Identification of Radil as a Ras binding partner and putative activator
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100314
ISSN1083-351X
AutoresByeong Hyeok Choi, Ziyue Kou, Tania Colon, Chih‐Hong Chen, Yuan Chen, Wei Dai,
Tópico(s)Cancer Cells and Metastasis
ResumoRas 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. 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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 A.R. Puzon-McLaughlin W. Bobkov A.A. Ginsberg M.H. Structural basis of dimeric Rasip1 RA domain recognition of the Ras subfamily of GTP-binding proteins.Structure. 2016; 24: 2152-2162Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). DIL domain was originally identified fungi containing a stretch of sequences homologous to the cargo-binding domain of class V myosins. To date, the function of DIL domain remains largely unknown. Upon activation, cytosolic Radil is capable of translocating to the plasma membrane in a Rap1a-GTP–dependent manner (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). Overexpression of Radil causes focal adhesion kinase (FAK) activation and promotes cell adhesion and sustains elongated morphology (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, 21Liu L. Aerbajinai W. Ahmed S.M. Rodgers G.P. Angers S. Parent C.A. Radil controls neutrophil adhesion and motility through beta2-integrin activation.Mol. Biol. Cell. 2012; 23: 4751-4765Crossref PubMed Scopus (18) Google Scholar). In this report, we describe that Radil may function as a new branch in the Ras signaling network. Affinity pulldown coupled with mass spectrometry identified that Radil physically interacted with Ras proteins with KRas exhibiting the highest affinity. The physical interaction between Radil and Ras was influenced by the GTP-bound status of Ras. Radil knockdown led to reduced activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling axis, which is coupled with decreased cell proliferation, adhesion, and invasion. Moreover, Radil promotes expression of Vimentin, Zeb1, and Snail, transcription factors of the mesenchymal cells. Ras activities are directly mediated by various downstream components it interacts with (22Lynch S.J. Snitkin H. Gumper I. Philips M.R. Sabatini D. Pellicer A. The differential palmitoylation states of N-Ras and H-Ras determine their distinct Golgi subcompartment localizations.J. Cell Physiol. 2015; 230: 610-619Crossref PubMed Scopus (28) Google Scholar, 23Sasaki A.T. Carracedo A. Locasale J.W. Anastasiou D. Takeuchi K. Kahoud E.R. Haviv S. Asara J.M. Pandolfi P.P. Cantley L.C. Ubiquitination of K-Ras enhances activation and facilitates binding to select downstream effectors.Sci. Signal. 2011; 4: ra13Crossref PubMed Scopus (120) Google Scholar, 24Sung P.J. Tsai F.D. Vais H. Court H. Yang J. Fehrenbacher N. Foskett J.K. Philips M.R. Phosphorylated K-Ras limits cell survival by blocking Bcl-xL sensitization of inositol trisphosphate receptors.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 20593-20598Crossref PubMed Scopus (63) Google Scholar, 25Zhang H. Luo J. SUMO wrestling with Ras.Small GTPases. 2016; 7: 39-46Crossref PubMed Scopus (3) Google Scholar). To further elucidate the function of Ras in regulating cell proliferation and oncogenic transformation, we attempted to identify new molecular components that physically interacted with Ras. We ectopically expressed HRasV12 that was tagged with the Flag moiety. Through the use of affinity pulldown and mass spectrometry, we identified a number of candidate proteins that physically interacted with Flag-tagged HRas (Fig. 1, A and B). To ascertain which proteins were enriched in the KRas IP over the empty vector control, we calculated a fold change using the spectral counts. To be able to calculate a ratio for proteins that had only been identified in the sample and not the control, we imputed the data by adding 2 spectral counts to all proteins in both samples. As expected, HRas (the bait) was the highest scored proteins (Table S1). KRas was identified as the top-interacting protein (Fig. 1B). Among others, Radil is one of the top-interacting proteins (Fig. 1B, highlighted). We first focused on characterizing Radil as it contains interesting domains. Western blotting revealed that Radil expression varied among transformed cell lines with A549 (lung carcinoma) and HCT116 (colorectal carcinoma) exhibiting high levels of expression (Fig. 1C). Given that direct physical association between Ras and Radil has not been reported in the literature, we performed a series of experiments to confirm whether Radil is a bona fide Ras-interacting protein. We transfected HEK293T cells with Flag-Radil plasmid for 24 h, after which cells were collected and lysed for coimmunoprecipitation (Co-IP) analysis using the anti-Flag antibody. Flag-Radil was efficiently expressed and recovered by immunoprecipitation (Fig. 2A). Endogenous Ras proteins were enriched in the immunoprecipitates after blotting with a pan-Ras antibody. Endogenous KRas4B (KRas thereafter) was also pulled down with Flag-Radil, suggesting that Radil physically associates with KRas. As a reciprocal approach, we transfected HEK293T cells with a Flag-KRasV12 expression plasmid for 24 h, after which the cell lysates were immunoprecipitated with the Flag antibody. Co-IP analysis revealed that a significant amount of Radil was enriched in Flag-KRasV12 precipitates (Fig. 2B). These combined results thus indicate that Radil physically interacts with KRas. Radil protein consists of RA, DIL, and PDZ domains (Fig. 2C). The RA domain (RAΔ) has been found in several other Ras-interacting proteins including Rin1, RASSF5, RalGDS, PLCs, and Ras GEFs (26Rodriguez-Viciana P. Sabatier C. McCormick F. Signaling specificity by Ras family GTPases is determined by the full spectrum of effectors they regulate.Mol. Cell Biol. 2004; 24: 4943Crossref PubMed Scopus (251) Google Scholar). In fact, it is essential for mediating the interaction between Radil and Rap1, the latter being a small G-protein that plays an important role in the regulation of endothelial function (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). We then determined whether it was also required for mediating its interaction with Ras. We observed that whereas the full-length Radil was capable of pulling down both KRas and Rap1 (Fig. 2D), the deletion of the RAΔ completely abolished the interaction between Radil and Ras (and Rap1). Expression of Flag-Radil and Flag-RadilRAΔ was equally efficient (Fig. 2D). These results indicate that Radil physically interacts with Ras and that RAΔ of Radil is essential for mediating the interaction. Biochemically, Ras GTPases catalyze the hydrolysis of GTP to GDP. GTP-bound Ras is active, whereas GDP-bound one is inactive (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 test whether GTP/GDP-loading status affected the interaction between Radil and Ras, we transfected HEK293T cells for 24 h with a plasmid encoding Flag-HRas (WT HRas), constitutively active mutant (HRasV12 bound with GTP), or dominant negative mutant (HRasN17 incapable of GTP-binding). Co-IP with the Flag antibody followed by immunoblotting revealed that more Radil was associated with HRasV12 than with HRas (Fig. 3A), strongly suggesting that GTP binding affects the Radil–Ras interaction. Further supporting this notion, no Radil was detected in HRasN17 immumoprecipitates (Fig. 3A). Expression of transfected plasmids coding for various Ras proteins was comparable, and the immunoprecipitation was efficient as well. Therefore, these observations strongly suggest that Radil may positively regulate or mediate Ras activation and signaling. Ras binds to GTP, or GDP results in conformational 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 speculated that Radil may prefer a GTP-bound Ras conformation. To test this hypothesis, we simulated 3D conformation of Radil-RA–KRasGTP and Radil-RA–KRasGDP complexes using available software. We found that Radil displays a higher affinity to GTP-bound KRas than to GDP-bound one (Fig. 3B). Downstream effectors that mediate Ras activities are frequently isoform specific (27Nakhaeizadeh H. Amin E. Nakhaei-Rad S. Dvorsky R. Ahmadian M.R. The RAS-effector interface: Isoform-specific differences in the effector binding regions.PLoS One. 2016; 11e0167145Crossref PubMed Scopus (32) Google Scholar). To investigate whether Radil binds to different isoforms of Ras with a different affinity, we transfected HEK293T cells with a plasmid construct coding for Flag-tagged HRas, NRas, or KRas. Plasmids expressing constitutively active mutants (V12) of each isoform were also used as controls. We observed that Radil interacted with WT KRas more efficiently than with either WT NRas or WT HRas although KRas expression was very low compared with that of either NRas or HRas (Fig. 3C). Consistent with above observations, an enhanced interaction was detected between Radil and constitutively active Ras (HRasV12, KRasV12, and NRasV12), given that expression of WT Ras and active Ras of various isoforms was comparable (Fig. 3C). We next asked how KRas might mediate the interaction with Radil. Cells transfected with plasmids expressing GFP-KRas and/or Flag-Radil were lysed, and cell lysates were immunoprecipitated with the anti-Flag antibody. We observed that Rap1 and ArhGAP29 were present in Radil immunoprecipitates, indicating that Radil constitutively interacted with Rap1 and ArhGAP29 (Fig. 3D). Radil also strongly interacted with GFP–KRas as Radil immunoprecipitates contained a high level of GFP-KRas. Interestingly, the interaction between KRas and Radil suppressed the interaction between Radil and Rap1 or ArhGAP29, suggesting that KRas competes with Rap1 and ArhGAP29 for Radil. Given that the association between Radil and Ras is affected by the GTP-binding status of Ras, we hypothesized that Radil is involved in regulating Ras activity and signaling. To test this possibility, we generated stable cell line expressing inducible KRas (293FT/Flag-KRasV12). Upon doxycycline (Dox) treatment, expression of transfected FLAG-KRas was steadily induced, leading to the activation of downstream signaling components including p-MEK and p-ERK (Fig. 4A). We then asked the role of Radil in KRasV12-activated downstream activation. 293FT/Flag-KRasV12 cells were transfected with Radil siRNAs or control sRNAs and then treated with Dox for 12 or 24 h, after which cells were analyzed for expression of transfected KRas and endogenous Radil, as well as Ras downstream signaling. We observed that knocking down Radil had no significant effect on the activation/phosphorylation of MEK by ectopically expressed KRasV12 (Fig. 4B and Fig. S1D). To further elucidate the role of Radil in modulating Ras signaling, we generated stable cell line with inducible expression of Flag-Radil (293FT/Flag-Radil). Upon Dox treatment, Flag–Radil expression was rapidly and steadily induced (Fig. 4C). Increased expression of Flag–Radil was correlated with transient activation of MEK and ERK, peaking around 4 h after Dox treatment (Fig. 4C). Intriguingly, further increase in Radil expression was correlated with suppression of p-MEK and p-ERK. To elucidate the possible mechanism of Ras regulation by Radil, we measured the level of GTP-bound Ras after induced Radil expression. We observed that treatment with Dox for about 4 h induced Radil expression, which was coupled with increased GTP–Ras as well (Fig. 4D). On the other hand, continued induction of Radil deceased the level of GTP–Ras, strongly suggesting that Radil may regulate Ras activity in a concentration-dependent manner. We next determined whether knocking down the basal level of Radil affected Ras downstream signaling. Cells transfected with siRNAs to either control or Radil for 24 h after which cells were fed 20% fetal bovine serum (FBS) for various times. We observed that Radil downregulation compromised phosphorylation/activation of cRaf and MEK by growth factors in a time-dependent manner (Fig. 5 and Fig. S1C). These results strongly suggest that Radil is involved in regulating Ras downstream signaling. We have previously shown that KRas 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 determine whether Radil might be also involved in these processes, we knocked down Radil and/or KRas in A549 cells and then measured their proliferation rate. We observed that compared with the control cells, knocking down either Radil or KRas significantly reduced cell proliferation (Fig. 6A). Immunoblotting revealed that Radil knockdown greatly reduced expression of vimentin (Fig. 6B and Fig. S1B), a mesenchymal marker positively associated with motility and adhesion (28Kidd M.E. Shumaker D.K. Ridge K.M. The role of vimentin intermediate filaments in the progression of lung cancer.Am. J. Respir. Cell Mol. Biol. 2014; 50: 1-6PubMed Google Scholar, 29Konig K. Meder L. Kroger C. Diehl L. Florin A. Rommerscheidt-Fuss U. Kahl P. Wardelmann E. Magin T.M. Buettner R. Heukamp L.C. Loss of the keratin cytoskeleton is not sufficient to induce epithelial mesenchymal transition in a novel KRAS driven sporadic lung cancer mouse model.PLoS One. 2013; 8e57996Crossref PubMed Scopus (24) Google Scholar, 30Satelli A. Li S. Vimentin in cancer and its potential as a molecular target for cancer therapy.Cell. Mol. Life Sci. 2011; 68: 3033-3046Crossref PubMed Scopus (855) Google Scholar). Radil knockdown significantly reduced the expression of Snail and Zeb1, two master transcription factors for mesenchymal cells, with a concomitant increase of E-cadherin, an epithelial cell marker (Fig. 6B). As expected, knockdown of KRas also caused downregulation of vimentin and snail, which was correlated with upregulation of ZO-1, an epithelial cell marker (Fig. 6B). Further supporting EMT induced by Radil and KRas, we carried out cell invasion assays after downregulation of Radil and KRas. We observed that knockdown of Radil or KRas led to reduced cell invasion and that the effect of decreased cell invasion by downregulation of Radil and Ras was additive (Fig. 6, C and D). To further understand how Radil or KRas affects cell proliferation and invasion, as well as EMT, we measured cell adhesion and cytoskeleton organization after modulating either Radil or KRas expression. Cells transfected siRNAs to Radil, KRas, or both were fixed and stained with antibodies to vinculin (a component of focal adhesion [FA]) and phalloidin (staining for F-actin). Compared with control cells, Radil knockdown greatly reduced the number of FAs in the cells (Fig. 7A), which was associated with the loss of actin polymerization and a significantly reduced cell size. KRas knockdown also reduced the number of intracellular FAs and actin polymerization; it also affected the cell morphology although the cell size did not seem to be reduced as dramatically as it did with Radil siRNA (Fig. 7A). Kinase profiling revealed that expression of KRasV12 also activated FAK (Fig. S2), a protein kinase positively involved in integrin-mediated cell adhesion. Based on the present study, we propose the following model that depicts Radil's role in modulating KRas during cell proliferation, migration, and EMT (Fig. 7B). As a molecular component that mediates the function of growth factors, Radil interacts with and modulates KRas activity. Prolonged activation of KRas and its downstream signaling leads to enhanced expression of mesenchymal cell phenotypes coupled with suppression of epithelial cell phenotypes. We have identified Radil as a new Ras-interacting protein that plays an important role in regulating KRas activity in cell proliferation, migration, and EMT. We demonstrate that Radil interacts with all three isoforms of Ras including HRas, NRas, and KRas although it exhibits the strongest interaction with KRas. Moreover, Radil interacts with GTP-bound Ras more efficiently than it does with GDP-bound one, suggesting a possibility that Radil may be involved in regulating Ras activity. Supporting this, we show that induced expression of Radil leads to activation of MEK and ERK albeit transiently. In addition, knockdown of Radil compromises activation of Ras downstream components including cRaf and MEK1 by FBS. Intriguingly, high levels of Radil lead to reduced downstream signaling (Fig. 4C). This could be due to feedback control of KRas activity by a downstream component(s). Alternatively, high levels of intracellular Radil could promote its interaction with and activation of Rap1, also a small GTPase. Competition between Rap1 and Ras proteins for intracellular GTP would diminish the activation of KRas signaling. Supporting this, we observe that continued induction of Radil reduces the amount of GTP-bound Ras (Fig. 4D). Radil-mediated activation of Ras signaling is likely associated with its ability in promoting cell adhesion, migration, and EMT. (i) Radil knockdown decreases cell proliferation and cell invasion; (ii) Radil kno
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