5 g of pCMV5-G 13 wild type, pCMV5-G 13Q226L, pCMV5G 13K204A, or pCMV5-G 13K204A/Q226L. Cells were harvested 24 h after transfection and lysed in 500 l of ice-cold IP buffer (20 mM Tris-HCl, pH 7.5, 1 mM DTT, 100 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 5 M GDP, 10 mM Na3VO4, 10 mM -glycerophosphate, 0.7% Triton X-100, 16 g/ml phenylmethylsulfonyl fluoride, 16 g/ml Ntosyl-L-phenylalanine-chloromethyl ketone, 16 g/ml N-tosyl-L-lysine-chloromethyl ketone, 3.2 g/ml leupeptin, and 3.2 g/ml lima bean trypsin inhibitor). To indicated samples, 30 M AlCl3 and 5 mM NaF (AlF4 ) were added. Soluble lysates (100,000g, 20 min, 4°C) were first precleared by incubation with protein G-Sepharose for 30 min at 4°C, and then incubated for 1 h at 4°C with protein G-Sepharose coupled to monoclonal anti-myc antibody (9E10; Covance, Richmond, CA). Beads were pelleted (10,000g, 5 min, 4°C) and washed three times with IP buffer (with or without AlF4 ). Finally, beads were boiled in SDS-PAGE sample buffer. Protein samples either bound to beads or in total lysates were resolved by SDS-PAGE. Immunoblot analyses were performed using specific antibodies raised against either the myc epitope tag (9E10) or G 13 (B-859; Singer et al., 1994). Expression and Purification of Recombinant Proteins. Recombinant baculoviruses were prepared and amplified using the Bac-to-Bac system (Invitrogen). Mutant G 13K204A was purified from the membranes of Sf9 cells coinfected with baculoviruses encoding G 13K204A, 1, and His62, using methods described previously for purification of wild-type G 13 (Kozasa, 1999). Glu-Glutagged p115RhoGEF or His6-LARG were expressed and purified from Sf9 cells using either anti-Glu-Glu (Covance) immunoaffinity or nickel-NTA (QIAGEN, Valencia, CA) immobilized metal affinity chromatography, respectively (Suzuki et al., 2003). Recombinant His6-RhoA used in RhoGEF assays was purified from Sf9 cells infected with baculovirus encoding His6-RhoA. Cell pellets from 1 liter of culture were resuspended in 200 ml of lysis buffer (20 mM NaHEPES, pH 7.4, 10 mM 2-mercaptoethanol, 50 mM NaCl, 1 mM MgCl2, 10 M GDP, and proteinase inhibitors) and lysed by nitrogen cavitation for 30 min at 4°C. Lysates were centrifuged (1000g, 15 min, 4°C), and the supernatant was extracted with 1% cholate for 1 h on ice. After ultracentrifugation (100,000g, 30 min, 4°C), the supernatant was loaded onto a 1-ml nickel-NTA column equilibrated with 10 volumes of buffer A (lysis buffer supplemented with 1% cholate). 1030 Nakamura et al. at A PE T Jornals on A uust 6, 2017 m oharm .aspeurnals.org D ow nladed from The column was washed with 30 volumes of buffer B (lysis buffer supplemented with 400 mM NaCl, 10 mM imidazole, and 0.5% cholate). Recombinant His6-RhoA was eluted in 5 fractions of 1 volume of buffer C (lysis buffer supplemented with 100 mM NaCl, 100 mM imidazole, and 1% cholate). Peak elution fractions containing His6-RhoA were pooled and exchanged to buffer D (20 mM Na-HEPES, pH 7.4, 1 mM DTT, 100 mM NaCl, 1 mM MgCl2, 1 M GDP, and 1% cholate) using a Centricon YM-10 unit (Millipore Corporation, Billerica, MA). Finally, octyl-D-glucopyranoside was added to a final concentration of 1% (Calbiochem, San Diego, CA). GTP S Binding Assays. GTP S binding to G 13 or G 13K204A (1 pmol) was measured at 30°C in binding buffer (50 mM Na-HEPES, pH 8.0, 1 mM EDTA, 1 mM DTT, 1.5 mM MgCl2, 0.05% C12E10, 10 M GTP S, and 2000 cpm/pmol [S]GTP S). Fifty-microliter aliquots were withdrawn at the indicated time points and mixed with wash buffer containing 10 mM MgSO4 to terminate reactions. Samples were applied to BA-85 filters (Schleicher & Schuell, Keene, NH) and filtered under vacuum. After washing three times, radioactivity remaining on filters was measured by liquid scintillation counting. The inhibitory effect of G on GTP S binding to G 13 wild-type or G 13K204A (2.5 pmol) was evaluated in the same buffer described above. Samples were incubated for 90 min at 30°C, either in the absence or presence of purified 1 2 (7.5 pmol). GTPase Assays. Single-turnover GTP hydrolysis activity of G 13 wild-type or G 13K204A mutant was assessed essentially as described previously (Kozasa et al., 1998). Thirty picomoles of recombinant, purified G 13 or G 13K204A protein was loaded with -[P]GTP (50–100 cpm/fmol) for 40 min at 30°C in the presence of 5 mM EDTA and 5 M GTP. Samples were rapidly gel filtered through Sephadex G-50 (Amersham Biosciences Inc., Piscataway, NJ) to remove unbound nucleotide and free P-labeled phosphate, and GTP hydrolysis at 15°C was monitored after the addition of -[P]GTP-labeled G protein to the reaction mixture (50 mM NaHEPES, pH 8.0, 1 mM DTT, 5 mM EDTA, 8 mM MgSO4, 1 mM GTP, 0.05% C12E10, and 100 nM either EE-p115-RhoGEF or His6-LARG). Fifty-microliter aliquots were taken at the indicated time points and mixed with 750 l of 5% (w/v) NoritA in 50 mM NaH2PO4. Radioactivity in the supernatants after centrifugation (1200g, 10 min, 4°C) was measured by liquid scintillation counting. RhoGEF Assays. G 13 or G 13K204A (1.5 pmol) was first incubated in the presence of 60 M AlCl3, 5 mM MgCl2, and 20 mM NaF for 15 min at 0°C, and then incubated with His6-RhoA (25 pmol) in the presence of indicated proteins at 30°C in binding buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 0.5 mM EDTA, 50 mM NaCl, 5 mM MgCl2, 0.05% C12E10, 10 M GTP S, and 500 cpm/pmol [S]GTP S), in a final reaction volume of 50 l. Binding reactions were terminated by addition of wash buffer containing 10 mM MgSO4, followed by filtration through BA-85 nitrocellulose filters. After washing three times, radioactivity remaining on filters was measured by liquid scintillation counting. Miscellaneous Procedures. Statistical significance was assigned based on results of t test analyses of data.