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• W2009159093 abstract "In normal cells the protein kinase PKR effects apoptosis in response to various extra and intracellular cues and can also function to suppress the neoplastic phenotype. Because most neoplastic cells are resistant to certain apoptotic cues, we reasoned that an early molecular event in carcinogenesis or leukemogenesis might be the inactivation of PKR by expression or activation of intracellular PKR inhibitors. Seeking novel PKR-modulating proteins we report here that nucleophosmin (NPM), a protein frequently overexpressed in a variety of human malignancies, binds to PKR, and inhibits its activation. Co-immunoprecipitation and in vitro binding experiments showed that NPM associated with PKR. Kinase assays demonstrated that recombinant NPM inhibited PKR activation in a dose-dependent manner. In addition, purified recombinant NPM was phosphorylated by activated PKR. Most importantly, overexpression of NPM suppressed PKR activity, enhanced protein synthesis, and inhibited apoptosis. Lymphoblasts from patients with Fanconi anemia (FA) expressed low levels of NPM, which correlated with high ground-state activation of PKR and cellular hypersensitivity to apoptotic cues, but enforced expression of NPM in these mutant cells reduced aberrant apoptotic responses. Inhibition of PKR by NPM may be one mechanism by which neoplastic clones evolve in sporadic malignancies and in neoplastic cells arising in the context of the cancer predisposition syndrome, Fanconi anemia. In normal cells the protein kinase PKR effects apoptosis in response to various extra and intracellular cues and can also function to suppress the neoplastic phenotype. Because most neoplastic cells are resistant to certain apoptotic cues, we reasoned that an early molecular event in carcinogenesis or leukemogenesis might be the inactivation of PKR by expression or activation of intracellular PKR inhibitors. Seeking novel PKR-modulating proteins we report here that nucleophosmin (NPM), a protein frequently overexpressed in a variety of human malignancies, binds to PKR, and inhibits its activation. Co-immunoprecipitation and in vitro binding experiments showed that NPM associated with PKR. Kinase assays demonstrated that recombinant NPM inhibited PKR activation in a dose-dependent manner. In addition, purified recombinant NPM was phosphorylated by activated PKR. Most importantly, overexpression of NPM suppressed PKR activity, enhanced protein synthesis, and inhibited apoptosis. Lymphoblasts from patients with Fanconi anemia (FA) expressed low levels of NPM, which correlated with high ground-state activation of PKR and cellular hypersensitivity to apoptotic cues, but enforced expression of NPM in these mutant cells reduced aberrant apoptotic responses. Inhibition of PKR by NPM may be one mechanism by which neoplastic clones evolve in sporadic malignancies and in neoplastic cells arising in the context of the cancer predisposition syndrome, Fanconi anemia. Sustained cellular proliferation and resistance to apoptotic cues are characteristic features of neoplastic cells. Ongoing protein synthesis is required for both of these processes, so factors that directly control mRNA translation also can directly or indirectly influence apoptotic activity. One such factor is the interferon (IFN) 1The abbreviations used are: IFN, interferon; NPM, nucleophosmin; PKR, interferon-inducible, double-stranded RNA; eIF-2α, eukaryotic initiation factor 2α; TNF-α, tumor necrosis factor-α; FA, Fanconi anemia; MEF, murine embryonic fibroblasts; GST, glutathione S-transferase; FCS, fetal calf serum.1The abbreviations used are: IFN, interferon; NPM, nucleophosmin; PKR, interferon-inducible, double-stranded RNA; eIF-2α, eukaryotic initiation factor 2α; TNF-α, tumor necrosis factor-α; FA, Fanconi anemia; MEF, murine embryonic fibroblasts; GST, glutathione S-transferase; FCS, fetal calf serum.-inducible, double-stranded RNA (dsRNA)-dependent protein kinase PKR (1Clemens M.J. Bommer U.A. Int. J. Biochem. Cell Biol. 1999; 31: 1-23Crossref PubMed Scopus (204) Google Scholar, 2Jagus R. Joshi B. Barber G.N. Int. J. Biochem. Cell Biol. 1999; 31: 123-138Crossref PubMed Scopus (175) Google Scholar), a pivotal antiviral response protein in eukaryotic cells (3Schneider R.J. Shenk T. Annu. Rev. Biochem. 1987; 56: 317-332Crossref PubMed Scopus (132) Google Scholar, 4Samuel C.E. Clin. Microbiol. Rev. 2001; 14: 778-809Crossref PubMed Scopus (2088) Google Scholar) that functions to inhibit mRNA translation. In the ground state PKR is unphosphorylated but by binding dsRNA the molecule dimerizes and autophosphorylates on multiple serine and threonine residues (5Srivastava S.P. Kumar K.U. Kaufman R.J. J. Biol. Chem. 1998; 273: 2416-2423Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 6Zhang F. Romano P.R. Nagamura-Inoue T. Tian B. Dever T.E. Mathews M.B. Ozato K. Hinnebusch A.G. J. Biol. Chem. 2001; 276: 24946-24958Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Phosphorylated PKR inactivates the eukaryotic initiation factor 2α subunit (eIF-2α) by phosphorylating it on Ser-51 (7Barber G.N. Cell Death Differ. 2001; 8: 113-126Crossref PubMed Scopus (425) Google Scholar). PKR has tumor suppressor activity (2Jagus R. Joshi B. Barber G.N. Int. J. Biochem. Cell Biol. 1999; 31: 123-138Crossref PubMed Scopus (175) Google Scholar). Enforced expression of dominant negative mutants of either PKR or eIF-2α induced malignant transformation of mouse cells (8Koromilas A.E. Roy S. Barber G.N. Katze M.G. Sonenberg N. Science. 1992; 257: 1685-1689Crossref PubMed Scopus (494) Google Scholar, 9Meurs E.F. Galabru J. Barber G.N. Katze M.G. Hovanessian A.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 232-236Crossref PubMed Scopus (414) Google Scholar, 10Barber G.N. Wambach M. Thompson S. Jagus R. Katze M.G. Mol. Cell Biol. 1995; 15: 3138-3146Crossref PubMed Scopus (139) Google Scholar, 11Donze O. Jagus R. Koromilas A.E. Hershey J.W. Sonenberg N. EMBO J. 1995; 14: 3828-3834Crossref PubMed Scopus (251) Google Scholar, 12Li S. Koromilas A.E. J. Biol. Chem. 2001; 276: 13881-13890Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Transforming Ras genes and the nuclear proto-oncogene c-Myc inhibit PKR activity (13Mundschau L.J. Faller D.V. J. Biol. Chem. 1992; 267: 23092-23098Abstract Full Text PDF PubMed Google Scholar, 14Raveh T. Hovanessian A.G. Meurs E.F. Sonenberg N. Kimchi A. J. Biol. Chem. 1996; 271: 25479-25484Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The functional tumor suppressor activity of PKR derives, at least in part, from its capacity to induce apoptosis. Because of its pro-apoptotic effects, overexpression of PKR in mouse, insect, and yeast cells causes growth inhibition and cell death (2Jagus R. Joshi B. Barber G.N. Int. J. Biochem. Cell Biol. 1999; 31: 123-138Crossref PubMed Scopus (175) Google Scholar, 8Koromilas A.E. Roy S. Barber G.N. Katze M.G. Sonenberg N. Science. 1992; 257: 1685-1689Crossref PubMed Scopus (494) Google Scholar, 15Chong K.L. Feng L. Schappert K. Meurs E. Donahue T.F. Friesen J.D. Hovanessian A.G. Williams B.R. EMBO J. 1992; 11: 1553-1562Crossref PubMed Scopus (289) Google Scholar). Indeed, inappropriate activation of PKR has been associated with certain disease states characterized by high level apoptotic activity. For example, activation, in the brain, of PKR and the PKR-like kinase PERK has been implicated in the pathogenesis of Alzheimer's, Parkinson's, and Huntington's diseases (16Peel A.L. Rao R.V. Cottrell B.A. Hayden M.R. Ellerby L.M. Bredesen D.E. Hum. Mol. Genet. 2001; 10: 1531-1538Crossref PubMed Google Scholar, 17Siman R. Flood D.G. Thinakaran G. Neumar R.W. J. Biol. Chem. 2001; 276: 44736-44743Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 18Ryu E.J. Harding H.P. Angelastro J.M. Vitolo O.V. Ron D. Greene L.A. J. Neurosci. 2002; 22: 10690-10698Crossref PubMed Google Scholar, 19Chang R.C. Suen K.C. Ma C.H. Elyaman W. Ng H.K. Hugon J. J. Neurochem. 2002; 83: 1215-1225Crossref PubMed Scopus (143) Google Scholar) and activation of PKR in bone marrow may play a role in the pathogenesis of bone marrow failure in children and adults with Fanconi anemia (20Pang Q. Keeble W. Diaz J. Christianson T.A. Fagerlie S. Rathbun R.K. Faulkner G.R. O'Dwyer M. Bagby G.C. Blood. 2001; 97: 1644-1652Crossref PubMed Scopus (55) Google Scholar). Taking into account the high prevalence of myeloid leukemia in Fanconi anemia, excessive activation of PKR in hematopoietic stem cells may also serve as a selective force for the emergence of adapted stem cells that have acquired the capacity to suppress PKR activation or expression, a somatic change that might account for clonal evolution and leukemia (21Lensch M.W. Rathbun R.K. Olson S.B. Jones G.R. Bagby Jr., G.C. Leukemia. 1999; 13: 1784-1789Crossref PubMed Scopus (56) Google Scholar). For these reasons, we sought to identify factors that bind to and inhibit PKR reasoning that enhanced expression of such factors could represent an early adaptive response of a stem cell in a highly apoptotic environment. We report here that the nucleolar protein nucleophosmin (NPM), known to be overexpressed in tumor cells, actively proliferating cells (22Feuerstein N. Chan P.K. Mond J.J. J. Biol. Chem. 1988; 263: 10608-10612Abstract Full Text PDF PubMed Google Scholar, 23Chan W.Y. Liu Q.R. Borjigin J Busch H. Rennert O.M. Tease L.A. Chan PK. Biochemistry. 1989; 28: 1033-1039Crossref PubMed Scopus (261) Google Scholar, 24Shields L.B. Gercel-Taylor C. Yashar C.M. Wan T.C. Katsanis W.A. Spinnato J.A. Taylor D.D. J. Soc. Gynecol. Investig. 1997; 4: 298-304Crossref PubMed Scopus (63) Google Scholar, 25Subong E.N. Shue M.J. Epstein J.I. Briggman J.V. Chan P.K. Partin A.W. Prostate. 1999; 39: 298-304Crossref PubMed Scopus (100) Google Scholar, 26You B.J. Huang I.J. Liu W.H. Hung Y.B. Chang J.H. Yung B.Y. Naunyn Schmiedebergs Arch. Pharmacol. 1999; 360: 683-690Crossref PubMed Scopus (24) Google Scholar), and stem cells (stemcell.princeton.edu ) is such a factor. We find that NPM expression is suppressed in Fanconi anemia cells, increases when these cells are complemented with the proper FA gene, modulates PKR activity in normal cells, and, in gain-of-function analyses, complements the apoptotic phenotype of FA cells. We suggest that the capacity of NPM to suppress PKR activation is one mechanism by which NPM promotes cell proliferation, suppresses apoptosis, and maintains states of transformation. Cell Culture and Treatments—Mouse embryo fibroblasts (MEF) from wild-type (PKR+/+) and PKR knockout (PKRo/o) mice (27Yang Y.L. Reis L.F. Pavlovic J. Aguzzi A. Schafer R. Kumar A. Williams B.R. Aguet M. Weissmann C. EMBO J. 1995; 14: 6095-6106Crossref PubMed Scopus (561) Google Scholar), were maintained in DMEM medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (FCS). Normal JY and mutant HSC536N lymphoblasts derived from a Fanconi anemia patient (FA-C) were maintained in RPMI media 1640 (Invitrogen) supplemented with 15% heat-inactivated FCS. HeLa cells and human normal fibroblasts were grown in Dulbecco's modified Eagle's medium with 10% FCS and αMEM with 20% FCS, respectively. The factor-dependent myeloid cell line MO7e and chronic myelogenous leukemia line K562 were maintained in various media in accordance to the requirements. MEF were transfected with 100 μg/ml of poly(I)·poly(C) (Amersham Biosciences) and lysed 2 h post-transfection. For treatments of lymphoblasts, cells were stimulated with recombinant human IFNγ (10 ng/ml) and TNFα (10 ng/ml) (R & D Systems, Minneapolis, MN) for the indicated time periods followed by immunoprecipitation or flow cytometric analysis. In Vitro dsRNA Binding Assays, NPM Isolation and Microsequencing, and Mass Spectrometry Analysis—Whole cell extracts (WCE) were prepared in Nonidet P-40 (Nonidet P-40) lysis buffer (1% Nonidet P-40, 20 mm Tris-HCl, pH 8.0, 137 mm NaCl, and 10% glycerol) supplemented with 1% aprotinin, 1 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 2 mm sodium orthovanadate. 500 μl of WCE containing 2 mg of total proteins were mixed with 50 μl of poly(I)·poly(C)-agarose (Amersham Biosciences) beads and rocked at 4 °C for 60 min. The beads were then washed three times with 500 μl of Nonidet P-40 lysis buffer. Proteins bound to beads were eluted from the beads by heating the samples at 94 °C for 5 min in 2× Laemmli SDS sample buffer, separated by SDS-PAGE, stained with Ponceau S, and subjected to immunoblot analysis. To isolate the 40-kDa protein for sequence analysis, WCE from ∼109 cells were passed through poly(I)·poly(C)-agarose columns. After three washes, the bound proteins were eluted in 6× Laemmli SDS sample buffer, resolved by SDS-PAGE and transferred to a polyvinylidene difluoride protein transfer and sequencing membrane (Schleicher & Schuell). The 40-kDa band was visualized by Ponseau S staining, excised, and subjected to protein microsequencing analysis (Shriners Hospital Peptide Sequencing Core Facility, Portland, OR). For mass spectrometry analysis, whole cell extracts from normal lymphoblasts were immunoprecipitated with polyclonal PKR antibody (Santa Cruz Biotechnology) as described below. The immunocomplexes were then separated by SDS-PAGE and visualized by Coomassie Blue stain. The excised protein bands were subjected to in-gel digestion. Protein identity following in-gel digestion was determined by atmospheric pressure matrix assisted laser desorption ionization mass mass spectrometry (AP-MALDI). Gel digests were purified using 10 μl disposable pipette tips filled at the tip with a single layer of C18 membrane material (Empore, 3M Bioanalytical, St. Paul, MN). Following binding of peptides by drawing the 10-μl digest through the membrane material five times, the sample was washed with 10 μl of 0.1% trifluoroacetic acid, and peptides directly eluted onto a MALDI plate in 1 μl of 10 mg/ml α-cyano-4-hydroxycinnamic acid dissolved in 50% acetonitrile, 0.1% trifluoroacetic acid. Spectra were then collected using an APMALDI source (Mass Technologies, Burtonsville, MD) and LCQ ion trap mass spectrometer (ThermoFinnigan, San Jose, CA). 50 microscans were collected in MS mode and used to perform data-dependant MS/MS on the 10 most abundant ions from each sample. Proteins were then identified using Sequest software (ThermoFinnigan) to correlate experimental MS/MS spectra with theoretical MS/MS spectra calculated from peptide sequences in a non-redundant data base of human proteins (National Center for Biotechnology Information, Bethesda, MD). Immunoprecipitation and Immunoblotting—Whole cell extracts (1 mg of total protein), prepared in 500 μl of Nonidet P-40 lysis buffer as described above, were precleared with 50 μl of 50% protein A-Sepharose suspension (Amersham Biosciences) for 1 h at 4 °C. Where indicated, the extracts were treated with RNase A (300 μg/ml) or DNase I (100 μg/ml) (Sigma-Aldrich) at 30 °C for 1 h before the preclearing step. After separation of the protein A-Sepharose the extract was incubated with anti-mPKR (a gift from John C. Bell), or anti-NPM (Santa Cruz Biotechnology) antibodies. Immunocomplexes were then bound to protein A-Sepharose beads, recovered by centrifugation, and washed with Nonidet P-40 lysis buffer. For immunoblotting, samples were heated in Laemmli SDS sample buffer, separated by SDS-PAGE and transferred onto nitrocellulose membranes. Immunoblots were incubated with the indicated primary antibodies for 2 h at room temperature. After washing, the blots were incubated with appropriate secondary antibodies for 1 h at room temperature, and developed by using an enhanced chemiluminescence kit (Amersham Biosciences). Plasmids—Plasmids used for in vitro transcription-translation were constructed by insertion of the full-length and human PKR or human NPM, amplified by PCR using Pfu DNA polymerase (Stratagen, La Jolla, CA), into the XhoI and BamHI sites of vector pCITE-2a (Novagen, Madison, WI) to create pCITE-PKR and pCITE-NPM, respectively. Deleted versions were prepared by performing PCR mutagenesis on pCITE-PKR and pCITE-NPM to give the derivatives pCITE-PKRΔDRBM, pCITE-PKRΔkd, pCITE-PKRLS4, pCITE-NPMΔRBD, and pCITE-NPMΔN. pT7-luciferase (Promega, Madison, WI) was used as control. Epitope-tagged PKR or NPM constructs were made by fusing the full-length PKR or NPM in-frame to the 3′-end of a 3×FLAG peptide sequence in the p3XFLAG-CMV vector (Sigma-Aldrich) to create pFLAG-PKR or pFLAG-NPM, respectively. FANCC cDNA was subcloned into the retroviral vector pLXSN as described previously (28Pang Q. Fagerlie S. Christianson T.A. Keeble W. Faulkner G. Diaz J. Rathbun R.K. Bagby G.C. Mol. Cell Biol. 2000; 20: 4724-4735Crossref PubMed Scopus (96) Google Scholar). FLAG-tagged NPM retroviral construct pLXSN-FLAG-NPM was created by insertion of the FLAG-NPM fragment into the XhoI and BamHI sites of pLXSN. Plasmid pEGST-PKR/λPP, which was used to co-express GST-PKR and lambda phosphatase (λ-PPase) in Escherichia coli (29Matsui T. Tanihara K. Date T. Biochem. Biophys, Res. Commun. 2001; 284: 798-807Crossref PubMed Scopus (30) Google Scholar), was obtained from T. Date. GST-NPM was constructed by subcloning the full-length NPM into the BamHI and EcoRI sites of pGEX-2T (Amersham Biosciences). All constructs were verified by sequence analysis. GST-PKR Fusion Protein Purification and in Vitro Binding Assays— Expression and purification of the GST-PKR fusion proteins were performed according to the protocol as described previously (29Matsui T. Tanihara K. Date T. Biochem. Biophys, Res. Commun. 2001; 284: 798-807Crossref PubMed Scopus (30) Google Scholar). For binding, GST-PKR fusion proteins bound to glutathione-Sepharose beads were incubated with WCE (1 mg of total protein) prepared in Nonidet P-40 lysis buffer. After incubation at 30 °C for 20 min, the beads were recovered, washed, and analyzed by SDS-PAGE and immunoblotting analysis using antibodies against human PKR and NPM (Santa Cruz Biotechnology). In Vitro Protein Interactions—[35S]methionine-labeled or unlabeled (cold) full-length PKR, NPM, and their truncated derivatives were synthesized in vitro using the TnT wheat-germ coupled transcription/translation system (Promega, Madison, WI). All in vitro translated products were treated with RNase A (300 μg/ml) at 30 °C for 1 h before use in binding assays. For binding, 30 μl of [35S]methionine-labeled proteins was incubated with equal amounts of cold proteins, as indicated, at 30 °C for 15 min. The mixtures were immunoprecipitated with anti-PKR or anti-NPM antibodies as described above, and analyzed by SDS-PAGE and autoradiography. In Vitro Kinase Assays—The indicated amounts of GST-PKR and GST-NPM, suspended in kinase buffer (20 mm Tris-HCl, pH 7.5, 50 mm KCl, 2 mm MgCl2,2mm MnCl2, 0.1 mm ATP, 5% glycerol, and a mixture of protease inhibitors including 1% aprotinin, 1 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride), were incubated with or without eIF2α at 30 °C for 10 min. Then 5 μg/ml of poly(I)·poly(C) and 10 μCi of [γ-32P]ATP were added, and the mixtures were incubated for an additional 10 min. The phosphorylated proteins were analyzed by SDS-PAGE and autoradiography. Transfection and Transduction—dsRNA transfections were carried out as previously described (20Pang Q. Keeble W. Diaz J. Christianson T.A. Fagerlie S. Rathbun R.K. Faulkner G.R. O'Dwyer M. Bagby G.C. Blood. 2001; 97: 1644-1652Crossref PubMed Scopus (55) Google Scholar). PKR nullizygous (PKRo/o) MEFs were transiently transfected with the empty vector p3XFLAG-CMV (2 μg), pFLAG-PKR (1 μg), or indicated amounts of pFLAG-NPM, along with a pEGFP plasmid (BD Biosciences CloneTech, Palo Alto, CA) that served as an internal control for transfection efficiency. Mock and transfected cells were treated with dsRNA followed by detection for the exogenously expressed proteins, examination of apoptotic morphology, and analyses of the phosphorylation of PKR and eIF2α and the rates of protein synthesis. The Moloney (pLXSN) retroviral vectors encoding FANCC or NPM were transduced into HSC536N lymphoblasts by a previously described protocol (28Pang Q. Fagerlie S. Christianson T.A. Keeble W. Faulkner G. Diaz J. Rathbun R.K. Bagby G.C. Mol. Cell Biol. 2000; 20: 4724-4735Crossref PubMed Scopus (96) Google Scholar). Sets of isogenic lines were selected for G418 (300 μg/ml) resistance, and subjected to analyses for PKR activity and apoptosis. Analysis of Cell Viability and Apoptosis—Cell viability was measured by trypan blue exclusion analysis. To quantify apoptotic cells, we used a polyclonal antibody to the active form of caspase 3 in a flow cytometric assay to detect cells in the early stages of apoptosis. The flow cytometric assays were performed by the procedure described elsewhere (20Pang Q. Keeble W. Diaz J. Christianson T.A. Fagerlie S. Rathbun R.K. Faulkner G.R. O'Dwyer M. Bagby G.C. Blood. 2001; 97: 1644-1652Crossref PubMed Scopus (55) Google Scholar). In Vivo 32P and 35S Labeling of Proteins—Cells were starved for 60 min in phosphate-free medium containing 10% dialyzed fetal bovine serum, and were treated with 100 μg/ml of poly(I)·poly(C) or combination of IFNγ and TNFα (10 ng/ml each) for the indicated time periods. Labeling was performed in the same medium by addition of [32P]orthophosphate (150 μCi/ml, PerkinElmer Life Sciences). After labeling for 3 h, whole cell lysates were prepared as described above and were subjected to immunoprecipitation with antibodies specific for mPKR or eIF-2α. [32P]phosphate-incorporated immunocomplexes were analyzed by SDS-PAGE followed by autoradiography. Immunoblot analysis was performed on these samples to determine the quantity of PKR or eIF-2α precipitated by the respective antibody. For 35S labeling, 0.5 × 106 MEF were seeded in a 60-mm dish and cultured overnight. Cells were rinsed with methionine-cysteine-free Dulbecco's modified Eagle's medium and treated with dsRNA and IFNγ where indicated. Labeling was again performed in the same medium containing 50 μCi/ml of [35S]methionine-cysteine labeling mix (PerkinElmer Life Sciences) and incubated at 37 °C for 60 min. Whole cell lysates were prepared in Nonidet P-40 lysis buffer and protein concentration was determined. Equal amounts of protein were analyzed on SDS-PAGE followed by autoradiography. Protein synthesis was measured by the incorporation of [35S]methionine and [35S]cysteine into trichloroacetic acid-precipitable proteins. Induced NPM Binding to dsRNA Columns in Normal Lymphoblasts—We recently showed that mutant FA-C cells were hypersensitive to IFNγ and dsRNA, that the dsRNA-binding protein kinase PKR was constitutively activated in mutant FA-C cells and that its activation in response to a combination of IFN γ and dsRNA was exaggerated in FA-C cells (20Pang Q. Keeble W. Diaz J. Christianson T.A. Fagerlie S. Rathbun R.K. Faulkner G.R. O'Dwyer M. Bagby G.C. Blood. 2001; 97: 1644-1652Crossref PubMed Scopus (55) Google Scholar). Seeking to identify cellular factor(s) that might modulate PKR activity in normal hematopoietic cells but not cells with FANCC mutations, we applied dsRNA affinity chromatography assays to normal and FA-C (HSC536N) lymphoblast extracts and obtained one protein of ∼40 kDa that exhibited increased binding to dsRNA in normal lymphoblasts treated with IFNγ and TNFα (Fig. 1A, compare lane 5 with lane 6). While we recovered similar amounts of PKR from whole cell extracts of normal and FA-C lymphoblasts (data not shown), we found that significantly higher amounts of this 40-kDa protein co-eluted with PKR in extracts of normal lymphoblasts than those of FA-C cells (Fig. 1A, compare lane 6 with lane 8). We suspected that this 40-kDa protein might be functionally related to the IFN/TNF hypersensitivity of the mutant FA cells. The 40-kDa band was isolated from a preparative SDS-PAGE. Microsequence analysis revealed that the four peptides derived from proteolytic digestion of the 40-kDa protein were found to be identical to predicted sequences of the nucleolar phosphoprotein nucleophosmin (NPM) (30Schmidt-Zachmann M.S. Franke W.W. Chromosoma. 1988; 96: 417-426Crossref PubMed Scopus (74) Google Scholar), with peptide 1 identical to residues 33–42, peptide 2 to residues 213–221, peptide 3 to residues 251–257, and peptide 4 to residues 268–274 (Fig. 1B). Subsequently, we performed immunoprecipitation experiments with these whole lymphoblast extracts using anti-PKR antibody to isolate potential PKR-interacting proteins in hematopoietic cells. As assessed by Coomassie Blue staining of the SDS-PAGE of anti-PKR immunoprecipitates there were six unique bands (as compared with IgG control), which were then removed from the gel and subjected to mass spectrometric analysis. NPM was one of the proteins that co-immunoprecipitated with PKR (Fig. 1C). NPM was identified by tandem mass spectrometric analysis of peptides produced from in-gel digestion (Fig. 1C, middle and bottom panels). The 3 major ions in the MS spectra of the peptide digest were selected for MS/MS analysis. The top ranking matches for the MS/MS spectra of the two most intense ions with m/z values of 1819.8 and 1835.7 were for peptide 278–291 of NPM (Xcorr values of 2.14 and 1.5, respectively). Interestingly, among these six potential PKR-interacting proteins, five are involved in protein synthesis. 2S. Vanderwerf, H. Carlson, and G. Bagby, manuscript in preparation. NPM Binds to GST-PKR—To confirm that NPM associated with PKR, we performed an in vitro binding assay with GST-PKR fusion proteins and tested the bound products by immunoblotting using an anti-NPM antibody. Because stimulation of the lymphoblasts with IFNγ and TNFα increased substantially the NPM protein recovered from the dsRNA column, we also asked whether treatment of IFNγ and TNFα might enhance interaction between PKR and NPM. We thus used whole cell lysates from the normal lymphoblasts stimulated with both IFNγ and TNFα in the binding assays. As shown in Fig. 1D, NPM did bind to GST-PKR (top, lanes 5 and 6) but not to GST alone (lanes 3 and 4). Treatments with IFNγ and TNFα only marginally increased the association of NPM with GST-PKR (compare lane 5 with lane 6). Reprobing the blot with antibody to PKR revealed similar amounts of GST-PKR in the binding reactions (Fig. 1D, bottom). Note that there is an additional lower band revealed by anti-PKR antibody in GST-PKR pulldown samples, which runs at approximately the position of endogenous PKR (Fig. 1D, bottom, lanes 5 and 6). We suspected the bacterially expressed GST-PKR could have dimerized with the endogenous PKR in the whole cell extracts of the normal lymphoblasts. We confirmed this by incubating GST-PKR (twice the amount we used in the pull-down) with phosphate-buffered saline buffer and demonstrating the absence of the lower band (lane 7). Association of Endogenous PKR and NPM in MEFs and Human Cell Lines—Given that binding of NPM to dsRNA increased after treatment of normal lymphoblasts with IFNγ and TNFα and that ground state and cytokine induced PKR activation is higher than normal in FA-C lymphoblasts, (Ref. 20Pang Q. Keeble W. Diaz J. Christianson T.A. Fagerlie S. Rathbun R.K. Faulkner G.R. O'Dwyer M. Bagby G.C. Blood. 2001; 97: 1644-1652Crossref PubMed Scopus (55) Google Scholar; see below), we reasoned that interaction of NPM with PKR might modulate PKR activation or activity. We first confirmed that NPM and PKR co-immunoprecipitated in lysates of MEF from wild-type (PKR+/+) but not from PKRo/o cells (27Yang Y.L. Reis L.F. Pavlovic J. Aguzzi A. Schafer R. Kumar A. Williams B.R. Aguet M. Weissmann C. EMBO J. 1995; 14: 6095-6106Crossref PubMed Scopus (561) Google Scholar) (Fig. 2A). Treatment of the cells with dsRNA slightly increased the association of NPM with PKR (compare lane 5 with lane 6). Since NPM binds both RNA and DNA (31Dumbar T.S. Gentry G.A. Olson M.O. Biochemistry. 1989; 28: 9495-9501Crossref PubMed Scopus (153) Google Scholar), we sought to assure that the NPM-PKR interaction was direct and not dependent upon the presence of nucleic acids by comparing the interaction between NPM and PKR in untreated and RNase- or DNase-treated lysates. The association of these two proteins was influenced by treatment with neither enzyme (Fig. 2B, lanes 3–8). To determine whether the interaction of NPM with PKR occurred in other cell types, we performed coimmunoprecipitation using lysates from HeLa, a normal human foreskin fibroblast line NFF-6, and the myeloid leukemic cell lines MO7e and K562. Association of NPM with PKR could be detected in all these cell types with particularly strong interactions observed in the leukemic cell lines MO7e and K562 cells (Fig. 2C). To demonstrate the specificity of the anti-PKR antibody used in co-immunoprecipitation experiments, we used purified recombinant GST-PKR and NPM proteins in an anti-PKR immunoprecipitation experiment in which the potential capacity of the anti-PKR antibody to bind to NPM directly has been ruled out. Specifically, the anti-PKR antibody did not precipitate NPM when purified recombinant NPM was incubated with the PKR antibody (Fig. 2D, lane 6), thus excluding the possibility that the PKR antibody interacts with NPM. NPM Interacts with PKR in Vitro—We performed in vitro binding assays using 35S-labeled proteins synthesized in a wheat germ-coupled transcription/translation system. Labeled PKR was coimmunoprecipitated by anti-NPM antibody when unlabeled NPM was present (Fig. 3A, lane 4). Conversely, NPM co-precipitated with PKR using an anti-PKR antibody in the presence of unlabeled PKR (lane 6). Increased amounts of dsRNA did not influence the interaction between the two proteins (lanes 7–9). To test whether dsRNA binding was required for the interaction, RNA binding-defective mutants of both proteins were used. It is known that PKR encodes two dsRNA-binding motifs (DRBM) in its N termin" @default.
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