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• W2085492709 abstract "Gα13, the α-subunit of the heterotrimeric G protein G13, has been shown to stimulate cell migration in addition to inducing oncogenic transformation. Cta, a Drosophila ortholog of G13, has been shown to be critical for cell migration leading to the ventral furrow formation in Drosophila embryos. Loss of Gα13 has been shown to disrupt cell migration associated with angiogenesis in developing mouse embryos. Whereas these observations point to the vital role of G13-orthologs in regulating cell migration, widely across the species barrier, the mechanism by which Gα13 couples to cytoskeleton and cell migration is largely unknown. Here we show that Gα13 physically interacts with Hax-1, a cytoskeleton-associated, cortactin-interacting intracellular protein, and this interaction is required for Gα13-stimulated cell migration. Hax-1 interaction is specific to Gα13, and this interaction is more pronounced with the mutationally or functionally activated form of Gα13 as compared with the wild-type Gα13. Expression of Hax-1 reduces the formation of actin stress fibers and focal adhesion complexes in Gα13-expressing NIH3T3 cells. Coexpression of Hax-1 also attenuates Gα13-stimulated activity of Rho while potentiating Gα13-stimulated activity of Rac. The presence of a quadnary complex consisting of Gα13, Hax-1, Rac, and cortactin indicates the role of Hax-1 in tethering Gα13 to the cytoskeletal component(s) involved in cell movement. Whereas the expression of Hax-1 potentiates Gα13-mediated cell movement, silencing of endogenous Hax-1 with Hax-1-specific small interfering RNAs drastically reduces Gα13-mediated cell migration. These findings, along with the observation that Hax-1 is overexpressed in metastatic tumors and tumor cell lines, suggest a novel role for the association of oncogenic Gα13 and Hax-1 in tumor metastasis. Gα13, the α-subunit of the heterotrimeric G protein G13, has been shown to stimulate cell migration in addition to inducing oncogenic transformation. Cta, a Drosophila ortholog of G13, has been shown to be critical for cell migration leading to the ventral furrow formation in Drosophila embryos. Loss of Gα13 has been shown to disrupt cell migration associated with angiogenesis in developing mouse embryos. Whereas these observations point to the vital role of G13-orthologs in regulating cell migration, widely across the species barrier, the mechanism by which Gα13 couples to cytoskeleton and cell migration is largely unknown. Here we show that Gα13 physically interacts with Hax-1, a cytoskeleton-associated, cortactin-interacting intracellular protein, and this interaction is required for Gα13-stimulated cell migration. Hax-1 interaction is specific to Gα13, and this interaction is more pronounced with the mutationally or functionally activated form of Gα13 as compared with the wild-type Gα13. Expression of Hax-1 reduces the formation of actin stress fibers and focal adhesion complexes in Gα13-expressing NIH3T3 cells. Coexpression of Hax-1 also attenuates Gα13-stimulated activity of Rho while potentiating Gα13-stimulated activity of Rac. The presence of a quadnary complex consisting of Gα13, Hax-1, Rac, and cortactin indicates the role of Hax-1 in tethering Gα13 to the cytoskeletal component(s) involved in cell movement. Whereas the expression of Hax-1 potentiates Gα13-mediated cell movement, silencing of endogenous Hax-1 with Hax-1-specific small interfering RNAs drastically reduces Gα13-mediated cell migration. These findings, along with the observation that Hax-1 is overexpressed in metastatic tumors and tumor cell lines, suggest a novel role for the association of oncogenic Gα13 and Hax-1 in tumor metastasis. Cell migration plays a vital role in different biological processes ranging from embryogenesis to immune response (1Franz C.M. Jones G.E. Ridley A.J. Dev. Cell. 2002; 2: 153-158Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 2Vicente-Manzanres M. Sancho D. Yanez-Mo M. Sanchez-Madrid F. Int. Rev. Cytol. 2002; 216: 233-289Crossref PubMed Scopus (55) Google Scholar). However, an aberrant activation of cell migration in neoplastic cells results in tumor metastasis. Cells migrate in response to different cues through the coordinated interactions of actin- and/or microtubule-associated cytoskeletal proteins (3Rodriguez O.C. Schaefer A.W. Mandato C.A. Forscher P. Bement W.M. Waterman-Storer C.M. Nat. Cell Biol. 2003; 5: 599-609Crossref PubMed Scopus (705) Google Scholar, 4Mitchison T.J. Cramer L.P. Cell. 1996; 84: 371-379Abstract Full Text Full Text PDF PubMed Scopus (1303) Google Scholar). G protein-coupled receptors and their cognate G proteins play a major role in regulating cell migration and chemokinesis (5Rossi D. Zlotnik A. Annu. Rev. Immunol. 2000; 18: 217-242Crossref PubMed Scopus (2092) Google Scholar). The G12 family of G proteins, defined by α-subunits Gα12 and Gα13, has been shown to activate novel signaling pathways involved in cell growth and neoplastic transformation (6Radhika V. Dhanasekaran N. Oncogene. 2001; 20: 1607-1614Crossref PubMed Scopus (122) Google Scholar). Two lines of evidence indicate that the α-subunit of G13, Gα13, is primarily involved in the regulation of cell migration (7Parks S. Wieschaus E. Cell. 1991; 64: 447-458Abstract Full Text PDF PubMed Scopus (244) Google Scholar, 8Offermanns S. Mancino V. Revel J.-P. Simon M.I. Science. 1997; 275: 533-536Crossref PubMed Scopus (287) Google Scholar, 9Gu J.L. Muller S. Mancino V. Offermanns S. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9352-9357Crossref PubMed Scopus (100) Google Scholar). The first is the observation that Cta, an ortholog of Gα13, is critically required for cell movement during Drosophila embryogenesis (7Parks S. Wieschaus E. Cell. 1991; 64: 447-458Abstract Full Text PDF PubMed Scopus (244) Google Scholar). The second is the finding that Gα13-null (Gα13-/-) fibroblasts show the loss of chemokinetic response to thrombin or lysophosphatidic acid receptor-mediated cell movement (8Offermanns S. Mancino V. Revel J.-P. Simon M.I. Science. 1997; 275: 533-536Crossref PubMed Scopus (287) Google Scholar, 9Gu J.L. Muller S. Mancino V. Offermanns S. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9352-9357Crossref PubMed Scopus (100) Google Scholar). Existing models of cell movement suggest that the initial movements in cell migration involve polymerization of filamentous actin at the leading edge forming membrane extensions (3Rodriguez O.C. Schaefer A.W. Mandato C.A. Forscher P. Bement W.M. Waterman-Storer C.M. Nat. Cell Biol. 2003; 5: 599-609Crossref PubMed Scopus (705) Google Scholar, 4Mitchison T.J. Cramer L.P. Cell. 1996; 84: 371-379Abstract Full Text Full Text PDF PubMed Scopus (1303) Google Scholar). Subsequent adhesion at the leading edge is followed by the translocation of the cell body by the forward flow of the cytosol. Finally, whereas the leading edge of the cell is still attached to the substratum, the rear-end is detached and retracted into the cell body thereby effecting cell movement. All of these distinct phases of cell movement, with the exception of cytosolic translocation, involve temporal and spatial regulation of actin polymerization and depolymerization. Although, many different proteins control actin polymerization, several lines of evidence indicate that the interaction between cortactin and actin-related proteins 2/3 plays a critical role in actin polymerization leading to cell movement (10Patel A.S. Schechter G.L. Wasilenko W.J. Somers K.D. Oncogene. 1998; 16: 3227-3232Crossref PubMed Scopus (126) Google Scholar, 11Uruno T. Liu J. Zhang P. Fan Y-X. Egile C. Li R. Mueller S.C. Zhan X. Nat. Cell Biol. 2001; 3: 259-266Crossref PubMed Scopus (452) Google Scholar, 12Uruno T. Zhang P. Liu J. Hao J.J. Zhan X. Biochem. J. 2003; 371: 485-493Crossref PubMed Scopus (65) Google Scholar). Cortactin is a major substrate for tyrosine kinases such as Src, Fer, and Syk, and is usually present in the cytosol. Upon growth factor stimulation, it is translocated by activated Rac-1 to cell periphery where it interacts with F-actin and stimulates actin-related protein 2/3-mediated actin polymerization (13Weed S.A. Du Y. Parsons J.T. J. Cell Sci. 1998; 111: 2433-2443PubMed Google Scholar). As a result, tyrosine kinases as well as small GTPases converge on cortactin to stimulate actin polymerization and consequent cell movement. Although Gα13 has been known to be involved in the regulation of thrombin and lysophosphatidic acid-receptor-stimulated cell migration of fibroblasts (8Offermanns S. Mancino V. Revel J.-P. Simon M.I. Science. 1997; 275: 533-536Crossref PubMed Scopus (287) Google Scholar, 9Gu J.L. Muller S. Mancino V. Offermanns S. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9352-9357Crossref PubMed Scopus (100) Google Scholar), the mechanism by which Gα13 stimulates cell movement or the identity of the signaling components involved in Gα13-mediated cytokinesis is largely unknown. Therefore, we sought to identify the signaling components involved in Gα13-mediated cell movement. Here we demonstrate that Gα13 physically associates with intracellular protein Hax-1, which has been previously identified as a cortactin-interacting protein (14Suzuki Y. Demoliere C. Daisuke K. Takeshita H. Deuschle U. Watanabe T. J. Immunol. 1997; 158: 2736-2744PubMed Google Scholar) and that Hax-1 promotes Gα13-mediated cell migration. Furthermore, we show that Gα13 and Hax-1 exists in a complex consisting of Rac and cortactin. We also demonstrate that the coexpression of Hax-1 enhances Gα13-mediated Rac activity while inhibiting Rho activity, both of which can promote cell movement. These results describe for the first time a critical role for Hax-1 in cell movement mediated by Gα13. Plasmids, Strains, and Cells—The yeast expression vector pLexA-Gα13ED was constructed by ligating the PCR-derived cDNA insert encoding amino acids 221–347 of Gα13 into pBTM116 vector. Expression vector pcDNA3-Hax-1 was constructed by ligating the EcoRI-SpeI-excised Hax-1 insert from pME18S-Hax-1 into the EcoRI and XbaI site of pcDNA3 vector. C-terminal S-protein-tagged Hax-1 was constructed by replacing the stop codon of Hax-1 with the coding sequence for the S-tag (KETAAAKFERQHMDS) followed by a stop codon. The resultant Hax-1-S-tag was cloned into the pcDNA3 vector. Cell lines Gα13QL- and Gα13WT-NIH3T3 have been previously described (31Dermott J.M. Ha J.H. Lee C.H. Dhanasekaran N. Oncogene. 2004; 23: 226-232Crossref PubMed Scopus (24) Google Scholar). All the constructs were verified by sequencing. Transfection of NIH3T3 cells was carried out using the calcium phosphate method as previously described (32Dermott J.M. Dhanasekaran N. Methods Enzymol. 2002; 344: 298-309Crossref PubMed Scopus (9) Google Scholar). COS-7 cells were transfected using FuGENE-6 reagent (Roche Applied Science) according to the manufacturer's protocol. Yeast Two-hybrid Screen—The yeast two-hybrid screen was performed in yeast strain L40 transformed with pLexA-Gα13ED and plasmid pGAD-GH containing an oligo(dT)-primed HeLa cell cDNA library (Clontech, Palo Alto, CA). Of the 4 × 106 transformants screened, 550 clones were found to grow in the absence of His. The His+ colonies were restreaked on SD-His/-Leu/-Trp plates and subjected to β-galactosidase activity using a filter assay. The resultant 454 His+Lacz+ colonies were grown in SD-Leu medium to enable the segregation of pLexA-Gα13ED. The Trp-Leu+ segregates were mated to yeast strain AMR70 that had been pre-transformed with the plasmid pLex-Lamin or pLexA-Gα13ED. The leu+ trp+ diploids were then assayed for transactivation of the lacZ reporter by a filter assay. The positive clones totaling 115 that showed β-galactosidase activity only in the presence of pLexA-Gα13ED were isolated and the library plasmids were recovered. The cDNA inserts of the library plasmids were amplified by PCR and 10 representative clones were further sequenced. The sequence analysis of the cDNA inserts revealed that they are five independent fusions of human Hax-1. Co-precipitation and Immunoblot Analysis—Co-precipitation studies were carried out with COS-7 cells using S-protein-agarose (Novagen, EMD Biosciences, Inc., Madison, WI) or antibodies specific to the protein of interest. At 24 h following transfection, cells were lysed and cell lysate protein (1 μg each) was incubated with 35 μl of S-protein-agarose for 4 h at 4 °C. After repeated washes with lysis buffer, the S-proteinagarose-bound proteins were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. Co-immunoprecipitation analyses were carried out by incubating cell lysate protein (1 μg each) with 1–5 μg of the respective antibodies for 4 h at 4 °C followed by the addition of 30 μl of 50% slurry of protein A-Sepharose (Amersham Biosciences). Antibodies to cortactin (05-180) and Rac (05-389) were from Upstate Signaling Solutions (Charlottesville, VA), whereas antibodies to the hemagglutinin epitope (2362) and Hax-1 (H65220) were from Cell Signaling (Beverly, MA), and BD Biosciences (San Jose, CA) respectively. Antibodies to S-epitope (SC-802), Gαs (sc-823), Gαq (sc-393), and Gα12 (sc-409) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibodies to Gαi (1521) were a kind gift from Dr. David Manning, University of Pennsylvania, Philadelphia, PA. For immunoblot and immunoprecipitation studies for Gα13WT and Gα13QL, rabbit polyclonal antibodies (AS1-89-2) raised against the C terminus of Gα13 were used. After washing the immunoprecipitates twice with lysis buffer, the immunoprecipitated proteins were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. Immunoblot analyses with specific antibodies were carried out following the previously published procedures (31Dermott J.M. Ha J.H. Lee C.H. Dhanasekaran N. Oncogene. 2004; 23: 226-232Crossref PubMed Scopus (24) Google Scholar). Co-localization of Hax-1 and Gα13—Cells were grown on coverslips for 48 h, fixed with 3% paraformaldehyde in PBS 1The abbreviations used are: PBS, phosphate-buffered saline; siRNA, short interfering RNA; WT, wild-type. for 10 min, permeablized by 0.05% Triton X-100 (10 min), blocked with 1% bovine serum albumin in PBS (30 min), and incubated with mouse monoclonal antibodies to Hax-1 or rabbit polyclonal antibodies to Gα13 (1:200) for 1 h at 25 °C. After washing, samples were incubated with 1:100 dilution of Alexa Fluor 488-labeled goat anti-mouse IgG or Texas Red-labeled goat anti-rabbit IgG (Molecular Probes, Eugene, OR) for 1 h at 25 °C to obtain immunofluorescent imaging of Hax-1 and Gα13, respectively. The coverslips containing the cells were washed with PBS, which were mounted on glass slides with 10 μl of Prolong Antifade reagent (Molecular Probes, Eugene, OR). The images were recorded and analyzed using an Olympus confocal microscope with a ×60/NA 1.4 plan-apochromat objective. Actin Staining—Actin staining was carried out using fluorescein isothiocyanate-labeled phalloidin (Sigma) following previously published methods (33Radhika V. Naik N.R. Advani S.H. Bhisey A.N. Cytometry. 2000; 42: 379-386Crossref PubMed Scopus (11) Google Scholar). Briefly, NIH3T3 cells/transfectants of interest were fixed on coverslips with 3% formaldehyde. After washing with PBS the cells were incubated at 37 °C in PBS containing 1 μm fluorescein isothiocyanate-phalloidin, 0.01% lysolecithin, and 0.05% bovine serum albumin for 10 min. At the end of the incubation, the cells were washed with PBS containing 2% bovine serum albumin and examined under a fluorescent microscope. Cell Migration Assay—Cell migration assay was carried out in cell culture inserts (PET membrane with 8-μm pores, BD Biosciences). The cell culture inserts containing 3 × 104 cells in 200 μl of serum-free Dulbecco's modified Eagle's medium was placed in the well of the companion plate containing 500 μl of Dulbecco's modified Eagle's medium with 5% serum per well. Cells were incubated at 37 °C for 3 h. Non-migrating cells on the inner side of the inserts were removed with a cotton swab and the migrated cells on the underside of the insert were fixed and stained with Hemacolor (EMD Chemicals Inc., Gibbstown, NJ). Photomicrographs of three random fields were taken and enumerated to calculate the average number of cells that had migrated. Silencing Hax-1 with Short Interfering RNAs (siRNA)—siRNA targeting mouse Hax-1, coding regions 188–208 bases (5′-AATTCGGTTTCAGCTTCAGCC-3′), was synthesized and purified (Qiagen Inc., Valencia, CA). An equimolar pool containing four different nonspecific control siRNA duplexes (number D-0011206-13-05, Dharmacon, Inc., Lafayette, CO) were used as control. Cells were transfected with 20 μm siRNA at 24-h intervals using TransMessenger reagent (Qiagen Inc.) for 96 h. Rac/Rho Activation Assay—NIH3T3 cells stably expressing Gα13WT or Gα13QL were transiently transfected with pCDNA3-Hax-1. At 48 h following transfection, cells were lysed and the Rho or Rac pull-down assay was carried out according to the manufacturer's protocol (Upstate Signaling Solutions, Charlottesville, VA). Activated GTP-bound Rho was pulled-down from 1 mg of lysate protein using 20 μg of Rhotekin-RBD-(7–89)-agarose beads. The pulled-down Rho was identified by immunoblot analysis using anti-Rho (-A, -B, and -C). Active GTP-bound Rac was assayed by pulling down active Rac using 10 μg of PAK-PBD-(67–150)-agarose beads from 1 mg of lysates. The pulled-down Rac-GTP was identified by immunoblot analysis using anti-Rac antibody. Interaction of Gα13 with Hax-1—To identify novel signaling proteins that interact with Gα13, a yeast two-hybrid screen was carried out in which the effector interacting domain of Gα13 spanning amino acids 221–347, deduced from the crystal structures of the Gαt and GαI (15Noel J.P. Hamm H.E. Sigler P.B. Nature. 1993; 366: 654-663Crossref PubMed Scopus (706) Google Scholar), was used as bait in a human HeLa cell cDNA library. Analyses of a set of transformants that were positive for Gα13 interaction identified Hax-1 (HS-1 associated protein X-1) as a Gα13-interacting protein. Previous studies have identified Hax-1 as an intracellular, 35-kDa HS-1 or cortactin-interacting protein (14Suzuki Y. Demoliere C. Daisuke K. Takeshita H. Deuschle U. Watanabe T. J. Immunol. 1997; 158: 2736-2744PubMed Google Scholar, 16Gallagher A.R. Cedzich A. Gretz N. Somlo S. Witzgall R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4017-4022Crossref PubMed Scopus (162) Google Scholar). Sequence analyses of Hax-1 inserts rescued from these transformants revealed that Hax-1 coding sequences of varying lengths interact with Gα13 (Fig. 1A). The interactions between these Hax-1 inserts and Gα13 were also verified using β-galactosidase activity of the LacZ reporter gene (Fig. 1B). Sequence alignment of the different Gα13-interacting Hax-1 fragments indicated that amino acids 176–247 of Hax-1 was sufficient for its interaction with Gα13 (Fig. 1C). To examine the in vivo interaction between Gα13 and Hax-1, co-immunoprecipitation studies were carried out in COS-7 cells that were cotransfected with an expression vector containing a cDNA insert encoding S-epitope-tagged Hax-1 and a vector containing an insert encoding wild-type Gα13 (Gα13WT) or its activated mutant (Gα13QL). Examination of Gα13 immunoprecipitates for the presence of Hax-1 indicated that Hax-1 was coimmunoprecipitated with Gα13 (Fig. 2A). Similarly, examination of Hax-1 immunoprecipitates by immunoblot analysis showed that Gα13 was coimmunoprecipitated with Hax-1, thereby confirming the physical interaction between these two proteins. Although both Gα13WT and its activated mutant Gα13QL could be seen to interact with Hax-1, the interaction between Gα13QL and Hax-1 is more pronounced than that of the wild-type (Fig. 2A). Furthermore, pretreatment of Gα13WT transfectants with aluminum fluoride for 15 min, which is known to convert the unstimulated wild-type α-subunit to an active conformation (17Plonk S.G. Park S-K. Exton J.H. J. Biol. Chem. 1998; 273: 4823-4826Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 18Yuan J. Slice L.W. Rozengurt E. J. Biol. Chem. 2001; 276: 38619-38627Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), drastically enhanced the interaction of Hax-1 with Gα13WT, demonstrating that the active configuration of Gα13 more avidly interacts with Hax-1 (Fig. 2B). Co-immunoprecipitation analyses to investigate whether Hax-1 interacts with the α-subunits of other G proteins subfamilies represented by Gαs, Gαi, and Gαq, indicated that Hax-1 failed to interact with any of these α-subunits (Fig. 2C). Interestingly, a similar analysis to determine the interaction between Hax-1 and Gα12, the α-subunit closely related to Gα13, indicated that Hax-1 does not interact with Gα12 thereby indicating the specificity of Gα13-Hax-1 interaction (Fig. 2C). These results are significant in light of the observations that only Gα13, but not Gα12, is involved in the cell migratory response (9Gu J.L. Muller S. Mancino V. Offermanns S. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9352-9357Crossref PubMed Scopus (100) Google Scholar). The interaction between Gα13 and Hax-1 can also be observed in NIH3T3 cells stably expressing Gα13QL in which Hax-1 is transiently expressed. Double immunofluorescent labeling of Gα13 and Hax-1 followed by image-merging analysis indicated the colocalization of Gα13 and Hax-1 in NIH3T3 cells, further confirming their interaction in a cell-type independent manner (Fig. 2D). These findings, taken together with the previous observations, that Gα13 regulates cell migratory response (8Offermanns S. Mancino V. Revel J.-P. Simon M.I. Science. 1997; 275: 533-536Crossref PubMed Scopus (287) Google Scholar, 9Gu J.L. Muller S. Mancino V. Offermanns S. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9352-9357Crossref PubMed Scopus (100) Google Scholar) and Hax-1 interacts with cortactin (16Gallagher A.R. Cedzich A. Gretz N. Somlo S. Witzgall R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4017-4022Crossref PubMed Scopus (162) Google Scholar), which promotes cell migration (10Patel A.S. Schechter G.L. Wasilenko W.J. Somers K.D. Oncogene. 1998; 16: 3227-3232Crossref PubMed Scopus (126) Google Scholar, 11Uruno T. Liu J. Zhang P. Fan Y-X. Egile C. Li R. Mueller S.C. Zhan X. Nat. Cell Biol. 2001; 3: 259-266Crossref PubMed Scopus (452) Google Scholar, 12Uruno T. Zhang P. Liu J. Hao J.J. Zhan X. Biochem. J. 2003; 371: 485-493Crossref PubMed Scopus (65) Google Scholar, 13Weed S.A. Du Y. Parsons J.T. J. Cell Sci. 1998; 111: 2433-2443PubMed Google Scholar), suggested to us the interesting possibility that Hax-1 is involved in linking Gα13 to cell migration-associated actin cytoskeletal motor. Cytoskeletal Changes in Gα13-Hax-1 Co-transfectants—Previous studies from others, as well as us, have also shown that Gα13 stimulates the formation of focal adhesion complexes through the small GTPase Rho (19Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 20Hooley R. Yu C-Y. Symons M. Barber D.L. J. Biol. Chem. 1996; 271: 6152-6158Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). It has also been demonstrated that Gα13 stimulates signaling pathways regulated by the small GTPase Rac (17Plonk S.G. Park S-K. Exton J.H. J. Biol. Chem. 1998; 273: 4823-4826Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Whereas Rho-stimulated stress fiber and focal adhesion complex formations are often associated with cell adhesion, Rac-stimulated membrane ruffling and lamellipodia formation are associated with cell protrusion during cell movement (21Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5199) Google Scholar, 22Ridley A.J. J. Cell Sci. 2001; 114: 2713-2722Crossref PubMed Google Scholar). Although these two responses seem to contradict each other, cell migration often involves a sequential as well as spatio-temporal regulation of adhesion, de-adhesion, and protrusion mechanisms involving both of these GTPases (3Rodriguez O.C. Schaefer A.W. Mandato C.A. Forscher P. Bement W.M. Waterman-Storer C.M. Nat. Cell Biol. 2003; 5: 599-609Crossref PubMed Scopus (705) Google Scholar, 4Mitchison T.J. Cramer L.P. Cell. 1996; 84: 371-379Abstract Full Text Full Text PDF PubMed Scopus (1303) Google Scholar). Therefore, it is possible that Gα13 can transmit adhesion and protrusion signals to the actin cytoskeleton by recruiting different signaling components in a context-specific manner. If such a differential signaling for cell movement is promoted by Gα13-Hax-1 interaction, a decrease in stress fiber formation with a concomitant increase in actin-rich structures at the leading edges of the cell, a characteristic feature of cell migration, should be observed. This was investigated by analyzing the actin organization in cells expressing Gα13QL and Hax-1 (Fig. 3). Consistent with previous findings (19Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 20Hooley R. Yu C-Y. Symons M. Barber D.L. J. Biol. Chem. 1996; 271: 6152-6158Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), actin staining showed that NIH3T3 cells expressing Gα13QL as well as the control group showed extensive stress fiber formations. However, upon expression of Hax-1, cells expressing Gα13QL showed a drastic reduction in the number of stress fibers along with the formation of actin-rich structures within the periphery of the membrane ruffles. It should be noted here that the observed actin reorganization is analogous to the one stimulated by Ras during cell migration, wherein Ras promotes Rac-mediated lamellipodia formation with a reduction in actin stress fibers so that the cells can repetitively adhere, de-adhere, and move (23Vial E. Sahai E. Marshall C.J. Cancer Cell. 2003; 4: 6779Abstract Full Text Full Text PDF Scopus (337) Google Scholar). Differential Effects of Hax-1 on Gα13-mediated Activation of Rho-GTPases—Because stress fiber formation has been shown to be directly correlated with Rho activity (19Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 20Hooley R. Yu C-Y. Symons M. Barber D.L. J. Biol. Chem. 1996; 271: 6152-6158Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) and the co-expression of Gα13QL and Hax1 showed a reduction in stress fiber formation (Fig. 3), we sought to investigate whether the co-expression of Hax-1 would suppress Gα13QL-mediated Rho activation. Whereas the expression of Gα13QL stimulated Rho activity confirming previous results (19Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 20Hooley R. Yu C-Y. Symons M. Barber D.L. J. Biol. Chem. 1996; 271: 6152-6158Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 24Kozasa T. Jiang X. Hart M.J. Sterweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (736) Google Scholar, 25Chikumi H. Fukuhara S. Gutkind J.S. J. Biol. Chem. 2002; 277: 12463-12473Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), co-expression of Hax-1 drastically attenuated the Rho activity stimulated by Gα13QL (Fig. 4A). Although, the mechanism(s) through which Hax-1 inhibits Rho activity remains to be resolved, it is possible that Hax-1 sequesters Gα13 thereby preventing it from interacting with the Rho-guanine-nucleotide exchange factor in stimulating Rho. Based on the formation of actin-rich structures in these cells, often associated with Rac (26Gohla A. Harhammer R. Schultz G. J. Biol. Chem. 1998; 273: 4653-4659Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar), it can be deduced that the co-expression of Hax-1 would potentiate Gα13QL-mediated Rac activation. To test this postulate, we investigated whether the coexpression of Hax-1 modulated any changes in Gα13QL-stimulated Rac activity. In contrast to its effect on Rho activity, Hax-1 potentiated Gα13QL-stimulated Rac activity in these cells. An analysis of Rac activity indicated that while Rac is stimulated by the expression of Gα13QL in NIH3T3 cells (Fig. 4B), coexpression of Hax further enhanced such Gα13QL-stimulated Rac activity. Thus, the co-expression of Hax-1 appears to differentially modulate the ability of Gα13 to stimulate Rho and Rac. Although the mechanism(s) through which Hax-1 potentiates Gα13-mediated Rac activation remains unknown, the finding that Hax-1 enhances Gα13 stimulation of Rac is of great physiological significance in the context of cell movement regulated by Gα13. Because it has been shown that activated Rac is involved in translocating cortactin to membrane ruffles where cortactin stimulates actin-related protein 2/3-mediated actin polymerization (10Patel A.S. Schechter G.L. Wasilenko W.J. Somers K.D. Oncogene. 1998; 16: 3227-3232Crossref PubMed Scopus (126) Google Scholar, 11Uruno T. Liu J. Zhang P. Fan Y-X. Egile C. Li R. Mueller S.C. Zhan X. Nat. Cell Biol. 2001; 3: 259-266Crossref PubMed Scopus (452) Google Scholar, 12Uruno T. Zhang P. Liu J. Hao J.J. Zhan X. Biochem. J. 2003; 371: 485-493Crossref PubMed Scopus (65) Google Scholar, 13Weed S.A. Du Y. Parsons J.T. J. Cell Sci. 1998; 111: 2433-2443PubMed Google Scholar), we investigated the possibility that Gα13-Hax-1 interaction plays a critical role in Rac-mediated translocation of cortactin and subsequent membrane ruffling. Because the actin-rich membrane ruffling precedes the formation of cell protrusion en route to cell migration, these findings suggest that Gα13 and Hax-1 promote the critical actin cytoskeletal reorganization necessary for cell migration. Role of Hax-1 in Gα13-mediated Cell Movement—To investigate the role of Hax-1 in Gα13-mediated cell migration, Hax-1 was transiently expressed in NIH3T3 cells stably expressing Gα13QL. When these cells were analyzed for migration, the results indicated that Hax-1 increased the migration of Gα13QL-NIH3T3 cells by 3-fold (Fig. 5, A and B). Because lysophosphatidic acid and lysophosphatidic acid-like agonists in the serum can activate wild-type Gα13 (Gα13WT) through their cognate receptors (26Gohla A. Harhammer R. Schultz G. J. Biol. Chem. 1998; 273: 4653-4659Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar), we tested whether Hax-1 promotes receptor-stimulated migration of NIH3T3-Gα13WT toward 5% serum. The results clearly demonstrated that Hax-1 increased the migration of these cells over the vector controls by 2.5-fold (Fig. 5, A and B). Expression of Hax-1 in migrated cells was verified by monitoring the fluorescence of the co-transfected pCEFL-GFP in NIH3T3-Gα13WT and NIH3T3-Gα13QL cells (Fig. 5C). Further analysis was carried out to test the effects of inhibiting Hax-1 on Gα13-mediated cell migration. siRNAs specific to Hax-1 or nonspecific control siRNAs were transfected into NIH3T3-Gα13WT cells and the migrations of these transfectants toward 5% serum were analyzed. Silencing of the endogenous Hax-1 by Hax-1-siRNA reduced the migration of NIH3T3-Gα13WT cells (Fig. 6A), whereas there was no effect with control siRNA. Similar results were obtained with NIH3T3-Gα13QL cells in which Hax-1 was silenced by siRNA (Fig. 6A). Quantification of these results indicated that Hax-1 siRNA reduced migration of NIH3T3-Gα13WT as well as NIH3T3-Gα13QL cells by 60% (Fig. 6B). The extent of reduction in cell migration upon silencing Hax-1 can be correlated with the siRNA-mediated reduction in the Hax-1 expression levels (Fig. 6B, lower panel). Quantification of the immunoblots showed that Hax-1 protein expression was reduced to 50–65% by 96 h after transfection. These results strongly indicate that Hax-1 plays a critical role in Gα13-mediated cell migration. Quadnary Complex Consisting of Gα13, Hax-1, Cortactin, and Rac—In this context, it should be noted that cortactin, the previously known binding partner of Hax-1 is critically involved in actin polymerization and cell movement (10Patel A.S. Schechter G.L. Wasilenko W.J. Somers K.D. Oncogene. 1998; 16: 3227-3232Crossref PubMed Scopus (126) Google Scholar, 11Uruno T. Liu J. Zhang P. Fan Y-X. Egile C. Li R. Mueller S.C. Zhan X. Nat. Cell Biol. 2001; 3: 259-266Crossref PubMed Scopus (452) Google Scholar, 12Uruno T. Zhang P. Liu J. Hao J.J. Zhan X. Biochem. J. 2003; 371: 485-493Crossref PubMed Scopus (65) Google Scholar, 13Weed S.A. Du Y. Parsons J.T. J. Cell Sci. 1998; 111: 2433-2443PubMed Google Scholar). Whereas it has been shown that the cortactin/HS1 interacting site of Hax-1 is located around amino acid 114 (14Suzuki Y. Demoliere C. Daisuke K. Takeshita H. Deuschle U. Watanabe T. J. Immunol. 1997; 158: 2736-2744PubMed Google Scholar), the core domain of Hax-1 involved in Gα13 interaction spans a region encompassing amino acids 176–247 (Fig. 1). As the Hax-1 domains involved in cortactin interaction and Gα13 interactions are non-overlapping, it can be envisioned that Hax-1 interacts with both Gα13 and cortactin in promoting cell movement. Thus, Hax-1, through its interaction with cortactin on the one hand and Gα13 with the other, is likely to be involved in tethering Gα13 to the membrane-ruffling site, which is involved in the formation of cell protrusion. In such an event, Hax-1 would form a physical complex involving both Gα13 and cortactin. To investigate the presence of such complex, COS-7 cells were transfected with either S-epitope-tagged Hax-1 and Gα13QL (Fig. 7A). Examination of S-tagged Hax-1 precipitates for the presence of Gα13 and cortactin indicated that Gα13 and cortactin were coimmunoprecipitated with Hax-1 (Fig. 7B). Likewise, the analyses of Gα13 immunoprecipitates for the presence of Hax-1 and cortactin indicated that Hax-1 and cortactin were coimmunoprecipitated along with Gα13 (Fig. 7C). Because Rac, unlike Rho, is involved in the translocation of cortactin (13Weed S.A. Du Y. Parsons J.T. J. Cell Sci. 1998; 111: 2433-2443PubMed Google Scholar) and Rac activity is enhanced in Gα13-Hax-1 cotransfectants, we also examined the presence of Rac in these immunoprecipitates. Results indicated that Rac was coimmunoprecipitated along with Gα13 as well as Hax-1 (Fig. 7, B and C). Together these results indicate the presence of a quadnary complex involving Gα13, Hax-1, cortactin, and Rac. In light of the critical roles played by Rac and cortactin in cell protrusion and migration, it can be inferred that Hax-1 association with Gα13 brings them closer to the active site of cell protrusion, where the stimulation of Rac activity by Gα13 would have a more pronounced effect on cell movement. Considering the observations that 1) Gα13 stimulates the activation of Rac, which is potentiated by Hax-1 (Fig. 4); 2) Rac stimulates the translocation of cortactin (10Patel A.S. Schechter G.L. Wasilenko W.J. Somers K.D. Oncogene. 1998; 16: 3227-3232Crossref PubMed Scopus (126) Google Scholar, 13Weed S.A. Du Y. Parsons J.T. J. Cell Sci. 1998; 111: 2433-2443PubMed Google Scholar); 3) cortactin stimulates actin polymerization leading to lamellipodia formation (11Uruno T. Liu J. Zhang P. Fan Y-X. Egile C. Li R. Mueller S.C. Zhan X. Nat. Cell Biol. 2001; 3: 259-266Crossref PubMed Scopus (452) Google Scholar, 12Uruno T. Zhang P. Liu J. Hao J.J. Zhan X. Biochem. J. 2003; 371: 485-493Crossref PubMed Scopus (65) Google Scholar, 13Weed S.A. Du Y. Parsons J.T. J. Cell Sci. 1998; 111: 2433-2443PubMed Google Scholar); 4) Hax-1 promotes Gα13-mediated cell motility (Figs. 5 and 6); and 5) Gα13 is in complex with Hax-1, cortactin, and Rac (Fig. 7), it is reasonable to conclude that both Gα13 and Hax-1 are part of a signaling complex involved in cell motility. It is likely that such proximal positioning of Gα13, Rac, Hax-1, and cortactin stimulates cell migration by countering Gα13-Rho-mediated cell adhesion (Fig. 8). The studies presented here demonstrate for the first time a novel interaction between Gα13 and the cytoskeleton-associated protein Hax-1, thereby identifying a cytoskeletal signaling locus for Gα13 in cell movement. Based on the results, it can be envisioned that Hax-1 plays a central role in Gα13-mediated cell motility by affecting three distinct, but closely related, signaling loci. First, by associating with Gα13, Hax-1 sequesters Gα13 from activating Rho and associated cell adhesion pathways. Second, through the potentiation of Gα13-stimulated Rac activity, Hax-1 promotes Rac-mediated translocation of cortactin to the periphery where cortactin along with actin-related proteins induces cell protrusion and migration. Finally, by tethering Gα13 to cortactin in a complex containing Rac, Hax-1 provides a signaling nexus that facilitates the dynamic and uninterrupted transmission of signals from Gα13 to cortactin through Rac to promote cell protrusion and migration. Our present study does not address the mechanism(s) through which Hax-1 potentiates Gα13-mediated Rac activation. However, it is interesting to note that Hax-1 contains the characteristic PXXXP motif (amino acids 198–202, PXXXP motif flanked by PXXP motifs on either side), which is known to be the binding motif for the PIX family of Rac/CDC42 guanine-nucleotide exchange factors (27Manser E. Loo T.H. Koh C.G. Zhao Z.S. Chen X.Q. Tan L. Tan I. Leung T. Lim L. Mol. Cell. 1998; 2: 183-192Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar). Therefore, it is possible that Hax-1 potentiates Rac activation by bringing Gα13 closer to a specific Rac-guanine-nucleotide exchange factor. Although our initial studies did not identify such interaction between PIX-β and Hax-1, 2J. H. Ha and N. Dhanasekaran, unpublished data. it is possible that a closely related guanine-nucleotide exchange factor may interact with Hax-1 through this site or other PXXP sites that are present in Hax-1. Further studies should define the mechanisms through which Hax-1 potentiates Gα13-stimulated activation of Rac. Previous studies have shown that Gα13 is critically required for the development of mouse embryos as Gα13-/- embryos are resorbed by day 10.5 (8Offermanns S. Mancino V. Revel J.-P. Simon M.I. Science. 1997; 275: 533-536Crossref PubMed Scopus (287) Google Scholar). The lethality of the Gα13-/- genotype is ascribed to the loss of Gα13-mediated cell motility associated with embryonic angiogenesis. It is intriguing to note that Gα12, although it shares 67% amino acid identity with Gα13 (6Radhika V. Dhanasekaran N. Oncogene. 2001; 20: 1607-1614Crossref PubMed Scopus (122) Google Scholar), failed to compensate for the loss of Gα13 in these embryos. Because both Gα12 and Gα13 can be stimulated by the same receptors to activate similar cellular responses, the molecular basis for the differential effect on cell motility remained elusive. In this context, the results presented here demonstrate that Hax-1 specifically interacts with Gα13 but not Gα12, and that such interaction is critical for Gα13-mediated cell motility that provides a molecular basis for such unique and differential signaling by Gα13. Further studies should define the role of Gα13-Hax-1 complexes in physiological responses that require cell movement or cytoskeletal changes mediated by Gα13. In light of the recent findings that Hax-1 is differentially expressed in hypoxic tumor progression (28Jiang Y. Zhang W. Keichii K. Klco J.M. Martin St T.B. Dufault M.R. Madden S.L. Kaelin W.G. Nacht M. Mol. Cancer Res. 2003; 1: 453-462PubMed Google Scholar) and overexpressed in many different metastatic tumors as indicated by SAGE analysis (28Jiang Y. Zhang W. Keichii K. Klco J.M. Martin St T.B. Dufault M.R. Madden S.L. Kaelin W.G. Nacht M. Mol. Cancer Res. 2003; 1: 453-462PubMed Google Scholar, 29Velculescu V.E. Zhang L. Vogelstein B. Kinzler K.W. Science. 1995; 270: 484-487Crossref PubMed Scopus (3575) Google Scholar, 30Mirmohammadsadegh A. Tartler U. Michel G. Baer A. Walz M. Wolf R. Ruzicka T. Hengge U.R. J. Investig. Dermatol. 2003; 120: 1045-1051Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), it is more likely that Gα13-Hax-1 interaction plays a critical role in the metastatic phenotype of these tumors. We are grateful to Dr. R. A. Vaillancourt for yeast strains and yeast vectors and Dr. T. Watanabe for Hax-1 full-length cDNA. Helpful discussions and critical reading of the manuscript by Rashmi Kumar and Kimia Kashef are gratefully acknowledged." @default.
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