c-Jun Is a JNK-independent Coactivator of the PU.1 Transcription Factor
1999; Elsevier BV; Volume: 274; Issue: 8 Linguagem: Inglês
10.1074/jbc.274.8.4939
ISSN1083-351X
AutoresGerhard Behre, Alan J. Whitmarsh, Matthew P. Coghlan, Trang Hoang, Christopher L. Carpenter, Dong‐Er Zhang, Roger J. Davis, Daniel G. Tenen,
Tópico(s)Acute Myeloid Leukemia Research
ResumoThe ETS domain transcription factor PU.1 is necessary for the development of monocytes and regulates, in particular, the expression of the monocyte-specific macrophage colony-stimulating factor (M-CSF) receptor, which is critical for monocytic cell survival, proliferation, and differentiation. The bZIP transcription factor c-Jun, which is part of the AP-1 transcription factor complex, is also important for monocytic differentiation, but the monocyte-specific M-CSF receptor promoter has no AP-1 consensus binding sites. We asked the question of whether c-Jun could promote the induction of the M-CSF receptor by collaborating with PU.1. We demonstrate that c-Jun enhances the ability of PU.1 to transactivate the M-CSF receptor promoter as well as a minimal thymidine kinase promoter containing only PU.1 DNA binding sites. c-Jun does not directly bind to the M-CSF receptor promoter but associates via its basic domain with the ETS domain of PU.1. Consistent with our observation that AP-1 binding does not contribute to c-Jun coactivation is the observation that the activation of PU.1 by c-Jun is blocked by overexpression of c-Fos. Phosphorylation of c-Jun by c-Jun NH2-terminal kinase on Ser-63 and -73 does not alter the ability of c-Jun to enhance PU.1 transactivation. Activated Ras enhances the transcriptional activity of PU.1 by up-regulating c-Jun expression without changing the phosphorylation pattern of PU.1. The activation of PU.1 by Ras is blocked by a mutant c-Jun protein lacking the basic domain. The expression of this mutant form of c-Jun also completely blocks 12-O-tetradecanoylphorbol-13-acetate-induced M-CSF receptor promoter activity during monocytic differentiation. We propose therefore that c-Jun acts as a c-Jun NH2-terminal kinase-independent coactivator of PU.1, resulting in M-CSF receptor expression and development of the monocytic lineage. The ETS domain transcription factor PU.1 is necessary for the development of monocytes and regulates, in particular, the expression of the monocyte-specific macrophage colony-stimulating factor (M-CSF) receptor, which is critical for monocytic cell survival, proliferation, and differentiation. The bZIP transcription factor c-Jun, which is part of the AP-1 transcription factor complex, is also important for monocytic differentiation, but the monocyte-specific M-CSF receptor promoter has no AP-1 consensus binding sites. We asked the question of whether c-Jun could promote the induction of the M-CSF receptor by collaborating with PU.1. We demonstrate that c-Jun enhances the ability of PU.1 to transactivate the M-CSF receptor promoter as well as a minimal thymidine kinase promoter containing only PU.1 DNA binding sites. c-Jun does not directly bind to the M-CSF receptor promoter but associates via its basic domain with the ETS domain of PU.1. Consistent with our observation that AP-1 binding does not contribute to c-Jun coactivation is the observation that the activation of PU.1 by c-Jun is blocked by overexpression of c-Fos. Phosphorylation of c-Jun by c-Jun NH2-terminal kinase on Ser-63 and -73 does not alter the ability of c-Jun to enhance PU.1 transactivation. Activated Ras enhances the transcriptional activity of PU.1 by up-regulating c-Jun expression without changing the phosphorylation pattern of PU.1. The activation of PU.1 by Ras is blocked by a mutant c-Jun protein lacking the basic domain. The expression of this mutant form of c-Jun also completely blocks 12-O-tetradecanoylphorbol-13-acetate-induced M-CSF receptor promoter activity during monocytic differentiation. We propose therefore that c-Jun acts as a c-Jun NH2-terminal kinase-independent coactivator of PU.1, resulting in M-CSF receptor expression and development of the monocytic lineage. The ETS domain transcription factor PU.1 is preferentially expressed in myeloid and B cells (1Chen H.M. Zhang P. Voso M.T. Hohaus S. Gonzalez D.A. Glass C.K. Zhang D.-E. Tenen D.G. Blood. 1995; 85: 2918-2928Crossref PubMed Google Scholar, 2Klemsz M.J. McKercher S.R. Celada A. Van Beveren C. Maki R.A. Cell. 1990; 61: 113-124Abstract Full Text PDF PubMed Scopus (759) Google Scholar) and plays a pivotal role in their development (3Tenen D.G. Hromas R. Licht J.D. Zhang D.-E. Blood. 1997; 90: 489-519Crossref PubMed Google Scholar, 4Shivdasani R.A. Orkin S.H. Blood. 1996; 87: 4025-4039Crossref PubMed Google Scholar). Indeed, mice deficient in PU.1 display a complete block in development of monocytes, macrophages, and B cells (5Scott E.W. Simon M.C. Anastasi J. Singh H. Science. 1994; 265: 1573-1577Crossref PubMed Scopus (1282) Google Scholar, 6McKercher S.R. Torbett B.E. Anderson K.L. Henkel G.W. Vestal D.J. Baribault H. Klemsz M. Feeney A.J. Wu G.E. Paige C.J. Maki R.A. EMBO J. 1996; 15: 5647-5658Crossref PubMed Scopus (934) Google Scholar). During hematopoietic development, PU.1 mRNA is expressed at low levels in murine embryonic stem cells and human CD34+ stem cells and is specifically up-regulated upon myeloid differentiation, and down-regulated upon erythroid differentiation (7Voso M.T. Burn T.C. Wulf G. Lim B. Leone G. Tenen D.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7932-7936Crossref PubMed Scopus (139) Google Scholar, 8Cheng T. Shen H. Giokas D. Gere J. Tenen D.G. Scadden D.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13158-13163Crossref PubMed Scopus (154) Google Scholar). PU.1 regulates the expression of almost all characterized myeloid genes, including growth factor receptors, and in particular directs the monocyte-specific expression of the macrophage colony-stimulating factor (M-CSF) 1The abbreviations M-CSFmacrophage colony-stimulating factorTPA12-O-tetradecanoylphorbol-13-acetatebpbase pair(s)GSTglutathione S-transferaseTKthymidine kinase receptor (9Zhang D.-E. Hetherington C.J. Chen H.M. Tenen D.G. Mol. Cell. Biol. 1994; 14: 373-381Crossref PubMed Scopus (53) Google Scholar,10Reddy M.A. Yang B.S. Yue X. Barnett C.J.K. Ross I.L. Sweet M.J. Hume D.A. Ostrowski M.C. J. Exp. Med. 1994; 180: 2309-2319Crossref PubMed Scopus (111) Google Scholar). Thus, PU.1-deficient hematopoietic cells display minimal expression of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor receptors and no detectable M-CSF receptors (11Anderson K.L. Smith K.A. Conners K. McKercher S.R. Maki R.A. Torbett B.E. Blood. 1998; 91: 3702-3710Crossref PubMed Google Scholar, 12Iwama A. Zhang P. Darlington G.J. Maki R.A. Tenen D.G. Nucleic Acids Res. 1998; 26: 3034-3043Crossref PubMed Scopus (86) Google Scholar). macrophage colony-stimulating factor 12-O-tetradecanoylphorbol-13-acetate base pair(s) glutathione S-transferase thymidine kinase The M-CSF receptor is critical for monocytic cell survival, proliferation, and differentiation (3Tenen D.G. Hromas R. Licht J.D. Zhang D.-E. Blood. 1997; 90: 489-519Crossref PubMed Google Scholar, 13Sherr C.J. Blood. 1990; 75: 1-12Crossref PubMed Google Scholar). M-CSF is known to augment monocyte survival and, therefore, to allow macrophage differentiation (14Lagasse E. Weissman I.L. Cell. 1997; 89: 1021-1031Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). The responsiveness of hematopoietic progenitor cells to M-CSF is regulated at the level of M-CSF receptor expression (15Olweus J. Thompson P.A. Lund-Johansen F. Blood. 1996; 88: 3741-3754Crossref PubMed Google Scholar). Although the important role of the M-CSF receptor for the development of monocytes has been clearly demonstrated, little is known about the signaling molecules or protein-protein interactions that modulate the effect of PU.1 to regulate the M-CSF receptor promoter activity (3Tenen D.G. Hromas R. Licht J.D. Zhang D.-E. Blood. 1997; 90: 489-519Crossref PubMed Google Scholar, 16Hume D.A. Yue X. Ross I.L. Favot P. Lichanska A. Ostrowski M.C. Mol. Reprod. Dev. 1997; 46: 46-52Crossref PubMed Scopus (26) Google Scholar). c-Jun belongs to the bZIP group of DNA binding proteins and is a component of AP-1 transcription factor complexes (17Bohmann D. Bos T.J. Admon A. Nishimura T. Vogt P.K. Tjian R. Science. 1987; 238: 1386-1392Crossref PubMed Scopus (953) Google Scholar). c-Jun forms homodimers or can heterodimerize with other Jun family members or with other bZIP proteins including members of the Fos and ATF/cAMP response element-binding protein (CREB) families (18Whitmarsh A.J. Davis R.J. J. Mol. Med. 1996; 75: 589-607Crossref Scopus (1397) Google Scholar, 19van Dam H. Duyndam M. Rottier R. Bosch A. de Vries-Smits L. Herrlich P. Zanteman A. Angel P. van der Eb A.J. EMBO J. 1993; 12: 479-487Crossref PubMed Scopus (343) Google Scholar). AP-1 has been shown to be involved in many cellular processes including proliferation, differentiation, apoptosis, and stress responses (18Whitmarsh A.J. Davis R.J. J. Mol. Med. 1996; 75: 589-607Crossref Scopus (1397) Google Scholar). In particular, there is evidence that c-Jun plays a role in monocytic differentiation. c-Jun mRNA is up-regulated upon monocyte differentiation of bipotential myeloid cell lines (20Lord K.A. Abdollahi A. Hoffman-Liebermann B. Liebermann D.A. Mol. Cell. Biol. 1993; 13: 841-851Crossref PubMed Scopus (175) Google Scholar, 21Mollinedo F. Gajate C. Tugores A. Flores I. Naranjo J.R. Biochem. J. 1993; 294: 137-144Crossref PubMed Scopus (62) Google Scholar, 22Gaynor R. Simon K. Koeffler P. Blood. 1991; 77: 2618-2623Crossref PubMed Google Scholar), while stable transfection of c-Jun into myeloid cell lines results in partial differentiation (23Szabo E. Preis L.H. Birrer M.J. Cell Growth Differ. 1994; 5: 439-446PubMed Google Scholar,24Li J. King I. Sartorelli A.C. Cell Growth Differ. 1994; 5: 743-751PubMed Google Scholar). Although c-Jun and PU.1 are both pivotal for monocytic development, it is still unclear whether c-Jun is involved in the regulation of the M-CSF receptor, which is critical for monocyte survival, proliferation, and differentiation. It has been shown that during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced monocytic differentiation of U937 cells, c-Jun and M-CSF receptor mRNA expression increases (25Hass R. Prudovsky I. Kruhoffer M. Leuk. Res. 1997; 21: 589-594Crossref PubMed Scopus (29) Google Scholar). However, the monocyte-specific M-CSF receptor promoter (9Zhang D.-E. Hetherington C.J. Chen H.M. Tenen D.G. Mol. Cell. Biol. 1994; 14: 373-381Crossref PubMed Scopus (53) Google Scholar, 26Zhang D.-E. Fujioka K.I. Hetherington C.J. Shapiro L.H. Chen H.M. Look A.T. Tenen D.G. Mol. Cell. Biol. 1994; 14: 8085-8095Crossref PubMed Google Scholar, 27Zhang D.-E. Hetherington C.J. Meyers S. Rhoades K.L. Larson C.J. Chen H.M. Hiebert S.W. Tenen D.G. Mol. Cell. Biol. 1996; 16: 1231-1240Crossref PubMed Google Scholar) contains no AP-1 consensus binding sites. As c-Jun and the regulation of the M-CSF receptor by PU.1 play important roles in monocytic differentiation, we hypothesized that c-Jun might be involved in the regulation of the M-CSF receptor, not by binding to AP-1 sites, but possibly via a novel mechanism. Therefore, we asked the question of whether c-Jun modulates the ability of PU.1 to transactivate the human monocyte specific M-CSF receptor promoter. Monkey kidney CV-1 cells (ATCC CCL-70; American Type Culture Collection, Rockville, MD) were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% calf serum (HyClone, Logan, UT). Murine embryonal carcinoma F9 cells (ATCC CRL-1720; American Type Culture Collection) and human kidney 293T cells (kindly provided by John Blenis, Harvard Medical School, Boston, MA) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (HyClone). U937 cells (ATCC CRL 1593; American Type Culture Collection) were maintained in RPMI 1640 medium (Life Technologies) supplemented with 10% fetal bovine serum, and differentiated with 2 × 10−7m TPA (Sigma) (stock solution: 1 × 10−3m in Me2SO) or vehicle only. The human monocyte-specific M-CSF receptor promoter ranging from bp −88 to +71 with respect to the major monocytic transcription start site (9Zhang D.-E. Hetherington C.J. Chen H.M. Tenen D.G. Mol. Cell. Biol. 1994; 14: 373-381Crossref PubMed Scopus (53) Google Scholar, 26Zhang D.-E. Fujioka K.I. Hetherington C.J. Shapiro L.H. Chen H.M. Look A.T. Tenen D.G. Mol. Cell. Biol. 1994; 14: 8085-8095Crossref PubMed Google Scholar) was subcloned in the firefly luciferase vector pXP2 (28Nordeen S.K. BioTechniques. 1988; 6: 454-458PubMed Google Scholar). pTK with PU.1 sites is a dimer of both PU.1 sites from the granulocyte colony-stimulating factor receptor promoter from bp +28 to +54 (29Smith L.T. Hohaus S. Gonzalez D.A. Dziennis S.E. Tenen D.G. Blood. 1996; 88: 1234-1247Crossref PubMed Google Scholar) subcloned into pTK81luc, a pXP2-based luciferase construct with a TATA box only as a minimal promoter (28Nordeen S.K. BioTechniques. 1988; 6: 454-458PubMed Google Scholar). pTK with mutated PU.1 sites is a dimer of both mutated PU.1 sites from the granulocyte colony-stimulating factor receptor promoter from bp +28 to +54 (primers: 5′-TCG AGT GGT TTC ACA AAC TTT TGT TGA CGA GAG-3′ and 5′-TCG ACT CTC GTC AAC AAA AGT TTG TGA AAC CAC-3′) subcloned into pTK81luc and was constructed as described for pTK with PU.1 sites (29Smith L.T. Hohaus S. Gonzalez D.A. Dziennis S.E. Tenen D.G. Blood. 1996; 88: 1234-1247Crossref PubMed Google Scholar). As an internal control plasmid for co-transfection assays, the pRL-null construct driving a Renilla luciferase gene (Promega, Madison, WI) was used (30Behre G. Smith L.T. Tenen D.G. Biotechniques. 1999; 26: 24-28Crossref PubMed Scopus (69) Google Scholar). The PU.1 mutants pcDNA1-PU.1/Δ1–133, pcDNA1-PU.1/Δ1–100, and pcDNA1-PU.1/Δ1–70 (31Shin M.K. Koshland M.E. Genes Dev. 1993; 7: 2006-2015Crossref PubMed Scopus (122) Google Scholar) were kindly provided by Marian Koshland (University of California, Berkeley, CA). The PU.1 deletion mutants pECE-PU.1/Δ119–160 and pECE-PU.1/Δ8–32 and PU.1 serine to alanine mutants pECE-PU.1-S41A/S45A and pECE-PU.1-S148A (32Klemsz M.J. Maki R.A. Mol. Cell. Biol. 1996; 16: 390-397Crossref PubMed Scopus (82) Google Scholar) were a gift from Richard Maki (the Burnham Institute, La Jolla, CA and Neurocrine Biosciences, San Diego, CA) and Michael Klemsz (Indiana University Medical Center, Indianapolis, IN). pEBG, a mammalian GST expression vector using the strong constitutive EF1α promoter, was kindly provided by Bruce Mayer (Harvard Medical School, Boston, MA). In order to subclone PU.1 into pEBG, the PvuI/EcoRI fragment of murine pECE-PU.1 (2Klemsz M.J. McKercher S.R. Celada A. Van Beveren C. Maki R.A. Cell. 1990; 61: 113-124Abstract Full Text PDF PubMed Scopus (759) Google Scholar), a gift from Michael Klemsz and Richard Maki, was first subcloned into theSmaI/EcoRI-cut vector pBluescript KS+/− (Stratagene, La Jolla, CA). Then the NaeI/NotI fragment of pBS-PU.1 was subcloned into thePmlI/NotI fragment of pEBG. The bacterial GST expression vector pGEX-2TK-PU.1 has been described previously (33Hagemeier C. Bannister A.J. Cook A. Kouzarides T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1580-1584Crossref PubMed Scopus (273) Google Scholar). Human pS3H-c-Jun containing wild-type c-Jun (34Smeal T. Binetruy B. Mercola D.A. Birrer M. Karin M. Nature. 1991; 354: 494-496Crossref PubMed Scopus (699) Google Scholar), pS3H-c-Jun-S63A/S73A containing serine to alanine mutations in amino acid residues 63 and 73 of the human c-Jun cDNA (34Smeal T. Binetruy B. Mercola D.A. Birrer M. Karin M. Nature. 1991; 354: 494-496Crossref PubMed Scopus (699) Google Scholar), and murine pSV40-c-Fos (35Deng T. Karin M. Nature. 1994; 371: 171-175Crossref PubMed Scopus (318) Google Scholar) were kindly provided by Jianmin Tian and Michael Karin (University of California, San Diego). Murine pSV-SPORT1-c-Jun, pUC18-c-Jun/Δ1–87, pUC18-c-Jun/Δ6–199, pSV-SPORT1-c-Jun/ΔLZ lacking amino acids 281–313, and pSV-SPORT1-c-Jun/ΔBD lacking amino acids 251–276 were described previously (36Paradis P. MacLellan W.R. Belaguli N.S. Schwartz R.J. Schneider M.D. J Biol. Chem. 1996; 271: 10827-10833Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). pSP65-c-Jun and pSP65-c-Fos (37Hu E. Mueller E. Oliviero S. Papaioannou V.E. Johnson R. Spiegelman B.M. EMBO J. 1994; 13: 3094-3103Crossref PubMed Scopus (169) Google Scholar) were a gift from Elisabetta Mueller and Bruce Spiegelman (Dana Farber Cancer Institute, Boston, MA). Human activated pMT3-Ha-Ras(L61) (38Feig L.A. Mol. Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (679) Google Scholar, 39Feig L.A. Cooper G.M. Mol. Cell. Biol. 1988; 8: 2472-2478Crossref PubMed Scopus (155) Google Scholar) and inactive pMT3-Ha-Ras(N17) (38Feig L.A. Mol. Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (679) Google Scholar, 39Feig L.A. Cooper G.M. Mol. Cell. Biol. 1988; 8: 2472-2478Crossref PubMed Scopus (155) Google Scholar) were kindly provided by Larry Feig (Tufts University, Boston, MA). pcDNA3-Flag-MEKK1 was constructed by subcloning Flag-MEKK1 (40Yan M. Dai T. Deak J.C. Kyriakis J.M. Zon L.I. Woodgett J.R. Templeton D.J. Nature. 1994; 372: 798-800Crossref PubMed Scopus (660) Google Scholar) into the EcoRI andEcoRV sites of pcDNA3 (Invitrogen, Carlsbad, CA). pcDNA3/EGFP (enhanced green fluorescence protein) was kindly provided by Joseph Sodroski (Dana Farber Cancer Center Institute, Boston, MA). CV-1 cells, F9 cells, or 293T cells were transfected using LipofectAMINE Plus (Life Technologies) as described by the manufacturer. U937 cells were transiently transfected by electroporation as described previously (9Zhang D.-E. Hetherington C.J. Chen H.M. Tenen D.G. Mol. Cell. Biol. 1994; 14: 373-381Crossref PubMed Scopus (53) Google Scholar). Firefly luciferase activities from the constructs pM-CSFR, pXP2, and pTK with PU.1 sites and pTK with mutated PU.1 sites and Renilla luciferase activity from the internal control plasmid pRL-null were determined 24 h after the initiation of the transfection protocols using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activities were normalized to the Renilla luciferase values of pRL-null. Results are given as means ± S.E. of at least six independent experiments. The following DNA concentrations of the reporter constructs and expression plasmids were used for LipofectAMINE Plus transfections: 0.3 μg of the human monocyte-specific M-CSF receptor promoter in pXP2, pXP2, the TK promoter with PU.1 sites, and the TK promoter with mutated PU.1 sites; 0.05 μg of the internal control plasmid pRL-null; 0.5 μg of pEBG-PU.1; 0.2 μg of the other expression plasmids for PU.1 and PU.1 mutants; 0.25 μg of Ras(L61), Ras(N17), and MEKK1; 0.1 μg of c-Jun, c-Jun mutants, and c-Fos; and the same concentrations of the empty expression vectors as controls, respectively. For electroporation, 10 μg of the firefly luciferase reporter constructs, 5 μg of expression plasmids, and 1 μg of the internal control plasmid were used. pRL-null was chosen as internal control plasmid, because it was not transactivated by Ras (30Behre G. Smith L.T. Tenen D.G. Biotechniques. 1999; 26: 24-28Crossref PubMed Scopus (69) Google Scholar) or by PU.1, c-Jun, c-Fos, or MEKK1 in CV-1, F9, 293T, or U937 cells (data not shown). Electrophoretic mobility shift assays were performed as described previously (9Zhang D.-E. Hetherington C.J. Chen H.M. Tenen D.G. Mol. Cell. Biol. 1994; 14: 373-381Crossref PubMed Scopus (53) Google Scholar, 26Zhang D.-E. Fujioka K.I. Hetherington C.J. Shapiro L.H. Chen H.M. Look A.T. Tenen D.G. Mol. Cell. Biol. 1994; 14: 8085-8095Crossref PubMed Google Scholar,27Zhang D.-E. Hetherington C.J. Meyers S. Rhoades K.L. Larson C.J. Chen H.M. Hiebert S.W. Tenen D.G. Mol. Cell. Biol. 1996; 16: 1231-1240Crossref PubMed Google Scholar, 29Smith L.T. Hohaus S. Gonzalez D.A. Dziennis S.E. Tenen D.G. Blood. 1996; 88: 1234-1247Crossref PubMed Google Scholar). As a positive control for c-Jun binding, a double-stranded AP-1 probe from the collagenase promoter (5′-AAT TCG CTT GAT GAC TCA GCC GGA A-3′) was labeled with Klenow polymerase and [α-32P]dCTP (NEN Life Science Products) and incubated with 0.1 μg/μl of double-stranded poly(dI-dC) (Sigma) with 1 μl of in vitro translated c-Jun or c-Fos. In some experiments, a 100-fold molar excess of the AP-1 probe was added as specific unlabeled competitor. Similarly, a double-stranded M-CSF receptor promoter oligonucleotide extending from position bp −88 to +71 with respect to the major transcription start site (9Zhang D.-E. Hetherington C.J. Chen H.M. Tenen D.G. Mol. Cell. Biol. 1994; 14: 373-381Crossref PubMed Scopus (53) Google Scholar, 26Zhang D.-E. Fujioka K.I. Hetherington C.J. Shapiro L.H. Chen H.M. Look A.T. Tenen D.G. Mol. Cell. Biol. 1994; 14: 8085-8095Crossref PubMed Google Scholar) was Klenow-labeled with [α-32P]dCTP and incubated with 0.1 μg/μl double-stranded poly(dI-dC) with 1 μl of in vitro translated PU.1, c-Jun, or c-Fos. In some experiments, a 100-fold molar excess of specific unlabeled competitor was added: an oligonucleotide with the PU.1 DNA binding site in the CD11b promoter (41Pahl H.L. Scheibe R.J. Zhang D.-E. Chen H.M. Galson D.L. Maki R.A. Tenen D.G. J. Biol. Chem. 1993; 268: 5014-5020Abstract Full Text PDF PubMed Google Scholar), 3′-AGC CTA CTT CTC CTT TTC TGC CCT TCT TTG-5′, to compete for PU.1, and the AP-1 probe described above to compete for c-Jun. Protein interaction assays were performed as described previously (27Zhang D.-E. Hetherington C.J. Meyers S. Rhoades K.L. Larson C.J. Chen H.M. Hiebert S.W. Tenen D.G. Mol. Cell. Biol. 1996; 16: 1231-1240Crossref PubMed Google Scholar, 42Petrovick M.S. Hiebert S.W. Friedman A.D. Hetherington C.J. Tenen D.G. Zhang D.-E. Mol. Cell. Biol. 1998; 18: 3915-3925Crossref PubMed Google Scholar). c-Jun and c-Fos werein vitro transcribed and translated using the TNT Reticulocyte Lysate System (Promega) and labeled with [35S]methionine (NEN Life Science Products). 1 μl of labeled in vitro translated c-Jun or c-Fos was mixed with 1 μg of bacterially expressed GST-PU.1 or equivalent amounts of GST or glutathione-agarose beads (Sigma) for 1 h at 4 °C in NETN buffer (20 mm Tris (pH 8.0), 200 mm NaCl, 1 mm EDTA, and 0.5% Nonidet P-40). GST-PU.1 was recovered using glutathione-agarose beads, washed seven times with NETN buffer, and separated by 10% SDS-polyacrylamide gel electrophoresis. Prior to autoradiography, the gel was stained with Coomassie Brilliant Blue (Bio-Rad) to verify that the protein concentrations of GST-PU.1 and GST were the same in all lanes. To detect changes in the phosphorylation pattern of PU.1 upon stimulation with activated Ras in vivo, 0.5 μg of pEBG-PU.1 either with 0.25 μg of activated Ras(L61) or with inactive Ras(N17) was transfected into 293T cells using LipofectAMINE Plus (Life Technologies). 3 h after transfection, cells were starved in serum-free Dulbecco's modified Eagle's medium. After 18 h, cells were placed into serum-free and phosphate-free Dulbecco's modified Eagle's medium (Life Technologies) for 30 min before they were metabolically labeled with [32P]orthophosphate (2.5 mCi/ml). After 4 h, cells were lysed with radioimmunoprecipitation assay buffer containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.15 m NaCl, 5 mm EDTA, and 50 mm Tris (pH 8.0) and supplemented with aprotinin, phenylmethylsulfonyl fluoride, pepstatin A, leupeptin, antipain, and chymostatin as protease inhibitors (Sigma) and sodium pyrophosphate, sodium fluoride, and sodium vanadate as phosphatase inhibitors (Sigma). In parallel plates, 0.3 μg of the M-CSF receptor promoter was co-transfected in 293T cells, and luciferase activities were determined to ensure that Ras enhances the transactivation function of PU.1 in the particular experiment used inin vivo labeling and subsequent phosphoamino acid analysis and phosphopeptide mapping. GST-PU.1 was isolated from the radioimmunoprecipitation assay lysates using glutathione-agarose beads (Sigma), washed four times with radioimmunoprecipitation assay buffer, separated on 10% SDS-polyacrylamide gels, and transferred to Immobilon-P membrane (Millipore Corp., Bedford, MA) for phosphoamino acid analysis or nitrocellulose (Bio-Rad) for phosphopeptide mapping. After transfer, the 69-kDa GST-PU.1 protein bands were excised. For phosphoamino acid analysis, the samples were boiled at 100 °C for 1 h with 6n HCl (Pierce), and the presence of serine, threonine, or tyrosine phosphorylation was determined as described (43Boyle W.J. van D.G. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar). To determine the phosphorylated protein residues of PU.1, GST-PU.1 protein bands were digested with 1-chloro-3-tosylamido-7-amino-2-heptanone-treated chymotrypsin (Worthington) and endoproteinase Glu-C (V8 protease) (Boehringer Mannheim) and processed for phosphopeptide mapping as described previously (43Boyle W.J. van D.G. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar). 24 h after the start of transfection, cells were lysed with radioimmunoprecipitation assay buffer. Equal amounts of total protein were separated on 10% SDS-polyacrylamide gels and transferred to Immobilon-P membrane (Millipore). Membranes were incubated with anti-c-Jun antibody (catalog no. SC-45; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-PU.1 antibody (catalog no. SC-352; Santa Cruz Biotechnology), or anti-β-tubulin antibody as an internal control (catalog no. 1111876; Boehringer Mannheim) for 60 min and then with Protein A-horseradish peroxidase conjugate (Amersham, Buckinghamshire, United Kingdom) for 45 min. For U937 cells, an anti-M-CSF receptor antibody (catalog no. SC-692, Santa Cruz Biotechnology) was used. Signals were detected with ECL Western blotting detection reagents (Amersham). In parallel plates, the M-CSF receptor promoter construct was co-transfected, and luciferase activities were determined to ensure that Ras enhances the transactivation function of PU.1 in the particular experiment used for Western blot analysis of c-Jun expression or PU.1 expression and that the transfection efficacy was the same (less than 10% difference between plates) in the particular experiment. Differences in protein expression were quantitated by ImageQuant software (Molecular Dynamics). Since c-Jun and the regulation of the M-CSF receptor by PU.1 are both important for monocytic development, we asked the question of whether c-Jun enhances the ability of PU.1 to transactivate the M-CSF receptor promoter. CV-1 cells, which contain c-Jun (Fig.4 B), were transfected with a plasmid containing the human monocyte-specific M-CSF receptor promoter (9Zhang D.-E. Hetherington C.J. Chen H.M. Tenen D.G. Mol. Cell. Biol. 1994; 14: 373-381Crossref PubMed Scopus (53) Google Scholar, 26Zhang D.-E. Fujioka K.I. Hetherington C.J. Shapiro L.H. Chen H.M. Look A.T. Tenen D.G. Mol. Cell. Biol. 1994; 14: 8085-8095Crossref PubMed Google Scholar) cloned upstream of the luciferase reporter gene along with expression plasmids for PU.1 and c-Jun, and reporter gene expression was determined 24 h post-transfection. Transfection of a c-Jun expression construct significantly enhanced the ability of PU.1 to transactivate the M-CSF receptor promoter (Fig. 1 A). Moreover, in c-Jun-deficient F9 cells, PU.1 weakly transactivated the M-CSF receptor promoter (2 fold), while co-expression of c-Jun with PU.1 led to robust transactivation (33-fold) (Fig. 1 B). The cooperation of c-Jun with PU.1 is therefore important for M-CSF receptor promoter activity.Figure 1Effect of c-Jun on the ability of PU.1 to transactivate the M-CSF receptor promoter and a minimal TK promoter containing only PU.1 DNA binding sites. A, CV-1 cells were transfected with the human monocyte-specific M-CSF receptor promoter or the promoterless vector pXP2 and with the expression plasmids pECE-PU.1 and pSV-SPORT1-c-Jun. Luciferase activities were determined 24 h after transient transfection with LipofectAMINE Plus and normalized to the activities of the internal control plasmid pRL-null. B, c-Jun-deficient F9 cells were transfected as described for Fig. 1 A. C, F9 cells were transfected with a minimal TK promoter with PU.1 sites or a minimal TK promoter with mutated PU.1 sites and with the expression plasmids for PU.1 and c-Jun.View Large Image Figure ViewerDownload (PPT) We next asked the following questions: (a) whether the binding of PU.1 to DNA was necessary for its activation by c-Jun and (b) whether a PU.1 binding site alone was sufficient for the c-Jun-enhanced PU.1 activation. We observed enhanced PU.1 transactivation mediated by c-Jun using a reporter construct containing four PU.1 binding sites cloned upstream of a minimal TK promoter (Fig.1 C). In control experiments, no effect of c-Jun on PU.1 activity was observed when the PU.1 binding sites were mutated (Fig.1 C). These data indicate that PU.1 binding to DNA is necessary for its activation by c-Jun and that PU.1 binding sites are sufficient to mediate this effect. To elucidate the mechanism by which c-Jun augments the transcriptional activity of PU.1, we performed experiments to determine whether the activation of PU.1 by c-Jun required the binding of c-Jun·AP-1 complexes to the M-CSF receptor promoter. Since there are no AP-1 consensus sites in the human monocyte-specific M-CSF receptor promoter from bp −88 to +71 with respect to the major monocytic transcription start site (9Zhang D.-E. Hetherington C.J. Chen H.M. Tenen D.G. Mol. Cell. Biol. 1994; 14: 373-381Crossref PubMed Scopus (53) Google Scholar, 26Zhang D.-E. Fujioka K.I. Hetherington C.J. Shapiro L.H. Chen H.M. Look A.T. Tenen D.G. Mol. Cell. Biol. 1994; 14: 8085-8095Crossref PubMed Google Scholar) (Fig.1, A and B) or in the TK promoter containing PU.1 sites (Fig. 1 C), our data suggested that c-Jun augmentation
Referência(s)