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SHP1 Protein-tyrosine Phosphatase Inhibits gp91PHOXand p67PHOX Expression by Inhibiting Interaction of PU.1, IRF1, Interferon Consensus Sequence-binding Protein, and CREB-binding Protein with Homologous Cis Elements in the CYBB andNCF2 Genes

2001; Elsevier BV; Volume: 276; Issue: 41 Linguagem: Inglês

10.1074/jbc.m103381200

ISSN

1083-351X

Autores

Bryan Kautz, Renu Kakar, Ebenezer David, Elizabeth A. Eklund,

Tópico(s)

Galectins and Cancer Biology

Resumo

The CYBB and NCF2 genes encode the phagocyte respiratory burst oxidase proteins, gp91PHOX and p67PHOX. Previously, we identified homologous CYBB and NCF2 cis elements that are necessary for lineage-specific transcription during late myeloid differentiation. We determined that these homologous cis elements are activated by PU.1, IRF1, interferon consensus sequence-binding protein (ICSBP), and the CREB-binding protein (CBP). Since expression of PU.1 and ICSBP is lineage-restricted, our investigations identified a mechanism of lineage-specific CYBB and NCF2transcription. Since PU.1, IRF1, ICSBP, and CBP are expressed in undifferentiated myeloid cells, our investigations did not determine the mechanism of differentiation stage-specific CYBB andNCF2 transcription. In the current investigations, we determine that SHP1 protein-tyrosine phosphatase (SHP1-PTP) inhibits gp91PHOX and p67PHOX expression, in undifferentiated myeloid cell lines, by decreasing interaction of PU.1, IRF1, ICSBP, and CBP with the CYBB and NCF2genes. We also determine that IRF1 and ICSBP are tyrosine-phosphorylated during interferon γ differentiation of myeloid cell lines, and we identify IRF1 and ICSBP tyrosine residues that are necessary for CYBB and NCF2transcription. Therefore, these investigations identify a novel mechanism by which SHP1-PTP antagonizes myeloid differentiation and determine that tyrosine phosphorylation of IRF1 and ICSPB mediates stage-specific transcriptional activation in differentiating myeloid cells. The CYBB and NCF2 genes encode the phagocyte respiratory burst oxidase proteins, gp91PHOX and p67PHOX. Previously, we identified homologous CYBB and NCF2 cis elements that are necessary for lineage-specific transcription during late myeloid differentiation. We determined that these homologous cis elements are activated by PU.1, IRF1, interferon consensus sequence-binding protein (ICSBP), and the CREB-binding protein (CBP). Since expression of PU.1 and ICSBP is lineage-restricted, our investigations identified a mechanism of lineage-specific CYBB and NCF2transcription. Since PU.1, IRF1, ICSBP, and CBP are expressed in undifferentiated myeloid cells, our investigations did not determine the mechanism of differentiation stage-specific CYBB andNCF2 transcription. In the current investigations, we determine that SHP1 protein-tyrosine phosphatase (SHP1-PTP) inhibits gp91PHOX and p67PHOX expression, in undifferentiated myeloid cell lines, by decreasing interaction of PU.1, IRF1, ICSBP, and CBP with the CYBB and NCF2genes. We also determine that IRF1 and ICSBP are tyrosine-phosphorylated during interferon γ differentiation of myeloid cell lines, and we identify IRF1 and ICSBP tyrosine residues that are necessary for CYBB and NCF2transcription. Therefore, these investigations identify a novel mechanism by which SHP1-PTP antagonizes myeloid differentiation and determine that tyrosine phosphorylation of IRF1 and ICSPB mediates stage-specific transcriptional activation in differentiating myeloid cells. interferon γ SHP1 protein-tyrosine phosphatase protein-tyrosine phosphatase protein-tyrosine kinase base pair electrophoretic mobility shift assay chloramphenicol acetyltransferase CREB-binding protein polyacrylamide gel electrophoresis glutathione S-transferase interferon consensus sequence-binding protein interferon regulatory factors Myeloid differentiation is characterized by sequential acquisition of phagocyte functions, a process requiring sequential activation of myeloid-specific genes (1Tenen D.G. Hromas R. Licht J.D. Zhang D.-E. Blood. 1997; 90: 489-516Crossref PubMed Google Scholar). We have been studying transcription of theCYBB and NCF2 genes (2Eklund E.A. Skalnik D.G. J. Biol. Chem. 1995; 270: 8267-8273Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 3Eklund E.A. Luo W. Skalnik D.G. J. Immunol. 1996; 15: 2418-2430Google Scholar, 4Eklund E.A. Kakar R. J. Biol. Chem. 1997; 272: 9344-9355Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 5Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar, 7Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). These genes, which encode the respiratory burst oxidase proteins gp91PHOX and p67PHOX, are actively transcribed in myeloid cells that have differentiated beyond the promyelocyte stage (8Royer-Pokora B. Kunkle L.M. Monaco A.P. Goff S.C. Newburger P.E. Baehner R.L. Cole F.S. Curnutte J.T. Orkin S.H. Nature. 1986; 322: 32-38Crossref PubMed Scopus (651) Google Scholar, 9Leto T.L. Lomax K.J. Volpp B.D. Nunio H. Sechler J.M.G. Nauseef W.M. Clark R. Gallin J.I. Malech H.L. Science. 1990; 248: 727-730Crossref PubMed Scopus (345) Google Scholar). In mature phagocytes, CYBB and NCF2 transcription is increased by IFNγ,1lipopolysaccharide, and tumor necrosis factor α during the inflammatory response (10Newburger P.E. Ezekowitz R.A.B. Whitney C. Wright J. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5215-5220Crossref PubMed Scopus (121) Google Scholar). Therefore, CYBB andNCF2 transcription is lineage-specific and differentiation stage-specific and increases during phagocyte activation. SinceCYBB and NCF2 transcription occurs simultaneously, we hypothesize that common transcription factors regulate expression of these two genes (6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar, 7Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Previous investigations (7Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 11Neufeld E.J. Skalnik D.G. Lievens P.M. Orkin S.H. Nat. Genet. 1992; 1: 50-55Crossref PubMed Scopus (190) Google Scholar) have identified positive and negative cis elements that regulate CYBB and NCF2transcription. In undifferentiated myeloid cells, CYBBtranscription is repressed by interaction of HoxA10 and Pbx1a with three negative cis elements in the CYBB promoter. Consistent with our hypothesis, several homologous sequences in theNCF2 gene also interact in vitro with HoxA10 and Pbx1a (7Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). We found that HoxA10 is tyrosine-phosphorylated during IFNγ-induced differentiation of myeloid cell lines, and tyrosine phosphorylation decreases HoxA10 DNA binding affinity (7Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Therefore, post-translational modification of HoxA10 is one mechanism that regulates differentiation stage-specific gp91PHOX and p67PHOX expression (7Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). We also identified homologous, positive cis elements in theCYBB and NCF2 genes that are necessary for lineage-specific, IFNγ-induced transcription (5Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). These cis elements (referred to as "HAF1" elements for "hematopoiesis-associated factors") are proximate to transcription start sites in the two genes. We found that the HAF1 elements are activated by PU.1 (an ETS protein) and two interferon regulatory factors (IRFs), interferon regulatory factor 1 (IRF1) and the interferon consensus sequence-binding protein (ICSBP) (5Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). These proteins cooperate to recruit the CREB-binding protein (CBP) to the proximal CYBB and NCF2 promoters (6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). In vitro, PU.1 binds the HAF1 elements as a monomer, as a heterodimer with either IRF1 or ICSBP (the "HAF1" complex), or as a multiprotein complex with IRF1 and ICSBP and CBP (the "HAF1a" complex) (6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). Although the HAF1 cis elements contain composite ETS/IRF consensus sequences, IRF1 and ICSBP do not bind these elements in the absence of PU.1 (6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). This suggests that the IRF proteins interact with both the HAF1-DNA sequence and PU.1 protein domains. Additionally, PU.1, IRF1, and ICSBP do not interact with CBP in the absence of a HAF1 DNA-binding site (6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). This suggests that CBP interacts with PU.1, IRF1, and/or ICSBP domains that are brought into proximity by DNA binding. Since expression of PU.1 and ICSBP is restricted to myeloid and B-cells, our previous investigations (5Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar) identified a mechanism that contributes to lineage-restricted CYBB andNCF2 transcription. However, these investigations did not determine the mechanism by which PU.1, IRF1, and ICSBP participate in differentiation stage-specific transcription. By in vitroDNA binding assays, we determined that access to the HAF1 elements is not sterically hindered by binding of HoxA10/Pbx1a to theCYBB and NCF2 repressor elements, in undifferentiated myeloid cells (2Eklund E.A. Skalnik D.G. J. Biol. Chem. 1995; 270: 8267-8273Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 3Eklund E.A. Luo W. Skalnik D.G. J. Immunol. 1996; 15: 2418-2430Google Scholar). Additionally, we determined that IFNγ treatment of myeloid cell lines does not increase the abundance of PU.1, IRF1, ICSBP, or CBP. This is consistent with previous results (1Tenen D.G. Hromas R. Licht J.D. Zhang D.-E. Blood. 1997; 90: 489-516Crossref PubMed Google Scholar, 12Petrovick M.S. Heibert S.W. Friedman A.D. Hetherington C.J. Tenen D.G. Zhang D.-E. Mol. Cell. Biol. 1998; 18: 3915-3929Crossref PubMed Google Scholar, 13Gabriele L. Phung J. Fukumoto J. Segal D. Wang I.M. Giannakakou P. Giese N.A. Ozato K. Morse H.C. J. Exp. Med. 1999; 190: 411-421Crossref PubMed Scopus (102) Google Scholar) indicating that PU.1 and ICSBP regulate transcription at multiple points during myelopoiesis. Based on these observations, we hypothesize that post-translational modification of proteins that participate in the HAF1a DNA-protein complex occurs during myelopoiesis and alters the HAF1a protein-DNA interaction. Previously, other investigators (14Kozlowski M. Mlinaric-Rascan I. Feng G.S. Shen R. Pawson T. Siminovitch K.A. J. Exp. Med. 1993; 178: 2157-2163Crossref PubMed Scopus (212) Google Scholar, 15Tapley P. Shevde N.K. Schweitzer P.A. Gallina M. Christianson S.W. Lin I.L. Stein R.B. Shultz L.D. Rosen J. Lamb P. Exp. Hematol. 1997; 25: 122-131PubMed Google Scholar, 16Dong Q. Siminovitch K.A. Fialkow L. Fukushima T. Downey G.P. J. Immunol. 1999; 162: 3220-3230PubMed Google Scholar) demonstrated that SHP1 protein-tyrosine phosphatase (SHP1-PTP) antagonizes phagocyte differentiation and activation. In the current investigations, we demonstrate that SHP1-PTP activity decreases CYBB andNCF2 transcription, in undifferentiated myeloid cell lines. Consistent with this, SHP1-PTP decreases DNA binding of the HAF1a protein complex and tyrosine phosphorylation of IRF1 and ICSBP. We also determine that activation of the HAF1 cis elements is dependent upon tyrosine phosphorylation of conserved tyrosine residues in IRF1 and ICSBP. Therefore, these investigations identify modulation of transcription factor interaction with the CYBB andNCF2 genes as a mechanism by which SHP1-PTP regulates phagocyte differentiation and activation. These investigations also demonstrate that tyrosine phosphorylation of IRF1 and ICBP is necessary for these proteins to activate oxidase gene transcription during myeloid differentiation. CYBBpromoter sequences have been described previously (17Skalnik D.G. Strauss E.C. Orkin S.H. J. Biol. Chem. 1991; 266: 16736-16744Abstract Full Text PDF PubMed Google Scholar). NCF2genomic clone was obtained from T. Leto (National Institutes of Health, Bethesda) (18Kenney R.T. Malech H.L. Epstein N.D. Roberts R.L. Leto T.L. Blood. 1993; 82: 3739-3744Crossref PubMed Google Scholar). NCF2 reporter gene constructs were described previously (6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). CYBB and NCF2 genomic sequences were subcloned into the reporter gene vector pCATE (Promega, Madison, WI) as described (6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). Artificial promoter/reporter constructs with multiple copies of the CYBB or NCF2 HAF1 cis elements have been described previously (5Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar) using the minimal promoter/reporter vector, p-TATACAT (19Scholer H.R. Balling R. Hazopoulos A.K. Suzuki N. Gruss P. EMBO J. 1989; 8: 2551-2558Crossref PubMed Scopus (286) Google Scholar) (obtained from Dr. A. Kraft, University of Colorado, Denver, CO). The cDNA for human PU.1 was obtained from M. Klemsz (Indiana University, Indianapolis) and subcloned in to the mammalian expression vector pSRα (20Klemsz M. McKercher S.R. Celada A. Van Beveren C. Maki R.A. Cell. 1990; 61: 113-124Abstract Full Text PDF PubMed Scopus (840) Google Scholar, 21Takebe Y. Seki M. Fujisajwa J.-I. Hoy P. Yokota K. Arai K.-I. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar). The human ICSBP cDNA, obtained from B.-Z. Levi (Technicon, Haifa, Israel), and the human IRF1 cDNA, obtained from R. Pine (New York University Medical Center, New York, NY), were subcloned into the mammalian expression vectors pcDNAamp and pcDNA3.1his (Invitrogen, San Diego, CA). The pcDNA3.1his vector expresses fusion proteins with the "xpress" and 6× histidine epitope tags. The cDNAs for mutant IRF1 (mutation of tyrosine 109 to phenylalanine) and ICSBP (mutation of tyrosine 95 to phenylalanine) were generated by site-directed mutagenesis, using the CLONTECH "Quickchange" protocol. Mutant cDNAs were sequenced to verify mutagenesis and to determine that no other mutations had been introduced. Y109F IRF1 and Y95F ICSBP were also subcloned into the pcDNAamp and pcDNA3.1his vectors. The cDNAs for SHP1-PTP and CS453-SHP1-PTP (dominant negative), subcloned into the pSRα vector, were obtained from Dr. Stuart Frank (Birmingham Veterans Affairs Hospital and the Department of Medicine, University of Alabama, Birmingham) (23Somani A.K. Bignon J.S. Mills G.B. Siminovitch K.A. Branch D.R. J. Biol. Chem. 1997; 272: 21113-21122Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Oligonucleotides were synthesized by the Core Facility of the Comprehensive Cancer Center, University of Alabama, Birmingham, or by the Oligonucleotide Core Facility at the Riley Children's Hospital of Indiana, Indianapolis. The oligonucleotides used are as follows:CYBB promoter HAF1 cis element from −32 to −69 bp (cybbhaf) (2Eklund E.A. Skalnik D.G. J. Biol. Chem. 1995; 270: 8267-8273Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), 5′-ctgctgttttcatttcctcattggaagaagaagcatag-3′;NCF2 intron 1 HAF1 cis element from 160 to 190 bp 5′ of the ATG (ncf2haf) (6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar), 5′-ccaaaaggtgggacatttcctgtggatttgc-3′; CCAAT box from the α-globin gene (urccaat) (24Larrick J.W. Anderson S.J. Koren H.S. J. Immunol. 1980; 125: 6-14Crossref PubMed Google Scholar), 5′-ccgggctccgcgccagccaatgagcgccgcgg-3′. All cell lines were of human origin. The myelomonocytic cell line U937 (24Larrick J.W. Anderson S.J. Koren H.S. J. Immunol. 1980; 125: 6-14Crossref PubMed Google Scholar) was obtained from Andrew Kraft (University of Colorado, Denver, CO). The promyelocytic cell line PLB985 (25Tucker K.A. Lilly M.B. Heck L. Rado T.A. Blood. 1987; 71: 372-378Crossref Google Scholar) was obtained from Tom Rado (University of Alabama, Birmingham). Cell lines were maintained and differentiated as described (6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). U937 cells were treated with 200 or 1,000 units per ml human recombinant IFNγ (Roche Molecular Biochemicals). Nuclear extract proteins were prepared by the method of Dignamet al. (26Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1993; 11: 1475-1479Crossref Scopus (9815) Google Scholar) with protease and inhibitors, as described previously (6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). In some experiments, protein-tyrosine phosphatase inhibitor was added to the buffers, as described (0.1 μmNaVO3) (5Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). Oligonucleotides probes were prepared, and EMSA and antibody supershift assays were performed, as described (5Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). EMSA were performed as previously described previously (5Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In some experiments, the nuclear proteins were preincubated with Yopprotein-tyrosine phosphatase or purified SHP1 protein for 30 min at 30 °C, as described by the manufacturer's instructions (New England Biolabs, Worchester, MA). Control samples were similarly incubated in the absence of protein-tyrosine phosphatase. In other experiments, EMSA binding reactions were preincubated for 4 h on ice with anti-xpress and anti-His6 antibodies prior to addition of radiolabeled probe (0.5 μg each, Santa Cruz Biotechnology, Santa Cruz, CA). To generate stable transfectant pools, U937 cells (32 × 106) were transfected with 50 μg of plasmid: SHP1/pSRα, CS453-SHP1/pSRα, or control pSRα; IRF1/pcDNA3.1his, Y109F IRF1/pcDNA3.1his, ICSBP/pcDNA3.1his, Y95F ICSBP/pcDNA3.1his, or control pcDNA3.1his. Cells were incubated in Dulbecco's modified Eagle's medium, 10% fetal calf serum, 1% penicillin/streptomycin, 1 mg/ml geneticin (G418) to select for stable transfectants. Expression of the various proteins was verified by Western blots of cell lysates, according to standard procedures, with commercially available antibodies. Cells were transfected by electroporation, as described (5Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Stable U937 transfectants (with SHP1/pSRα, CS453-SHP1/pSRα, or pSRα) were co-transfected with 50 μg of pCATE constructs and 15 μg p-CMV/β-galactosidase (CLONTECH, Palo Alto, CA). In other experiments, U937 cells were transfected with 70 μg of p-TATACAT or p-cybbhafTATACAT; 30 μg of pSRα or PU.1/pSRα; 30 μg of IRF1/pcDNAamp, Y109F IRF1/pcDNAamp, ICSBP/pcDNAamp, Y95F ICSBP/pcDNAamp, or control pcDNAamp; or 15 μg each of various combinations of IRF1/pcDNAamp or Y109F IRF1/pcDNAamp with ICSBP/pcDNAamp or Y95F ICSBP/pcDNAamp; and 15 μg of p-CMV/β-galactosidase. Transfectants were incubated for 24 h at 37 °C, 5% CO2, followed by 24 h with or without IFNγ (1,000 units/ml). Preparation of cell extracts, β-galactosidase, and chloramphenicol acetyltransferase assays (CAT) assays were as described (27Seed B. Sheen J.-Y. Gene (Amst.). 1988; 67: 271-275Crossref PubMed Scopus (895) Google Scholar, 28Sambrook H. Fritch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Total cellular RNA was extracted (29Chomszynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159PubMed Google Scholar) from U937 cells with or without 48 h of IFNγ treatment (200 units/ml). Northern blots were performed with 10 or 20 μg of RNA, as described (30Ausubel F.M. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1998: 2.15-2.18Google Scholar). For analysis of RNA stability, U937 cells were treated with actinomycin D (10 μg/ml). Cells were harvested at various times, and RNA was extracted for Northern blot. Autoradiographs of Northern blots were analyzed by densitometry to determine mRNA abundance at various times after actinomycin D treatment. Results were normalized to mRNA abundance at t = 0. U937 stable transfectants with SHP1, CS453-SHP1, or control vector were lysed in RIPA buffer, as described (in the presence of SDS) (6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). Cell lysates (200 μg) were immunoprecipitated for 4 h at 4 °C with anti-SHP1 antibody (2.0 μg, Santa Cruz Biotechnology, Santa Cruz, CA) or irrelevant control antibody (anti-mouse IgG), and immunoprecipitates were collected with staphylococcus protein A-Sepharose, as described previously (6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). For PTP assays, staphylococcus protein A-Sepharose beads were washed extensively in RIPA buffer (with no SDS) and resuspended in Tyr(P) assay buffer (25 mm HEPES, pH 7.2, 50 mm NaCl, 5 mmdithiothreitol, 2.5 mm EDTA). Phosphopeptide RRLIEDAEpYAARG (where pY is Tyr(P)) (2 nm) was added, and the samples were incubated for 15 min at room temperature. Assays were performed according to Harder et al. (31Harder K.W. Owen P. Wong L.K. Aebersold R. Clark-Lewis I. Jirik F.R. Biochem. J. 1994; 298: 395-401Crossref PubMed Scopus (187) Google Scholar) using a commercially available kit (PTP Assay Kit 1, Upstate Biotechnology, Inc., Lake Placid, NY). In other experiments, immunoprecipitated SHP1 (or control immunoprecipitate) was eluted with pH 4.0 buffer, neutralized with 1m Tris, pH 9.5 (as per the manufacturer, Amersham Pharmacia Biotech), and incubated with nuclear proteins or in vitrotranslated proteins. Immunoprecipitation experiments were performed with 200 μg of nuclear proteins extracted from U937 cells, with or without 48 h IFNγ incubation. Nuclear proteins were diluted into RIPA buffer, with protease and phosphatase inhibitors, as described previously (6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar), and incubated with either 1 μl of anti-phosphotyrosine antibody (4G10, Upstate Biotechnology Inc., Lake Placid, NY), anti-ICSBP antibody (goat anti-human ICSBP, Santa Cruz Biotechnology), anti-IRF1 antibody (mouse anti-human IRF1, Santa Cruz Biotechnology), or irrelevant antibody (mouse anti-rabbit IgG or goat anti-rabbit IgG, Santa Cruz Biotechnology) for 4 h at 4 °C, followed by 1 h of incubation with 15 μl of 50% staphylococcus protein A-Sepharose bead slurry. In some experiments, immunoprecipitations were performed in the presence of SDS to prevent co-immunoprecipitation of other proteins. Beads were washed with RIPA buffer (no SDS), proteins eluted in SDS sample buffer, separated on 12% SDS-PAGE, and transferred to nitrocellulose. Blots were probed with antibodies as indicated under "Results," and proteins were detected by chemiluminescence according to the manufacturer's instructions (Amersham Pharmacia Biotech). Immunoprecipitation of in vitro translated proteins was similarly performed. In vitro transcription and translation of ICSBP, IRF-1, Y95F ICSBP, and Y109F IRF1 (sub-cloned into pcDNAamp) were performed using the Promega In Vitro Transcription System and rabbit reticulocyte lysate In Vitro Translation System, according to the manufacturer's instructions (Promega, Madison, WI). JM109 Escherichia coli cells transformed with PU.1/pGEX2, IRF1/pGEX1, or control pGEX2 were grown to log phase, supplemented to 0.1 mmisopropyl-1-thio-β-d-galactopyranoside, and incubated for 3 h at 37 °C with shaking. The cells were harvested and resuspended in HN buffer (20 mm HEPES, pH 7.4, 0.1m NaCl, 2 mm MgCl2, 0.1 mm EDTA, 0.5% Nonidet P-40, 0.1% Triton X-100, 2 mm phenylmethylsulfonyl fluoride, 5 mm NaF), and sonicated on ice. Debris was removed by centrifugation, and the lysate was incubated 30 min at 4 °C with glutathione-agarose beads (Sigma) and washed extensively with HN buffer. The beads were preincubated for 30 min at 4 °C with 5 μl of control rabbit reticulocyte lysate and then for 1 h with 20 μl of [35S]methionine-labeled and in vitrotranslated protein and washed extensively in HN buffer. Proteins were eluted with SDS-PAGE sample buffer and separated on 12% SDS-PAGE, and an autoradiograph was performed. Previously, we determined that lineage and differentiation stage-specificCYBB and NCF2 transcription requires intact HAF1 cis elements in the two genes (2Eklund E.A. Skalnik D.G. J. Biol. Chem. 1995; 270: 8267-8273Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). We also determined that overexpression of PU.1, IRF1, ICSBP, and CBP activates artificial promoter constructs with multiple copies of the CYBB orNCF2 HAF1 elements in myeloid cell line transfectants (6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). Additionally, we found that this interaction is increased by IFNγ-induced differentiation of the transfectants. Consistent with the functional data, PU.1, IRF1, ICSBP, and CBP bind to theCYBB and NCF2 cis elements in EMSA (5Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). Three protein complexes bind in vitro to the CYBB andNCF2 HAF1 cis elements as follows: PU.1 monomer; PU.1 heterodimer with IRF1 or ICSBP (the "HAF1" complex); and a multiprotein complex that includes PU.1, IRF1, ICSBP, and CBP (the "HAF1a" complex) (see Fig. 1) (5Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). Since IRF1 and ICSBP binding to the HAF1 cis elements requires PU.1 (6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar), and since ICSBP and IRF1 are able to interact with each other (32Sharf R. Meraro D. Azreil A. Thornton A.M. Ozato K. Petricoin E.F. Larner A.C. Schaper R. Hauser H. Levi B.-Z. J. Biol. Chem. 1997; 272: 9785-9792Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), three of the following interactions are possible: PU.1 interacts directly with IRF1 and IRF1 binds ICSBP; PU.1 interacts directly with ICSBP and ICSBP binds IRF1; or PU.1 interacts simultaneously with IRF1 and ICSBP. We found that DNA binding of the three proteins creates a binding site for CBP (6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). In our previous investigations (5Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 6Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar), IFNγ differentiation of myeloid cell lines did not increase in vitro binding of either PU.1, the HAF1 complex, or the HAF1a complex to theCYBB and NCF2 cis elements. Therefore, our transfection data indicate that the HAF1 cis elements are necessary forCYBB and NCF2 transcription during IFNγ-induced differentiation in myeloid cell lines. In contrast, our binding assays indicate that in vitro protein interaction with the HAF1 elements is not altered by IFNγ-induced differentiation of myeloid cell lines. To resolve this discrepancy, we re-evaluated the conditions under which we were analyzing in vitro DNA-protein interactions. We first noted the HAF1a complex in EMSA with the CYBB HAF1 cis element and myeloid nuclear proteins that were isolated in the presence of protein phosphatase inhibitors (sodium orthovanadate and sodium fluoride) (5Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In these previous experiments, phosphatase inhibitors were added to all buffers used for nuclear protein isolation, including a 10-min incubation in pre-lysis buffer (according to Dignam et al. (26Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1993; 11: 1475-1479Crossref Scopus (9815) Google Scholar)). Therefore, our previous investigations assumed that protein phosphatase activity is equivalent in IFNγ-differentiated and -undifferentiated myeloid cell lines. If IFNγ treatment decreases net protein phosphatase activity, this assumption would be incorrect. In that case, treatment of the cells with protein phosphatase inhibitors might obscure IFNγ-induced alterations in protein phosphorylation and therefore IFNγ-induced alteration in protein-protein or protein-DNA interactions. To investigate this, we determined whether protein-tyrosine phosphatase (PTP) activity influen

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