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• W2014091091 abstract "We have reported previously that PDK1 physically interacts with STRAP, a transforming growth factor-β (TGF-β) receptor-interacting protein, and enhances STRAP-induced inhibition of TGF-β signaling. In this study we show that PDK1 coimmunoprecipitates with Smad proteins, including Smad2, Smad3, Smad4, and Smad7, and that this association is mediated by the pleckstrin homology domain of PDK1. The association between PDK1 and Smad proteins is increased by insulin treatment but decreased by TGF-β treatment. Analysis of the interacting proteins shows that Smad proteins enhance PDK1 kinase activity by removing 14-3-3, a negative regulator of PDK1, from the PDK1-14-3-3 complex. Knockdown of endogenous Smad proteins, including Smad3 and Smad7, by transfection with small interfering RNA produced the opposite trend and decreased PDK1 activity, protein kinase B/Akt phosphorylation, and Bad phosphorylation. Moreover, coexpression of Smad proteins and wild-type PDK1 inhibits TGF-β-induced transcription, as well as TGF-β-mediated biological functions, such as apoptosis and cell growth arrest. Inhibition was dose-dependent on PDK1, but no inhibition was observed in the presence of an inactive kinase-dead PDK1 mutant. In addition, confocal microscopy showed that wild-type PDK1 prevents translocation of Smad3 and Smad4 from the cytoplasm to the nucleus, as well as the redistribution of Smad7 from the nucleus to the cytoplasm in response to TGF-β. Taken together, our results suggest that PDK1 negatively regulates TGF-β-mediated signaling in a PDK1 kinase-dependent manner via a direct physical interaction with Smad proteins and that Smad proteins can act as potential positive regulators of PDK1. We have reported previously that PDK1 physically interacts with STRAP, a transforming growth factor-β (TGF-β) receptor-interacting protein, and enhances STRAP-induced inhibition of TGF-β signaling. In this study we show that PDK1 coimmunoprecipitates with Smad proteins, including Smad2, Smad3, Smad4, and Smad7, and that this association is mediated by the pleckstrin homology domain of PDK1. The association between PDK1 and Smad proteins is increased by insulin treatment but decreased by TGF-β treatment. Analysis of the interacting proteins shows that Smad proteins enhance PDK1 kinase activity by removing 14-3-3, a negative regulator of PDK1, from the PDK1-14-3-3 complex. Knockdown of endogenous Smad proteins, including Smad3 and Smad7, by transfection with small interfering RNA produced the opposite trend and decreased PDK1 activity, protein kinase B/Akt phosphorylation, and Bad phosphorylation. Moreover, coexpression of Smad proteins and wild-type PDK1 inhibits TGF-β-induced transcription, as well as TGF-β-mediated biological functions, such as apoptosis and cell growth arrest. Inhibition was dose-dependent on PDK1, but no inhibition was observed in the presence of an inactive kinase-dead PDK1 mutant. In addition, confocal microscopy showed that wild-type PDK1 prevents translocation of Smad3 and Smad4 from the cytoplasm to the nucleus, as well as the redistribution of Smad7 from the nucleus to the cytoplasm in response to TGF-β. Taken together, our results suggest that PDK1 negatively regulates TGF-β-mediated signaling in a PDK1 kinase-dependent manner via a direct physical interaction with Smad proteins and that Smad proteins can act as potential positive regulators of PDK1. Transforming growth factor-β (TGF-β) 2The abbreviations used are: TGF-β, transforming growth factor-β; Smad, Sma and Mad-related protein; TβR, transforming growth factor-β receptor; STRAP, serine-threonine kinase receptor-associated protein; SGK, serum/glucocorticoid regulated kinase; PI3K, phosphatidylinositol 3-kinase; PKB/Akt, protein kinase B; GST, glutathione S-transferase; PAI-1, plasminogen activator inhibitor-1; GFP, green fluorescent protein; PtdIns, phosphatidylinositol; WT, wild type; KD, kinase dead; siRNA, small interfering RNA; CMV, cytomegalovirus; PH, pleckstrin homology; re.Smad, recombinant Smad.2The abbreviations used are: TGF-β, transforming growth factor-β; Smad, Sma and Mad-related protein; TβR, transforming growth factor-β receptor; STRAP, serine-threonine kinase receptor-associated protein; SGK, serum/glucocorticoid regulated kinase; PI3K, phosphatidylinositol 3-kinase; PKB/Akt, protein kinase B; GST, glutathione S-transferase; PAI-1, plasminogen activator inhibitor-1; GFP, green fluorescent protein; PtdIns, phosphatidylinositol; WT, wild type; KD, kinase dead; siRNA, small interfering RNA; CMV, cytomegalovirus; PH, pleckstrin homology; re.Smad, recombinant Smad. plays a critical role in the modulation of a wide variety of biological and developmental processes (1Derynck R. Feng X.H. Biochim. Biophys. Acta. 1997; 1333: 105-150Crossref PubMed Scopus (507) Google Scholar, 2Massague J. Attisano L. Wrana J.L. Trends Cell Biol. 1994; 4: 172-178Abstract Full Text PDF PubMed Scopus (526) Google Scholar). The diverse cellular responses elicited by TGF-β are triggered by activation of TGF-β receptors (type I and II), which are serine/threonine kinases. TGF-β receptors subsequently propagate signals through phosphorylation of intracellular signaling mediators referred to as Smads (3Piek E. Heldin C.H. ten Dijke P. FASEB J. 1999; 13: 2105-2124Crossref PubMed Scopus (737) Google Scholar, 4Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (475) Google Scholar, 5Massague J. Wotton D. EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar). There are three functional classes of Smad proteins (6Derynck R. Zhang Y. Feng X.H. Cell. 1998; 95: 737-740Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar), the receptor-regulated Smads (R-Smads), the common Smads (Co-Smads), and the inhibitory Smads (I-Smads). The R-Smads are directly phosphorylated and activated by the type I TGF-β receptor and undergo homotrimerization and heterodimerization with a Co-Smad (Smad 4). The activated heteromeric Smad complexes are translocated into the nucleus and cooperate with other nuclear cofactors to regulate the transcription of target genes (7Shi Y. Massague J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4739) Google Scholar). Smad-mediated signaling may be simple but it is under the control of a number of Smad-interacting proteins. Several lines of evidence have demonstrated the existence of cellular Smad regulators that interact with Smads to control the subcellular localization and the rate of R-Smad association with the TGF-β receptor and subsequent phosphorylation at the plasma membrane or in the cytoplasm or nucleus. Several of these Smad regulators have been identified, including the FYVE domain protein SARA (8Tsukazaki T. Chiang T.A. Davison A.F. Attisano L. Wrana J.L. Cell. 1998; 95: 779-791Abstract Full Text Full Text PDF PubMed Scopus (784) Google Scholar), microtubules (9Dong C. Li Z. Alvarez Jr., R. Feng X.H. Goldschmidt-Clermont P.J. Mol. Cell. 2000; 5: 27-34Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar), Daxx (10Perlman R. Schiemann W.P. Brooks M.W. Lodish H.F. Weinberg R.A. Nat. Cell Biol. 2001; 8: 708-714Crossref Scopus (301) Google Scholar), the truncated receptor-like molecule BAMBI (11Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massague J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (557) Google Scholar), the ubiquitin ligase Smurf1 (12Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. Nature. 1999; 400: 687-693Crossref PubMed Scopus (674) Google Scholar), the integral inner nuclear membrane protein MAN1 (13Pan D. Estevez-Salmeron L.D. Stroschein S.L. Zhu X. He J. Zhou S. Luo K. J. Biol. Chem. 2005; 280: 15992-16001Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), and I-Smads (14Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 15Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (577) Google Scholar, 16Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.-E. Heldin C.-H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1546) Google Scholar), Smad6 and Smad7. Thus, identification and characterization of additional Smad-interacting molecules should provide greater insight into the regulation of Smad-mediated signaling. In addition, growth factor- and insulin-mediated signaling pathways modulate TGF-β signaling through a physical interaction between PKB/Akt and Smad3 (17Remy I. Montmarquette A. Michnick S.W. Nat. Cell Biol. 2004; 6: 358-365Crossref PubMed Scopus (330) Google Scholar), suggesting a possible cross-talk between TGF-β- and PI3K/PDK1-mediated signaling pathways.The 3-phosphoinositide-dependent protein kinase-1 (PDK1) is a member of the protein kinase A, G, and C subfamily of protein kinases with a PH domain that binds phosphoinositides such as PtdIns(3,4)P2 and PtdIns(3,4,5)P3 for its activity and phosphorylates Thr-308 of PKB/Akt. Phosphorylation on both Thr-308 and Ser-473 is required for maximal activation of PKB/Akt (18Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar, 19Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1180) Google Scholar, 20Sabassov dos D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5157) Google Scholar). Furthermore, these residues are independently phosphorylated by PDK1 (for Thr-308) and PDK2 (for Ser-473). Emerging evidence indicates that PDK1 kinase activity is controlled by several cellular proteins that interact with PDK1, including Hsp90 (21Fujita N. Sato S. Ishida A. Tsuruo T. J. Biol. Chem. 2002; 277: 10346-10353Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), 14-3-3 (22Sato S. Fujita N. Tsuruo T. J. Biol. Chem. 2002; 277: 39360-39367Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), protein kinase C-related kinase 2 (23Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar), and STRAP (24Seong H.-A. Jung H. Choi H.-S. Kim K.-T. Ha H. J. Biol. Chem. 2005; 280: 42897-42908Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). These observations strongly suggest that the PDK1-interacting proteins can regulate PDK1 activity. In this study, we show that there are direct physical and functional interactions between PDK1 and Smad proteins (Smad2, -3, -4, and -7) and that these interactions may play an important role in the regulation of both the PDK1 and Smad activities involved in PI3K/PDK1- and TGF-β-mediated signaling.MATERIALS AND METHODSCell Culture and Plasmids—293T, HepG2, Hep3B, HaCaT, and HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen). The Myc-tagged human wild-type and kinase-dead PDK1 plasmids were obtained as described previously (24Seong H.-A. Jung H. Choi H.-S. Kim K.-T. Ha H. J. Biol. Chem. 2005; 280: 42897-42908Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). FLAG-tagged Smad2, Smad3, Smad4, and Smad7 were a kind gift from Dr. R. Derynck (University of California, San Francisco). The B42-Smad3 constructs, B42-MH1(L), B42-MH1, B42-MH2(L), and B42-MH2, and the p21-Luc reporter plasmid were kindly provided by Dr. H-S. Choi (Chonnam National University, Kwangju, Korea). The p3TP-Lux reporter plasmid was a kind gift from Dr. J. Massague (Memorial Sloan-Kettering Cancer Center, New York). Two deletion constructs, FLAG-PDK1(PH) and FLAG-PDK1(CA), were generated by PCR using the full-length PDK1 cDNA as the template as described previously (24Seong H.-A. Jung H. Choi H.-S. Kim K.-T. Ha H. J. Biol. Chem. 2005; 280: 42897-42908Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). To generate four Smad3 deletion constructs, FLAG-MH1(L), FLAG-MH1, FLAG-MH2(L), and FLAG-MH2, the four B42-Smad3 plasmids were digested with EcoRI and XhoI, and the EcoRI/XhoI fragments were cloned into pFLAG-PAG (25Jung H. Kim T. Chae H.-Z. Kim K.-T. Ha H. J. Biol. Chem. 2001; 276: 15504-15510Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), a proliferation-associated gene cDNA cloned into the pFLAG-CMV-2 vector, cut with EcoRI and SalI.Reagents—Porcine TGF-β1 and anti-Smad7 antibody were purchased from R&D Systems (Minneapolis, MN). The anti-GST and anti-FLAG (M2) antibodies have been described previously (26Kim T. Jung H. Min S. Kim K.-T. Ha H. FEBS Lett. 1999; 460: 363-368Crossref PubMed Scopus (10) Google Scholar). The anti-PDK1, anti-Smad4, anti-14-3-3 θ, anti-histone H2B, anti-CDK4, and anti-cyclin D1 antibodies used for immunoprecipitation and immunoblotting were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Smad3 was obtained from Upstate Inc. (Charlottesville, VA). Anti-phospho-Ser/Thr was purchased from Abcam plc (Cambridge, UK). The anti-Smad2, anti-Akt, anti-Bad, anti-phospho-Akt (Thr-308), and anti-phospho-Bad (Ser-136) antibodies were obtained from Cell Signaling Technology (Beverly, MA). Insulin, wortmannin, isopropyl β-d-thiogalactopyranoside, dithiothreitol, aprotinin, phenylmethylsulfonyl fluoride, hydroxyurea, propidium iodide, RNase A, and the anti-β-actin antibody were purchased from Sigma. Polyvinylidene difluoride membrane was obtained from Millipore Corp. (Bedford, MA). The Alexa Fluor-594 anti-mouse and Alexa Fluor-488 anti-rabbit secondary antibodies were obtained from Molecular Probes. [γ-32P]ATP was purchased from PerkinElmer Life Sciences.Transient Transfection, in Vivo Interaction Assay, and Western Blot Analysis—Cells were transfected with appropriate plasmids using WelFect-Ex™ Plus (WelGENE, Daegu, Korea), according to the manufacturer's instructions. After culturing overnight, the transfected cells were incubated in the presence or absence of TGF-β1 (100 pm) for 20 h. Cells were then washed and solubilized with lysis buffer containing 0.1% Nonidet P-40 as described (27Seong H.-A. Kim K.-T. Ha H. J. Biol. Chem. 2003; 278: 9655-9662Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Detergent-insoluble materials were removed by centrifugation, and the cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) and then washed three times with the lysis buffer. For Western blotting, coprecipitates or whole cell extracts were resolved by SDS-PAGE. For immunoprecipitations, cell lysates were incubated with protein A-Sepharose that had been conjugated to the appropriate antibodies (anti-Myc, anti-PDK1, anti-Smad3, anti-Smad4, anti-Akt, and anti-Bad). The immunoprecipitated proteins were electrophoresed and blotted onto polyvinylidene difluoride membranes. The membranes were immunoblotted with the indicated antibodies and then developed using an ECL detection system according to the manufacturer's instructions (Amersham Biosciences).PDK1 Kinase Assay—To estimate PDK1-dependent serum/glucocorticoid-regulated kinase (SGK) phosphorylation in vitro, 293T cells transiently transfected with the appropriate plasmids were washed three times with ice-cold phosphate-buffered saline (PBS) and solubilized with 100 μl of lysis buffer (20 mm Hepes, pH 7.9, 10 mm EDTA, 0.1 m KCl, and 0.3 m NaCl). The cleared lysates were mixed with glutathione-Sepharose beads and rotated for 2 h at 4 °C. After washing the precipitate three times with lysis buffer, and then twice with kinase buffer (50 mm Hepes, pH 7.4, 1 mm dithiothreitol, and 10 mm MgCl2), the precipitate was incubated with 5 μCi of [γ-32P]ATP at 37 °C for 15 min in the presence of kinase buffer containing 500 ng of recombinant SGK (Upstate). The reactions were separated by electrophoresis and visualized by autoradiography.Small Interfering RNA (siRNA) Treatment—siRNAs and their complementary RNA strands were synthesized by SamChully Pharm. Ltd. (Seoul, Korea). The sequences used were as follows: PDK1 siRNA(a) targeting a coding region (amino acids 420-425) of the human PDK1 (24Seong H.-A. Jung H. Choi H.-S. Kim K.-T. Ha H. J. Biol. Chem. 2005; 280: 42897-42908Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar); PDK1 siRNA(b) (5′-GAGACCUCGUGGAGAAACU-3′) corresponding to a coding region (amino acids 310-316) of the human PDK1; Smad2 siRNA (5′-GCAGAACUAUCUCCUACUATT-3′) corresponding to a coding region (amino acids 247-253) of the human Smad2 (GenBank™ accession number AF027964); Smad3 siRNA (a, 5′-GUGAGUUCGCCUUCAAUAUTT-3′;b, 5′-ACCUAUCCCCGAAUCCGAUTT-3′) corresponding to coding regions (a, amino acids 109-115; b, amino acids 206-212) of the human Smad3 (GenBank™ accession number BC050743); Smad7 siRNA (a, 5′-GCUCAAUUCGGACAACAAGTT-3′;b, 5′-GUUUCUCCAUCAAGGCUUUTT-3′) corresponding to coding regions (a, amino acids 300-306; b, amino acids 363-369) of the human Smad7 (GenBank™ accession number NM005904); and a nonspecific control siRNA (28Yao K. Shida S. Selvakumaran M. Zimmerman R. Simon E. Schick J. Hass N.B. Balke M. Ross H. Johnson S.W. O'Dwyer P.J. Clin. Cancer Res. 2005; 11: 7264-7272Crossref PubMed Scopus (37) Google Scholar) (5′-GCGCGGGGCACGUUGGUGUTT-3′). The sense and antisense oligonucleotides for each siRNA were mixed and heated at 90 °C for 2 min, annealed at 30 °C for 1 h, and transfected into 293T, Hep3B, HaCaT, or HeLa cells using the WelFect-Ex™ Plus method. Cell lysates were collected 48 h post-transfection and analyzed by immunoblottings to confirm the down-regulation of target proteins.Luciferase Reporter Assay—HepG2 cells were transfected using WelFect-Ex™ Plus with the p3TP-Lux or p21-Luc reporter plasmids, along with each expression vector as indicated. The cells were harvested 48 h post-transfection, and luciferase activity was measured using the Promega dual luciferase assay kit according to the manufacturer's instructions. Light emission was determined with a VICTOR™ luminometer (1420 luminescence counter, PerkinElmer Life Sciences). The total DNA concentration was kept constant by supplementing with empty vector DNA. The data were normalized to the expression levels of a cotransfected β-galactosidase reporter control, and experiments were repeated at least four times.Cell Death Assay—The number of HeLa or HaCaT cells undergoing apoptosis after treatment with TGF-β1 (HeLa, 10 ng/ml for 20 h; HaCaT, 2 ng/ml for 20 h) was quantified using the GFP system, as described previously (24Seong H.-A. Jung H. Choi H.-S. Kim K.-T. Ha H. J. Biol. Chem. 2005; 280: 42897-42908Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Cells grown on sterile coverslips were transfected with pEGFP, an expression vector encoding GFP, together with the indicated expression vectors. The cells were treated with TGF-β1, at 24 h post-transfection. The cells were fixed with ice-cold 100% methanol, washed three times with PBS, and then stained with a bisbenzimide (Hoechst 33258). The coverslips were washed with PBS, then mounted on glass slides using Gelvatol, and visualized using a fluorescence microscope (Leica DM IRB, Germany). The percentage of apoptotic cells was calculated as the number of GFP-positive cells with apoptotic nuclei divided by the total number of GFP-positive cells.Preparation of Recombinant Proteins—Recombinant glutathione S-transferase (GST) fusion vectors containing Smad3 and Smad4 were constructed by subcloning the cDNA fragments of Smad3 and Smad4 into pGEX4T-1 (Amersham Biosciences) and purified by affinity chromatography on glutathione-Sepharose 4B columns (Amersham Biosciences) as described previously (25Jung H. Kim T. Chae H.-Z. Kim K.-T. Ha H. J. Biol. Chem. 2001; 276: 15504-15510Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar).FACS Analysis—HaCaT cells (2 × 105/60-mm dish) transfected with the indicated combinations of plasmid vectors (empty vector, PDK1, Smad3, and Smad7) and siRNA duplexes (Smad3, Smad7, PDK1, and control siRNAs) were washed with ice-cold PBS and then synchronized in G0/G1 by treating with hydroxyurea (2 mm) for 20 h. The fraction of cells in each stage of the cell cycle was analyzed after 10% serum treatment for 24 h in the presence or absence of TGF-β1 (2 ng/ml). Trypsinized cells were washed twice with ice-cold PBS and incubated at 37 °C for 30 min with a solution (1 mm Tris-HCl, pH 7.5) containing 50 μg/ml propidium iodide and 1 mg/ml RNase A. The cells in each phase of the cell cycle were identified using the Mod-FitLT version 3.0 (PMac) program. Flow cytometry analysis was performed using FACSCalibur-S system (BD Biosciences).Indirect Immunofluorescence—Hep3B cells were plated and transfected with FLAG-Smads (Smad3, Smad4, and Smad7) and/or Myc-tagged wild-type and kinase-dead PDK1 constructs on sterile coverslips, placed on ice, and washed three times with ice-cold PBS prior to fixation with 4% paraformaldehyde for 10 min at room temperature. Cells were then washed with PBS, treated with 0.2% Triton X-100, and rewashed with PBS. The cells were incubated with mouse anti-FLAG (M2), diluted 1:1000 in PBS, or rabbit anti-Myc, diluted 1:200 in PBS, for 2 h at 37 °C. The cells were then washed three times with PBS and incubated with Alexa Fluor-594 anti-mouse or Alexa Fluor-488 anti-rabbit secondary antibodies, diluted 1:1000 in PBS, at 37 °C for 1 h. The coverslips were washed three times with PBS and then mounted on glass slides using Gelvatol. Proteins were visualized using a Leica Dmire2 confocal microscopy (Germany).RESULTSIdentification of PDK1 as a Smad-interacting Protein—We have found previously that STRAP, a TGF-β receptor interacting protein, physically interacts with PDK1 in mammalian cells (24Seong H.-A. Jung H. Choi H.-S. Kim K.-T. Ha H. J. Biol. Chem. 2005; 280: 42897-42908Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In addition, STRAP inhibits TGF-β signaling by stabilizing the TGF-β receptor-Smad7 complex, and STRAP itself binds to Smad proteins such as Smad2, Smad3, and Smad7 (29Datta P.K. Moses H.L. Mol. Cell. Biol. 2000; 20: 3157-3167Crossref PubMed Scopus (148) Google Scholar). Based on these data, we reasoned that PDK1 might interact with Smad proteins, as well as with STRAP, in intact cells. To examine whether PDK1 directly binds to Smad proteins, we performed in vivo binding assays and coimmunoprecipitation experiments using overexpressed or endogenous proteins in 293T cells. The interaction of FLAG-tagged Smad proteins with a Myc-PDK1 fusion protein was analyzed by immunoprecipitation with an anti-Myc antibody, followed by immunoblotting with an anti-FLAG antibody. Smad2, -3, -4, and -7 were detected in the immunoprecipitate when coexpressed with Myc-PDK1 (Fig. 1A), indicating that PDK1 physically interacts with Smad proteins in cells. To confirm the interaction of PDK1 with Smad proteins in vivo, we next performed coimmunoprecipitation experiments with endogenous PDK1 and exogenous FLAG-tagged Smad proteins (Fig. 1B). Endogenous PDK1 was immunoprecipitated with an anti-PDK1 antibody from cell lysates, and the binding of Smad proteins was subsequently analyzed by immunoblotting with an anti-FLAG antibody. Smad proteins were present in the PDK1 immunoprecipitate (upper panel), but not in immunoprecipitates from control lysates of cells transfected with empty vector alone (CMV). Moreover, to examine the interaction between the two endogenous proteins, immunoprecipitation of endogenous PDK1 using an anti-PDK1 antibody was performed, and the binding of the endogenous Smad proteins (Smad2, -3, and -7) was subsequently analyzed by immunoblotting with the indicated anti-Smad antibodies. As shown in Fig. 1C, endogenous PDK1 physically interacted with the endogenous Smad proteins used in 293T cells. We have further analyzed this association using other cell lines, including Hep3B cells and SK-N-BE2C cells (27Seong H.-A. Kim K.-T. Ha H. J. Biol. Chem. 2003; 278: 9655-9662Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), a human neuroblastoma line, and we confirmed that this association could occur in vivo (data not shown). To determine whether the Smad proteins can be substrates for PDK1 in vitro, recombinant Smad proteins (re.Smad3 and -4) were expressed in Escherichia coli and purified and then used as substrates in a PDK1 kinase assay. Extracts from 293T cells expressing GST-PDK1 and FLAG-STRAP were purified with glutathione-Sepharose beads, and incubated with [γ-32P]ATP to allow phosphorylation of the recombinant Smad proteins. As shown in Fig. 1D, the Smad proteins were phosphorylated by PDK1 when the in vitro kinase assays were performed using re.Smad3 and -4 as substrates. However, phosphorylation of the recombinant Smad proteins was not detected in the absence of PDK1 (data not shown). In addition, we observed that the coexpression of STRAP, a potential positive regulator of PDK1 (24Seong H.-A. Jung H. Choi H.-S. Kim K.-T. Ha H. J. Biol. Chem. 2005; 280: 42897-42908Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), significantly increased the phosphorylation of Smad proteins by PDK1 (Fig. 1D, 2nd versus 3rd lane and 4th versus 5th lane). Similar results showing that the Smad proteins can be phosphorylated by PDK1 were also observed in vivo using cells coexpressing PDK1 and Smad2, -3, -4, or -7 instead of the recombinant Smad proteins (data not shown). To further confirm that the phosphorylation of Smad proteins by PDK1 occurs in vivo, we compared PDK1-mediated phosphorylation of Smad3/4 in cells expressing wild-type (WT) PDK1 or a kinase-dead (KD) PDK1 mutant or in the presence of a PDK1 siRNA. Expression of wild-type PDK1 produced a higher level of Smad3/4 phosphorylation, compared with cells expressing either kinase-dead PDK1 or a PDK1-specific siRNA (Fig. 1E). Taken together, our results indicate that PDK1 directly interacts with Smad proteins in vivo and that Smad proteins can be substrates for PDK1.Mapping of the PDK1 and Smad Protein Domains Involved in the PDK1-Smad Complex Formation—To establish which regions of PDK1 are necessary for association with the Smad proteins, we generated two PDK1 deletion constructs FLAG-PDK1(PH), comprising the carboxyl-terminal pleckstrin homology (PH) domain (amino acids 411-556), and FLAG-PDK1(CA), harboring the catalytic domain (amino acids 67-359), as described previously (24Seong H.-A. Jung H. Choi H.-S. Kim K.-T. Ha H. J. Biol. Chem. 2005; 280: 42897-42908Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and we examined whether these constructs were able to interact with Smad proteins. Wild-type FLAG-PDK1 and FLAG-PDK1(PH), which lacks the catalytic domain of PDK1, interacted with Smad2, -3, -4, and -7 when the proteins were coexpressed in 293T cells (Fig. 2A). However, FLAG-PDK1(CA), which contains only the catalytic domain, was unable to do so (Fig. 2A, top panel), indicating that the interaction with Smad proteins is mediated via the carboxyl-terminal PH domain of PDK1. Next, to examine which region of Smad3 was required for binding of PDK1 in vivo, we generated four FLAG-tagged Smad3 deletion constructs (Fig. 2B, upper panel). The FLAG-MH1 (amino acids 1-136), FLAG-MH1(L) (amino acids 1-231), FLAG-MH2 (amino acids 231-425), and FLAG-MH2(L) (amino acids 136-425) constructs were expressed in 293T cells and used for in vivo binding assays with GST-PDK1. The binding of PDK1 to the Smad3 deletion constructs (MH1(L), MH2, and MH2(L)) was readily detectable (Fig. 2B, lower top panel). However, PDK1 binding to the MH1 construct was not detected (Fig. 2B). These results suggest that the PDK1 PH domain binds to the Smad3 Linker/MH2 region.FIGURE 2Mapping of the binding site involved in PDK1-Smad complex formation. A, mapping of PDK1 domains involved in Smads binding. The structure of the WT PDK1 is depicted with the relative locations of its catalytic domain (CA) and PH domain. The numbers indicate amino acid residues, and the amino acid numbers of the domain boundaries are indicated (upper panel). 293T cells were cotransfected with GST alone or GST-Smads (Smad2, -3, -4, and -7), together with FLAG-PDK1(WT), FLAG-PDK1(CA), and FLAG-PDK1(PH), and purified with glutathione-Sepharose beads (GST purification). The amount of PDK1(PH) and PDK1(WT) bound to Smad proteins was determined by Western blot (WB) analysis using an anti-FLAG antibody (lower, top panel). The same stripped blot was re-probed with an anti-GST antibody to determine the expression of GST fusion proteins in the coprecipitates (lower, middle panel), and the expression of FLAG-tagged PDK1 protein in total cell lysates was analyzed by Western analysis using the anti-FLAG antibody (lower, bottom panel, Lysate). B, mapping of Smad3 domains involved in PDK1 binding. 293T cells were transiently transfected with vector alone (GST), or GST-PDK1, in combination with the indicated FLAG-Smad3 deletion constructs, FLAG-MH1, FLAG-MH1(L), FLAG-MH2, and FLAG-MH2(L), and cell lysates were purified with glutathione-Sepharose beads (GST purification). The complex formation between PDK1 and Smad3 deletion constructs was determined by immunoblotting with the anti-FLAG antibody (lower, top panel). Expression levels of FLAG-Smad3 deletion constructs were confirmed by Western blot analysis of total cell extracts using the anti-FLAG antibody (lower, bottom panel). These experiments were independently performed at least four times with similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)PDK1-Smads Complex Formation Is Regulated by Insulin and TGF-β—We" @default.
• W2014091091 created "2016-06-24" @default.
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• W2014091091 title "3-Phosphoinositide-dependent PDK1 Negatively Regulates Transforming Growth Factor-β-induced Signaling in a Kinase-dependent Manner through Physical Interaction with Smad Proteins" @default.
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• W2014091091 cites W1519258930 @default.
• W2014091091 cites W1553444759 @default.
• W2014091091 cites W157700276 @default.
• W2014091091 cites W1589859980 @default.
• W2014091091 cites W1666089077 @default.
• W2014091091 cites W1726025741 @default.
• W2014091091 cites W1975244118 @default.
• W2014091091 cites W1983628734 @default.
• W2014091091 cites W1987095400 @default.
• W2014091091 cites W1991088949 @default.
• W2014091091 cites W1991821808 @default.
• W2014091091 cites W1992506198 @default.
• W2014091091 cites W2007044718 @default.
• W2014091091 cites W2011915114 @default.
• W2014091091 cites W2014590807 @default.
• W2014091091 cites W2018434369 @default.
• W2014091091 cites W2019788712 @default.
• W2014091091 cites W2020989705 @default.
• W2014091091 cites W2021484300 @default.
• W2014091091 cites W2030464812 @default.
• W2014091091 cites W2030572220 @default.
• W2014091091 cites W2030573603 @default.
• W2014091091 cites W2030720692 @default.
• W2014091091 cites W2033887719 @default.
• W2014091091 cites W2035312783 @default.
• W2014091091 cites W2037787006 @default.
• W2014091091 cites W2041823687 @default.
• W2014091091 cites W2046867705 @default.
• W2014091091 cites W2054271691 @default.
• W2014091091 cites W2075776349 @default.
• W2014091091 cites W2080032926 @default.
• W2014091091 cites W2087307986 @default.
• W2014091091 cites W2092344861 @default.
• W2014091091 cites W2109998353 @default.
• W2014091091 cites W2122146830 @default.
• W2014091091 cites W2131936396 @default.
• W2014091091 cites W2132008353 @default.
• W2014091091 cites W2133193835 @default.
• W2014091091 cites W2149715294 @default.
• W2014091091 cites W2154568166 @default.
• W2014091091 cites W2160364430 @default.
• W2014091091 cites W2162814275 @default.
• W2014091091 cites W3141873631 @default.
• W2014091091 cites W4238567307 @default.
• W2014091091 cites W4296927411 @default.
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