Abstract: The basic domain‐leucine zipper protein, activating transcription factor 4 (ATF4), was recently shown to control key aspects of osteoblast biology. ATF4 regulates the timely onset of osteoblast differentiation, the synthesis of type I collagen, and the transcription of the osteocalcin and RANKL (receptor activator of NFκ‐B ligand) genes. Accordingly, the levels and activity of ATF4 are under tight control through mechanisms that include protein stability and phosphorylation. We have uncovered yet another mode of inhibition of ATF4 through its interaction with the leucine zipper protein FIAT (Factor Inhibiting ATF4‐mediated Transcription, also described as γ‐taxilin). FIAT/γ‐taxilin localizes to the nucleus in osteoblasts and dimerizes with ATF4 to form inactive dimers, because it does not contain a DNA‐binding basic domain moiety. The interaction of FIAT/γ‐taxilin with ATF4 thus inhibits ATF4‐mediated transcription. Transgenic mice overexpressing FIAT/γ‐taxilin show osteopenia and reduced expression of the ATF4 target gene, osteocalcin. Interestingly, FIAT/γ‐taxilin also interacts with the transcriptional co‐activator αNAC (Nascent polypeptide associated complex And Coactivator alpha), suggesting alternative, non‐mutually exclusive mechanisms contributing to the inhibition of ATF4‐dependent osteocalcin gene transcription by FIAT/γ‐taxilin.
Overexpression of the integrin-linked kinase (ILK) was shown to increase c-Jun-dependent transcription. We now show that this effect of ILK involves the c-Jun transcriptional coactivator, nascent polypeptide-associated complex and coactivator α (α-NAC). ILK phosphorylated α-NAC on residue Ser-43 upon adhesion of cells to fibronectin. Co-expression of constitutively active ILK with α-NAC led to the nuclear accumulation of the coactivator. Conversely, α-NAC remained in the cytoplasm of cells transfected with a dominant-negative ILK mutant, and a mutated α-NAC at phosphoacceptor position Ser-43 (S43A) also localized outside of the nucleus. The S43A α-NAC mutant could not potentiate the effect of ILK on c-Jun-dependent transcription. We conclude that ILK-dependent phosphorylation of α-NAC induced the nuclear accumulation of the coactivator and that phosphorylation of α-NAC by ILK is required for the potentiation of c-Jun-mediated responses by the kinase. The results represent one of the rare examples of a transcriptional coactivator shuttling between the cytosol and the nucleus. Overexpression of the integrin-linked kinase (ILK) was shown to increase c-Jun-dependent transcription. We now show that this effect of ILK involves the c-Jun transcriptional coactivator, nascent polypeptide-associated complex and coactivator α (α-NAC). ILK phosphorylated α-NAC on residue Ser-43 upon adhesion of cells to fibronectin. Co-expression of constitutively active ILK with α-NAC led to the nuclear accumulation of the coactivator. Conversely, α-NAC remained in the cytoplasm of cells transfected with a dominant-negative ILK mutant, and a mutated α-NAC at phosphoacceptor position Ser-43 (S43A) also localized outside of the nucleus. The S43A α-NAC mutant could not potentiate the effect of ILK on c-Jun-dependent transcription. We conclude that ILK-dependent phosphorylation of α-NAC induced the nuclear accumulation of the coactivator and that phosphorylation of α-NAC by ILK is required for the potentiation of c-Jun-mediated responses by the kinase. The results represent one of the rare examples of a transcriptional coactivator shuttling between the cytosol and the nucleus. Integrin-mediated interactions of cells with components of the extracellular matrix affect many aspects of cell function, including survival, proliferation, differentiation, and migration, by ultimately regulating gene transcription (1Mondal K. Lofquist A.K. Watson J.M. Morris J.S. Price L.K. Haskill J.S. Biochem. Soc. Trans. 1995; 23: 460-464Crossref PubMed Scopus (12) Google Scholar, 2Danen E.H. Sonnenberg A. J. Pathol. 2003; 200: 471-480Crossref PubMed Scopus (100) Google Scholar). Following engagement with extracellular matrix components, integrin receptors signal via multiple downstream effectors (3Hynes R. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6955) Google Scholar), including integrin-linked kinase (ILK) 1The abbreviations used are: ILK, integrin-linked kinase; AP-1, activating protein-1; α-NAC, nascent polypeptide-associated complex and coactivator α; NLS, nuclear localization sequence; GSK, glycogen synthase kinase. (4Hannigan G.E. Leung-Hagesteijn C. Fitz-Gibbon L. Coppolino M.G. Radeva G. Filmus J. Bell J.C. Dedhar S. Nature. 1996; 379: 91-96Crossref PubMed Scopus (970) Google Scholar, 5Wu C. Dedhar S. J. Cell Biol. 2001; 155: 505-510Crossref PubMed Scopus (351) Google Scholar). The kinase activity of ILK is stimulated in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner (6Delcommenne M. Tan C. Gray V. Rue L. Woodgett J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (950) Google Scholar) following binding of extracellular matrix components to integrin receptors (5Wu C. Dedhar S. J. Cell Biol. 2001; 155: 505-510Crossref PubMed Scopus (351) Google Scholar). Unregulated ILK expression promotes anchorage-independent growth, fibronectin matrix assembly, and tumorigenesis (4Hannigan G.E. Leung-Hagesteijn C. Fitz-Gibbon L. Coppolino M.G. Radeva G. Filmus J. Bell J.C. Dedhar S. Nature. 1996; 379: 91-96Crossref PubMed Scopus (970) Google Scholar, 7Radeva G. Petrocelli T. Behrend E. Leung-Hagesteijn C. Filmus J. Slingerland J. Dedhar S. J. Biol. Chem. 1997; 272: 13937-13944Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 8Wu C. Keightley S.Y. Leung-Hagesteijn C. Radeva G. Coppolino M. Goicoechea S. McDonald J.A. Dedhar S. J. Biol. Chem. 1998; 273: 528-536Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). These results confirm the key role of ILK as an effector of downstream integrin signaling and as an important regulator of cellular activity. The net effect of stimulating the ILK signaling cascade is to modulate gene transcription. ILK activation or constitutive ILK expression has been shown to stimulate cyclin D1 transcription (7Radeva G. Petrocelli T. Behrend E. Leung-Hagesteijn C. Filmus J. Slingerland J. Dedhar S. J. Biol. Chem. 1997; 272: 13937-13944Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 9D'Amico M. Hulit J. Amanatullah D.F. Zafonte B.T. Albanese C. Bouzahzah B. Fu M. Augenlicht L.H. Donehower L.A. Takemaru K.I. Moon R.T. Davis R. Lisanti M. Shtutman M. Zhurinsky J. Ben-Ze'ev A. Troussard A.A. Dedhar S. Pestell R.G. J. Biol. Chem. 2000; 275: 32649-32657Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 10Persad S. Troussard A.A. McPhee T.R. Mulholland D.J. Dedhar S. J. Cell Biol. 2001; 153: 1161-1174Crossref PubMed Scopus (207) Google Scholar). Upon activation, ILK phosphorylates its downstream effectors, which include protein kinase B/Akt and glycogen synthase kinase 3 (6Delcommenne M. Tan C. Gray V. Rue L. Woodgett J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (950) Google Scholar). Activation or overexpression of ILK leads to translocation of β-catenin to the nucleus, where it functions as a coactivator of lymphoid enhancer factor-1/T cell factor-dependent transcription (11Novak A. Hsu S.C. Leung-Hagesteijn C. Radeva G. Papkoff J. Montesano R. Roskelley C. Grosschedl R. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4374-4379Crossref PubMed Scopus (406) Google Scholar). Overexpression of ILK has also been shown to potentiate homodimeric c-Jun activating protein-1 (AP-1)-dependent transcription (12Troussard A.A. Tan C. Yoganathan T.N. Dedhar S. Mol. Cell. Biol. 1999; 19: 7420-7427Crossref PubMed Scopus (140) Google Scholar). The Jun proteins, members of the AP-1 family of transcription factors, regulate a wide variety of cellular processes including cell proliferation, differentiation, apoptosis, and oncogenesis (13Hartl M. Bader A.G. Bister K. Curr. Cancer Drug Targets. 2003; 3: 41-55Crossref PubMed Scopus (56) Google Scholar). Jun proteins function as dimeric transcription factors that bind AP-1 regulatory elements in the promoter and/or enhancer regions of numerous genes (14Curran T. Franza Jr., B.R. Cell. 1988; 55: 395-397Abstract Full Text PDF PubMed Scopus (1313) Google Scholar). Jun family members (c-Jun, JunB, JunD) can homodimerize as well as form heterodimers among themselves or with partners of the Fos or activating transcription factor families (15Kerppola T.K. Curran T. Mol. Cell. Biol. 1993; 13: 5479-5489Crossref PubMed Scopus (140) Google Scholar, 16Hai T. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3720-3724Crossref PubMed Scopus (1119) Google Scholar). The dimeric complexes bind DNA on AP-1 sites with high affinity and cAMP response elements with low affinity (16Hai T. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3720-3724Crossref PubMed Scopus (1119) Google Scholar). Jun proteins interact with coactivators to potentiate transcription. The following proteins were characterized as coactivators of AP-1-mediated transcription: CBP (cAMP-response element-binding protein (CREB)-binding protein) (17Bannister A.J. Oehler T. Wilhelm D. Angel P. Kouzarides T. Oncogene. 1995; 11: 2509-2514PubMed Google Scholar), JAB-1 (jun-activation domain-binding protein 1) (18Claret F.X. Hibi M. Dhut S. Toda T. Karin M. Nature. 1996; 383: 453-457Crossref PubMed Scopus (409) Google Scholar), SRC-1 (steroid receptor coactivator-1) (19Lee S.K. Kim H.J. Na S.Y. Kim T.S. Choi H.S. Im S.Y. Lee J.W. J. Biol. Chem. 1998; 273: 16651-16654Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), ASC-2 (activating signal cointegrator-2) (20Lee S.-K. Na S.-Y. Jung S.-Y. Jhun B.H. Cheong J. Meltzer P.S. Lee Y.C. Lee J.W. Mol. Endocrinol. 2000; 14: 915-925Crossref PubMed Scopus (60) Google Scholar), and α-NAC (nascent polypeptide associated complex and coactivator α) (21Quelo I. Akhouayri O. Prud'homme J. St-Arnaud R. Biochemistry. 2004; 43: 2906-2914Crossref PubMed Scopus (35) Google Scholar, 22Quelo I. Hurtubise M. St-Arnaud R. Gene Expr. 2002; 10: 255-262Crossref PubMed Scopus (22) Google Scholar, 23Moreau A. Yotov W.V. Glorieux F.H. St-Arnaud R. Mol. Cell. Biol. 1998; 18: 1312-1321Crossref PubMed Scopus (75) Google Scholar). The α-NAC protein, first described as involved in translational control (24Wiedmann B. Sakai H. Davis T.A. Wiedmann M. Nature. 1994; 370: 434-440Crossref PubMed Scopus (324) Google Scholar), was also shown to function as a transcriptional coactivator by potentiating the activity of the chimeric Gal4-VP16 activator (25Yotov W.V. Moreau A. St-Arnaud R. Mol. Cell. Biol. 1998; 18: 1303-1311Crossref PubMed Scopus (87) Google Scholar) and of c-Jun homodimers (21Quelo I. Akhouayri O. Prud'homme J. St-Arnaud R. Biochemistry. 2004; 43: 2906-2914Crossref PubMed Scopus (35) Google Scholar, 22Quelo I. Hurtubise M. St-Arnaud R. Gene Expr. 2002; 10: 255-262Crossref PubMed Scopus (22) Google Scholar, 23Moreau A. Yotov W.V. Glorieux F.H. St-Arnaud R. Mol. Cell. Biol. 1998; 18: 1312-1321Crossref PubMed Scopus (75) Google Scholar). α-NAC provides a protein bridge between sequence-specific DNA binding transcription factors and the basal transcriptional machinery by contacting the general transcription factor TATA binding protein (25Yotov W.V. Moreau A. St-Arnaud R. Mol. Cell. Biol. 1998; 18: 1303-1311Crossref PubMed Scopus (87) Google Scholar). This stabilizes the transcription factors on their cognate response elements and results in enhanced transcription rates (23Moreau A. Yotov W.V. Glorieux F.H. St-Arnaud R. Mol. Cell. Biol. 1998; 18: 1312-1321Crossref PubMed Scopus (75) Google Scholar). To exert its coactivation function, α-NAC enters the nucleus (21Quelo I. Akhouayri O. Prud'homme J. St-Arnaud R. Biochemistry. 2004; 43: 2906-2914Crossref PubMed Scopus (35) Google Scholar, 22Quelo I. Hurtubise M. St-Arnaud R. Gene Expr. 2002; 10: 255-262Crossref PubMed Scopus (22) Google Scholar, 25Yotov W.V. Moreau A. St-Arnaud R. Mol. Cell. Biol. 1998; 18: 1303-1311Crossref PubMed Scopus (87) Google Scholar, 26Franke J. Reimann B. Hartmann E. Kohlerl M. Wiedmann B. J. Cell Sci. 2001; 114: 2641-2648Crossref PubMed Google Scholar), and the subcellular localization of the protein is regulated at several levels. Phosphorylation by GSK3β impacts on the half-life of α-NAC, and inhibition of the kinase leads to stabilization and nuclear accumulation of the coactivator (21Quelo I. Akhouayri O. Prud'homme J. St-Arnaud R. Biochemistry. 2004; 43: 2906-2914Crossref PubMed Scopus (35) Google Scholar) in a manner analogous to β-catenin (27Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2172) Google Scholar). The export of α-NAC from the nucleus also appears phosphorylation-dependent. 2I. Quélo and R. St-Arnaud, unpublished results. Until now, the signal(s) that initiate the α-NAC cascade and tag the coactivator for entry into the nucleus have remained unidentified. In a search for ILK targets, an interaction between ILK and α-NAC was observed. The similarities between the signal transduction pathways involving both molecules, namely the GSK3β intermediate and the potentiation of c-Jun-mediated transcription, prompted us to evaluate whether signaling by ILK impacts on the subcellular localization of α-NAC and its coactivating function. We report that ILK phosphorylates α-NAC on residue serine 43 in vitro and in living cells. This serves as the signal for entry of α-NAC into the nucleus and leads to maximal coactivation of c-Jun-dependent transcription by the α-NAC protein. Plasmids and Constructs—The FLAG epitope was inserted into the pSI mammalian expression vector (Promega, Madison, WI) to yield the pSI-Flag plasmid. The cDNAs encoding wild-type or mutated α-NAC (see Fig. 2A) were inserted in-frame into pSI-Flag to yield the pSI-NAC-Flag expression vectors. Full-length α-NAC (wild type) and mutant cDNAs were also sub-cloned in-frame at their C termini with the intein-chitin binding domain of the pTYB2 expression vector (New England Biolabs Ltd., Mississauga, ON, Canada) to give pTYB2-NAC plasmids. 3Subcloning details and vector maps are available on request. Coimmunoprecipitation—COS-7 cells were transfected with the pSINAC-Flag and pcDNA3.1/V5-His-ILK (12Troussard A.A. Tan C. Yoganathan T.N. Dedhar S. Mol. Cell. Biol. 1999; 19: 7420-7427Crossref PubMed Scopus (140) Google Scholar) expression vectors in the combinations indicated in the legend to Fig. 1. Forty-eight hours post-transfection, the cells were lysed in 2× lysis buffer (100 mm Tris-Cl, pH 7.4, 300 mm NaCl, 2 mm EDTA, 2 mm EGTA, 2% Triton X-100) with inhibitors of proteases (5 μg/ml leupeptin, aprotinin, pepstatin A, and 1 mm phenylmethylsulfonyl fluoride). The cell lysates were diluted with H2O to reach 1× lysis buffer. They were incubated overnight at 4 °C with Sepharose beads conjugated to an anti-Flag antibody (anti-Flag M2 affinity gel, Sigma) or to an unrelated anti-glutathione-S-transferase antibody. The beads were extensively washed in 1× lysis buffer and resuspended in SDS-sample buffer. Immunoprecipitates were run on 12% SDS-PAGE and transferred to polyvinylidene difluoride. The blots were probed with an anti-ILK antibody (Upstate Biotechnology, Inc., Lake Placid, NY) and then probed with an anti-rabbit secondary antibody conjugated to horseradish peroxidase. Proteins were detected by chemifluorescence with ECL Plus Western blotting detection reagents (Amersham Biosciences). The blot was subsequently stripped and reprobed with the anti-α-NAC antibody (55Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar). In Vitro Kinase Assays—Wild-type His-tagged ILK was purified from COS-7 cells stably transfected with the pcDNA3.1/V5-His-ILK expression vector (12Troussard A.A. Tan C. Yoganathan T.N. Dedhar S. Mol. Cell. Biol. 1999; 19: 7420-7427Crossref PubMed Scopus (140) Google Scholar). Briefly, confluent cells were rinsed in phosphate-buffered saline, lysed in MCAC-0 buffer (50 mm NaH2PO4, pH 8.0, 500 mm NaCl, 10% glycerol, 1 μg/ml each of the aprotinin, pepstatin, and leupeptin antiproteases, 1 mm phenylmethanesulfonyl fluoride), and sonicated following 30 min of incubation on ice. The total cell extract was incubated for 2.5 h at 4 °C with 0.5 ml of nickel-nitrilotriacetic acid slurry (Qiagen Canada Inc., Mississauga, ON) in an Econocolumn (Bio-Rad). The nickel-nitrilotriacetic acid resin was then rinsed with 6 ml of MCAC-0 buffer, followed by 3.5 ml of MCAC-20 buffer (MCAC buffer with 20 mm imidazole). The His-ILK protein was eluted from the column with 1 × 2 ml and then with 1 × 0.5 ml of MCAC-200 buffer (200 mm imidazole). It was then pooled, dialyzed against dialysis buffer (20 mm Hepes, pH 7.9, 100 mm KCl, 10% glycerol, 1 μg/ml each of the aprotinin, pepstatin, and leupeptin antiproteases, 1 mm phenylmethanesulfonyl fluoride), and concentrated to 100 ng/μl using a Centricon 10 column (Amicon, Inc., Beverley, MA). The recombinant α-NAC proteins from pTYB2-NAC plasmids were produced and purified in Escherichia coli following the manufacturer's procedure (New England Biolabs Ltd.). For in vitro kinase assays, 2 μg of the recombinant α-NAC proteins were incubated for 15 min at 30 °C in ILK buffer (50 mm Hepes, pH 7.0, 10 mm MnCl2, 10 mm MgCl2, 2 mm NaF) with 200 ng of purified ILK and 5 μCi of [γ-32P]ATP, and then were resolved by SDS-PAGE on a 12% gel. The dried gel was autoradiographed on X-AR film (Eastman Kodak Co.). In Vivo Phosphorylation Assays—COS-7 African green monkey kidney cells were maintained in low glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in 5% CO2 and transiently transfected with the GenePorter transfection reagent (5 μl/μg DNA) according to the manufacturer's procedure (Gene Therapy System, San Diego, CA). Cells were transfected with the wild-type pSI-NAC-Flag vector alone or in combination with constitutively active (ILK S343D) or dominant-negative (ILK S343A) ILK expression vectors (12Troussard A.A. Tan C. Yoganathan T.N. Dedhar S. Mol. Cell. Biol. 1999; 19: 7420-7427Crossref PubMed Scopus (140) Google Scholar). One sample was transfected with the α-NAC phosphoacceptor mutant S43A, and one sample was transfected with pSI-NAC-Flag and treated for 1 h prior to permeabilization with 50 μm of the specific ILK inhibitor, KP392 (56Persad S. Attwell S. Gray V. Mawji N. Deng J.T. Leung D. Yan J. Sanghera J. Walsh M.P. Dedhar S. J. Biol. Chem. 2001; 276: 27462-27469Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). At 48 h post-transfection, the transfected COS-7 cells were permeabilized with 0.6 unit/ml streptolysin O (Sigma-Aldrich) and labeled for 1 h with 50 μCi of [γ-32P]ATP (Amersham Biosciences), as described by Carter (29Carter N.A. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 4. John Wiley & Sons, Inc., New York1997: 18.18.11-18.18.19Google Scholar). The cells were lysed in 2× lysis buffer (100 mm Tris-Cl, pH 7.4, 300 mm NaCl, 2 mm EDTA, 2 mm EGTA, 2% Triton X-100) with inhibitors of phosphatases (2 mm β-glycerophosphate, 2 mm orthovanadate, 5 mm sodium pyrophosphate) and of proteases (5 μg/ml leupeptin, aprotinin, pepstatin A, and 1 mm phenylmethylsulfonyl fluoride). The cell lysates were diluted with H2O to reach 1× lysis buffer. The radiolabeled cell extracts were incubated overnight at 4 °C with anti-Flag M2 affinity gel (Sigma). The affinity gels were washed extensively in 1× lysis buffer and resuspended in SDS-sample buffer in the absence of dithiothreitol. Immunoprecipitates were run on 12% SDS-PAGE. The gel was subsequently dried and exposed at -80 °C. The intensity of the signals was quantified using a Typhoon 8600 PhosphorImager (Amersham Biosciences). To control for protein expression levels, one-third of the lysates were run onto a 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was blocked and incubated with the anti-NAC antibody (55Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar). After washes, the membrane was incubated with the anti-rabbit secondary antibody conjugated to horseradish peroxidase (Amersham Biosciences). The signal was revealed with the ECL Plus kit (Amersham Biosciences) and quantified with the Typhoon PhosphorImager. Phosphorylation signals were normalized to the protein expression levels, and the relative signal calculated for wild-type α-NAC-Flag was arbitrarily assigned the value of 100%. Normalized phosphorylation signals for the other samples were expressed as a percentage of the α-NAC-Flag signal. Phospho-Ser-43-specific Antibody—A peptide corresponding to α-NAC residues 35-46 was synthesized with a phosphoserine residue at relative position 43, coupled to ovalbumin, and used to raise rabbit polyclonal antibodies following standard protocols. The antiserum was depleted of nonspecific immunoglobulins by purification against the corresponding unphosphorylated peptide (57Bangalore L. Tanner A.J. Laudano A.P. Stern D.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11637-11641Crossref PubMed Scopus (38) Google Scholar) coupled to SulfoLink gel (Pierce). Adhesion Assays—Confluent plates of COS-7 cells were trypsinized, and the trypsin was inhibited with 1 mg of trypsin inhibitor (Sigma). Cells were resuspended in serum-free Dulbecco's modified Eagle's medium at 9 × 105 cells/ml. Tissue culture plates (p100) were coated for 15 min with 2 ml of a fresh solution of fibronectin at 20 μg/ml; the fibronectin solution was then aspirated and the plates were left to air dry. Cells (2 ml) were added and left to adhere for the indicated times. Control cells were left in suspension for the duration of the experiment. Plates were washed three times with phosphate-buffered saline, and the adherent cells were recovered in 0.4 ml of 2× lysis buffer. The samples were subsequently diluted to 1× with water. An equal volume of SDS-PAGE sample buffer was added, and the samples were migrated on a 12% SDS-PAGE gel, transferred to polyvinylidene difluoride, and probed with the phospho-Ser-43-specific antibody (1:100 dilution). After washes, the membrane was incubated with the anti-rabbit secondary antibody conjugated to horseradish peroxidase (Amersham Biosciences). The signal was revealed with the ECL Plus kit (Amersham Biosciences). The membrane was stripped according to the manufacturer's protocol and reprobed with the anti-NAC antibody (55Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar). Immunofluorescence—COS-7 cells were transfected as described above with the constitutively active (ILK S343D) or dominant-negative (ILK S343A) ILK expression vectors (12Troussard A.A. Tan C. Yoganathan T.N. Dedhar S. Mol. Cell. Biol. 1999; 19: 7420-7427Crossref PubMed Scopus (140) Google Scholar) alone or in combination with the wild-type pSI-NAC-Flag vector or an expression vector for the Flag-epitope-tagged α-NAC phosphoacceptor mutant S43A. At 24 h post-transfection, the cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Following blocking with 1% blocking reagent (Roche Molecular Biochemicals) supplemented with 0.2% Tween 20, the cells were incubated with the anti-Flag M2 antibody (1:200; Sigma) and a monoclonal anti-V5 tag antibody (1:200; Invitrogen). The cells were then incubated for 1-2 h at room temperature with a fluorescein isothiocyanate-conjugated anti-rabbit IgG secondary antibody (dilution 1:500) to reveal the V5-tagged ILK molecules and a rhodamine-conjugated anti-mouse IgG secondary antibody (dilution 1:500) to detect Flag-tagged recombinant α-NAC proteins. Cover-slips were mounted in Vectashield (with 4′, 6-diamidino-2-phenylindole) mounting medium (Vector Laboratories). All results were visualized on a Leica DM-R microscope at ×400 and digital images were acquired using a Leica DC300F digital camera with the Leica IM50 image management software. Transcription Assays—The COS-7 cells were transiently transfected as described above. Plasmids used were from the PathDetect c-Jun trans-reporting system (Stratagene, La Jolla, CA): 500 ng of pFR-Luc reporter plasmid containing five Gal4 binding sites fused to the luciferase gene, 50 ng of pFA2c-Jun (c-Jun-Gal4 DNA binding domain fusion), 500 ng of expression vectors for wild-type α-NAC or the S43A phosphoacceptor point mutant, together with 500 ng of the constitutively active form of ILK (pcDNA3.1/V5-His-ILK S343D) (12Troussard A.A. Tan C. Yoganathan T.N. Dedhar S. Mol. Cell. Biol. 1999; 19: 7420-7427Crossref PubMed Scopus (140) Google Scholar). Negative controls included pFC2-DNA binding domain (expressing the unfused Gal4 DNA binding domain) and corresponding empty vectors. Transfections were carried out for 48 h in Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum. Subsequently, the cells were lysed for 20 min in the reporter gene assay lysis buffer (Roche Molecular Biochemicals), and 20 μl of cell lysate was used for single luciferase reporter assays following the manufacturer's procedure (Promega) and analyzed with a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). All assays were normalized with protein concentrations determined by a Bradford assay, and the presence of the transfected α-NAC proteins was controlled by immunoblotting with the anti-Flag M2 antibody (data not shown). Statistical analysis was performed using analysis of variance and the Tukey posttest. p < 0.05 was accepted as significant. α-NAC Interacts with ILK in Mammalian Cells—The interaction of α-NAC and ILK was first detected using an ILK bait in the yeast two-hybrid protein interaction assay (data not shown) (28Leung-Hagesteijn C. Mahendra A. Naruszewicz I. Hannigan G.E. EMBO J. 2001; 20: 2160-2170Crossref PubMed Scopus (116) Google Scholar). We confirmed that the two proteins interacted in mammalian cells using co-immunoprecipitation (Fig. 1). COS-7 cells were transiently transfected with expression vectors for ILK and Flag epitope-tagged α-NAC, and the recombinant α-NAC protein was immunoprecipitated with an antibody directed against the epitope tag. Under the conditions used, ILK was co-immunoprecipitated with α-NAC-Flag (Fig. 1, lane 3). ILK was not immunoprecipitated when an unrelated antibody was used (lane 1), and the anti-Flag antibody did not immunoprecipitate ILK when α-NAC-Flag was transfected alone (lane 2). These results show that α-NAC interacts with ILK in mammalian cells. α-NAC Is an ILK Substrate—To determine whether ILK could phosphorylate α-NAC, the wild-type α-NAC protein, deletion and point mutants (Fig. 2A) were produced and purified in E. coli using pTYB2-based expression vectors, which yield recombinant proteins devoid of an associated fusion moiety. The recombinant proteins were used for in vitro kinase assays with wild-type His-tagged ILK purified from COS-7 cells stably transfected with the pcDNA3.1/V5-His-ILK expression vector (12Troussard A.A. Tan C. Yoganathan T.N. Dedhar S. Mol. Cell. Biol. 1999; 19: 7420-7427Crossref PubMed Scopus (140) Google Scholar). The purified ILK preparation was devoid of contaminants that could be phosphorylated by ILK (Fig. 2B, lane 1), and ILK did not phosphorylate the negative control maltose-binding protein purified by the same method as the α-NAC mutants (lane 9). The results of the kinase assay show that α-NAC was a substrate of ILK in vitro (Fig. 2B, lane 2). Deleting residues 179-215 (mutant Δ179-215, lane 3), 4-25 (mutant Δ4-25, lane 4), 46-69 (mutant Δ46-69, lane 6), or 69-80 (mutant Δ69-80, lane 7) did not affect phosphorylation of the recombinant proteins by ILK (Fig. 2B). Deleting residues 4-45 from the α-NAC protein, however, resulted in an almost complete lack of phosphorylation of mutant Δ4-45 by ILK (lane 5), suggesting that the ILK phosphoacceptor site within the α-NAC protein was located between residues 26 and 45. Edman sequencing of recombinant α-NAC kinased by ILK in the presence of [γ-32P]ATP showed that the radioactive tracer eluted at cycle 43, a serine residue (not shown). A point mutation was engineered to replace residue Ser-43 by an alanine residue (mutant S43A). The recombinant S43A point mutant was poorly phosphorylated by ILK in the in vitro kinase assay (Fig. 2B, lane 8), confirming that it is the main ILK phosphoacceptor site within the α-NAC protein sequence. We next tested whether α-NAC was a substrate of ILK in cells (Fig. 2C). Wild-type or mutant α-NAC-Flag in COS-7 cells was transfected in the presence or absence of ILK expression vectors or ILK inhibitors, and metabolic labeling of the intact cells was performed with [γ-32P]ATP after permeabilization. Labeling of phosphoproteins in permeabilized, intact cells is a powerful experimental approach to study protein kinase-catalyzed phosphorylation reactions and identify relevant substrates (29Carter N.A. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 4. John Wiley & Sons, Inc., New York1997: 18.18.11-18.18.19Google Scholar). The phosphorylation levels were measured by PhosphorImager and controlled for protein expression levels (data not shown). Wild-type α-NAC is phosphorylated in intact cells (21Quelo I. Akhouayri O. Prud'homme J. St-Arnaud R. Biochemistry. 2004; 43: 2906-2914Crossref PubMed Scopus (35) Google Scholar), and this level of phosphorylation was arbitrarily set at 100% (Fig. 2C). Co-transfecting a constitutively active ILK molecule (12Troussard A.A. Tan C. Yoganathan T.N. Dedhar S. Mol. Cell. Biol. 1999; 19: 7420-7427Crossref PubMed Scopus (140) Google Scholar) with α-NAC increased the phosphorylation of α-NAC (Fig. 2C, ILK S343D). Mutating residue Ser-43 to an alanine (mutant S43A) significantly reduced the phosphorylation level of the protein in cells. The reduction in phosphorylation observed in mutant S43A matched the inhibition observed in the presence of an ILK inhibitor (KP392) or in the presence of a dominant-negative ILK mutant (ILK S343A) (12Troussard A.A. Tan C. Yoganathan T.N. Dedhar S. Mol. Cell. Biol. 1999; 19: 7420-7427Crossref PubMed Scopus (140) Google Scholar) (Fig. 2C). Taken together, these data confirm that α-NAC was a substrate of ILK in living mammalian cells. Induction of α-NAC Phosphorylation upon Cell Adhesion—We raised an antibody directed against the ILK-phosphorylated form of α-NAC (Ser-43-phosphorylated) and used it to assay the phosphorylation status of the coactivator following engagement of integrin receptors by extracellular matrix components. Fig. 3 shows that α-NAC was not phosphorylated on residue Ser-43 when cells were maintained in suspension. Upon adhesion of the cells to fibronectin, there was a transient increase in Ser-43-phosphorylated α-NAC that peaked at 40 min postadhesion (Fig. 3, lanes 2-5). The total amount of α-NAC did not vary whether the cells were in suspension or adhered to fibronectin (Fig. 3, lowe
Abstract The catalytic subunit of DNA‐dependent protein kinase (DNA‐PKcs) is a pleiotropic enzyme involved in DNA repair, cell cycle control, and transcription regulation. A potential role for DNA‐PKcs in the regulation of osteoblastogenesis remains to be established. We show that pharmacological inhibition of DNA‐PKcs kinase activity or gene silencing of Prkdc (encoding DNA‐PKcs) in murine osteoblastic MC3T3‐E1 cells and human adipose‐derived mesenchymal stromal cells markedly enhanced osteogenesis and the expression of osteoblast differentiation marker genes. Inhibition of DNA‐PKcs inhibited cell cycle progression and increased osteogenesis by significantly enhancing the bone morphogenetic protein 2 response in osteoblasts and other mesenchymal cell types. Importantly, in vivo pharmacological inhibition of the kinase enhanced bone biomechanical properties. Bones from osteoblast‐specific conditional Prkdc ‐knockout mice exhibited a similar phenotype of increased stiffness. In conclusion, DNA‐PKcs negatively regulates osteoblast differentiation, and therefore DNA‐PKcs inhibitors may have therapeutic potential for bone regeneration and metabolic bone diseases.
