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Protease-activated Receptor-1 (PAR1) Acts via a Novel Gα13-Dishevelled Axis to Stabilize β-Catenin Levels

2010; Elsevier BV; Volume: 285; Issue: 20 Linguagem: Inglês

10.1074/jbc.m109.072843

ISSN

1083-351X

Autores

Hagit Turm, Myriam Maoz, Vered Katz, Yongjun Yin, Stefan Offermanns, Rachel Bar-Shavit,

Tópico(s)

Cancer-related gene regulation

Resumo

We have previously shown a novel link between hPar-1 (human protease-activated receptor-1) and β-catenin stabilization. Although it is well recognized that Wnt signaling leads to β-catenin accumulation, the role of PAR1 in the process is unknown. We provide here evidence that PAR1 induces β-catenin stabilization independent of Wnt, Fz (Frizzled), and the co-receptor LRP5/6 (low density lipoprotein-related protein 5/6) and identify selective mediators of the PAR1-β-catenin axis. Immunohistological analyses of hPar1-transgenic (TG) mouse mammary tissues show the expression of both Gα12 and Gα13 compared with age-matched control counterparts. However, only Gα13 was found to be actively involved in PAR1-induced β-catenin stabilization. Indeed, a dominant negative form of Gα13 inhibited both PAR1-induced Matrigel invasion and Lef/Tcf (lymphoid enhancer factor/T cell factor) transcription activity. PAR1-Gα13 association is followed by the recruitment of DVL (Dishevelled), an upstream Wnt signaling protein via the DIX domain. Small interfering RNA-Dvl silencing leads to a reduction in PAR1-induced Matrigel invasion, inhibition of Lef/Tcf transcription activity, and decreased β-catenin accumulation. It is of note that PAR1 also promotes the binding of β-arrestin-2 to DVL, suggesting a role for β-arrestin-2 in PAR1-induced DVL phosphorylation dynamics. Although infection of small interfering RNA-LRP5/6 or the use of the Wnt antagonists, SFRP2 (soluble Frizzled-related protein 2) or SFRP5 potently reduced Wnt3A-mediated β-catenin accumulation, no effect was observed on PAR1-induced β-catenin stabilization. Collectively, our data show that PAR1 mediates β-catenin stabilization independent of Wnt. We propose here a novel cascade of PAR1-induced Gα13-DVL axis in cancer and β-catenin stabilization. We have previously shown a novel link between hPar-1 (human protease-activated receptor-1) and β-catenin stabilization. Although it is well recognized that Wnt signaling leads to β-catenin accumulation, the role of PAR1 in the process is unknown. We provide here evidence that PAR1 induces β-catenin stabilization independent of Wnt, Fz (Frizzled), and the co-receptor LRP5/6 (low density lipoprotein-related protein 5/6) and identify selective mediators of the PAR1-β-catenin axis. Immunohistological analyses of hPar1-transgenic (TG) mouse mammary tissues show the expression of both Gα12 and Gα13 compared with age-matched control counterparts. However, only Gα13 was found to be actively involved in PAR1-induced β-catenin stabilization. Indeed, a dominant negative form of Gα13 inhibited both PAR1-induced Matrigel invasion and Lef/Tcf (lymphoid enhancer factor/T cell factor) transcription activity. PAR1-Gα13 association is followed by the recruitment of DVL (Dishevelled), an upstream Wnt signaling protein via the DIX domain. Small interfering RNA-Dvl silencing leads to a reduction in PAR1-induced Matrigel invasion, inhibition of Lef/Tcf transcription activity, and decreased β-catenin accumulation. It is of note that PAR1 also promotes the binding of β-arrestin-2 to DVL, suggesting a role for β-arrestin-2 in PAR1-induced DVL phosphorylation dynamics. Although infection of small interfering RNA-LRP5/6 or the use of the Wnt antagonists, SFRP2 (soluble Frizzled-related protein 2) or SFRP5 potently reduced Wnt3A-mediated β-catenin accumulation, no effect was observed on PAR1-induced β-catenin stabilization. Collectively, our data show that PAR1 mediates β-catenin stabilization independent of Wnt. We propose here a novel cascade of PAR1-induced Gα13-DVL axis in cancer and β-catenin stabilization. IntroductionPAR1 (protease-activated receptor-1) is the first identified and prototype member of an established protease-activated receptor family. In addition to the traditional role of PAR1 in thrombosis, hemostasis, and vascular biology, its role in tumor biology is currently emerging (1Nierodzik M.L. Karpatkin S. Cancer Cell. 2006; 10: 355-362Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 2Arora P. Cuevas B.D. Russo A. Johnson G.L. Trejo J. Oncogene. 2008; 27: 4434-4445Crossref PubMed Scopus (100) Google Scholar, 3Even-Ram S. Uziely B. Cohen P. Grisaru-Granovsky S. Maoz M. Ginzburg Y. Reich R. Vlodavsky I. Bar-Shavit R. Nat. Med. 1998; 4: 909-914Crossref PubMed Scopus (404) Google Scholar). We have established a novel link between hPar1 and β-catenin stabilization, both in transgenic mouse mammary glands and in a wide spectrum of tumor cell lines (4Yin Y.J. Katz V. Salah Z. Maoz M. Cohen I. Uziely B. Turm H. Grisaru-Granovsky S. Suzuki H. Bar-Shavit R. Cancer Res. 2006; 66: 5224-5233Crossref PubMed Scopus (32) Google Scholar). Protease-activated receptors are part of a large seven-transmembrane-spanning G protein-coupled receptor (GPCR) 2The abbreviations used are: GPCRG protein-coupled receptorWTwild typeMEFmouse embryo fibroblastMMTVmurine mammary tumor virussiRNAsmall interfering RNADNdominant negativeIPimmunoprecipitationLPAlysophosphatidic acidTGtransgenic. family (5Déry O. Corvera C.U. Steinhoff M. Bunnett N.W. Am. J. Physiol. 1998; 274: C1429-C1452Crossref PubMed Google Scholar, 6Macfarlane S.R. Seatter M.J. Kanke T. Hunter G.D. Plevin R. Pharmacol. Rev. 2001; 53: 245-282PubMed Google Scholar), shown to couple to Gαi/o, Gαq, or Gα12/13 within the same cell type (7Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2103) Google Scholar). Of the 16 Gα genes found in the mammalian genome, the Gα12 subfamily is of particular interest to cancer biologists. Gα12 and its sister family member, Gα13, are the only heterotrimeric G proteins that are capable of transforming fibroblasts when overexpressed in their wild-type (WT) form (8Chan A.M. Fleming T.P. McGovern E.S. Chedid M. Miki T. Aaronson S.A. Mol. Cell Biol. 1993; 13: 762-768Crossref PubMed Scopus (144) Google Scholar, 9Xu N. Bradley L. Ambdukar I. Gutkind J.S. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 6741-6745Crossref PubMed Scopus (174) Google Scholar, 10Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5185) Google Scholar). Recent studies have demonstrated that Gα12 is markedly up-regulated in adenocarcinoma of the breast and have identified Gα12 protein as an important regulator of breast and prostate cancer invasion (11Kelly P. Moeller B.J. Juneja J. Booden M.A. Der C.J. Daaka Y. Dewhirst M.W. Fields T.A. Casey P.J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8173-8178Crossref PubMed Scopus (130) Google Scholar, 12Kelly P. Stemmle L.N. Madden J.F. Fields T.A. Daaka Y. Casey P.J. J. Biol. Chem. 2006; 281: 26483-26490Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The Gα12 protein subunit also plays a role in disrupting cadherin-β-catenin interaction and in the down-regulation of the extracellular cell-cell adhesive function of cadherins (13Kaplan D.D. Meigs T.E. Casey P.J. J. Biol. Chem. 2001; 276: 44037-44043Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar).Although an association between PAR1 and β-catenin stabilization has been demonstrated (4Yin Y.J. Katz V. Salah Z. Maoz M. Cohen I. Uziely B. Turm H. Grisaru-Granovsky S. Suzuki H. Bar-Shavit R. Cancer Res. 2006; 66: 5224-5233Crossref PubMed Scopus (32) Google Scholar), the path connecting PAR1 to β-catenin is unknown. Here we set out to elucidate the molecular mechanism and identify selective partners that mediate this association. β-Catenin accumulation is a well established core process of the Wnt signaling pathway. Wnt proteins are secreted glycoprotein ligands that bind to members of the Fz (Frizzled) class of heptahelical GPCR and low density lipoprotein receptor-related protein (LRP5/6) (14Cong F. Schweizer L. Varmus H. Development. 2004; 131: 5103-5115Crossref PubMed Scopus (270) Google Scholar, 15Liu G. Bafico A. Aaronson S.A. Mol. Cell Biol. 2005; 25: 3475-3482Crossref PubMed Scopus (64) Google Scholar), known to regulate cell fate and play a central role in both embryonic development (16Logan C.Y. Nusse R. Annu. Rev. Cell Dev. Biol. 2004; 20: 781-810Crossref PubMed Scopus (4180) Google Scholar) and oncogenicity (17Bienz M. Clevers H. Cell. 2000; 103: 311-320Abstract Full Text Full Text PDF PubMed Scopus (1299) Google Scholar, 18Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar). The canonical Wnt signaling path has been shown to require Gαo and Gαq, which centrally regulate the stability of intracellular β-catenin (19Feigin M.E. Malbon C.C. J. Cell Sci. 2007; 120: 3404-3414Crossref PubMed Scopus (27) Google Scholar, 20Liu X. Rubin J.S. Kimmel A.R. Curr. Biol. 2005; 15: 1989-1997Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 21Malbon C.C. Sci. STKE. 2005; 2005: pe35PubMed Google Scholar). In the absence of Wnt signaling, a complex of proteins comprising the scaffold protein axin, the APC (adenomatous polyposis coli) tumor suppressor protein, GSK-3β (glycogen synthase kinase 3β), and CKI (casein kinase I) assemble to comprise the destruction machinery of β-catenin. Once the complex is assembled, GSK-3β phosphorylates β-catenin that is now being tagged for ubiquitination and degradation by the proteasomal system (22Giles R.H. van Es J.H. Clevers H. Biochim. Biophys. Acta. 2003; 1653: 1-24Crossref PubMed Scopus (1323) Google Scholar, 23Kikuchi A. Kishida S. Yamamoto H. Exp. Mol. Med. 2006; 38: 1-10Crossref PubMed Scopus (169) Google Scholar). Upon Wnt activation, the tagging is eliminated, and hence, β-catenin is no longer detected for degradation. Accumulation of β-catenin in the cytoplasm further leads to its translocation to the nucleus, where it acts as a co-transcription factor with Lef/Tcf (lymphoid enhancer factor/T-cell factor family) and activates wnt target genes downstream (22Giles R.H. van Es J.H. Clevers H. Biochim. Biophys. Acta. 2003; 1653: 1-24Crossref PubMed Scopus (1323) Google Scholar, 23Kikuchi A. Kishida S. Yamamoto H. Exp. Mol. Med. 2006; 38: 1-10Crossref PubMed Scopus (169) Google Scholar). DVL (Dishevelled) is an upstream Wnt signaling protein that mediates all known Wnt pathways, including the canonical, non-canonical, and planar cell polarity pathways (24Malbon C.C. Wang H.Y. Curr. Top. Dev. Biol. 2006; 72: 153-166Crossref PubMed Scopus (96) Google Scholar, 25Wharton Jr., K.A. Dev. Biol. 2003; 253: 1-17Crossref PubMed Scopus (263) Google Scholar, 26Wallingford J.B. Habas R. Development. 2005; 132: 4421-4436Crossref PubMed Scopus (372) Google Scholar). Three DVL isoforms are found in mammalian species (e.g. DVL1 to -3) (27Pizzuti A. Amati F. Calabrese G. Mari A. Colosimo A. Silani V. Giardino L. Ratti A. Penso D. Calzà L. Palka G. Scarlato G. Novelli G. Dallapiccola B. Hum. Mol. Genet. 1996; 5: 953-958Crossref PubMed Scopus (61) Google Scholar, 28Greco T.L. Sussman D.J. Camper S.A. Mamm. Genome. 1996; 7: 475-476Crossref PubMed Scopus (15) Google Scholar, 29Semënov M.V. Snyder M. Genomics. 1997; 42: 302-310Crossref PubMed Scopus (66) Google Scholar). High DVL1 protein expression is detected in breast and cervical squamous cell carcinoma as well as in prostate cancer (30Okino K. Nagai H. Hatta M. Nagahata T. Yoneyama K. Ohta Y. Jin E. Kawanami O. Araki T. Emi M. Oncol. Rep. 2003; 10: 1219-1223PubMed Google Scholar, 31Mizutani K. Miyamoto S. Nagahata T. Konishi N. Emi M. Onda M. Tumori. 2005; 91: 546-551Crossref PubMed Scopus (44) Google Scholar). Similarly, elicited levels of DVL3 have been demonstrated in malignant mesotheliomas and lung cancer (32Uematsu K. He B. You L. Xu Z. McCormick F. Jablons D.M. Oncogene. 2003; 22: 7218-7221Crossref PubMed Scopus (284) Google Scholar, 33Uematsu K. Kanazawa S. You L. He B. Xu Z. Li K. Peterlin B.M. McCormick F. Jablons D.M. Cancer Res. 2003; 63: 4547-4551PubMed Google Scholar). DVL is composed of several structural domains: a DIX binding domain located at the N terminus, a PDZ domain in the midregion, and a DEP domain located about midway between the PDZ domain and the C terminus of DVL (34Boutros M. Mlodzik M. Mech. Dev. 1999; 83: 27-37Crossref PubMed Scopus (234) Google Scholar, 35Strovel E.T. Sussman D.J. Exp. Cell Res. 1999; 253: 637-648Crossref PubMed Scopus (17) Google Scholar). Although the DIX domain enables the dimerization of DVL with other members of the family as well as with axin (36Kishida S. Yamamoto H. Hino S. Ikeda S. Kishida M. Kikuchi A. Mol. Cell Biol. 1999; 19: 4414-4422Crossref PubMed Google Scholar), the PDZ domain provides a docking site for the C terminus of Fz receptors (37Wong H.C. Bourdelas A. Krauss A. Lee H.J. Shao Y. Wu D. Mlodzik M. Shi D.L. Zheng J. Mol. Cell. 2003; 12: 1251-1260Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). The linker between DIX and PDZ provides an essential phosphorylation site for CKI (38Sakanaka C. Leong P. Xu L. Harrison S.D. Williams L.T. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 12548-12552Crossref PubMed Scopus (187) Google Scholar). The DEP domain regulates Rho GTPases in the planar polarity pathway (39Ponting C.P. Bork P. Trends Biochem. Sci. 1996; 21: 245-246Abstract Full Text PDF PubMed Scopus (143) Google Scholar) and is also involved in Fz-induced translocation of DVL1 to the plasma membrane in the canonical pathway (40Pan W.J. Pang S.Z. Huang T. Guo H.Y. Wu D. Li L. Cell Res. 2004; 14: 324-330Crossref PubMed Scopus (55) Google Scholar). Upon Wnt signaling, Fz, together with its co-receptor LRP5/6, recruits DVL via the PDZ domain, leaving the DIX domain free to bind axin-GSK3β proteins (41Zeng X. Huang H. Tamai K. Zhang X. Harada Y. Yokota C. Almeida K. Wang J. Doble B. Woodgett J. Wynshaw-Boris A. Hsieh J.C. He X. Development. 2008; 135: 367-375Crossref PubMed Scopus (352) Google Scholar).In the present study, we identified the selective involvement of Gα13 and DIX-DVL as mediators of PAR1-induced β-catenin stabilization. Our data point to a novel Gα13-DVL axis in cancer, independent (at least upstream of DVL) of Wnt-elicited β-catenin stabilization.DISCUSSIONIn the present paper, we identified a novel pathway of PAR1-induced β-catenin stabilization. Although both Gα12/13 G-protein family members are abundantly overexpressed in hPar1-TG mouse mammary glands as compared with age-matched WT counterparts, dissection between the family members demonstrates the selective functional involvement of activated Gα13. DVL, an upstream junction protein of Wnt signaling, is recruited to Gα13. Once associated with activated Gα13, DVL is dislocated from the cytoplasmic pools and becomes connected with the cell membrane compartment. DVL association with activated Gα13 is shown to be mediated specifically via the DIX domain. The fact that DVL binds to the activated form of Gα13 but not to the inactive form suggests a high affinity interaction of DIX-DVL with the GTP-bound form of Gα13 but not with Gα13-GDP. The critical contribution of the Gα13-DVL axis in PAR1-induced β-catenin stabilization and oncogenicity is highlighted by the potent inhibition of PAR1-induced Lef/Tcf transcription activity, abrogation of Matrigel invasion, and lack of PAR1-elicited β-catenin levels obtained in the presence of either a dominant negative form of Gα13 or siRNA-silenced DVL. PAR1-induced β-catenin stabilization takes place regardless of whether LRP5/6 co-receptors are knocked down or in the presence of the Wnt antagonists SFRP2 and -5. This specifies PAR1-stimulated β-catenin stabilization via the axis of Gα13-DVL. However, it still remains to be determined whether PAR1, a member of GPCR family, acts in an unknown manner to communicate with the Fz receptor via DVL as a mobile scaffold protein for β-catenin stabilization.Other GPCRs have been reported to stimulate β-catenin stabilization and proliferation of colon cancer cells. Most notable of these are the lysophosphatidic acid (LPA) receptors (e.g. LPA1, LPA2, and LPA3) (51Yang M. Zhong W.W. Srivastava N. Slavin A. Yang J. Hoey T. An S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 6027-6032Crossref PubMed Scopus (161) Google Scholar) and EP2 receptor activated by the cyclooxygenase (COX-2) proinflammatory metabolite prostaglandin E2 (52Castellone M.D. Teramoto H. Williams B.O. Druey K.M. Gutkind J.S. Science. 2005; 310: 1504-1510Crossref PubMed Scopus (759) Google Scholar, 53Fujino H. Regan J.W. J. Biol. Chem. 2001; 276: 12489-12492Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The LPA2 receptor couples to Gα12/13, and LPA3 couples to Gαq; both receptors function to trigger massive β-catenin stabilization and cell proliferation via protein kinase C. The signaling route of COX-2 and EP2 is suggested to involve PI3K and the protein kinase Akt, initiated by the direct association of Gαs with the "regulator of G-protein signaling" (RGS) domain of axin. Altogether, these pathways were shown to converge on the Wnt signaling route to induce cytoplasmic β-catenin accumulation, nuclear localization, and enhanced Lef/Tcf transcriptional activities.Each of the Gα12/13 subunits exhibits a different path. Knock-out of the Gα13 gene causes lethality in mouse embryos at midgestation, a phenotype that largely resembles that of the PAR1 knock-out mice (54Offermanns S. Mancino V. Revel J.P. Simon M.I. Science. 1997; 275: 533-536Crossref PubMed Scopus (286) Google Scholar). In contrast, Gα12−/− mice appear viable and grow normally. In addition, during vascular development, a selective and critical role for Gα13 in endothelial cells has been demonstrated (55Ruppel K.M. Willison D. Kataoka H. Wang A. Zheng Y.W. Cornelissen I. Yin L. Xu S.M. Coughlin S.R. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 8281-8286Crossref PubMed Scopus (79) Google Scholar). It therefore appears that Gα13 plays a significant role in PAR1-induced developmental processes. It remains to be determined whether a similar role is played by Gα13 in PAR1-induced tumor biology. Here, we assign a prime function for Gα13 in PAR1-mediated β-catenin stabilization. Recent studies have underscored the role of Gα12 family signaling in both breast and prostate cancer progression (11Kelly P. Moeller B.J. Juneja J. Booden M.A. Der C.J. Daaka Y. Dewhirst M.W. Fields T.A. Casey P.J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8173-8178Crossref PubMed Scopus (130) Google Scholar, 12Kelly P. Stemmle L.N. Madden J.F. Fields T.A. Daaka Y. Casey P.J. J. Biol. Chem. 2006; 281: 26483-26490Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Unexpectedly, we found direct binding of DVL with activated Gα13. The distinct role of DVL is even more complex because DVL was recently found localized in the nucleus, acting along with the β-catenin Lef/Tcf transcription complex to trigger Wnt target gene expression (56Gan X.Q. Wang J.Y. Xi Y. Wu Z.L. Li Y.P. Li L. J. Cell Biol. 2008; 180: 1087-1100Crossref PubMed Scopus (180) Google Scholar). DVL protein binds to the intracellular portion of the Fz transmembrane receptors as well as to axin. Although we found that PAR1 interacts via Gα13 and induces DVL1 membrane translocation, leading to β-catenin stabilization, recent studies in Drosophila revealed that Wnt signaling transduces its signals via Gαo. It was shown that the βγ subunits of Gαo bind and relocalize DVL to the plasma membrane, whereas the α-subunit of Gαo interacts with and recruits axin to the plasma membrane, leading to β-catenin stabilization (57Egger-Adam D. Katanaev V.L. Dev. Dyn. 2010; 239: 168-183Crossref PubMed Google Scholar, 58Angers S. Thorpe C.J. Biechele T.L. Goldenberg S.J. Zheng N. MacCoss M.J. Moon R.T. Nat. Cell Biol. 2006; 8: 348-357Crossref PubMed Scopus (300) Google Scholar). Although binding of DVL to the C-terminal portion of Fz is mediated via the PDZ domain (37Wong H.C. Bourdelas A. Krauss A. Lee H.J. Shao Y. Wu D. Mlodzik M. Shi D.L. Zheng J. Mol. Cell. 2003; 12: 1251-1260Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar), many other DVL functions are mediated via the DIX domain. The highly conserved DIX domain is located within the N-terminal region of DVL and is essential for its signaling activity (59Axelrod J.D. Miller J.R. Shulman J.M. Moon R.T. Perrimon N. Genes Dev. 1998; 12: 2610-2622Crossref PubMed Scopus (535) Google Scholar). Interestingly, only two other proteins contain a close relative of the DIX domain: axin, in its C-terminal domain, as well as a less well known protein, Ccd1, which is found exclusively in vertebrates (60Shiomi K. Uchida H. Keino-Masu K. Masu M. Curr. Biol. 2003; 13: 73-77Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 61Luo W. Zou H. Jin L. Lin S. Li Q. Ye Z. Rui H. Lin S.C. J. Biol. Chem. 2005; 280: 5054-5060Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). DVL has been shown to have a strong tendency to form self-associating protein assemblies in the cytoplasm that are visible as puncta and are mediated via the DIX domain. Overall, the DIX domain is well known to mediate oligomerization and binding to axin (36Kishida S. Yamamoto H. Hino S. Ikeda S. Kishida M. Kikuchi A. Mol. Cell Biol. 1999; 19: 4414-4422Crossref PubMed Google Scholar, 62Hsu W. Zeng L. Costantini F. J. Biol. Chem. 1999; 274: 3439-3445Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 63Julius M.A. Schelbert B. Hsu W. Fitzpatrick E. Jho E. Fagotto F. Costantini F. Kitajewski J. Biochem. Biophys. Res. Commun. 2000; 276: 1162-1169Crossref PubMed Scopus (54) Google Scholar). The phosphorylation of DVL is another critical step in the regulation of DVL-mediated downstream signal transduction. The best studied serine/threonine kinases are casein kinase I ϵ and δ (CKI ϵ/δ). CKI ϵ/δ are positive regulators of the Wnt signaling pathway, phosphorylating DVL in response to Wnt signaling at the SXXS motif, located at sites 139 and 142 of DVL, respectively. In fact, this site is present beyond the DIX domain, residing within the linker region between the DIX and PDZ (e.g. the DIX domain resides between 10–94) (64Schwarz-Romond T. Fiedler M. Shibata N. Butler P.J. Kikuchi A. Higuchi Y. Bienz M. Nat. Struct. Mol. Biol. 2007; 14: 484-492Crossref PubMed Scopus (302) Google Scholar, 65Schwarz-Romond T. Metcalfe C. Bienz M. J. Cell Sci. 2007; 120: 2402-2412Crossref PubMed Scopus (168) Google Scholar). Whether activation of PAR1 leads to stimulation of the traditional Wnt-elicited kinases and/or others remains to be determined. We demonstrate that PAR1 activation causes DVL phosphorylation (Fig. 6c). It is therefore postulated that the activation of PAR1 stimulates DVL binding (to Gα13) and activation (e.g. serine phosphorylation). It was recently demonstrated that β-arrestin binds DVL in the vicinity of the CKI ϵ/δ phosphorylation sites following Wnt signaling. Indeed, inhibition of Wnt signaling by CKI inhibitors reduced the binding of β-arrestin to DVL and β-catenin stabilization (50Bryja V. Gradl D. Schambony A. Arenas E. Schulte G. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 6690-6695Crossref PubMed Scopus (122) Google Scholar). β-Arrestins are known to regulate GPCR desensitization as well as signaling (49Reiter E. Lefkowitz R.J. Trends Endocrinol. Metab. 2006; 17: 159-165Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar). The phosphorylation of PAR1 is critical for its rapid termination, mediated most likely via GRK4 and GRK5 (GPCR kinases) (66Ishii K. Chen J. Ishii M. Koch W.J. Freedman N.J. Lefkowitz R.J. Coughlin S.R. J. Biol. Chem. 1994; 269: 1125-1130Abstract Full Text PDF PubMed Google Scholar, 67Tiruppathi C. Yan W. Sandoval R. Naqvi T. Pronin A.N. Benovic J.L. Malik A.B. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 7440-7445Crossref PubMed Scopus (81) Google Scholar), regulated via β-arrestin-1 (68Paing M.M. Stutts A.B. Kohout T.A. Lefkowitz R.J. Trejo J. J. Biol. Chem. 2002; 277: 1292-1300Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). We demonstrate here that PAR1 activation also promotes the association of β-arrestin-2 with DVL. We suggest that activation of PAR1 leads to receptor desensitization and internalization mediated via β-arrestin-1 (68Paing M.M. Stutts A.B. Kohout T.A. Lefkowitz R.J. Trejo J. J. Biol. Chem. 2002; 277: 1292-1300Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), whereas β-arrestin-2 is free to act as a signaling protein that associates with DVL to elicit β-catenin stabilization. It is postulated that β-arrestin identifies phosphorylated (primed) DVL and subsequently recruits other kinases for the completion of DVL activation and dynamics. In this regard, activation of PAR1 resembles DVL activation by Wnt3A stimulation. Furthermore, although we demonstrate that Wnt antagonist SFRPs do not impair PAR1-induced β-catenin stabilization, this does not exclude the possibility that there is cross-talk between GPCRs (e.g. Fz and PAR1) mediated by a junction-labile cytoplasmic protein. It is possible that PAR1-induced β-catenin levels converge with the traditional canonical Wnt pathway downstream of DVL.One should keep in mind that Wnts act through both Fz and LRP receptors to bring them into proximity (14Cong F. Schweizer L. Varmus H. Development. 2004; 131: 5103-5115Crossref PubMed Scopus (270) Google Scholar). It is further believed that Wnt stimulates the β-catenin pathway by relocating axin to the plasma membrane, thereby inactivating its function as a tumor suppressor protein. It is still not clear whether Wnt-induced axin membrane translocation is a prerequisite for its dissociation from the β-catenin degradation complex. In addition, although we still have gaps in our understanding of the β-catenin stabilization pathway, it appears that two separate axes exist: the LRP-axin axis and the Fz-DVL phosphorylation axis. These two branches ultimately converge at a later stage, eventually resulting in β-catenin stabilization and nuclear translocation (69Cadigan K.M. Liu Y.I. J. Cell Sci. 2006; 119: 395-402Crossref PubMed Scopus (395) Google Scholar). In the Wnt-induced Fz-LRP6 receptor complex, Fz recruits DVL through the PDZ domain, leaving the DVL-DIX domain accessible for further interactions. DVL-DIX then recruits the axin-GSK3β complex via oligomerization and puncta formation (64Schwarz-Romond T. Fiedler M. Shibata N. Butler P.J. Kikuchi A. Higuchi Y. Bienz M. Nat. Struct. Mol. Biol. 2007; 14: 484-492Crossref PubMed Scopus (302) Google Scholar), thus promoting GSK3β-induced LRP phosphorylation. Axin is therefore required for LRP6 phosphorylation and activation. Supporting evidence for the distinct routes of axin-LRP and Fz-DVL is provided by several studies showing that truncated LRP5/6 activates β-catenin independently of DVL (14Cong F. Schweizer L. Varmus H. Development. 2004; 131: 5103-5115Crossref PubMed Scopus (270) Google Scholar, 15Liu G. Bafico A. Aaronson S.A. Mol. Cell Biol. 2005; 25: 3475-3482Crossref PubMed Scopus (64) Google Scholar, 70Li L. Mao J. Sun L. Liu W. Wu D. J. Biol. Chem. 2002; 277: 5977-5981Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 71Schweizer L. Varmus H. BMC Cell Biol. 2003; 4: 4Crossref PubMed Scopus (113) Google Scholar). This suggests that it can recruit axin to the plasma membrane independently of DVL. The relative role of the PAR1 Gα13-DVL axis in leading to β-catenin stabilization remains to be evaluated. Whether it can be bypassed via the axin-LRP path, shown to provide a route distinct from the Fz-DVL phosphorylation axis, is yet unknown.We demonstrated here a new pathway for DVL that associates via the DIX domain with Gα13, which is recruited to the C-terminal tail of PAR1. Overall, we propose an original Wnt-independent pathway for the PAR1-induced Gα13-DVL axis. This pathway provides new cellular targets central in cancer progression. IntroductionPAR1 (protease-activated receptor-1) is the first identified and prototype member of an established protease-activated receptor family. In addition to the traditional role of PAR1 in thrombosis, hemostasis, and vascular biology, its role in tumor biology is currently emerging (1Nierodzik M.L. Karpatkin S. Cancer Cell. 2006; 10: 355-362Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 2Arora P. Cuevas B.D. Russo A. Johnson G.L. Trejo J. 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Science. 1998; 279: 509-514Crossref PubMed Scopus (5185) Google Scholar). Recent studies have demonstrated that Gα12 is markedly up-regulated in adenocarcinoma of the breast and have identified Gα12 protein as an important regulator of breast and prostate cancer invasion (11Kelly P. Moeller B.J. Juneja J. Booden M.A. Der C.J. Daaka Y. Dewhirst M.W. Fields T.A. Casey P.J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8173-8178Crossref PubMed Scopus (130) Google Scholar, 12Kelly P. Stemmle L.N. Madden J.F. Fields T.A. Daaka Y. Casey P.J. J. Biol. Chem. 2006; 281: 26483-26490Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The Gα12 protein subunit also plays a role in disrupting cadherin-β-catenin interaction and in the down-regulation of the extracellular cell-cell adhesive function of cadherins (13Kaplan D.D. Meigs T.E. Casey P.J. J. Biol. Chem. 2001; 276: 44037-44043Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar).Although an association between PAR1 and β-catenin stabilization has been demonstrated (4Yin Y.J. Katz V. Salah Z. Maoz M. Cohen I. Uziely B. Turm H. Grisaru-Granovsky S. Suzuki H. Bar-Shavit R. Cancer Res. 2006; 66: 5224-5233Crossref PubMed Scopus (32) Google Scholar), the path connecting PAR1 to β-catenin is unknown. Here we set out to elucidate the molecular mechanism and identify selective partners that mediate this association. β-Catenin accumulation is a well established core process of the Wnt signaling pathway. Wnt proteins are secreted glycoprotein ligands that bind to members of the Fz (Frizzled) class of heptahelical GPCR and low density lipoprotein receptor-related protein (LRP5/6) (14Cong F. Schweizer L. Varmus H. Development. 2004; 131: 5103-5115Crossref PubMed Scopus (270) Google Scholar, 15Liu G. Bafico A. Aaronson S.A. Mol. Cell Biol. 2005; 25: 3475-3482Crossref PubMed Scopus (64) Google Scholar), known to regulate cell fate and play a central role in both embryonic development (16Logan C.Y. Nusse R. Annu. Rev. Cell Dev. Biol. 2004; 20: 781-810Crossref PubMed Scopus (4180) Google Scholar) and oncogenicity (17Bienz M. Clevers H. Cell. 2000; 103: 311-320Abstract Full Text Full Text PDF PubMed Scopus (1299) Google Scholar, 18Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar). The canonical Wnt signaling path has been shown to require Gαo and Gαq, which centrally regulate the stability of intracellular β-catenin (19Feigin M.E. Malbon C.C. J. Cell Sci. 2007; 120: 3404-3414Crossref PubMed Scopus (27) Google Scholar, 20Liu X. Rubin J.S. Kimmel A.R. Curr. Biol. 2005; 15: 1989-1997Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 21Malbon C.C. Sci. STKE. 2005; 2005: pe35PubMed Google Scholar). In the absence of Wnt signaling, a complex of proteins comprising the scaffold protein axin, the APC (adenomatous polyposis coli) tumor suppressor protein, GSK-3β (glycogen synthase kinase 3β), and CKI (casein kinase I) assemble to comprise the destruction machinery of β-catenin. Once the complex is assembled, GSK-3β phosphorylates β-catenin that is now being tagged for ubiquitination and degradation by the proteasomal system (22Giles R.H. van Es J.H. Clevers H. Biochim. Biophys. Acta. 2003; 1653: 1-24Crossref PubMed Scopus (1323) Google Scholar, 23Kikuchi A. Kishida S. Yamamoto H. Exp. Mol. Med. 2006; 38: 1-10Crossref PubMed Scopus (169) Google Scholar). Upon Wnt activation, the tagging is eliminated, and hence, β-catenin is no longer detected for degradation. Accumulation of β-catenin in the cytoplasm further leads to its translocation to the nucleus, where it acts as a co-transcription factor with Lef/Tcf (lymphoid enhancer factor/T-cell factor family) and activates wnt target genes downstream (22Giles R.H. van Es J.H. Clevers H. Biochim. Biophys. Acta. 2003; 1653: 1-24Crossref PubMed Scopus (1323) Google Scholar, 23Kikuchi A. Kishida S. Yamamoto H. Exp. Mol. Med. 2006; 38: 1-10Crossref PubMed Scopus (169) Google Scholar). DVL (Dishevelled) is an upstream Wnt signaling protein that mediates all known Wnt pathways, including the canonical, non-canonical, and planar cell polarity pathways (24Malbon C.C. Wang H.Y. Curr. Top. Dev. Biol. 2006; 72: 153-166Crossref PubMed Scopus (96) Google Scholar, 25Wharton Jr., K.A. Dev. Biol. 2003; 253: 1-17Crossref PubMed Scopus (263) Google Scholar, 26Wallingford J.B. Habas R. Development. 2005; 132: 4421-4436Crossref PubMed Scopus (372) Google Scholar). Three DVL isoforms are found in mammalian species (e.g. DVL1 to -3) (27Pizzuti A. Amati F. Calabrese G. Mari A. Colosimo A. Silani V. Giardino L. Ratti A. Penso D. Calzà L. Palka G. Scarlato G. Novelli G. Dallapiccola B. Hum. Mol. Genet. 1996; 5: 953-958Crossref PubMed Scopus (61) Google Scholar, 28Greco T.L. Sussman D.J. Camper S.A. Mamm. Genome. 1996; 7: 475-476Crossref PubMed Scopus (15) Google Scholar, 29Semënov M.V. Snyder M. Genomics. 1997; 42: 302-310Crossref PubMed Scopus (66) Google Scholar). High DVL1 protein expression is detected in breast and cervical squamous cell carcinoma as well as in prostate cancer (30Okino K. Nagai H. Hatta M. Nagahata T. Yoneyama K. Ohta Y. Jin E. Kawanami O. Araki T. Emi M. Oncol. Rep. 2003; 10: 1219-1223PubMed Google Scholar, 31Mizutani K. Miyamoto S. Nagahata T. Konishi N. Emi M. Onda M. Tumori. 2005; 91: 546-551Crossref PubMed Scopus (44) Google Scholar). Similarly, elicited levels of DVL3 have been demonstrated in malignant mesotheliomas and lung cancer (32Uematsu K. He B. You L. Xu Z. McCormick F. Jablons D.M. Oncogene. 2003; 22: 7218-7221Crossref PubMed Scopus (284) Google Scholar, 33Uematsu K. Kanazawa S. You L. He B. Xu Z. Li K. Peterlin B.M. McCormick F. Jablons D.M. Cancer Res. 2003; 63: 4547-4551PubMed Google Scholar). DVL is composed of several structural domains: a DIX binding domain located at the N terminus, a PDZ domain in the midregion, and a DEP domain located about midway between the PDZ domain and the C terminus of DVL (34Boutros M. Mlodzik M. Mech. Dev. 1999; 83: 27-37Crossref PubMed Scopus (234) Google Scholar, 35Strovel E.T. Sussman D.J. Exp. Cell Res. 1999; 253: 637-648Crossref PubMed Scopus (17) Google Scholar). Although the DIX domain enables the dimerization of DVL with other members of the family as well as with axin (36Kishida S. Yamamoto H. Hino S. Ikeda S. Kishida M. Kikuchi A. Mol. 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Upon Wnt signaling, Fz, together with its co-receptor LRP5/6, recruits DVL via the PDZ domain, leaving the DIX domain free to bind axin-GSK3β proteins (41Zeng X. Huang H. Tamai K. Zhang X. Harada Y. Yokota C. Almeida K. Wang J. Doble B. Woodgett J. Wynshaw-Boris A. Hsieh J.C. He X. Development. 2008; 135: 367-375Crossref PubMed Scopus (352) Google Scholar).In the present study, we identified the selective involvement of Gα13 and DIX-DVL as mediators of PAR1-induced β-catenin stabilization. Our data point to a novel Gα13-DVL axis in cancer, independent (at least upstream of DVL) of Wnt-elicited β-catenin stabilization.

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