Activation of SHP2 Protein-tyrosine Phosphatase Increases HoxA10-induced Repression of the Genes Encoding gp91PHOX and p67PHOX
2006; Elsevier BV; Volume: 282; Issue: 4 Linguagem: Inglês
10.1074/jbc.m608642200
ISSN1083-351X
AutoresStephan Lindsey, Weiqi Huang, Hao Wang, Elizabeth Horvath, Chunliu Zhu, Elizabeth A. Eklund,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoThe CYBB and NCF2 genes encode the phagocyte oxidase proteins gp91PHOX and p67PHOX, respectively. These genes are transcribed after the promyelocyte stage of differentiation, and transcription continues until cell death. In undifferentiated myeloid cells, homologous cis-elements in the CYBB and NCF2 genes are repressed by the homeodomain transcription factor HoxA10. During cytokine-induced myelopoiesis, tyrosine phosphorylation of HoxA10 decreases binding affinity for the CYBB and NCF2 cis-elements. This abrogates HoxA10-induced transcriptional repression as differentiation proceeds. Therefore, mechanisms involved in differentiation stage-specific HoxA10 tyrosine phosphorylation are of interest because HoxA10 phosphorylation modulates myeloid-specific gene transcription. In this study, we found that HoxA10 is a substrate for SHP2 protein-tyrosine phosphatase in undifferentiated myeloid cells. In contrast, HoxA10 is a substrate for a constitutively active mutant form of SHP2 in both undifferentiated and differentiating myeloid cells. Expression of such SHP2 mutants results in persistent HoxA10 repression of CYBB and NCF2 transcription during myelopoiesis. Both HoxA10 overexpression and activating SHP2 mutations have been described in human myeloid malignancies. Therefore, our results suggest that these mutations could cooperate, leading to decreased myeloid-specific gene transcription and functional differentiation block in myeloid cells with both defects. The CYBB and NCF2 genes encode the phagocyte oxidase proteins gp91PHOX and p67PHOX, respectively. These genes are transcribed after the promyelocyte stage of differentiation, and transcription continues until cell death. In undifferentiated myeloid cells, homologous cis-elements in the CYBB and NCF2 genes are repressed by the homeodomain transcription factor HoxA10. During cytokine-induced myelopoiesis, tyrosine phosphorylation of HoxA10 decreases binding affinity for the CYBB and NCF2 cis-elements. This abrogates HoxA10-induced transcriptional repression as differentiation proceeds. Therefore, mechanisms involved in differentiation stage-specific HoxA10 tyrosine phosphorylation are of interest because HoxA10 phosphorylation modulates myeloid-specific gene transcription. In this study, we found that HoxA10 is a substrate for SHP2 protein-tyrosine phosphatase in undifferentiated myeloid cells. In contrast, HoxA10 is a substrate for a constitutively active mutant form of SHP2 in both undifferentiated and differentiating myeloid cells. Expression of such SHP2 mutants results in persistent HoxA10 repression of CYBB and NCF2 transcription during myelopoiesis. Both HoxA10 overexpression and activating SHP2 mutations have been described in human myeloid malignancies. Therefore, our results suggest that these mutations could cooperate, leading to decreased myeloid-specific gene transcription and functional differentiation block in myeloid cells with both defects. The murine and human HOX genes are divided into four paralog groups (A–D) on four different chromosomes. These genes encode highly conserved homeodomain transcription factors that are expressed cephlad to caudad during embryogenesis (1Eklund E.A. Curr. Opin. Hematol. 2006; 13: 67-73Crossref PubMed Scopus (26) Google Scholar). Additionally, HOX gene transcription is differentiation stage-specific during definitive hematopoiesis (2Acampora D. D'Esposito M. Faiella A. Pannese M. Migliaccio E. Morelli F. Stornaiuolo A. Nitro V. Simeone A. Boncinelli A. Nucleic Acids Res. 1989; 17: 10385-10400Crossref PubMed Scopus (281) Google Scholar). For example, the HOX1–4 genes are maximally expressed in hematopoietic stem cells (3Sauvageau G. Lansdorp P.M. Eaves C.J. Hogge D.E. Dragowska W.H. Reid D.S. Largman C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12223-12227Crossref PubMed Scopus (441) Google Scholar). In contrast, HoxA5–13 (also referred to as the ABD HOXA genes) are maximally expressed in committed myeloid progenitors (3Sauvageau G. Lansdorp P.M. Eaves C.J. Hogge D.E. Dragowska W.H. Reid D.S. Largman C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12223-12227Crossref PubMed Scopus (441) Google Scholar). Differentiation stage-specific expression suggests that Hox proteins may regulate progression of myelopoiesis. Indeed, a number of studies indicate the importance of HoxA proteins for normal myeloid development (5Kasper L.H. Brindle P.K. Schnabel C.A. Pritchard C.E. Cleary M.L. Van Deursen J.M. Mol. Cell. Biol. 1999; 19: 764-776Crossref PubMed Scopus (267) Google Scholar, 6Kroon E. Krosl J. Thorsteindottir U. Banban S. Buchberg A.M. Sauvageau G. EMBO J. 1998; 17: 3714-3725Crossref PubMed Scopus (551) Google Scholar, 7Thorsteindottir U. Sauvageau G. Hough M.R. Dragowska W. 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This result suggests possible functional redundancy between these proteins. In contrast, overexpression of various Abd HoxA proteins results in significant abnormalities in murine bone marrow transplantation experiments. For example, mice transplanted with HoxA10-overexpressing bone marrow rapidly develop leukocytosis, characterized by circulating mature phagocytes (7Thorsteindottir U. Sauvageau G. Hough M.R. Dragowska W. Lansdorp P.M. Lawrence H.J. Largman C. Humphries R.K. Mol. Cell. Biol. 1997; 17: 495-505Crossref PubMed Scopus (296) Google Scholar). This myeloproliferative disorder evolves to clonal acute myeloid leukemia (AML) 2The abbreviations used are: AML, acute myeloid leukemia; PTP, proteintyrosine phosphatase; GST, glutathione S-transferase; EMSAs, electrophoretic mobility shift assays; IFN-γ, interferon-γ; GM-CSF, granulocytemacrophage colony-stimulating factor; IL-3, interleukin-3; M-CSF, macrophage colony-stimulating factor; NBT, nitro blue tetrazolium; PMA, phorbol 12-myristate 13-acetate; ICSBP, interferon consensus sequence-binding protein. over several months. Therefore, HoxA10 overexpression does not induce differentiation block and therefore is not adequate to induce AML. In this model, the time lag between myeloproliferation and AML is hypothesized to permit accumulation of additional genetic mutations; however, such cooperating mutations have not been identified. A study of bone marrow samples from human subjects with AML also suggests a role for HoxA10 overexpression in leukemogenesis (1Eklund E.A. Curr. Opin. Hematol. 2006; 13: 67-73Crossref PubMed Scopus (26) Google Scholar). Expression of the entire group of Abd HoxA proteins has been demonstrated in human AML bone marrow samples from subjects with translocations involving the MLL (mixed lineage leukemia) gene (8Kawagoe H. Humphries R.K. Blair A. Sutherland H.J. Hogge D.E. Leukemia (Basingstoke). 1999; 13: 687-698Crossref PubMed Scopus (183) Google Scholar). In murine transplantation experiments, expression of such leukemia-associated MLL fusion proteins induces myeloproliferation, which progresses to blast crisis over several months (14Corral J. Lavenir I. Impey H. Warren A.J. Forster A. Larson T.A. Bell S. McKinzie A.N. King G. Rabbitts T.H. Cell. 1996; 85: 853-861Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 15DiMartino J.F. Miller T. Ayton P.M. Landewe T. Hess J.L. Cleary M.L. Shilatifard A. Blood. 2000; 96: 3887-3893Crossref PubMed Google Scholar, 16DiMartino J.F. Ayton P.M. Chen E.H. Naftzger C.C. Young B.D. Cleary M.L. Blood. 2002; 99: 3780-3785Crossref PubMed Scopus (113) Google Scholar, 17Aynton P.M. Cleary M.L. Genes Dev. 2003; 17: 2298-2307Crossref PubMed Scopus (377) Google Scholar, 18So C.W. Karsunky H. Wong P. Weissman I.L. Cleary M.L. Blood. 2004; 103: 3192-3199Crossref PubMed Scopus (133) Google Scholar, 19Kumar A.R. Hudson W.A. Chen W. Nishiuchi R. Yao A. Kersey J.H. Blood. 2004; 103: 1823-1828Crossref PubMed Scopus (111) Google Scholar). Although such studies suggest that Abd HoxA proteins play important roles in normal and malignant hematopoiesis, relatively few genuine HoxA target genes have been identified. In previous studies, we found that HoxA10 represses transcription of the genes encoding two phagocyte respiratory burst oxidase proteins: gp91PHOX and p67PHOX (the CYBB and NCF2 genes, respectively) (20Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 21Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar). These genes are actively transcribed after the promyelocyte stage of myelopoiesis, and transcription continues until cell death (22Royer-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 (638) Google Scholar, 23Leto T.L. Lomax K.J. Volpp B.D. Nunio H. Sechler J.M. Nauseef W.M. Clark R.A. Gallin J.I. Malech H.L. Science. 1990; 248: 727-730Crossref PubMed Scopus (331) Google Scholar). We found that CYBB and NCF2 transcription is repressed in undifferentiated myeloid cells by interaction of HoxA10, Pbx1, and HDAC2 with homologous cis-elements in these genes (24Lu Y. Goldenberg I. Bei L. Andrejic J. Eklund E.A. J. Biol. Chem. 2003; 278: 47792-47802Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Pbx1 is dispensable for repression activity, which involves direct interaction of HDAC2 with a novel HoxA10 repression domain. Pbx1 is hypothesized to increase the DNA binding affinity of Hox proteins in such circumstances (25Chang C.P. Brocchieri L. Shen W.F. Largman C. Cleary M.L. Mol. Cell. Biol. 1996; 16: 1734-1745Crossref PubMed Scopus (251) Google Scholar). Additional studies suggest that HoxA10 also represses transcription of the genes encoding lactoferrin and neutrophil collagenase in undifferentiated myeloid cells (26Khanna-Gupta A. Zibello T. Kolla S. Neufeld E.J. Berliner N. Blood. 1997; 90: 2784-2795Crossref PubMed Google Scholar, 27Lawson N.D. Khanna-Gupta A. Berliner N. Blood. 1998; 91: 2517-2524Crossref PubMed Google Scholar). Therefore, HoxA10 represses transcription of multiple genes that confer the mature myeloid phenotype. HoxA10 binding to the CYBB and NCF2 cis-elements decreases during myeloid differentiation in vitro and in vivo (20Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 21Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar). However, HoxA10 protein abundance is relatively constant during differentiation of committed myeloid progenitor cells. In a previous study, we found that HoxA10 is tyrosine-phosphorylated during cytokine-induced myelopoiesis (28Eklund E.A. Goldenberg I. Lu Y. Andrejic J. Kakar R. J. Biol. Chem. 2002; 277: 36878-36888Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). We also found that phosphorylation of two tyrosine residues in the HoxA10 homeodomain decreases binding affinity for the CYBB and NCF2 promoters (21Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar, 28Eklund E.A. Goldenberg I. Lu Y. Andrejic J. Kakar R. J. Biol. Chem. 2002; 277: 36878-36888Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Therefore, cytokine-induced post-translational modification regulates HoxA10 repression of myeloid-specific gene transcription. These results have implications for the role of HoxA10 in leukemogenesis. Specifically, HoxA10 overexpression might be expected to block transcription of genes conferring the mature myeloid phenotype, resulting in differentiation block and blast crisis. However, overexpressed HoxA10 does not repress transcription of such genes because it is tyrosine-phosphorylated in response to hematopoietic cytokines (20Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 21Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar, 28Eklund E.A. Goldenberg I. Lu Y. Andrejic J. Kakar R. J. Biol. Chem. 2002; 277: 36878-36888Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Therefore, differentiation block by overexpressed HoxA10 requires inhibition of cytokine-induced tyrosine phosphorylation. This could be due to decreased kinase activity or increased phosphatase activity. The time to acquire such genetic lesions might explain the lag between HoxA10-induced myeloproliferation and development of AML in the murine model. Although the HoxA10 kinase has not been identified, we found that Jak2 activation is sufficient for HoxA10 tyrosine phosphorylation (29Kakar R. Kautz B. Eklund E.A. J. Leukocyte Biol. 2005; 77: 120-127Crossref PubMed Scopus (30) Google Scholar). Additionally, we determined previously that SHP1 protein-tyrosine phosphatase (PTP) is involved in maintaining HoxA10 in a non-tyrosine-phosphorylated state in immature myeloid cells (28Eklund E.A. Goldenberg I. Lu Y. Andrejic J. Kakar R. J. Biol. Chem. 2002; 277: 36878-36888Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). During cytokine-induced differentiation, SHP1 PTP activity decreases, increasing HoxA10 tyrosine phosphorylation. However, leukemia-associated activating mutants of SHP1 have not been identified. In contrast, activating mutations in the closely related SHP2 PTP have been identified in samples from human subjects with myelodysplastic syndromes, AML, or juvenile chronic myelomonocytic leukemia (30Tartaglia M. Niemeyer C.M. Fragale A. Song X. Buechner J. Jung A. Hahlen K. Hasle H. Licht J.D. Gelb B.D. Nat. Genet. 2003; 34: 148-150Crossref PubMed Scopus (847) Google Scholar, 31Loh M.L. Vattikuti S. Schubbert S. Reynolds M.G. Carlson E. Lieuw K.H. Cheng J.W. Lee C.M. Stokoe D. Bonifas J.M. Curtiss N.P. Gotlib J. Meshinchi S. Le Beau M.M. Emanuel P.D. Shannon K.M. Blood. 2004; 103: 2325-2331Crossref PubMed Scopus (357) Google Scholar). In the resting state, the catalytic domain in SHP2 is masked. Activation occurs when phosphotyrosine residues in a substrate or another protein interact with an SH2 (Src homology 2) domain in SHP2, resulting in a conformational change. Previously described leukemia-associated mutations induce a conformational change that unmasks the catalytic domain, resulting in constitutive activation (32O'Reilly A.M. Pluskey S. Shoelson S.E. Neel B.G. Mol. Cell. Biol. 2000; 20: 299-311Crossref PubMed Scopus (101) Google Scholar). Mice transplanted with bone marrow expressing such SHP2 PTP mutants develop a myeloproliferative disorder, but not AML (33Mohi M.G. Williams I.R. Dearolf C.R. Chan G. Kutok J.L. Cohen S. Morgan K. Boulton C. Shigematsu H. Keilhack H. Akashi K. Gilliland D.G. Neel B.G. Cancer Cell. 2005; 7: 179-191Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 34Schubbert S. Lieuw K. Rowe S.L. Lee C.M. Li X. Loh M.L. Clapp D.W. Shannon K.M. Blood. 2005; 106: 311-317Crossref PubMed Scopus (129) Google Scholar). In this study, we investigated whether HoxA10 is a substrate for leukemia-associated activating SHP2 mutants in differentiating myeloid cells. We also investigated the impact of expressing such SHP2 mutants on repression of CYBB and NCF2 transcription by overexpressed HoxA10 as a model for disease progression in myeloid malignancy. The goal of this study was to determine whether HoxA10 overexpression and activating SHP2 PTP mutants might be cooperating genetic defects in the multistep process leading from myeloproliferation to acute myeloid leukemia. Protein Expression Vectors—The human HoxA10 cDNA was obtained from Dr. C. Largman (University of California, San Francisco) (35Lowney P. Corral J. Detmer K. Le Beau M.M. Deaven L. Lawrence H.J. Largman C. Nucleic Acids Res. 1991; 19: 3443-3449Crossref PubMed Scopus (69) Google Scholar). This cDNA sequence represents the major transcript in mammalian hematopoietic cells, encoding a 393-amino acid 55-kDa protein. HoxA10 cDNA sequence was subcloned into the pcDNAamp vector for in vitro translation, pSRα for expression in mammalian cells (36Takebe Y. Seiki M. Fujisajwa J. Hoy P. Yokota K. Arai K. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar), and pMSCV (Stratagene, La Jolla, CA) for expression in murine bone marrow cells. The cDNA for SHP2 PTP was obtained from Dr. Stuart Frank (University of Alabama, Birmingham, AL). A leukemia-associated activating SHP2 mutant (E76K) was generated by site-directed mutagenesis using the Stratagene QuikChange protocol as described (37Huang W.Q. Saberwal G. Horvath E. Lindsey S. Eklund E.A. Mol. Cell. Biol. 2006; 26: 6311-6332Crossref PubMed Scopus (37) Google Scholar). Mutant clones were sequenced on both strands to verify that only the intended mutations had been introduced. Wild-type and E76K SHP2 were subcloned into the pMSCVneo vector (Stratagene) for retroviral production. These cDNAs were also subcloned into the pcDNAamp vector for in vitro protein translation. Wild-type SHP1 and SHP2 were subcloned into the pGEX1 vector (Amersham Biosciences) for expression in Escherichia coli as a fusion protein with glutathione S-transferase (GST). Reporter Constructs—Artificial promoter-reporter constructs were generated in the minimal promoter-reporter vector pTATACAT (obtained from Dr. Andrew Kraft, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC) as described previously (38Scholer H.R. Balling R. Hazopoulos A.K. Suzuki N. Gruss P. EMBO J. 1989; 8: 2551-2558Crossref PubMed Scopus (281) Google Scholar). A construct was generated with four copies of the negative cis-elements repressed by HoxA10 from the CYBB (–94 to –134 bp) or NCF2 (–600 to –637 bp) promoter subcloned into this vector in the forward direction (referred to as p-cybbTATACAT and p-ncf2TATACAT, respectively) (20Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 21Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar). Oligonucleotides were synthesized by the Core Facility of the Robert H. Lurie Comprehensive Cancer Center at Northwestern University as follows: dscybbA10 (the Hox/Pbx-binding sequence from the CYBB promoter, –94 to –134 bp), 5′-ttcagttgaccaatgattattagccaattttctgataaaa-3′, and dsncf2A10 (a homologous sequence from the NCF2 promoter, –600 to –637 bp). In these oligonucleotides, the HoxA10 core is in boldface, the Pbx core is in italics, and ccaat boxes are underlined. Complementary single-stranded oligonucleotides were annealed and used in electrophoretic mobility shift assays (EMSAs) or subcloned to generate artificial promoter constructs as described (20Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 21Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar). The human myelomonocytic cell line U937 (39Larrick J.W. Anderson S.J. Koren H.S. J. Immunol. 1980; 125: 6-14Crossref PubMed Google Scholar) was obtained from Dr. Andrew Kraft. Cells were maintained and differentiated as described (20Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 21Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar, 28Eklund E.A. Goldenberg I. Lu Y. Andrejic J. Kakar R. J. Biol. Chem. 2002; 277: 36878-36888Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). For differentiation experiments, U937 cells were treated for 48 h with 500 units/ml human recombinant interferon-γ (IFN-γ) (Roche Applied Science) (20Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Transfections for Promoter Analysis—U937 cells were cultured and transfected as described previously (20Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 21Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar). Cells (32 × 106/sample) were transfected with vector expressing HoxA10 (pSRα/HoxA10) or empty control vector; wild-type SHP2 (pSX/SHP2), SHP2 with a leukemia-associated activating mutation (pSX/E76K SHP2), a dominant-negative form of SHP2 (pSX/C463S SHP2), or empty control vector; the minimal promoter-reporter vector pTATACAT with four copies of the CYBB –94 to –134 bp sequence (p-cybbTATACAT), four copies of the NCF2 –600 to –637 bp sequence (p-ncf2TATACAT), or the empty pTATACAT control vector; and pCMV-β-galactosidase (to control for transfection efficiency). Transfectants were harvested 48 h after transfection with and without incubation with human recombinant IFN-γ (500 units/ml). Lysates were analyzed for chloramphenicol acetyltransferase and β-galactosidase activities as described (20Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Transfections for Stable Pools Overexpressing SHP2—U937 cells were transfected with empty expression vector or vector overexpressing wild-type or E76K SHP2 as described above. After 24 h, the medium was supplemented with G418 to 1 mg/ml, and stable transfectant pools were selected over 7–14 days as described previously (20Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Stable transfectant pools were selected instead of clones to compensate for possible integration site effects. All experiments were repeated with at least two independent transfectant pools for each construct. Bone marrow mononuclear cells were obtained from the femurs of wild-type mice. Bipotential myeloid progenitor cells were cultured (at a concentration of 2 × 105 cells/ml) for 48 h in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, 10 ng/ml murine granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D Systems, Minneapolis, MN), and 5 ng/ml murine recombinant IL-3 (R&D Systems). After retroviral transduction (see below), cells were either maintained in GM-CSF and interleukin-3 (IL-3; myeloid progenitor cells) or switched to Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, and 10 ng/ml murine recombinant macrophage colony-stimulating factor (M-CSF; R&D Systems) for 96 h (monocyte differentiation). Cells were harvested, and cell lysates were used in Western blot experiments as described below. High titer murine stem cell retroviral supernatants were produced using the pMSCVneo vector and the PT67 cell line following the instructions of Stratagene. Filtered retroviral supernatants were used immediately or stored at –80 °C. Transductions of murine bone marrow myeloid progenitor cells were performed as described previously (37Huang W.Q. Saberwal G. Horvath E. Lindsey S. Eklund E.A. Mol. Cell. Biol. 2006; 26: 6311-6332Crossref PubMed Scopus (37) Google Scholar). Briefly, cells were harvested, and 4.0 × 106 cells were plated in 3 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 10 ng/ml GM-CSF, and 5 ng/ml IL-3. An equal volume of retroviral supernatant was added to each dish, and Polybrene was added to a final concentration of 6 μg/ml. Cells were incubated for 8 h at 37°C and 5% CO2 and then diluted 3-fold with medium supplemented as described above. Cells were incubated overnight, and the procedure was repeated the next day. The day after transduction, G418 was added to 250 ng/ml. Cells were selected in antibiotics for 48 h and then treated with cytokines as indicated. Each experiment was repeated at least three times. Expression of transduced proteins was independently verified by Western blotting for each experiment. Nuclear extract proteins were isolated from U937 cells by the method of Dignam et al. (40Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1993; 11: 1475-1479Crossref Scopus (9586) Google Scholar) with protease inhibitors as described (41Kautz B. Kakar R. David E. Eklund E.A. J. Biol. Chem. 2001; 276: 37868-37878Abstract Full Text Full Text PDF PubMed Google Scholar). In some experiments, U937 cells were differentiated with 500 units/ml IFN-γ before nuclear protein isolation. Oligonucleotide probes were prepared, and EMSAs and antibody supershift assays were performed as described (20Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 21Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar). Rabbit anti-HoxA10 polyclonal antibody was generated against a unique peptide (Covance, Inc.) and has been described previously (20Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 21Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar, 28Eklund E.A. Goldenberg I. Lu Y. Andrejic J. Kakar R. J. Biol. Chem. 2002; 277: 36878-36888Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Irrelevant control anti-GST antibody was obtained from Santa Cruz Biotechnology, Inc. U937 cells were cultured with or without IFN-γ for 48 h as described above. Cells for chromatin immunoprecipitation were incubated with formaldehyde prior to lysis, and lysates were sonicated to generate chromatin fragments with an average size of 2.0 kb as described (42Zhu C. Saberwal G. Ly Y.F. Platanias L.C. Eklund E.A. J. Biol. Chem. 2004; 279: 50874-50885Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Lysates underwent immunoprecipitation with either antiserum to HoxA10 or preimmune serum as described (42Zhu C. Saberwal G. Ly Y.F. Platanias L.C. Eklund E.A. J. Biol. Chem. 2004; 279: 50874-50885Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Coprecipitated chromatin was analyzed by PCR for HoxA10-specific antibody coprecipitation of the CYBB and NCF2 gene promoters. For these experiments, input chromatin was used as a positive control, and chromatin precipitated by preimmune serum was used as a negative control. PCR products were analyzed by acrylamide gel electrophoresis. The identity of the PCR product was verified by subcloning into a plasmid vector, followed by dideoxy sequencing. Western Blotting of Lysate Proteins from Murine Bone Marrow Cells—Murine bone marrow cells were lysed by boiling in 2× SDS sample buffer. Lysate proteins (50 μg) were separated by SDS-PAGE and transferred to nitrocellulose according to standard techniques. Western blots were serially probed with antibodies to gp91PHOX, p67PHOX, SHP2, HoxA10, and glyceraldehyde-3-phosphate dehydrogenase (to control for loading). Anti-phosphotyrosine Immunoprecipitation and Western Blotting—U937 cells were lysed, and lysates were immunoprecipitated under denaturing conditions with HoxA10 antiserum or preimmune serum control. Murine bone marrow-derived myeloid cells were lysed and immunoprecipitated under denaturing conditions with antibody to phosphotyrosine (clone 4G10, Upstate, Charlottesville, VA) or irrelevant control antibody (anti-GST tag) as described previously (28Eklund E.A. Goldenberg I. Lu Y. Andrejic J. Kakar R. J. Biol. Chem. 2002; 277: 36878-36888Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Precipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose as described above. Western blots of U937 proteins were serially probed with antibodies to phosphotyrosine and HoxA10. Blots of murine myeloid cell proteins were probed with HoxA10-specific antibody. In vitro transcribed HoxA10 and E76K SHP2 mRNAs were generated from linearized template DNA using the Riboprobe system (Promega Corp.) according to the manufacturer's instructions. In vitro translated proteins were generated in rabbit reticulocyte lysate (Promega Corp.) according to the manufacturer's instructions. Control (unprogrammed) lysates were generated in similar reactions in the absence of input RNA. For these experiments, HoxA10 (but not E76K SHP2) was radiolabeled by including [35S]methionine in the translation reaction. For dephosphorylation assays, in vitro translated HoxA10 was incubated for 30 min at 30 °C with either in vitro translated E76K SHP2 or control lysates. The HoxA10 tyrosine phosphorylation state was determined by immunoprecipitating the reaction with anti-phosphotyrosine or irrelevant control antibody under denaturing conditions as described previously (28Eklund E.A. Golden
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