A Role for Mixed Lineage Kinases in Regulating Transcription Factor CCAAT/Enhancer-binding Protein-β-dependent Gene Expression in Response to Interferon-γ
2005; Elsevier BV; Volume: 280; Issue: 26 Linguagem: Inglês
10.1074/jbc.m413661200
ISSN1083-351X
AutoresSanjit K. Roy, Jon D. Shuman, Leonidas C. Platanias, Paul Shapiro, Sekhar P. Reddy, Peter F. Johnson, Dhananjaya V. Kalvakolanu,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoTranscription factor CCAAT/enhancer-binding protein-β (C/EBP-β) regulates a variety of cellular functions in response to exogenous stimuli. We have reported earlier that C/EBP-β induces gene transcription through a novel interferon (IFN)-response element called γ-IFN-activated transcriptional element. We show here that IFN-γ-induced, C/EBP-β/γ-IFN-activated transcriptional element-dependent gene expression is regulated by mixed lineage kinases (MLKs), members of the mitogen-activated protein kinase kinase kinase family. MLK3 appears to activate C/EBP-β in response to IFN-γ by a mechanism involving decreased phosphorylation of a specific phosphoacceptor residue, Ser64, within the transactivation domain. Decreased phosphorylation of Ser64 was independent of IFN-γ-stimulated ERK1/2 activation and did not require the ERK phosphorylation site Thr189 located in regulatory domain 2 of C/EBP-β. Together these studies provide the first evidence that MLK3 is involved in IFN-γ signaling and identify a novel mechanism of transcriptional activation by IFN-γ. Transcription factor CCAAT/enhancer-binding protein-β (C/EBP-β) regulates a variety of cellular functions in response to exogenous stimuli. We have reported earlier that C/EBP-β induces gene transcription through a novel interferon (IFN)-response element called γ-IFN-activated transcriptional element. We show here that IFN-γ-induced, C/EBP-β/γ-IFN-activated transcriptional element-dependent gene expression is regulated by mixed lineage kinases (MLKs), members of the mitogen-activated protein kinase kinase kinase family. MLK3 appears to activate C/EBP-β in response to IFN-γ by a mechanism involving decreased phosphorylation of a specific phosphoacceptor residue, Ser64, within the transactivation domain. Decreased phosphorylation of Ser64 was independent of IFN-γ-stimulated ERK1/2 activation and did not require the ERK phosphorylation site Thr189 located in regulatory domain 2 of C/EBP-β. Together these studies provide the first evidence that MLK3 is involved in IFN-γ signaling and identify a novel mechanism of transcriptional activation by IFN-γ. Interferons regulate the antiviral, antitumor, and immune responses by inducing the transcription of a number of IFN 1The abbreviations used are: IFN, interferon; ISG, IFN-stimulated gene; ISGF, ISG factor; C/EBP, CAAAT/enhancer-binding protein; CEP, CEP-11004, an inhibitor of MLKs; ERK, extracellular signal-regulated kinase; GATE, γ-IFN-activated transcriptional element; IP, immunoprecipitation; IRF, IFN gene-regulatory factor; MEF, mouse embryo fibroblast; MAPK, mitogen-activated protein kinase; MLK, mixed lineage kinase; STAT, signal transducer and activator of transcription; JNK, c-Jun N-terminal kinase; HA, hemagglutinin; GFP, green fluorescent protein; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MEKK, MEK kinase; TAD, transactivation domain; siRNA, short hairpin interfering RNA; Luc, luciferase; pIRE, palindromic IFN-response element. -stimulated genes (ISGs). IFN-γ regulates a number of diverse processes including cell growth and innate and specific immune responses (1Boehm U. Klamp T. Groot M. Howard J.C. Annu. Rev. Immunol. 1997; 15: 749-795Crossref PubMed Scopus (2495) Google Scholar). Although STAT1 is a critical primary regulator of IFN-γ-induced responses, several ISGs are critically dependent on other transcription factors. A STAT1-independent regulation of some genes by IFN-γ has been shown (2Gil M.P. Bohn E. O'Guin A.K. Ramana C.V. Levine B. Stark G.R. Virgin H.W. Schreiber R.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6680-6685Crossref PubMed Scopus (294) Google Scholar, 3Ramana C.V. Gil M.P. Han Y. Ransohoff R.M. Schreiber R.D. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6674-6679Crossref PubMed Scopus (209) Google Scholar). irf9 is an IFN-γ inducible gene that codes for a DNA-binding protein. In the IFN-α signal transduction pathway the IRF9 protein forms a trimeric transcription factor, ISGF3, in association with the STAT1 and STAT2 proteins to drive gene expression (4Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3383) Google Scholar, 5Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3380) Google Scholar). IFN-γ potently induces the expression of IRF9 to augment IFN-α/β-induced transcription (6Levy D.E. Lew D.J. Decker T. Kessler D.S. Darnell Jr., J.E. EMBO J. 1990; 9: 1105-1111Crossref PubMed Scopus (173) Google Scholar, 7Bandyopadhyay S.K. Kalvakolanu D.V. Sen G.C. Mol. Cell. Biol. 1990; 10: 5055-5063Crossref PubMed Scopus (55) Google Scholar). Indeed down-regulation of irf9 expression by viral products provides an escape route against the antiviral action of IFNs (8Kalvakolanu D.V. Trends Microbiol. 1999; 7: 166-171Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Interestingly the irf9 promoter lacks STAT-binding elements (9Weihua X. Kolla V. Kalvakolanu D.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 103-108Crossref PubMed Scopus (56) Google Scholar). Instead a novel regulatory element (termed GATE) and its cognate transcription factors control irf9 expression. We have shown earlier that the bZIP transcription factor C/EBP-β, a regulator of acute phase responses and cell differentiation (10Akira S. Kishimoto T. Adv. Immunol. 1997; 65: 1-46Crossref PubMed Google Scholar, 11Poli V. J. Biol. Chem. 1998; 273: 29279-29282Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar), binds to GATE and induces IFN-regulated transcription (12Roy S.K. Wachira S.J. Weihua X. Hu J. Kalvakolanu D.V. J. Biol. Chem. 2000; 275: 12626-12632Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). C/EBP-β (nuclear factor induced by interleukin-6, liver-activated protein, C/EBP-related protein 2, nuclear factor myeloid) (10Akira S. Kishimoto T. Adv. Immunol. 1997; 65: 1-46Crossref PubMed Google Scholar, 11Poli V. J. Biol. Chem. 1998; 273: 29279-29282Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 13Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-28548Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar) responds to a number of extracellular stimuli including interleukin-6, interleukin-1, tumor necrosis factor-α, and lipopolysaccharide (10Akira S. Kishimoto T. Adv. Immunol. 1997; 65: 1-46Crossref PubMed Google Scholar, 11Poli V. J. Biol. Chem. 1998; 273: 29279-29282Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar) and is necessary for regulating several processes including carbohydrate metabolism, lipid storage, Th1 immune responses, macrophage-mediated antibacterial and antitumor defenses, and female fertility (11Poli V. J. Biol. Chem. 1998; 273: 29279-29282Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 13Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-28548Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar, 14Tanaka T. Akira S. Yoshida K. Umemoto M. Yoneda Y. Shirafuji N. Fujiwara H. Suematsu S. Yoshida N. Kishimoto T. Cell. 1995; 80: 353-361Abstract Full Text PDF PubMed Scopus (470) Google Scholar, 15Sterneck E. Tessarollo L. Johnson P.F. Genes Dev. 1997; 11: 2153-2162Crossref PubMed Scopus (346) Google Scholar). C/EBP-β deficiency causes a lymphoproliferative disorder (16Screpanti I. Romani L. Musiani P. Modesti A. Fattori E. Lazzaro D. 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Phosphorylation of several serine and threonine residues distributed across the backbone of C/EBP-β by different protein kinases appears to contribute to its diverse actions. However, the ligand-inducible factors and the corresponding phosphorylation sites controlling its phosphorylation are not fully understood. Our recent studies have shown that mitogen-activated protein kinases (MAPKs) play a critical role in regulating C/EBP-β- and GATE-dependent gene expression. This occurs at least in part by phosphorylation of C/EBPβ on Thr189 by ERK1/2 (20Hu J. Roy S.K. Shapiro P.S. Rodig S.R. Reddy S.P. Platanias L.C. Schreiber R.D. Kalvakolanu D.V. J. Biol. Chem. 2001; 276: 287-297Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 21Roy S.K. Hu J. Meng Q. Xia Y. Shapiro P.S. Reddy S.P. Platanias L.C. Lindner D.J. Johnson P.F. Pritchard C. Pages G. Pouyssegur J. Kalvakolanu D.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7945-7950Crossref PubMed Scopus (80) Google Scholar). Here we show a role for mixed lineage kinases (MLKs), a subgroup of upstream kinases that regulate the MAPK family, in controlling C/EBP-β-dependent transcription. MLKs function as MAPK kinase kinases and have been implicated in the activation of c-Jun N-terminal kinase (JNK) and stress-activated protein kinase kinase 1 and transcription factor NF-κB. Some MLKs also activate p38 MAPK. Interestingly instead of stimulating phosphorylation, MLKs exerted a positive effect on the transactivation domain of C/EBP-β by promoting decreased phosphorylation at a specific serine residue in the transactivation domain. Thus, our studies identified a novel IFN-γ effector pathway involving MLKs and C/EBPβ. Reagents—Murine IFN-γ (Pestka Biomedical Laboratories); SB202190 (Calbiochem); antibodies specific for phospho-ERK1/2; actin (Sigma); native ERK2; and hemagglutinin (HA) epitope tag, transcriptional co-activator p300, and C/EBP-β (Santa Cruz Biotechnology) were used in these studies. MLK inhibitor CEP-11004 (henceforth referred to as CEP) was generously provided by Cephalon Inc., West Chester, PA. An antibody that detects a phospho-Thr189 form (Thr(P)189) of C/EBP-β was purchased from Cell Signaling Technology Inc. An antibody that specifically detects the phospho-Ser64 form (Ser(P)64) of C/EBP-β has been described elsewhere (22Shuman J.D. Sebastian T. Kaldis P. Copeland T.D. Zhu S. Smart R.C. Johnson P.F. Mol. Cell. Biol. 2004; 24: 7380-7391Crossref PubMed Scopus (67) Google Scholar). Affinity-purified rabbit polyclonal antibodies specific for the native (Santa Cruz Biotechnology) and activated (BIOSOURCE) forms of MLK3 were used in some experiments. In response to extracellular stimuli, the two critical threonine (Thr277) and serine (Ser281) residues located in the activation loop of MLK3 are phosphorylated (23Gallo K.A. Johnson G.L. Nat. Rev. Mol. Cell. Biol. 2002; 3: 663-672Crossref PubMed Scopus (456) Google Scholar). The antibody against the activated MLK3 isoform detects the diphosphorylated (Thr(P)277/Ser(P)281) form of the MLK3 protein. GFP-specific antibodies were from Clontech. Cell Culture and Plasmids—The murine macrophage cell line RAW (RAW264.7) was grown in RPMI 1640 medium with 5% fetal bovine serum. Isogenic mouse embryonic fibroblasts (MEFs) derived from wild type, mekk1–/– (24Xia Y. Makris C. Su B. Li E. Yang J. Nemerow G.R. Karin M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5243-5248Crossref PubMed Scopus (232) Google Scholar) and cebpb–/– (21Roy S.K. Hu J. Meng Q. Xia Y. Shapiro P.S. Reddy S.P. Platanias L.C. Lindner D.J. Johnson P.F. Pritchard C. Pages G. Pouyssegur J. Kalvakolanu D.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7945-7950Crossref PubMed Scopus (80) Google Scholar) mice were described earlier. These cells were cultured in Dulbecco's modified Eagle's medium with 2% fetal bovine serum during IFN-γ treatment. The murine ISGF3γ (p48) reporter construct, P4, was described earlier (9Weihua X. Kolla V. Kalvakolanu D.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 103-108Crossref PubMed Scopus (56) Google Scholar). In this construct a 74-bp element of murine p48 gene promoter, encompassing GATE, was cloned upstream of the SV40 early promoter driving luciferase. Mutagenesis of the GATE sequence in this construct caused a loss of IFN-γ response and C/EBP-β binding (12Roy S.K. Wachira S.J. Weihua X. Hu J. Kalvakolanu D.V. J. Biol. Chem. 2000; 275: 12626-12632Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). A wild type and a catalytically inactive dominant negative mutant (K114R) of Mlk3 cloned in pcDNA3.0 vector was described earlier (25Leung I.W. Lassam N. J. Biol. Chem. 1998; 273: 32408-32415Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The native irf9 promoter (A6-Luc) and the corresponding GATE mutant were described earlier (9Weihua X. Kolla V. Kalvakolanu D.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 103-108Crossref PubMed Scopus (56) Google Scholar). A mammalian expression vector with the transactivation domain (TAD) (amino acids 22–109) of C/EBP-β and its corresponding S64A mutant fused to the DNA binding domain of transcription factor GAL4 were described previously (26Williams S.C. Baer M. Dillner A.J. Johnson P.F. EMBO J. 1995; 14: 3170-3183Crossref PubMed Scopus (200) Google Scholar). The G-E1b-Luc reporter contains five tandem copies of the GAL4 binding sites placed upstream of minimal E1b promoter and luciferase gene. A pcDNA3.0 vector expressing the HA epitope-tagged p300 was provided by Betsy Barnes, The Johns Hopkins University School of Medicine, Baltimore, MD. GFP-tagged MLK2 expression vector was described elsewhere (27Akbarzadeh S. Ji H. Frecklington D. Marmy-Conus N. Mok Y.F. Bowes L. Devereux L. Linsenmeyer M. Simpson R.J. Dorow D.S. J. Biol. Chem. 2002; 277: 36280-36287Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). siRNA sequences that can specifically knock down the expression of human and mouse MLK3 genes were based on the sequences described recently (28Chadee D.N. Kyriakis J.M. Cell Cycle. 2004; 3: 1227-1229Crossref PubMed Scopus (31) Google Scholar). The following double-stranded oligonucleotides bearing the target sequences were synthesized and cloned into the SalI and XbaI sites of the targeting vector pSuppressor-Neo (Imgenex, Inc.): mouse MLK3, 5′-TCGAGGGGCAGCGATGTCTGGAGCTTGAGTACTGAAGCTCCAGACATCGCTGCCCTTTTT-3′; and human MLK3, 5′-TCGAGGGGCAGTGACGTCTGGAGTTTGAGTACTGAAACTCCAGACGTCACTGCCCTTTTT-3′. Because of the differences in the primary sequences of mouse and human MLK3 mRNAs (underlined bases), these siRNAs target only the cognate mRNA for degradation in the cells (28Chadee D.N. Kyriakis J.M. Cell Cycle. 2004; 3: 1227-1229Crossref PubMed Scopus (31) Google Scholar). Gene Expression Analyses—Western blot analyses, transfection, β-galactosidase and luciferase assays, and SDS-PAGE analyses were performed as described earlier (9Weihua X. Kolla V. Kalvakolanu D.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 103-108Crossref PubMed Scopus (56) Google Scholar). The total amount of transfected DNA (1.0 μg) was kept constant by adding pBluescript SK DNA if required. In general, 0.4 μg of luciferase and 0.1 μg of C/EBP-β expression vector were used for transfection. A β-actin promoter-driven β-galactosidase reporter (0.2 μg) was used as an internal control for normalizing variations in transfection efficiency. ERK activation was monitored by Western blot analysis of cell extracts with a phospho-ERK1/2-specific antibody (Cell Signaling Technology Inc.). Anti-ERK2 antibodies were used for determining the total ERK in these samples. In some experiments, antibodies specific for the native and phosphorylated forms of MLK3 were used. Chromatin Immunoprecipitation Assays—These assays were performed as described earlier (29Morris A.C. Beresford G.W. Mooney M.R. Boss J.M. Mol. Cell. Biol. 2002; 22: 4781-4791Crossref PubMed Scopus (109) Google Scholar). Briefly cells (1 × 108) were stimulated with IFN-γ for 8 h, and chromatin was cross-linked using paraformaldehyde. Nuclei were isolated, and chromatin was sheared into ∼1–2-kb fragments using a Bronson sonicator fitted with a microtip probe. After removing the debris, soluble chromatin was subjected to IP with either C/EBP-β- or transcriptional co-activator p300-specific antibodies. After extensively washing the immunoprecipitated products, cross-links were reversed, and the DNA was extracted using phenol-chloroform. The resultant DNA was used for 32 cycles of PCR with the following primers specific for mouse irf9 promoter: 5′-AAGGTGCTACTGCTGACTGAGG-3′and 5′-AAGGGCGGACGTGAAGAAATGG-3′ (9Weihua X. Kolla V. Kalvakolanu D.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 103-108Crossref PubMed Scopus (56) Google Scholar). PCR with these primers yields a 443-bp product. DNA extracted from an aliquot of the initial soluble chromatin was used for input control. IFN-γ-induced Gene Expression through GATE Is Inhibited by MLK Inhibitor CEP-11004—To examine the role of MLKs in IFN-γ-regulated signaling pathways, we used a specific semisynthetic inhibitor, CEP. A previously defined concentration of CEP that specifically inhibited MLKs (30Maroney A.C. Finn J.P. Connors T.J. Durkin J.T. Angeles T. Gessner G. Xu Z. Meyer S.L. Savage M.J. Greene L.A. Scott R.W. Vaught J.L. J. Biol. Chem. 2001; 276: 25302-25308Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), but not other MAPKs, was used in this study. These experiments were performed using the murine macrophage cell line RAW264.7. Cells were transfected with the P4 reporter gene and then treated with the inhibitors prior to IFN-γ treatment. CEP strongly inhibited (Fig. 1A) GATE-driven transcription. Under these conditions, SB202190, a pan-p38 MAPK-specific inhibitor, did not inhibit IFN-γ-induced gene expression through GATE (data not shown). Similar to P4-Luc, expression of A6-Luc, a reporter bearing the native irf9 promoter but not its corresponding mutant lacking a functional GATE, was induced following IFN-γ treatment (Fig. 1B). Such induction was inhibited by CEP. Thus, the native IRF9 enhancer and minimal enhancer were sensitive to CEP inhibition. To determine whether CEP exerted a similar inhibitory effect on another IFN-γ-regulated enhancer element we used palindromic IFN-response element (pIRE)-luciferase, whose expression is dependent on IFN-γ-activated STAT1 binding to the pIRE. STAT1-dependent expression of pIRE-Luc was unaffected by CEP (Fig. 1C). Similarly CEP did not significantly inhibit epidermal growth factor-induced expression through an activator protein 1-responsive element (Fig. 1D). Together these results suggest that MLK activity is critical for IFN-γ-dependent induction of IRF9. To test whether CEP exhibited a similar inhibitory effect on the expression of the endogenous irf9 gene, we performed reverse transcription-PCR analysis of irf9 mRNA levels following various treatments (Fig. 2A). CEP strongly inhibited the IFN-γ-induced expression of irf9, whereas it had no significant effect on the basal expression of the gene. In contrast, expression of another IFN-γ-regulated mRNA, IRF8/IFN consensus sequence-binding protein, was not inhibited by CEP (Fig. 2B). This gene is dependent on STAT1 for its expression. The expression of glyceraldehyde-3-phosphate dehydrogenase mRNA was not affected by any of the treatments (Fig. 2C). These results demonstrate a specific inhibitory effect of CEP on a subset of IFN-induced genes. CEP Inhibits C/EBP-β-dependent Gene Expression through GATE—The above studies indicate that GATE-driven gene expression is inhibited by CEP. However, they did not identify the transcription factor that is subject to inhibition. Our previous studies have established C/EBP-β as a critical regulator of IFN-γ-induced gene expression through GATE (12Roy S.K. Wachira S.J. Weihua X. Hu J. Kalvakolanu D.V. J. Biol. Chem. 2000; 275: 12626-12632Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). To demonstrate that C/EBP-β is indeed the target of CEP inhibition, we performed a transient transfection assay with the P4 reporter in cebpb–/– MEFs in the presence of exogenous C/EBP-β. Co-transfection of C/EBP-β but not an empty vector conferred a strong induction of luciferase (Fig. 3A). As reported earlier, C/EBP-β also induced the basal expression of this promoter (12Roy S.K. Wachira S.J. Weihua X. Hu J. Kalvakolanu D.V. J. Biol. Chem. 2000; 275: 12626-12632Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). CEP potently inhibited C/EBP-β-dependent IFN-γ-induced expression of the reporter but did not affect basal expression. These differential effects on the promoter are not due to variable expression of the C/EBP-β protein because a comparable level of C/EBP-β protein was found between the samples (Fig. 3B). These data suggest that an IFN-γ-induced post-translational modification(s) of C/EBP-β may be inhibited by CEP. Because CEP is an inhibitor of the mixed lineage kinases (30Maroney A.C. Finn J.P. Connors T.J. Durkin J.T. Angeles T. Gessner G. Xu Z. Meyer S.L. Savage M.J. Greene L.A. Scott R.W. Vaught J.L. J. Biol. Chem. 2001; 276: 25302-25308Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), we next determined whether a dominant negative MLK3 inhibits GATE-dependent transcription. We chose to focus on MLK3 because it is a ubiquitously expressed enzyme and has been relatively well characterized compared with the other known members of its family (23Gallo K.A. Johnson G.L. Nat. Rev. Mol. Cell. Biol. 2002; 3: 663-672Crossref PubMed Scopus (456) Google Scholar). Vectors carrying wild type Mlk3 and a catalytically inactive mutant were co-expressed with the P4-luciferase reporter to determine their effects on IFN-γ-induced expression through GATE (Fig. 3D). Wild type Mlk3 significantly augmented the IFN-γ-stimulated expression of the reporter (3–4-fold), whereas the catalytically inactive mutant significantly (3-fold) inhibited it. Neither wild type nor mutant Mlk3 altered the basal expression of the reporter. Together with data in Figs. 1 and 2, these observations indicate that MLK3 plays an important role in regulating IFN-induced GATE-dependent transcription. We next checked the relevance of endogenous Mlk3 to IFN-γ-induced gene expression through GATE using siRNAs. Because the mouse and human MLK3 siRNA targets differ in their primary sequence they degrade their target mRNAs in a species-specific manner (31Chadee D.N. Kyriakis J.M. Nat. Cell Biol. 2004; 6: 770-776Crossref PubMed Scopus (136) Google Scholar). We first examined the specificities of these siRNAs in knocking down the expression of Mlk3. RAW cells were transfected with the pSuppressor-Neo vector expressing mouse or human MLK3-specific siRNAs. The mouse Mlk3 siRNA knocked down the expression of MLK3 protein (70%) compared with the empty vector control. As expected, the human MLK3 siRNA did not affect the MLK3 expression (Fig. 3F). A converse effect of mouse siRNA on the expression of human MLK3 was found in HeLa cells (data not shown). Having demonstrated the specificity of these siRNAs, we next tested their effects on IFN-γ-induced transcription in RAW cells. These siRNA vectors were co-transfected with two IFN-γ-inducible reporters: 1) P4-Luc and 2) pIRE-Luc. As mentioned earlier the former is dependent on C/EBP-β and the latter is dependent on STAT1 for their expression. As shown in Fig. 3G, co-expression of mouse MLK3 siRNA, but not the human MLK3 siRNA, significantly inhibited the IFN-γ-induced expression of the P4-Luc compared with vector control. In contrast, these siRNAs did not affect expression of the pIRE-Luc. These data clearly indicate an important role for MLK3 in IFN-γ-induced transcription driven by GATE (Fig. 3G). The MLK proteins are expressed in a tissue-specific manner (23Gallo K.A. Johnson G.L. Nat. Rev. Mol. Cell. Biol. 2002; 3: 663-672Crossref PubMed Scopus (456) Google Scholar). MLK3 protein is expressed ubiquitously. The irf9 gene is induced by IFN-γ in most cell types, consistent with the ubiquitous expression of MLK3. In contrast, the expression of MLK2 is restricted to skeletal muscle, brain, and testis. To test the specificity of MLKs, we examined whether co-expression of MLK2 would also augment IFN-γ-induced transcription. For this purpose a GFP-tagged MLK2 protein was co-expressed with the P4-Luc reporter. GFP-MLK2 did not significantly augment the IFN-γ-induced expression of the luciferase gene when compared with the control, GFP expression vector alone (Fig. 3H). Expression of GFP-tagged MK2 was comparable in the control and IFN-γ-treated cells (Fig. 3I). IFN-γ Activates MLK3—To address the issue of whether endogenous MLK3 is activated, we stimulated RAW cells with IFN-γ for various lengths of time and prepared the cellular lysates. The protein extracts were subjected to Western blot analyses with antibodies that can specifically detect the native and diphosphorylated (Thr(P)277/Ser(P)281) forms of MLK3. Like the other members of the MAPK family, MLK3 is activated by phosphorylation at two critical threonine and serine (Thr277/Ser281) residues present in the activation loop (23Gallo K.A. Johnson G.L. Nat. Rev. Mol. Cell. Biol. 2002; 3: 663-672Crossref PubMed Scopus (456) Google Scholar). Following phosphorylation, MLK3 gains its catalytic activity. These analyses showed a time-dependent increase in MLK3 phosphorylation following treatment with IFN-γ (Fig. 4A). As early as 30 min after stimulation with IFN-γ a significant rise in MLK3 phosphorylation occurred compared with the control. Such activation continued to increase up to 6 h and declined thereafter. The kinetics of MLK3 activation is consistent with slow activation of the irf9 gene in response to IFN-γ. We next tested whether CEP blocked the IFN-γ-induced activation of MLK3 using the phosphospecific antibodies described above (Fig. 4B). Cells were exposed to IFN-γ in the absence and presence of CEP. As expected, IFN-γ induced the activation of MLK3 protein. Incubation of cells with CEP blocked such activation. These differences in the activation of MLK3 are not due to different levels of MLK3 protein. We could not measure the activation of other MLKs for the following reasons. 1) Unlike MLK3, the expression of MLK2 is restricted to very few tissues, and 2) no native- or activation-specific antibodies are available for the other isoforms. CEP Does Not Inhibit the ERK-induced Phosphorylation at Thr189 of C/EBP-β—We have shown earlier that ERK1/2 signals play an important role in regulating the irf9 gene (20Hu J. Roy S.K. Shapiro P.S. Rodig S.R. Reddy S.P. Platanias L.C. Schreiber R.D. Kalvakolanu D.V. J. Biol. Chem. 2001; 276: 287-297Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Therefore, we examined whether CEP interfered with ERK1/2 activation in response to IFN-γ. Western blot analysis with antibodies specific for diphosphorylated forms of ERK1/2 showed that ERK1/2 were activated equivalently by IFN-γ in the presence and absence of CEP (Fig. 5A). Analysis of total ERK2 levels revealed a comparable expression (Fig. 5B). Thus, CEP did not inhibit ERK activation by IFN-γ. Because our earlier studies identified an important role for the Thr189 residue (a target for ERK1/2-induced phosphorylation), located in the regulatory domain 2 of C/EBP-β, in IFN-γ-induced gene expression (20Hu J. Roy S.K. Shapiro P.S. Rodig S.R. Reddy S.P. Platanias L.C. Schreiber R.D. Kalvakolanu D.V. J. Biol. Chem. 2001; 276: 287-297Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 21Roy S.K. Hu J. Meng Q. Xia Y. Shapiro P.S. Reddy S.P. Platanias L.C. Lindner D.J. Johnson P.F. Pritchard C. Pages G. Pouyssegur J. Kalvakolanu D.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7945-7950Crossref PubMed Scopus (80) Google Scholar), we next examined whether CEP affected the IFN-γ-induced phosphorylation of C/EBP-β at Thr189. An antibody that detects the phospho-Thr189 form of C/EBP-β was used as a tool for detecting changes in ligand-induced phosphorylation. The specificity of anti-Thr(P)189 antibody is shown in Fig. 5C. In this experiment protein lysates from cebpb–/– cells transfected with wild type (188GTPS191) and a corresponding alanine-substituted mutant (188GAAA191) of C/EBP-β and stimulated with IFN-γ were used for a Western blot analysis with anti-Thr(P)189. A significant increase in IFN-γ-induced phosphorylation at Thr189 was observed with the wild type but not with the mutant protein. This difference was not due to differential expression of the wild type and mutant proteins (Fig. 5D). In the next experiment we examined whether CEP affected phosphorylation at Thr189 in RAW cells (Fig. 5E). In these studies U0126, a known inhibitor of ERK1/2, was used as an additional control. As expected, IFN-γ induced phosphorylation of Thr189, which was blocked by U0126. In contrast to U0126, CEP had no effect on this process. These inhibitors did not affect either the basal phosphorylation or the expression (Fig. 5F) of C/EBP-β. Thus, CEP appears to target another step involved in IFN-γ-induced CEBP-β activation. IFN-γ Negatively Regulates the Phosphorylation through a Serine Residue at the N Terminus of C/EBP-β—To further gain insight into the domain of C/EBP-β affected by CEP, we examined the sequence of C/EBP-β for potential phosphorylation sites. Recent studies (22Shuman J.D. Sebastian T. Kaldis P. Copeland T.D. Zhu S. Smart R.C. Johnson P.F. Mol. Cell. Biol. 2004; 24: 7380-7391Crossref PubMed Scopus (67) Google Scholar) identified a new phosphoacceptor site,
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