NM23-H1 Tumor Suppressor Physically Interacts with Serine-Threonine Kinase Receptor-associated Protein, a Transforming Growth Factor-β (TGF-β) Receptor-interacting Protein, and Negatively Regulates TGF-β Signaling
2007; Elsevier BV; Volume: 282; Issue: 16 Linguagem: Inglês
10.1074/jbc.m609832200
ISSN1083-351X
AutoresHyun‐A Seong, Haiyoung Jung, Hyunjung Ha,
Tópico(s)Magnesium in Health and Disease
ResumoNM23-H1 is a member of the NM23/NDP kinase gene family and a putative metastasis suppressor. Previously, a screen for NM23-H1-interacting proteins that could potentially modulate its activity identified serine-threonine kinase receptor-associated protein (STRAP), a transforming growth factor (TGF)-β receptor-interacting protein. Through the use of cysteine to serine amino acid substitution mutants of NM23-H1 (C4S, C109S, and C145S) and STRAP (C152S, C270S, and C152S/C270S), we demonstrated that the association between these two proteins is dependent on Cys145 of NM23-H1 and Cys152 and Cys270 of STRAP but did not appear to involve Cys4 and Cys109 of NM23-H1, suggesting that a disulfide linkage involving Cys145 of NM23-H1 and Cys152 or Cys270 of STRAP mediates complex formation. The interaction was dependent on the presence of dithiothreitol or β-mercaptoethanol but not H2O2. Ectopic expression of wild-type NM23-H1, but not NM23-H1(C145S), negatively regulated TGF-β signaling in a dose-dependent manner, enhanced stable association between the TGF-β receptor and Smad7, and prevented nuclear translocation of Smad3. Similarly, wild-type NM23-H1 inhibited TGF-β-induced apoptosis and growth inhibition, whereas NM23-H1(C145S) had no effect. Knockdown of NM23-H1 by small interfering RNA stimulated TGF-β signaling. Coexpression of wild-type STRAP, but not STRAP(C152S/C270S), significantly stimulated NM23-H1-induced growth of HaCaT cells. These results suggest that the direct interaction of NM23-H1 and STRAP is important for the regulation of TGF-β-dependent biological activity as well as NM23-H1 activity. NM23-H1 is a member of the NM23/NDP kinase gene family and a putative metastasis suppressor. Previously, a screen for NM23-H1-interacting proteins that could potentially modulate its activity identified serine-threonine kinase receptor-associated protein (STRAP), a transforming growth factor (TGF)-β receptor-interacting protein. Through the use of cysteine to serine amino acid substitution mutants of NM23-H1 (C4S, C109S, and C145S) and STRAP (C152S, C270S, and C152S/C270S), we demonstrated that the association between these two proteins is dependent on Cys145 of NM23-H1 and Cys152 and Cys270 of STRAP but did not appear to involve Cys4 and Cys109 of NM23-H1, suggesting that a disulfide linkage involving Cys145 of NM23-H1 and Cys152 or Cys270 of STRAP mediates complex formation. The interaction was dependent on the presence of dithiothreitol or β-mercaptoethanol but not H2O2. Ectopic expression of wild-type NM23-H1, but not NM23-H1(C145S), negatively regulated TGF-β signaling in a dose-dependent manner, enhanced stable association between the TGF-β receptor and Smad7, and prevented nuclear translocation of Smad3. Similarly, wild-type NM23-H1 inhibited TGF-β-induced apoptosis and growth inhibition, whereas NM23-H1(C145S) had no effect. Knockdown of NM23-H1 by small interfering RNA stimulated TGF-β signaling. Coexpression of wild-type STRAP, but not STRAP(C152S/C270S), significantly stimulated NM23-H1-induced growth of HaCaT cells. These results suggest that the direct interaction of NM23-H1 and STRAP is important for the regulation of TGF-β-dependent biological activity as well as NM23-H1 activity. The NM23 family of genes is characterized by reduced expression in certain highly metastatic cell lines and tumors (1Steeg P.S. Bevilacqua G. Kopper L. Thorgeirsson U.P. Talmadge J.E. Liotta L.A. Sobel M.E. J. Natl. Cancer Inst. 1988; 80: 200-204Crossref PubMed Scopus (1294) Google Scholar). In humans, the eight NM23 genes that have been identified to date, NM23-H1, NM23-H2, NM23-H3, NM23-H4, NM23-H5, NM23-H6, NM23-H7, and NM23-H8, encode NDP kinases or homologous isoforms (2Ishikawa N. Shimada N. Takagi Y. Ishijima Y. Fukuda M. Kimura N. J. Bioenerg. Biomembr. 2003; 35: 7-18Crossref PubMed Scopus (21) Google Scholar). However, although NM23-H1 was initially identified as a putative metastasis suppressor, its enzymatic activity does not appear to be responsible for its function as a metastasis suppressor during tumor progression (3Michelotti E.F. Sanford S. Freije J.M.P. MacDonald N.J. Steeg P.S. Levens D. J. Biol. Chem. 1997; 272: 22526-22530Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Studies of NM23 family proteins in other species have provided evidence for their role in proliferation, differentiation, apoptosis, development, and endocytosis (4Kimura N. Shimada N. Fukuda M. Ishijima Y. Miyazaki H. Ishii A. Takagi Y. Ishikawa N. J. Bioenerg. Biomembr. 2003; 32: 309-315Crossref Scopus (68) Google Scholar). In Drosophila, for example, abnormal wing discs (awd) is an NDP kinase, and the killer-of-prune mutation of awd (awdk-pn) causes abnormalities in cell morphology and differentiation (5Timmons L. Shearn A. Adv. Genet. 1997; 35: 207-252Crossref PubMed Scopus (16) Google Scholar). Recently, both NM23-H1 and NM23-H2 have been reported to play a role in endocytosis (6Rochdi M.D. Laroche G. Dupre E. Giguere P. Lebel A. Watier V. Hamelin E. Lepine M.C. Dupuis G. Parent J.-L. J. Biol. Chem. 2004; 279: 18981-18989Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In addition, NM23 has been associated with the differentiation of human MDA-MB-435 breast carcinoma cells (7Howlett A.R. Petersen O.W. Steeg P.S. Bissell M.J. J. Natl. Cancer Inst. 1994; 86: 1838-1844Crossref PubMed Scopus (139) Google Scholar). The ability of NM23 family proteins to regulate such a diverse set of cellular processes has recently been linked to their ability to modulate signal transduction by a diverse set of growth factors, such as transforming growth factor-β1 (TGF-β1), 2The abbreviations used are: TGF, transforming growth factor; TβR, transforming growth factor-β receptor; STRAP, serine-threonine kinase receptor-associated protein; GST, glutathione S-transferase; PAI-1, plasminogen activator inhibitor-1; GFP, green fluorescent protein; DTT, dithiothreitol; siRNA, small interfering RNA; PBS, phosphate-buffered saline.2The abbreviations used are: TGF, transforming growth factor; TβR, transforming growth factor-β receptor; STRAP, serine-threonine kinase receptor-associated protein; GST, glutathione S-transferase; PAI-1, plasminogen activator inhibitor-1; GFP, green fluorescent protein; DTT, dithiothreitol; siRNA, small interfering RNA; PBS, phosphate-buffered saline. nerve growth factor, platelet-derived growth factor, and insulin-like growth factor-1 (8Otero A.S. J. Bioenerg. Biomembr. 2000; 32: 269-275Crossref PubMed Scopus (92) Google Scholar). However, the mechanism of regulation of these signaling pathways by NM23 family proteins is unknown. To date, NM23 family proteins have been shown to associate with several cellular proteins, including glyceraldehyde-3-phosphate dehydrogenase (9Engel M. Seifert M. Theisinger B. Seyfert U. Welter C. J. Biol. Chem. 1998; 273: 20058-20065Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), Hsc70 (70-kDa heat shock cognate protein) (10Leung S.M. Hightower L.E. J. Biol. Chem. 1997; 272: 2607-2614Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), telomere (11Nosaka K. Kawahara M. Masuda M. Satomi Y. Nishino H. Biochem. Biophys. Res. Commun. 1998; 243: 342-348Crossref PubMed Scopus (56) Google Scholar), RORα (retinoid acid receptor-related orphan receptor α)/RZRβ (retinoid Z receptor β) (12Paravicini G. Steinmayr E. Andre E. Becker-Andre M. Biochem. Biophys. Res. Commun. 1996; 227: 82-87Crossref PubMed Scopus (34) Google Scholar), Rad, a Ras-related small GTPase (13Zhu J. Tseng Y.-H. Kantor J.D. Rhodes C.J. Zetter B.R. Moyers J.S. Kahn C.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14911-14918Crossref PubMed Scopus (103) Google Scholar), creatine kinase and antioxidant protein (14Otero A.S. J. Biol. Chem. 1997; 272: 14690-14694Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), and thromboxane A2 receptor, a G protein-coupled receptor (6Rochdi M.D. Laroche G. Dupre E. Giguere P. Lebel A. Watier V. Hamelin E. Lepine M.C. Dupuis G. Parent J.-L. J. Biol. Chem. 2004; 279: 18981-18989Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). These results suggest that the identification of additional binding partners of NM23 proteins will provide greater insight into the regulation and biological function of NM23 family proteins. STRAP interacts with both PDK1 (3-phosphoinositide-dependent protein kinase-1) and TGF-β receptor. Through this interaction, STRAP positively regulates PDK1 and negatively regulates TGF-β signaling by stabilizing the association between TGF-β receptor and Smad7 (15Seong 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, 16Datta P.K. Moses H.L. Mol. Cell. Biol. 2000; 20: 3157-3167Crossref PubMed Scopus (148) Google Scholar). Here, we report that the physical association of NM23-H1 with STRAP in vivo is important for the negative regulation of TGF-β-mediated signaling as well as NM23-H1 tumor suppressor activity. Cell Culture, Cell Line Construction, and Reagents—293T, HeLa, HepG2, Hep3B, HaCaT, and HCT116 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen) as described previously (17Jung 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 (93) Google Scholar). Porcine TGF-β1 and anti-Smad7 antibody were purchased from R&D Systems (Minneapolis, MN). Anti-GST, anti-β-actin, anti-FLAG (M2), and anti-STRAP antibodies were described previously (15Seong 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). Anti-NM23-H1, anti-CDK4, anti-cyclin D1, anti-PAI-1, and anti-histone (H2B) antibodies used in immunoprecipitation and immunoblot analyses were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Alexa Fluor-594 anti-mouse secondary antibody and Alexa Fluor-488 anti-rabbit secondary antibody were purchased from Molecular Probes, Inc. (Eugene, OR). Propidium iodide, RNase A, isopropyl-β-d-thiogalactopyranoside, dithiothreitol (DTT), aprotinin, phenylmethylsulfonyl fluoride, hydroxyurea, and anti-His antibody were purchased from Sigma. Polyvinylidene difluoride membranes were obtained from Millipore Corp. (Bedford, MA). [γ-32P]ATP was purchased from PerkinElmer Life Sciences. DNA Construction and Plasmids—The NM23-H1 mutants were generated by the PCR method. In brief, pBluescript containing a full-length NM23-H1 (pBS-NM23-H1; GenBank™ accession number X73066) was used as the template for amplification with either the T3 (5′-ATTAACCCTCACTAAAG-3′) or T7 (5′-AATACGACTCACTATAG-3′) primer, in conjunction with one of the following mutant primers containing alterations in the nucleotide sequence of NM23-H1: for NM23-H1 Cys4 → Ser (NM23-H1(C4S)), sense 5′-ATGGCCAACTCAGAGCGTACC-3′, antisense 5′-GGTACGCTCTGAGTTGGCCAT-3′; for NM23-H1 Cys109 → Ser (NM23-H1(C109S)), sense 5′-GGAGACTTCTCGATACAAGTT-3′, antisense 5′-AACTTGTATCGAGAAGTCTCC-3′; for NM23-H1 Cys145 → Ser (NM23-H1(C145S)), sense 5′-TACACGAGCTCAGCTCCAGAAC-3′, antisense 5′-GTTCTGAGCTGAGCTCGTGTA-3′. The reaction parameters were 5 min at 94 °C, 1 cycle; 30 s at 94 °C, 30 s at 45–58 °C, and 1 min at 72 °C, 20 cycles; 7 min at 72 °C, 1 cycle. The amplified PCR products were separated by agarose gel electrophoresis, excised from the gel, and subjected to a second round of PCR amplification in the absence of primers using the following parameters: 5 min at 94 °C, one cycle; 30 s at 94 °C, 30 s at 45 °C, 1 min at 72 °C, five cycles; 7 min at 72 °C, one cycle. A third round of PCR amplification was then performed using T3 and T7 primers and the following parameters: 5 min at 94 °C, 1 cycle; 30 s at 94 °C, 30 s at 45 °C, and 1 min at 72 °C, 20 cycles; 7 min at 72 °C, 1 cycle. To generate FLAG epitope-tagged fusion proteins of NM23-H1 mutants, amplified PCR products were digested with EcoRV and XbaI and ligated into pFLAG-CMV2. A ClaI/NotI fragment of each mutant in pBluescript was ligated into the ClaI/NotI site of pEBG to generate glutathione S-transferase (GST) fusion proteins of NM23-H1 mutants. Site-directed mutagenesis of STRAP was carried out using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the following mutant primers containing alterations in the nucleotide sequence of STRAP (GenBank™ accession number BC000162): for STRAP Cys152 → Ser (STRAP(C152S)), sense 5′-AGCTCTGTGGTCGAGTGAGGATA-3′, antisense 5′-TAT CCTCACTCGACCACAGAGCT-3′; for STRAP Cys270 → Ser (STRAP(C270S)), sense 5′-TCCTATTCACTCAGTGAGATTTA-3′, antisense 5′-TAAATCTCACTGAGTGAATAGGA-3′. To generate the double substitution mutant of STRAP (STRAP(C152S/C270S)), pBS-STRAP(C270S) was used as the template, and the STRAP(C152S) sense and antisense primers were used for PCR amplification. To generate the STRAP deletion constructs (STRAP-WD1–3, -WD4, -WD4(C152S), -WD5, -WD6, -WD6(C270S), and -CT), PCR was carried out using full-length STRAP, STRAP-WD4–6, and STRAP(C152S/C270S) as templates. The forward primers for STRAP-WD1–3 (5′-GCGAATTCTGCTCTGGCCACACGCGA-3′), -WD4 (5′-GCGAATTCCCTAAGGAAATTAGTGGT-3′), -WD5 (5′-GCGAATTCCCAATTAAATCCTTTGAA-3′), -WD6 (5′-GCGAATTCTTAGAATCCTACAAGGGA-3′), and -CT (5′-GCAAGCTTGTGGTAGGAAAAACGTAT-3′) each contained an EcoRI and a HindIII site (underlined). The reverse primers for STRAP-WD1–3 (5′-GCCTCGAGCAAGTCATATATGCGTAA-3′), -WD4 (5′-GCCTCGAGCATAGTAGCATGATCCCA-3′), -WD5 (5′-GCCTCGAGTCCACTATTATAATCATA-3′), -WD6 (5′-GCCTCGAGTCCTACCACAGTTTGCCA-3′), and -CT (5′-GCCTCGAGGGCCTTAACATCAGGAGC-3′) each contained an XhoI site (underlined). All PCR products were confirmed by DNA sequencing of both strands (Bioneer Corp., Cheongwon, Korea). The p3TP-Lux reporter plasmid was a kind gift from Dr. J. Massague (Memorial Sloan-Kettering Cancer Center, New York). The p21-Luc and Smad7-Luc reporter plasmids were kindly provided by H-S. Choi (Chonnam National University, Kwangju, Korea). GST-tagged and FLAG-tagged STRAP plasmids were described previously (15Seong 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). Yeast Two-hybrid Screen—The yeast genetic screen for the isolation of NM23-H1-interacting proteins was carried out as described previously (17Jung 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 (93) Google Scholar). Transfection, in Vivo Binding Assay, and Native Polyacrylamide Gel Electrophoresis—293T, HeLa, HaCaT, Hep3B, or HepG2 cells were transfected with the indicated expression vectors using WelFect-Ex™ Plus (WelGENE, Daegu, Korea), according to the manufacturer's instructions. In vivo binding assays were performed as previously described (18Seong H.-A. Kim K.-T. Ha H. J. Biol. Chem. 2003; 278: 9655-9662Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). For native polyacrylamide gel electrophoresis, the procedure was the same as that of denaturing polyacrylamide gel electrophoresis, except that solutions did not contain SDS or β-mercaptoethanol, and samples were not boiled prior to loading. siRNA Experiment—The NM23-H1-specific siRNA oligonucleotides (a, 5′-GGAGAUCGGCUUGUGGUUUTT-3′;b, 5′-GCUUCCGAAGAUCUUCUCATT-3′) corresponding to two coding regions (a, amino acids 129–134; b, amino acids 43–48) of human NM23-H1, and a nonspecific control siRNA (5′-GCGCGGGGCACGUUGGUGUdTdT-3′) (19Yao 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) were synthesized at SamChully Pharmaceutical Ltd. (Seoul, Korea). The sense and antisense oligonucleotides for each siRNA were annealed as described previously (15Seong 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). HeLa, HepG2, or HaCaT cells (2 × 105 cells/well) were plated in 6-well flat-bottomed microplates (Nunc, Rochester, NY) the day before transfection. Cells were transfected with siRNA oligonucleotides using the WelFectEx™ Plus method. After 48 h of incubation with the siRNAs, cells were analyzed by immunoblot to confirm down-regulation of target proteins. Preparation of Recombinant Proteins—Recombinant GST- or His6-tagged human STRAP (wild type and the C152S, C270S, and C152S/C270S mutants) and NM23-H1 (wild type and the C4S, C109S, and C145S mutants) were generated by subcloning the corresponding cDNA fragments of NM23-H1 and STRAP into pGEX4T-1 (Amersham Biosciences) and pQE30 (Qiagen, Valencia, CA), respectively. Proteins were purified by affinity chromatography using glutathione-Sepharose 4B columns (Amersham Biosciences) or a His-Bind Resin column (Novagen, Madison, WI), as described previously (17Jung 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 (93) Google Scholar). Spectroscopic Measurement—The far-UV CD spectra were recorded over the range of 190–250 nm on a Jasco J-715 spectropolarimeter at the following settings: 10 millidegrees sensitivity, 0.2 nm resolution, 3 units accumulation, 1 s response, and scanning speed 100 nm/min. The protein was assayed at a concentration of 0.058 mg/ml in a 1-mm path length cylindrical quartz cell. Luciferase Reporter Assay—HepG2 cells were transiently cotransfected using the WelFect-Ex™ Plus method with p3TP-Lux, p21-Luc, or Smad7-Luc, along with the indicated expression vectors, and luciferase activity was monitored with a luciferase assay kit from Promega (Madison, WI), according to the manufacturer's instructions and as described previously (15Seong 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). Apoptosis Assay—Assays for apoptosis were performed as previously described (15Seong 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). Briefly, cells grown on sterile coverslips were transfected with an expression vector encoding green fluorescent protein (GFP), together with the indicated expression vectors. After 24 h, the cells were treated with TGF-β1 (10 ng/ml for HeLa, 2 ng/ml for HaCaT) for 20 h and then fixed with ice-cold 100% methanol and washed three times with PBS. The cells were then stained with a bisbenzimide (Hoechst 33258) and visualized under a fluorescence microscope as described previously (15Seong 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). The percentage of apoptotic cells was determined by counting the number of GFP-positive cells with apoptotic nuclei and dividing this by the total number of GFP-positive cells. Preparation of Nuclear Fractions—Hep3B cells (∼4 × 105 cells/60-mm dish) transfected with the indicated combinations of expression vectors (for Smad3, STRAP, wild-type NM23-H1, and NM23-H1(C145S)) were harvested and washed twice with PBS and then resuspended in 400 μl of buffer A (10 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm DTT, 0.2 mm phenylmethylsulfonyl fluoride). Cells were solubilized by pipetting, and the lysate was incubated on ice for 10 min and then centrifuged at 15,000 rpm for 6 min at 4 °C. The resulting supernatant (cytoplasmic fraction) was separated from the insoluble pellet. Pellets were resuspended by pipetting in 100 μl of buffer C (20 mm HEPES, pH 7.9, 1.5 mm MgCl2, 420 mm NaCl, 0.2 mm EDTA, 0.5 mm DTT, 0.2 mm phenylmethylsulfonyl fluoride, 20% glycerol) and then incubated on ice for 20 min. The suspension was centrifuged at 15,000 rpm for 6 min at 4 °C, and the resulting supernatant (nuclear fraction) was collected in a chilled microcentrifuge tube. Autophosphorylation and Phosphotransferase Assay of NM23-H1—To assay autophosphorylation of NM23-H1, ∼3 μg of purified recombinant wild-type or mutant NM23-H1 were incubated with 1 μm [γ-32P]ATP (0.2 mCi/ml) at room temperature for 10 min in a 20-μl reaction volume containing TMD buffer (20 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 1 mm DTT). The reaction was stopped by the addition of 1 volume of SDS-PAGE sample buffer (pH 8.8) and incubated at room temperature for an additional 10 min. Proteins were resolved by 15% SDS-PAGE, and the wet gel was subjected to autoradiography at -70 °C. To assay the phosphotransferase activity of NM23-H1, ∼3 μg of purified recombinant His-NM23-H1 were incubated with 5 μCi of [γ-32P]ATP for 15 min at room temperature in a final volume of 100 μl of TMD buffer. The reaction products were concentrated with a Centricon-30 (Millipore, Billerica, MA) to a final volume of less than 50 μl. Five μl of the concentrated sample were incubated with unlabeled wild-type or mutant NM23-H1 (12 μg) in a final volume of 30 μl of TMD buffer for 10 min at room temperature. The reaction was stopped by the addition of 0.5 volumes of SDS-PAGE sample buffer (pH 8.8), incubated at room temperature for an additional 10 min, and then resolved by 10% SDS-PAGE. The wet gel was subjected to autoradiography at -70 °C. Assay of NM23-H1 NDP Kinase Activity—The NDP kinase assay was performed in TMD buffer (20 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 1 mm DTT) as described previously (20Freije J.M. Blay P. MacDonald N.J. Manrow R.E. Steeg P.S. J. Biol. Chem. 1997; 272: 5525-5532Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). In brief, 3 μg of purified recombinant wild-type or mutant NM23-H1 were incubated with 1 μm [γ-32P]ATP (0.2 mCi/ml) and 100 μm GDP in TMD buffer in a final volume of 20 μl for 10 min at room temperature. The reaction was stopped by the addition of 20 μl of 50 mm EDTA (pH 8.0). The reaction mixture (2 μl) was spotted onto 20 × 20-cm polyethyleneimine-cellulose TLC plates (Merck), and reaction products were resolved by capillary action in 0.75 m KH2PO4, pH 3.65. The TLC plates were dried and exposed to film, and the formation of [γ-32P]GTP was visualized by autoradiography. Fluorescence-activated Cell Sorting Analysis—HaCaT cells (2 × 105 cells/60-mm dish) transiently transfected with the indicated expression vectors or siRNA duplexes (NM23-H1 or control siRNA) were washed with ice-cold PBS and then treated with hydroxyurea (2 mm) for 20 h to synchronize the cells in G0/G1. Cell cycle distribution was analyzed after treatment with 10% serum for 24 h in the presence or absence of TGF-β1 (2 ng/ml) as follows. Cells were trypsinized, washed twice with ice-cold PBS, and then 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. Flow cytometry was carried out using a FACSCalibur-S system (BD Biosciences), and the data were analyzed using the ModFitLT version 3.0 (PMac) program. Immunofluorescence Cytochemistry—Immunohistochemistry was performed as described previously (15Seong 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 brief, Hep3B cells growing on sterile coverslips were transfected with the indicated expression vectors. The coverslips were placed on ice, and cells were washed three times with ice-cold PBS and then incubated 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. Mouse anti-FLAG (M2) antibody diluted 1000-fold in PBS or rabbit anti-NM23-H1 antibody diluted 200-fold in PBS was applied for 2 h at 37 °C. The cells were then washed three times with PBS and then incubated with Alexa Fluor-594 anti-mouse secondary antibody or Alexa Fluor-488 anti-rabbit secondary antibody (1000-fold dilutions) at 37 °C for 1 h. Cell nuclei were stained with 4′,6′-diamidino-2-phenylindole. The coverslips were washed three times with PBS and then mounted on glass slides using Gelvatol. Immunoreactive proteins were visualized on a Leica Dmire2 confocal laser-scanning microscope (Germany). NM23-H1 Physically Interacts with STRAP in Vivo—To identify proteins that interacted with NM23-H1, we carried out a yeast two-hybrid screen using the full-length human NM23-H1 as a bait, as described previously (18Seong H.-A. Kim K.-T. Ha H. J. Biol. Chem. 2003; 278: 9655-9662Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Screening of a human HeLa cDNA library led to the identification of eight distinct positive clones (data not shown). Among them, one clone was found to encode STRAP, a TGF-β receptor-interacting protein (23Datta P.K. Chytil A. Gorska A.E. Moses H.L. J. Biol. Chem. 1998; 273: 34671-34674Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Based on this previous result, we investigated whether NM23-H1 interacted with STRAP in mammalian cells by performing a set of cotransfection experiments using GST- and FLAG-tagged eukaryotic expression vectors. FLAG-STRAP was detected in association with NM23-H1 only when coexpressed with GST-NM23-H1 (Fig. 1A, top). These data corroborated the results of the yeast two-hybrid screen and demonstrated that NM23-H1 physically interacts with STRAP in cells. To confirm the physical association between NM23-H1 and STRAP, we performed a coimmunoprecipitation analysis of endogenous proteins using an anti-STRAP antibody or preimmune IgG as a control. NM23-H1 was present in anti-STRAP immune complexes from all cell lines examined, including 293T, Hep3B, and SK-N-BE(2)C (18Seong H.-A. Kim K.-T. Ha H. J. Biol. Chem. 2003; 278: 9655-9662Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) cells (Fig. 1B), demonstrating that NM23-H1 physically interacts with STRAP in vivo. To determine the subcellular localization of NM23-H1 and STRAP, immunofluorescence confocal microscopy was performed using Hep3B cells transfected with expression vectors encoding FLAG-STRAP and FLAG-NM23-H1. Both STRAP and NM23-H1 were distributed mainly in the cytoplasm and colocalized with each other, as seen in the merged image in Fig. 1C. These results indicated that NM23-H1 interacts with and colocalizes with STRAP in vivo. Cysteine Residues of NM23-H1 and STRAP Participate in NM23-H1-STRAP Complex Formation—Protein-protein interactions are mediated by disulfide linkages as well as prototypical protein binding domains in the interacting proteins (17Jung 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 (93) Google Scholar). Although STRAP contains six WD40 repeat regions (23Datta P.K. Chytil A. Gorska A.E. Moses H.L. J. Biol. Chem. 1998; 273: 34671-34674Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), NM23-H1 lacks any of the typical protein binding domains that would potentially mediate its interaction with the WD40 domains of STRAP. We speculated that the interaction of NM23-H1 and STRAP is mediated by cysteine residues present in the two proteins. 293T cells were transiently transfected with expression vectors encoding FLAG-NM23-H1 or the FLAG-tagged NM23-H1 substitution mutants NM23-H1(C4S), NM23-H1(C109S), and NM23-H1(C145S), together with an expression vector for GST-tagged wild-type STRAP (GST-STRAP). GST-STRAP was precipitated using glutathione-Sepharose beads, and complex formation between NM23-H1 and STRAP was examined by immunoblot using an anti-FLAG antibody (Fig. 2A). There was a dramatic decrease in complex formation in cells expressing NM23-H1(C145S), compared with wild-type NM23-H1, whereas complex formation was not influenced in cells expressing NM23-H1(C4S) and NM23-H1(C109S) (Fig. 2A, top, lanes 5–8). These results suggested that Cys145 of NM23-H1 plays a critical role in the association of NM23-H1 with STRAP. The amount of STRAP and NM23-H1 in each sample was similar (Fig. 2A, middle and bottom), indicating that the observed difference in complex formation was not due to differences in STRAP and NM23-H1 expression levels. The WD40 repeats of STRAP are functional motifs that are generally involved in protein-protein interactions and the assembly of multiprotein complexes (24Komachi K. Johnson A.D. Mol. Cell. Biol. 1997; 17: 6023-6028Crossref PubMed Scopus (85) Google Scholar). To determine whether cysteine residues within the WD40 repeat regions of STRAP (Cys152 and Cys270) affected complex formation between NM23-H1 and STRAP, 293T cells were cotransfected with expression plasmids encoding wild-type and mutant forms of GST-NM23-H1 and FLAG-STRAP. Coexpression of NM23-H1(C145S) with either STRAP (C152S) or STRAP(C270S) resulted in a significant decrease in complex formation between NM23-H1 and STRAP (Fig. 2B, top, lanes 4 and 6), whereas coexpression of wild-type NM23-H1 with either STRAP (C152S) or STRAP(C270S) resulted in only a slight decrease in complex formation, as compared with the expression of wild-type NM23-H1 and wild-type STRAP (Fig. 2B, top, lane 1 versus lanes 3 and 5). These results indicated that both Cys152 and Cys270 of STRAP play an important role in its association with NM23-H1. In addition, mutation of Cys270 of STRAP, which is within the sixth WD40 repeat, had a somewhat stronger effect on complex formation compared with mutation of Cys152, which is within the fourth WD40 repeat (Fig. 2B, lane 1 versus lanes 3 and 5, lane 2 versus lanes 4 and 6). To further examine the roles of Cys152 and Cys270 of STRAP in its association with NM23-H1, we generated a double substitution mutant of STRAP, STRAP(C152S/C270S), and examined its binding properties in the in vivo binding assay. Expression of STRAP (C152S/C270S) dramatically inhibited complex
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