Identification of Radil as a Ras binding partner and putative activator

Ras genes are among the most frequently mutated oncogenes in human malignancies. To date, there are no successful anticancer drugs in the clinic that target Ras proteins or their pathways. Therefore, it is imperative to identify and characterize new components that regulate Ras activity or mediate its downstream signaling. To this end, we used a combination of affinity-pulldown and mass spectrometry to search for proteins that are physically associated with KRas. One of the top hits was Radil, a gene product with a Ras-association domain. Radil is known to be a downstream effector of Rap1, inhibiting RhoA signaling to regulate cell adhesion and migration. We demonstrate that Radil interacted with all three isoforms of Ras including HRas, NRas, and KRas, although it exhibited the strongest interaction with KRas. Moreover, Radil interacts with GTP-bound Ras more efficiently, suggesting a possibility that Radil may be involved in Ras activation. Supporting this, ectopic expression of Radil led to transient activation of mitogen-activated protein kinase kinase and extracellular signal-regulated kinase; Radil knockdown resulted in weakened activation of Ras downstream signaling components, which was coupled with decreased cell proliferation and invasion, and reduced expression of mesenchymal cell markers. Moreover, Radil knockdown greatly reduced the number of adhesion foci and depolymerized actin filaments, molecular processes that facilitate cancer cell migration. Taken together, our present studies strongly suggest that Radil is an important player for regulating Ras signaling, cell adhesion, and the epithelial–mesenchymal transition and may provide new directions for Ras-related anticancer drug development. Ras genes are among the most frequently mutated oncogenes in human malignancies. To date, there are no successful anticancer drugs in the clinic that target Ras proteins or their pathways. Therefore, it is imperative to identify and characterize new components that regulate Ras activity or mediate its downstream signaling. To this end, we used a combination of affinity-pulldown and mass spectrometry to search for proteins that are physically associated with KRas. One of the top hits was Radil, a gene product with a Ras-association domain. Radil is known to be a downstream effector of Rap1, inhibiting RhoA signaling to regulate cell adhesion and migration. We demonstrate that Radil interacted with all three isoforms of Ras including HRas, NRas, and KRas, although it exhibited the strongest interaction with KRas. Moreover, Radil interacts with GTP-bound Ras more efficiently, suggesting a possibility that Radil may be involved in Ras activation. Supporting this, ectopic expression of Radil led to transient activation of mitogen-activated protein kinase kinase and extracellular signal-regulated kinase; Radil knockdown resulted in weakened activation of Ras downstream signaling components, which was coupled with decreased cell proliferation and invasion, and reduced expression of mesenchymal cell markers. Moreover, Radil knockdown greatly reduced the number of adhesion foci and depolymerized actin filaments, molecular processes that facilitate cancer cell migration. Taken together, our present studies strongly suggest that Radil is an important player for regulating Ras signaling, cell adhesion, and the epithelial–mesenchymal transition and may provide new directions for Ras-related anticancer drug development. Ras proteins comprise a family of small GTPases that are involved in regulating a variety of biological processes including cell survival, proliferation, and migration (1Malumbres M. Barbacid M. RAS oncogenes: The first 30 years.Nat. Rev. Cancer. 2003; 3: 459Crossref PubMed Scopus (1346) Google Scholar, 2Campbell P.M. Der C.J. Oncogenic Ras and its role in tumor cell invasion and metastasis.Semin. Cancer Biol. 2004; 14: 105-114Crossref PubMed Scopus (215) Google Scholar, 3Pylayeva-Gupta Y. Grabocka E. Bar-Sagi D. RAS oncogenes: Weaving a tumorigenic web.Nat. Rev. Cancer. 2011; 11: 761Crossref PubMed Scopus (1101) Google Scholar). Ras proteins function as binary signaling switches with “on” and “off” states, which are largely controlled by GTP and GDP binding, respectively (4Muratcioglu S. Chavan T.S. Freed B.C. Jang H. Khavrutskii L. Freed R.N. Dyba M.A. Stefanisko K. Tarasov S.G. Gursoy A. Keskin O. Tarasova N.I. Gaponenko V. Nussinov R. GTP-dependent K-Ras dimerization.Structure. 2015; 23: 1325-1335Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 5Mor A. Philips M.R. Compartmentalized Ras/MAPK signaling.Annu. Rev. Immunol. 2006; 24: 771-800Crossref PubMed Scopus (315) Google Scholar). Therefore, the activity, subcellular localization, and stability of Ras proteins are tightly regulated in normal cells. Cell adhesion and motility play a pivotal role in normal development as they are critical for wound healing, stem cell homing, and immune cell trafficking. In normal cells, activated Ras increases cell migration, which is accompanied by extensive actin cytoskeleton remodeling (6Tse J.M. Cheng G. Tyrrell J.A. Wilcox-Adelman S.A. Boucher Y. Jain R.K. Munn L.L. Mechanical compression drives cancer cells toward invasive phenotype.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 911-916Crossref PubMed Scopus (333) Google Scholar, 7Mouneimne G. Hansen S.D. Selfors L.M. Petrak L. Hickey M.M. Gallegos L.L. Simpson K.J. Lim J. Gertler F.B. Hartwig J.H. Mullins R.D. Brugge J.S. Differential remodeling of actin cytoskeleton architecture by profilin isoforms leads to distinct effects on cell migration and invasion.Cancer Cell. 2012; 22: 615-630Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 8Yamaguchi H. Condeelis J. Regulation of the actin cytoskeleton in cancer cell migration and invasion.Biochim. Biophys. Acta. 2007; 1773: 642-652Crossref PubMed Scopus (785) Google Scholar). Ras induces changes in interactions between cells and the extracellular matrix (2Campbell P.M. Der C.J. Oncogenic Ras and its role in tumor cell invasion and metastasis.Semin. Cancer Biol. 2004; 14: 105-114Crossref PubMed Scopus (215) Google Scholar, 9Giehl K. Oncogenic Ras in tumour progression and metastasis.Biol. Chem. 2005; 386: 193-205Crossref PubMed Scopus (222) Google Scholar) and downregulates adhesion junctions including E-cadherin (9Giehl K. Oncogenic Ras in tumour progression and metastasis.Biol. Chem. 2005; 386: 193-205Crossref PubMed Scopus (222) Google Scholar). Constitutively activated Ras enhances the motility of transformed cells by altering cytoskeleton structures and deregulating gene expression of adhesion molecules (2Campbell P.M. Der C.J. Oncogenic Ras and its role in tumor cell invasion and metastasis.Semin. Cancer Biol. 2004; 14: 105-114Crossref PubMed Scopus (215) Google Scholar, 10Pollock C.B. Shirasawa S. Sasazuki T. Kolch W. Dhillon A.S. Oncogenic K-RAS is required to maintain changes in cytoskeletal organization, adhesion, and motility in colon cancer cells.Cancer Res. 2005; 65: 1244-1250Crossref PubMed Scopus (107) Google Scholar). Epithelial and mesenchymal cells display different features including morphologies, cell-to-cell adhesion, and migratory behaviors (11Bezdekova M. Brychtova S. Sedlakova E. Langova K. Brychta T. Belej K. Analysis of Snail-1, E-cadherin and claudin-1 expression in colorectal adenomas and carcinomas.Int. J. Mol. Sci. 2012; 13: 1632-1643Crossref PubMed Scopus (38) Google Scholar, 12Campbell K. Casanova J. A common framework for EMT and collective cell migration.Development. 2016; 143: 4291-4300Crossref PubMed Scopus (87) Google Scholar, 13Campbell K. Casanova J. Skaer H. Mesenchymal-to-epithelial transition of intercalating cells in Drosophila renal tubules depends on polarity cues from epithelial neighbours.Mech. Dev. 2010; 127: 345-357Crossref PubMed Scopus (22) Google Scholar). During development, cells often switch from a static state to a migratory state or vice versa. For example, wound healing often invokes collective cell migration and epithelial–mesenchymal transition (EMT) in high animals (12Campbell K. Casanova J. A common framework for EMT and collective cell migration.Development. 2016; 143: 4291-4300Crossref PubMed Scopus (87) Google Scholar, 13Campbell K. Casanova J. Skaer H. Mesenchymal-to-epithelial transition of intercalating cells in Drosophila renal tubules depends on polarity cues from epithelial neighbours.Mech. Dev. 2010; 127: 345-357Crossref PubMed Scopus (22) Google Scholar). Dysfunctional transition can affect cell growth and differentiation, leading to disease states. At the molecular level, epithelial and mesenchymal cells express distinct sets of gene products, many of which are directly involved in cell adhesion and/or migration (14Evdokimova V. Tognon C. Ng T. Ruzanov P. Melnyk N. Fink D. Sorokin A. Ovchinnikov L.P. Davicioni E. Triche T.J. Sorensen P.H. Translational activation of snail1 and other developmentally regulated transcription factors by YB-1 promotes an epithelial-mesenchymal transition.Cancer Cell. 2009; 15: 402-415Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). E-cadherin, ZO-1, and CK18 are highly expressed in epithelial cells, whereas Snail, Twist, N-cadherin, Zeb1, vimentin, and Claudin-1 are expressed, or highly enriched, in mesenchymal cells (12Campbell K. Casanova J. A common framework for EMT and collective cell migration.Development. 2016; 143: 4291-4300Crossref PubMed Scopus (87) Google Scholar, 14Evdokimova V. Tognon C. Ng T. Ruzanov P. Melnyk N. Fink D. Sorokin A. Ovchinnikov L.P. Davicioni E. Triche T.J. Sorensen P.H. Translational activation of snail1 and other developmentally regulated transcription factors by YB-1 promotes an epithelial-mesenchymal transition.Cancer Cell. 2009; 15: 402-415Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). EMT is regulated by many proteins including Ras and Y-box–binding protein-1 (14Evdokimova V. Tognon C. Ng T. Ruzanov P. Melnyk N. Fink D. Sorokin A. Ovchinnikov L.P. Davicioni E. Triche T.J. Sorensen P.H. Translational activation of snail1 and other developmentally regulated transcription factors by YB-1 promotes an epithelial-mesenchymal transition.Cancer Cell. 2009; 15: 402-415Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). We have previously shown that expression of KRasV12 in MCF7 cells induced expression of Snail and Claudin-1, which is suppressed by the SUMO-resistant counterpart (15Choi B.H. Philips M.R. Chen Y. Lu L. Dai W. K-Ras Lys-42 is crucial for its signaling, cell migration, and invasion.J. Biol. Chem. 2018; 293: 17574-17581Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), suggesting that Ras sumoylation plays an important role in EMT. Rap1 is a ubiquitously expressed small GTPase, which plays a key role in modulating cell adhesion, angiogenesis, and endothelial barrier functions (16Chrzanowska-Wodnicka M. Rap1 in endothelial biology.Curr. Opin. Hematol. 2017; 24: 248-255Crossref PubMed Scopus (26) Google Scholar, 17Shah S. Brock E.J. Ji K. Mattingly R.R. Ras and Rap1: A tale of two GTPases.Semin. Cancer Biol. 2019; 54: 29-39Crossref PubMed Scopus (45) Google Scholar). Radil is a downstream effector of Rap1, regulating integrin activation and controlling neutrophil chemotaxis (18Ahmed S.M. Theriault B.L. Uppalapati M. Chiu C.W. Gallie B.L. Sidhu S.S. Angers S. KIF14 negatively regulates Rap1a-Radil signaling during breast cancer progression.J. Cell Biol. 2012; 199: 951-967Crossref PubMed Scopus (48) Google Scholar, 19Smolen G.A. Schott B.J. Stewart R.A. Diederichs S. Muir B. Provencher H.L. Look A.T. Sgroi D.C. Peterson R.T. Haber D.A. A Rap GTPase interactor, RADIL, mediates migration of neural crest precursors.Genes Dev. 2007; 21: 2131-2136Crossref PubMed Scopus (32) Google Scholar). Radil consists of three notable domains including Ras-association (RA), dilute-containing (DIL), and PDZ domains (20Gingras W. of of the Ras of 2016; 24: Full Text Full Text PDF PubMed Scopus Google Scholar). was a of to the of To date, the function of largely Radil is of to the in a (18Ahmed S.M. Theriault B.L. Uppalapati M. Chiu C.W. Gallie B.L. Sidhu S.S. Angers S. KIF14 negatively regulates Rap1a-Radil signaling during breast cancer progression.J. Cell Biol. 2012; 199: 951-967Crossref PubMed Scopus (48) Google Scholar). of Radil adhesion kinase activation and promotes cell adhesion and (18Ahmed S.M. Theriault B.L. Uppalapati M. Chiu C.W. Gallie B.L. Sidhu S.S. Angers S. KIF14 negatively regulates Rap1a-Radil signaling during breast cancer progression.J. Cell Biol. 2012; 199: 951-967Crossref PubMed Scopus (48) Google Scholar, L. W. S.M. Angers S. Radil neutrophil adhesion and motility Biol. Cell. 2012; 23: PubMed Scopus Google Scholar). In this we that Radil may function as a new in the Ras signaling coupled with mass spectrometry that Radil physically interacted with Ras proteins with the The interaction between Radil and Ras was by the GTP-bound of Radil knockdown led to reduced activation of the mitogen-activated protein kinase kinase signal-regulated kinase signaling which is coupled with decreased cell proliferation, adhesion, and Moreover, Radil promotes expression of Zeb1, and Snail, transcription factors of the mesenchymal cells. Ras are directly by downstream components it interacts with H. Philips M.R. D. A. The of and their distinct Cell 2015; PubMed Scopus Google Scholar, A.T. A. D. K. S. J.M. of K-Ras enhances activation and to downstream 2011; PubMed Scopus Google Scholar, H. H. J. N. Philips M.R. K-Ras cell by of Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, H. J. with 2016; PubMed Scopus Google Scholar). To the function of Ras in regulating cell proliferation and we to identify new molecular components that physically interacted with We expressed that was with the the of and mass we a number of proteins that physically interacted with A and To which proteins in the the we a the To be to a for proteins that in the and the we the by to all proteins in was the proteins was as the protein Radil is of the proteins We first on Radil as it that Radil expression among transformed cell with and high of expression that between Ras and Radil in the we a of to Radil is a We cells with for which cells and for the was expressed and by Ras proteins in the with a was with suggesting that Radil physically with KRas. a we cells with a expression for which the cell with the that a of Radil was in that Radil physically interacts with KRas. Radil protein consists of and PDZ domains The in other proteins including and Ras P. C. by Ras family GTPases is by the of they Cell Biol. 2004; 24: PubMed Scopus Google Scholar). In it is for the interaction between Radil and Rap1, the a small that plays an important role in the of endothelial function S. Brock E.J. Ji K. Mattingly R.R. Ras and Rap1: A tale of two GTPases.Semin. Cancer Biol. 2019; 54: 29-39Crossref PubMed Scopus (45) Google Scholar). We it was required for its interaction with We that whereas the Radil was of and Rap1 the of the the interaction between Radil and Ras of and was that Radil physically interacts with Ras and that of Radil is for the Ras GTPases the of GTP to GTP-bound Ras is whereas is (1Malumbres M. Barbacid M. RAS oncogenes: The first 30 years.Nat. Rev. Cancer. 2003; 3: 459Crossref PubMed Scopus (1346) Google Scholar, 2Campbell P.M. Der C.J. Oncogenic Ras and its role in tumor cell invasion and metastasis.Semin. Cancer Biol. 2004; 14: 105-114Crossref PubMed Scopus (215) Google Scholar, 3Pylayeva-Gupta Y. Grabocka E. Bar-Sagi D. RAS oncogenes: Weaving a tumorigenic web.Nat. Rev. Cancer. 2011; 11: 761Crossref PubMed Scopus (1101) Google Scholar). To the interaction between Radil and we cells for with a with or of with the by that more Radil was associated with with strongly suggesting that GTP the this no Radil was in of for Ras proteins was and the was as Therefore, strongly suggest that Radil may regulate or mediate Ras activation and signaling. Ras to or GDP in changes (4Muratcioglu S. Chavan T.S. Freed B.C. Jang H. Khavrutskii L. Freed R.N. Dyba M.A. Stefanisko K. Tarasov S.G. Gursoy A. Keskin O. Tarasova N.I. Gaponenko V. Nussinov R. GTP-dependent K-Ras dimerization.Structure. 2015; 23: 1325-1335Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). We that Radil may a GTP-bound Ras To this we of and We that Radil a to GTP-bound to that mediate Ras are frequently H. E. S. R. M.R. The in the effector 2016; PubMed Scopus (32) Google Scholar). To Radil to different isoforms of Ras with a different we cells with a for HRas, NRas, or KRas. of used as We that Radil interacted with more with or although expression was with that of or with an interaction was between Radil and Ras and that expression of Ras and Ras of isoforms was We mediate the interaction with with and/or and cell with the We that Rap1 and present in Radil that Radil interacted with Rap1 and Radil strongly interacted with as Radil a high of the interaction between and Radil suppressed the interaction between Radil and Rap1 or suggesting that with Rap1 and for that the between Radil and Ras is by the of we that Radil is involved in regulating Ras activity and signaling. To this we cell expression of was leading to the activation of downstream signaling components including and We the role of Radil in downstream activation. cells with Radil or and with for or which cells for expression of and Radil, as as Ras downstream signaling. We that Radil no on the of by expressed KRasV12 and To the role of Radil in modulating Ras signaling, we cell with expression of expression was and induced expression of was with transient activation of and in Radil expression was with of and To the of Ras by Radil, we the of GTP-bound Ras induced Radil We that with for induced Radil which was coupled with as the other of Radil the of strongly suggesting that Radil may regulate Ras activity in a We the of Radil Ras downstream signaling. with to or Radil for which cells for We that Radil of and by growth factors in a and strongly suggest that Radil is involved in regulating Ras downstream signaling. We have previously shown that plays an important role in regulating cell migration, invasion, and EMT (15Choi B.H. Philips M.R. Chen Y. Lu L. Dai W. K-Ras Lys-42 is crucial for its signaling, cell migration, and invasion.J. Biol. Chem. 2018; 293: 17574-17581Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). To Radil be involved in we Radil and/or in cells and their proliferation We that with the cells, Radil or reduced cell proliferation that Radil knockdown greatly reduced expression of and a mesenchymal associated with motility and adhesion The role of in the progression of J. Cell Mol. Biol. Google Scholar, K. L. C. L. A. U. P. E. R. of the cytoskeleton is to epithelial mesenchymal transition in a cancer PubMed Scopus Google Scholar, A. S. in cancer and its as a molecular target for cancer Mol. Sci. 2011; PubMed Scopus Google Scholar). Radil knockdown reduced the expression of Snail and Zeb1, two transcription factors for mesenchymal cells, with a of E-cadherin, an epithelial cell knockdown of of and which was with of ZO-1, an epithelial cell EMT induced by Radil and KRas, we cell invasion of Radil and KRas. We that knockdown of Radil or led to reduced cell invasion and that the of decreased cell invasion by of Radil and Ras was and To Radil or cell proliferation and invasion, as as we cell adhesion and cytoskeleton modulating Radil or to Radil, KRas, or and with to of adhesion and for with cells, Radil knockdown greatly reduced the number of in the cells which was associated with the of actin and a reduced cell knockdown reduced the number of and actin it the cell although the cell to be reduced as as it with Radil that expression of KRasV12 activated a protein kinase involved in cell on the present we the that role in modulating during cell proliferation, migration, and EMT a molecular that mediates the function of growth Radil interacts with and activation of and its downstream signaling leads to expression of mesenchymal cell coupled with of epithelial cell We have Radil as a new protein that plays an important role in regulating activity in cell proliferation, migration, and EMT. We demonstrate that Radil interacts with all three isoforms of Ras including HRas, NRas, and although it the strongest interaction with KRas. Moreover, Radil interacts with GTP-bound Ras more it with suggesting a possibility that Radil may be involved in regulating Ras Supporting this, we that induced expression of Radil leads to activation of and In knockdown of Radil activation of Ras downstream components including and by high of Radil to reduced downstream signaling be to of activity by a downstream high of Radil its interaction with and activation of Rap1, a small between Rap1 and Ras proteins for GTP the activation of signaling. Supporting this, we that of Radil the of GTP-bound Ras activation of Ras signaling is associated with its in cell adhesion, migration, and EMT. Radil knockdown cell proliferation and cell Radil knockdown expression of mesenchymal cell including Zeb1, and Snail coupled with an in expression of epithelial cell Radil knockdown greatly the number of adhesion foci and actin which is with a decreased cell EMT is frequently associated with tumor as invasive and R. R.A. The of epithelial-mesenchymal 2009; PubMed Scopus Google Scholar, The EMT and 2011; PubMed Scopus Google Scholar). it is that expression and/or of Radil may play an important role in tumor development and studies suggest that Radil and Radil knockdown plays a more role in expression of mesenchymal genes including and Snail suggesting that Radil is a that drives cells toward the mesenchymal with that Snail expression of E-cadherin (11Bezdekova M. Brychtova S. Sedlakova E. Langova K. Brychta T. Belej K. Analysis of Snail-1, E-cadherin and claudin-1 expression in colorectal adenomas and carcinomas.Int. J. Mol. Sci. 2012; 13: 1632-1643Crossref PubMed Scopus (38) Google Scholar, M. A. in cancer invasion and Cell Sci. PubMed Scopus Google Scholar), we that Radil and/or leads to of E-cadherin Radil knockdown greatly the cell with actin as with cells with knockdown of is for and is to that Radil is a critical that mediates the of and interactions with including and Radil is as an effector of Rap1, which is a small GTPase regulating adhesion, adhesion, and actin Ras and of in and signaling, regulating in invasion and S. Brock E.J. Ji K. Mattingly R.R. Ras and Rap1: A tale of two GTPases.Semin. Cancer Biol. 2019; 54: 29-39Crossref PubMed Scopus (45) Google Scholar). In this we that Radil interacts with Rap1 the the is crucial for the interaction between Ras and Radil, we have that there is a between and Rap1 in with Supporting we demonstrate that expression the interaction between Rap1 and of Radil may the interaction between Radil and Rap1, Ras downstream signaling. At it and Rap1 or the of cell adhesion and migration. that is by KRasV12 it is to that Radil is involved in cell adhesion and migration of the between and The cell was from for was from was from and from Rap1, ZO-1, Snail, and from Cell Radil and from from the and from was from and cell from the in the with and of and of cell from the was in with and of and of and cell from in with and of and of cells in with and and including or HRas, NRas, and by Philips and by molecular and of the human Radil or was the and of For cells a of in In of the with the gene of and of the the with the the cells to from and in the the of cell was induced with for in the and The cell by for to by in the for with the and proteins to in the A of was by or cells with Radil or with The Radil, KRas, and from and from To we of Radil and in which by key of Radil or to in cells in an with a of for The was in a with Cell and in and the was the cells in to the was to be between and in all the of a cell in the with a of and the protein of protein from used for by on on with a used for of as as the of the or of the its of the of the molecular shown in and and and as to for cells or with a cells and the proteins with to The proteins an to The and to with The and as previously and an of the was with coupled to a in a as previously M. A.S. S. S. Philips M. M. B. A. mass spectrometry of as a new Natl. Acad. Sci. U. S. A. 2018; PubMed Scopus Google Scholar). The a human to for more for mass spectrometry and to for of was the Ras and the Ras was by of was with RAS or For with the the by the and expressed as of the of kinase was of from and to from used to the of cells to a the by the cells with and or cells and for cells from the of the a of with and with The was with an proliferation was cell a of in a Cell proliferation was and and cell to by for The was on an three with and with for by for the cells by for 30 with for example, and for cells and with an and for 30 was with a with a shown in or of the different in was three and the The as the was used for all A of was in the are to with the as as the mass spectrometry have in the with the number The that they have no of with the of this We to the for its The mass in by and the and Cancer from the Cancer B. H. C. and W. D. B. H. C. and W. D. B. H. C. H. T. M. and W. 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