P-Rex1 Links Mammalian Target of Rapamycin Signaling to Rac Activation and Cell Migration

Abstract

Polarized cell migration results from the transduction of extra-cellular cues promoting the activation of Rho GTPases with the intervention of multidomain proteins, including guanine exchange factors. P-Rex1 and P-Rex2 are Rac GEFs connecting Gβγ and phosphatidylinositol 3-kinase signaling to Rac activation. Their complex architecture suggests their regulation by protein-protein interactions. Novel mechanisms of activation of Rho GTPases are associated with mammalian target of rapamycin (mTOR), a serine/threonine kinase known as a central regulator of cell growth and proliferation. Recently, two independent multiprotein complexes containing mTOR have been described. mTORC1 links to the classical rapamycin-sensitive pathways relevant for protein synthesis; mTORC2 links to the activation of Rho GTPases and cytoskeletal events via undefined mechanisms. Here we demonstrate that P-Rex1 and P-Rex2 establish, through their tandem DEP domains, interactions with mTOR, suggesting their potential as effectors in the signaling of mTOR to Rac activation and cell migration. This possibility was consistent with the effect of dominant-negative constructs and short hairpin RNA-mediated knockdown of P-Rex1, which decreased mTOR-dependent leucine-induced activation of Rac and cell migration. Rapamycin, a widely used inhibitor of mTOR signaling, did not inhibit Rac activity and cell migration induced by leucine, indicating that P-Rex1, which we found associated to both mTOR complexes, is only active when in the mTORC2 complex. mTORC2 has been described as the catalytic complex that phosphorylates AKT/PKB at Ser-473 and elicits activation of Rho GTPases and cytoskeletal reorganization. Thus, P-Rex1 links mTOR signaling to Rac activation and cell migration. Polarized cell migration results from the transduction of extra-cellular cues promoting the activation of Rho GTPases with the intervention of multidomain proteins, including guanine exchange factors. P-Rex1 and P-Rex2 are Rac GEFs connecting Gβγ and phosphatidylinositol 3-kinase signaling to Rac activation. Their complex architecture suggests their regulation by protein-protein interactions. Novel mechanisms of activation of Rho GTPases are associated with mammalian target of rapamycin (mTOR), a serine/threonine kinase known as a central regulator of cell growth and proliferation. Recently, two independent multiprotein complexes containing mTOR have been described. mTORC1 links to the classical rapamycin-sensitive pathways relevant for protein synthesis; mTORC2 links to the activation of Rho GTPases and cytoskeletal events via undefined mechanisms. Here we demonstrate that P-Rex1 and P-Rex2 establish, through their tandem DEP domains, interactions with mTOR, suggesting their potential as effectors in the signaling of mTOR to Rac activation and cell migration. This possibility was consistent with the effect of dominant-negative constructs and short hairpin RNA-mediated knockdown of P-Rex1, which decreased mTOR-dependent leucine-induced activation of Rac and cell migration. Rapamycin, a widely used inhibitor of mTOR signaling, did not inhibit Rac activity and cell migration induced by leucine, indicating that P-Rex1, which we found associated to both mTOR complexes, is only active when in the mTORC2 complex. mTORC2 has been described as the catalytic complex that phosphorylates AKT/PKB at Ser-473 and elicits activation of Rho GTPases and cytoskeletal reorganization. Thus, P-Rex1 links mTOR signaling to Rac activation and cell migration. P-Rex1 and P-Rex2 are Rac guanine exchange factors connecting G protein-coupled receptors, through Gβγ and phosphatidylinositol 3-kinase, to Rac activation. In particular, P-Rex1 has been associated with the activation of Rac2, generating reactive oxygen species in neutrophils. P-Rex2 (showing two splice variants) is similarly regulated by Gβγ and phosphatidylinositol 3-kinase. Northern blot assays revealed a differential distribution of the two members of the P-Rex 3The abbreviations used are: P-Rex, phosphatidylinositol 3,4,5-trisphosphate- and Gβγ-regulated guanine-nucleotide exchange factor for Rac; GEF, guanine nucleotide exchange factor; mTOR, mammalian target of rapamycin; DH, Dbl homology; PDZ, PSD-95/Discs-large/ZO-1 homology; DEP, domain found in Dishevelled, Egl-10, and Pleckstrin; AKT, serine/threonine kinase with a pleckstrin homology domain; shRNA, short hairpin RNA; TIAM, T-lymphoma invasion and metastasis; EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; GST, glutathione S-transferase; HA, hemagglutinin 3-[(3-cholamidopropyl)dimethylammonio]-1-pro-panesulfonic acid; GFP, green fluorescent protein; S6K, S6 kinase. 3The abbreviations used are: P-Rex, phosphatidylinositol 3,4,5-trisphosphate- and Gβγ-regulated guanine-nucleotide exchange factor for Rac; GEF, guanine nucleotide exchange factor; mTOR, mammalian target of rapamycin; DH, Dbl homology; PDZ, PSD-95/Discs-large/ZO-1 homology; DEP, domain found in Dishevelled, Egl-10, and Pleckstrin; AKT, serine/threonine kinase with a pleckstrin homology domain; shRNA, short hairpin RNA; TIAM, T-lymphoma invasion and metastasis; EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; GST, glutathione S-transferase; HA, hemagglutinin 3-[(3-cholamidopropyl)dimethylammonio]-1-pro-panesulfonic acid; GFP, green fluorescent protein; S6K, S6 kinase. family, suggesting that they exert equivalent functions in different cellular populations (1Welch H.C. 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Although the participation of Rho guanine exchange factors in the activation of Rho GTPases and cytoskeletal dynamics elicited by mTORC2 is expected, their identity and mechanism of action remain to be revealed. Here we identified the Rac guanine exchange factors P-Rex1 and P-Rex2 as the putative catalytic components of mTORC2 critical for the activation of Rac and cell migration elicited by leucine through the rapamycin-insensitive mTOR complex 2. These results implicate that P-Rex family members link mTORC2 signaling to Rac activation and cell migration in response to the activation of the rapamycin-insensitive mTOR complex. Yeast Two-hybrid Screening—The DEP-DEP domains of P-Rex1 used as a to a with the both the and the indicated by Vazquez-Prado J. J. Gutkind J.S. 2004; PubMed Scopus Google Scholar). leucine, and The of the the DEP-DEP P-Rex1 domain with the mTOR domain was the and by the and and from D. Sabatini D.D. Ali S.M. Kim D.H. Guertin D.A. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Curr. Biol. 2004; 14: 1296-1302Abstract Full Text Full Text PDF PubMed Scopus (2131) Google Scholar). The for P-Rex1 to the which was on the of the hairpin mTOR was from P. from M. Hoshino 13Yoshizawa M. Kawauchi T. Sone M. Nishimura Y.V. Terao M. Chihama K. Nabeshima Y. Hoshino M. J. Neurosci. 2005; 25: 4406-4419Crossref PubMed Scopus (84) Google Scholar). Cell and and in containing fetal bovine according to the and indicated of leucine for in and in on with and in of and for of mTOR containing and assays was to the and for on by of followed by at of for was to the and for at of of protein was and for at with of and in protein The P-Rex1 and was from as described for in this was from and also from was used as a and both of of was P-Rex1 was with from M. Hoshino 13Yoshizawa M. Kawauchi T. Sone M. Nishimura Y.V. Terao M. Chihama K. Nabeshima Y. Hoshino M. J. Neurosci. 2005; 25: 4406-4419Crossref PubMed Scopus (84) Google Scholar). to and the used for the mTOR protein S6 protein and in the in with the indicated and and as described with rapamycin for the with and in of and containing and and to for protein of and for at with of and in of protein and a containing FBS, which was used as a was in the of a containing for in leucine, to the The two by a with a with on a and was with indicated with and and for this as described the rapamycin for and with rapamycin for was also to the cell on the and also to the to the in to be the migration In and in in the according to the for we in the E. in protein E. amino acids was used as containing the The in a at at for of of amino acids was followed by for The by of and containing In was with glutathione containing P-Rex1 domain P-Rex1 DEP-DEP domains, both as from with in with The complexes in by and to for and mTOR with P-Rex1 in Yeast and to that P-Rex1, we used the tandem DEP domains of P-Rex1 as in a a found that in P-Rex1 DEP-DEP with the including the kinase domain, of this was as indicated by the growth, in leucine, and of with the of P-Rex DEP-DEP and mTOR the of the composed of and and in mammalian the of P-Rex1 with mTOR, the of P-Rex1 the two tandem DEP domains and the of mTOR to the from the two the cell as and proteins, The cell with glutathione The of mTOR was associated with to not with used as mTOR also with P-Rex1 DEP-DEP as a protein this with the mTOR to and P-Rex1 DEP-DEP domains mTOR and P-Rex1 In both the and P-Rex1 with mTOR demonstrate a P-Rex1 and mTOR, in was in mTOR and P-Rex1 DEP-DEP domains P-Rex1 domain as both as P-Rex1 domains and with the mTOR was associated with P-Rex1 DEP-DEP The of both was also by mTOR, and P-Rex1, a from in which P-Rex1 was was in the blot to the of the to P-Rex1 to the mTOR and members of the P-Rex with mTOR and P-Rex1, The assays that mTOR not only with P-Rex1 also with and was with the Rho used as These with the demonstrate that P-Rex family of Rac including P-Rex1, and P-Rex2b, are to directly with mTOR, the possibility that this of GEFs in the activation of Rac and cytoskeletal events of of mTOR with The and was by from from in which P-Rex1 was was in the blot to the of the to was used as a mTOR was and P-Rex1 was found associated with and and are with the different P-Rex family with and P-Rex1, and assays cell and on and by of mTOR with the P-Rex family members was with the used as mTOR Rac via rapamycin-insensitive complex, has been found to the actin cytoskeleton through Rho GTPases (4Sarbassov D.D. Ali S.M. Kim D.H. Guertin D.A. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Curr. Biol. 2004; 14: 1296-1302Abstract Full Text Full Text PDF PubMed Scopus (2131) Google Scholar, 6Jacinto E. Loewith R. Schmidt A. Lin S. Ruegg M.A. Hall A. Hall M.N. Nat. Cell Biol. 2004; 6: 1122-1128Crossref PubMed Scopus (1668) Google Scholar). Thus, we to the of mTOR with P-Rex1 be in the described ability of mTORC2 to the activation of Rac. amino acids activate mTOR, we to as a to the of this protein in Rac activation and cell migration. we mTOR by leucine would the of Rac in The of Rac with the of leucine of and for These results with the activation of S6K, a mTOR by in the of in response to leucine, in from the cell TIAM, a Rac GEF, used as was to activation of Rac and the of the protein that mTOR with short has been to in E. Loewith R. Schmidt A. Lin S. Ruegg M.A. Hall A. Hall M.N. Nat. Cell Biol. 2004; 6: 1122-1128Crossref PubMed Scopus (1668) Google we the participation of mTOR in leucine-induced Rac mTOR was and the of Rac was leucine The ability of leucine to the activation of Rac was decreased in with mTOR as expected, the of induced by leucine, was also in in which mTOR was These results with the of mTOR by blot the participation of P-Rex1 in the ability of mTORC2 to Rac activation in response to leucine, two independent a dominant-negative P-Rex1 was and the of Rac was leucine P-Rex1 was used to the of P-Rex1 The activation of Rac induced by leucine was decreased by both and These results indicated a relevant participation of this in the signaling from mTOR to Rac. It is known that amino acids as leucine the mTORC1 complex by activation of mTOR and of the S6 protein D.H. Sarbassov D.D. Ali S.M. King J.E. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2328) Google Scholar, B. H. R. Z. S.M. Am. J. Physiol. 2003; PubMed Scopus Google Scholar). In these we to the of of the S6 leucine in and in with the mTOR the dominant-negative P-Rex1 and the to P-Rex1 The results a in the of S6 only in the with mTOR as to the with the dominant-negative P-Rex1 the P-Rex1 shRNA, in which the of S6 with the of leucine to the these results that S6 is independent of P-Rex1, the that P-Rex1 is active as a of mTORC2 and when associated to mTORC1. was to the of the DEP-DEP domains of P-Rex1 with to mTOR in the of both and mTOR was by the of the DEP domains of P-Rex1, which with the domains of this In P-Rex1 DEP domains inhibit the activation of Rac induced by leucine and this is with the reduced effect of Rac activation with the dominant-negative P-Rex1 P-Rex1 of mTOR mTORC1 and mTOR has been found in two different multiprotein complexes (4Sarbassov D.D. Ali S.M. Kim D.H. Guertin D.A. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Curr. 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Cell. 2003; 11: 895-904Abstract Full Text Full Text PDF PubMed Scopus (760) Google of which is to activate Rac and cytoskeletal by a we that P-Rex1 would be a of the to this In to this we the of P-Rex1 in mTOR complex complex with P-Rex1, GβL/mLST8, and mTORC1 and mTORC2 by of and P-Rex1 was found associated with both mTORC1 and mTORC2 and did not with protein with the homology domain of that was used as a mTOR Cell via of the actin cytoskeleton by mTORC2 has been described (4Sarbassov D.D. Ali S.M. Kim D.H. Guertin D.A. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Curr. Biol. 2004; 14: 1296-1302Abstract Full Text Full Text PDF PubMed Scopus (2131) Google Scholar, 6Jacinto E. Loewith R. Schmidt A. Lin S. Ruegg M.A. Hall A. Hall M.N. Nat. Cell Biol. 2004; 6: 1122-1128Crossref PubMed Scopus (1668) Google Scholar). recent in the of the complex in and cell S. Comer F.I. Sasaki A. McLeod I.X. Duong Y. Okumura K. Yates J.R. II I Parent C.A. Firtel R.A. Mol. Biol. Cell. 2005; 16: 4572-4583Crossref PubMed Scopus Google Scholar). we have demonstrated that leucine Rac via mTOR and we to leucine also cell migration on The ability of to in the of leucine was demonstrated a migration was with the dominant-negative P-Rex1 the to P-Rex1, and with also used as a with leucine fetal bovine as a and to for at The results a in the of when with leucine in the with dominant-negative P-Rex1 P-Rex1 a in the of only when with leucine and expected, in which mTOR was with to in response to a leucine of at with was for these if mTORC1 is relevant for the activity induced by leucine, we the migration of with rapamycin, inhibitor of the in indicated that only migration of was only decreased in of a of rapamycin, the induced by leucine was not by Rac activation was also the results in Rac activity leucine in with rapamycin with these results demonstrate that leucine cell migration and Rac activation in a involving the participation of the mTORC2 complex. a mechanism by which mTORC2 the actin cytoskeleton through the activation of Rho GTPases (4Sarbassov D.D. Ali S.M. Kim D.H. Guertin D.A. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Curr. Biol. 2004; 14: 1296-1302Abstract Full Text Full Text PDF PubMed Scopus (2131) Google Scholar, 5Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5176) Google Scholar). on we that by of ability to complexes with mTOR, P-Rex1 link mTORC2 activation to the of Rac signaling, to the regulation of the actin cytoskeleton and cell migration. This that have been identified as part of the mTORC2 complex, and (4Sarbassov D.D. Ali S.M. Kim D.H. Guertin D.A. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Curr. Biol. 2004; 14: 1296-1302Abstract Full Text Full Text PDF PubMed Scopus (2131) Google Scholar, 5Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5176) Google that both P-Rex1 and mTOR are for Rac activation and cell migration induced by leucine, and they are part of the signaling through independent pathways to be as P-Rex1 is also a of also in Rac as in cell migration in response to insulin-like growth factor (15Liu L. Li F. Cardelli J.A. Martin K.A. Blenis J. Huang S. Oncogene. 2006; 25: 7029-7040Crossref PubMed Scopus (163) Google P-Rex1 be a of complexes containing both mTORC1 and mTORC2 S. Loewith R. Oppliger W. Hall M.N. J. Biol. Chem. 2005; 280: 30697-30704Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 17Takahara T. Hara K. Yonezawa K. Sorimachi H. Maeda T. J. Biol. Chem. 2006; 281: 28605-28614Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 18Schmidt A. Kunz J. Hall M.N. Proc. Natl. Acad. Sci. U. S. 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