The Regulator of G Protein Signaling Domain of Axin Selectively Interacts with G

Axin, a negative regulator of the Wnt signaling pathway, contains a canonical regulator of G protein signaling (RGS) core domain. Herein, we demonstrate both in vitro and in cells that this domain interacts with the subunit of the heterotrimeric G protein G12 but not with the closely related G 13 or with several other heterotrimeric G proteins. Axin preferentially binds the activated form of G 12, a behavior consistent with other RGS proteins. However, unlike other RGS proteins, that of axin (axinRGS) does not affect intrinsic GTP hydrolysis by G 12. Despite its inability to act as a GTPase-activating protein, we demonstrate that in cells, axinRGS can compete for G 12 binding with the RGS domain of p115RhoGEF, a known G12-interacting protein that links G12 signaling to activation of the small G protein Rho. Moreover, ectopic expression of axinRGS specifically inhibits G 12 -directed activation of the Rho pathway in MDA-MB 231 breast cancer cells. These findings establish that the RGS domain of axin is able to directly interact with the subunit of heterotrimeric G protein G12 and provide a unique tool to interdict G 12-mediated signaling processes. Heterotrimeric GTP-binding regulatory proteins (G proteins) stimulate a wide variety of cellular signals as a result of their interactions with G protein coupled receptors (GPCRs). Upon ligand binding by a GPCR, the intracellular portion of the GPCR engages and induces a conformational change in the subunit of the G protein, causing it to release natively bound GDP and bind GTP (Cabrera-Vera et al., 2003). The newly activated GTP-bound subunit then dissociates from the subunit, and both molecules subsequently interact with downstream effectors to trigger a variety of cellular events. G protein signaling is terminated when GTP is hydrolyzed to GDP. Although G subunits possess an intrinsic GTPase activity of their own, a family of proteins called regulators of G protein signaling (RGS) has been shown to interact with activated G subunits and greatly enhance GTP hydrolysis (Berman et al., 1996; Hunt et al., 1996; Watson et al., 1996). RGS proteins are defined by a conserved 120-residue domain termed the “RGS box” (Siderovski et al., 1996). This region binds with high affinity to the transition state of G subunits, lowering the energy required for GTP hydrolysis to occur (Ross and Wilkie, 2000). In addition to their GTPasestimulating activities, some RGS proteins act as scaffolding molecules that hold signaling complexes together. So far, more than 30 mammalian RGS or RGS-like family members have been described and are divided into six subfamilies based on identifiable domains (Hollinger and Hepler, 2002). One subfamily of RGS proteins, the primary member of which is a protein termed axin, contains multiple domains that facilitate its critical role in the Wnt pathway (Zeng et al., 1997). In this pathway, axin acts as a scaffolding protein holding together a signaling complex involved in the breakdown of -catenin, the primary target of the canonical Wnt signaling pathway. Wnts are secreted glycoproteins that bind to the Frizzled (Fz) family of seven transmembrane-spanning receptors (Nelson and Nusse, 2004). In the absence of Wnt ligand, cytosolic -catenin is bound to a complex of proteins including axin, adenomatous polyposis coli (APC), and glycogen synthase kinase 3 , where it becomes phosphorylated, ubiquitinated, and directed to the proteosome for degradaThis work was supported by National Institutes of Health grant CA100869 (to P.J.C.) and the National Institutes of Health Clinical Scientist Development Award DK62833 (to T.A.F.). L.N.S. and T.A.F. contributed equally to this work. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.023705. ABBREVIATIONS: GPCR, G protein-coupled receptor; RGS, regulator of G protein signaling; APC, adenomatous polyposis coli; RhoGEF, Rho guanine nucleotide exchange factor; QL, mutationally activated G form; WT, wild-type; GFP, green fluorescent protein; DTT, dithiothreitol; GST, glutathione transferase; HEK, human embryonic kidney; LPX, polyoxyethylene 10 laurel ether; PAGE, polyacrylamide gel electrophoresis; GTP S, guanosine 5 -O-(3-thio)triphosphate; PIPES, piperazine-N,N -bis(2-ethanesulfonic acid); PHEM, PIPES/HEPES/EGTA/MgSO4; PBS, phosphatebuffered saline; GAP, GTPase-activating protein. 0026-895X/06/7004-1461–1468$20.00 MOLECULAR PHARMACOLOGY Vol. 70, No. 4 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 23705/3143489 Mol Pharmacol 70:1461–1468, 2006 Printed in U.S.A. 1461 at A PE T Jornals on A uust 1, 2017 m oharm .aspeurnals.org D ow nladed from tion. The integrity of the -catenin destruction complex is dependent, in large part, on an interaction between axin and APC (Munemitsu et al., 1995; Spink et al., 2000; Rubinfeld et al., 2001; Choi et al., 2004). Mutations in the APC gene are prevalent in cancers, especially in the colon, and often involve truncations in the region of APC that interacts with axin (Munemitsu et al., 1995; Korinek et al., 1997). In the presence of a Wnt signal, the -catenin destruction complex dissociates and -catenin accumulates in the cytosol, eventually reaching levels high enough to enter the nucleus. Once inside the nucleus, it acts as a transcriptional coactivator with members of the lymphoid enhancer factor/T cell factor family of transcription factors and activates genes important in cell growth and development (Nelson and Nusse, 2004). The RGS domain of axin is the site on this protein at which binding of APC occurs. A cocrystal structure of the RGS domain of axin with the axin-binding domain of APC has confirmed that the axin RGS domain is structurally very similar to other RGS proteins with confirmed G protein binding capacity (Spink et al., 2000). However, binding of APC used a face of the RGS domain distinct from that used for G binding by other RGS proteins (Spink et al., 2000). The first description of an interaction between axin and a G subunit—G s—has recently been reported (Castellone et al., 2005). Here we report that the RGS domain of axin also directly interacts with the subunit of G12 in an activationsensitive manner. Biochemical analysis comparing the axin RGS to the RGS domain of a known G12 effector, p115RhoGEF, suggests that G 12 binds axinRGS and p115RGS in a mutually exclusive fashion. Although axinRGS did not significantly accelerate GTP hydrolysis during in vitro GTPase activity assays, it did specifically prevent G12activated Rho-dependent cell rounding in MDA-MB 231 breast cancer cells expressing activated G 12. These data suggest that G 12 binds to the RGS domain of axin in a manner similar to that in which it interacts with the RGS domain of p115RhoGEF. Materials and Methods Materials. G 12 and G 13 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), G q antibody was a gift of Tom Gettys (Pennington Biomedical Research Center, Baton Rouge, LA), and G i antibody (p960) was described previously (Mumby and Gilman, 1991). The anti-axin rabbit polyclonal antibody and the anti-myc M2 monoclonal antibody were both purchased from Zymed (South San Francisco, CA), and the M1 anti-FLAG mouse monoclonal antibody was purchased from Sigma-Aldrich (St. Louis, MO). The anti-GFP antibody was purchased from Roche (Indianapolis, IN). Plasmid Constructs. cDNAs encoding all G forms used in this study, including mutationally activated (QL) and wild-type (WT) forms, were obtained from the Guthrie Research Institute (now the UMR cDNA Research Center, Rolla, MO). GFP-axin DIX, which encodes a fusion between green fluorescent protein (GFP) and axin lacking residues 873 to 956, was kindly provided by Harold Varmus (Sloan-Kettering Institute, NewYork, NY) and myc-p115RGS by Tohru Kozasa (University of Illinois, Chicago, IL). Two axin RGScontaining sequences were created by PCR: axinRGS (residues 81– 502) and axinRGSa (residues 83–211). The domains were subcloned into pGEX-KG and pGEX5X-1 vectors (Pharmacia, Peapack, NJ), respectively, between EcoRI and HindIII for GST-axinRGS and BamHI and EcoRI for GST-axinRGSa. The pcDNA 3.1 plasmid was purchased from Invitrogen (Carlsbad, CA) and the pEGFP plasmid was purchased from Clontech (Mountain View, CA). Protein Purification. GST fusion proteins used in G 12 binding experiments (GST-axinRGS, GST-axinRGSa, GST-p115, and GST alone) were made in the BL21DE3 strain of Escherichia coli (Novagen, La Jolla, CA). In brief, transformed bacterial colonies containing the indicated constructs were inoculated into 10 ml of media and grown overnight. After 16 h, the small cultures were used to inoculate 500 ml of media and grown at 37°C for 2 to 3 h until the optical density reached 0.5 to 0.6. At this point, the cells were induced with 0.5 mM isopropyl-D-thiogalactopyranoside (Teknova) and cultures were grown for an additional 2.5 h at 37°C. The cells were harvested by centrifugation at 6000g for 15 min, and the resulting pellet was resuspended in 2.5 ml of buffer A (2.3 M sucrose, 50 mM Tris-HCl, pH 7.7, 1 mM EDTA, and a mix of protease inhibitors: 23 g/ml phenylmethylsulfonyl fluoride, 11 g/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone, and 11 g/ml 1-chloro-3-tosylamido-7amino-2-heptanone) followed by dilution with 10 ml of buffer B (50 mM Tris-HCl, pH 7.7, 10 mM KCl, 1 mM EDTA, 1 mM DTT, and mix of protease inhibitors). The cells were then passed three times at 10,000 p.s.i. through a microfluidizer (Microfluidics Corporation, Newton, MA). Cell lysates were cleared by centrifugation at 30,000g for 30 min at 4°C, and the resulting supernatants were incubated with glutathione-Sepharose 4B beads (Amersham Biosciences, Piscataway, NJ) with continuous rocking for 2 h. Beads with bound GST protein were washed with buffer B, and the protein was eluted from the beads by incubation in 50 mM HEPES and 20 mM reduced glutathione three times for 10 min each at 21°C. The eluent was then dialyzed into 50 mM HEPES, 1 mM EDTA, and 1 mM DTT and