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• W2090757676 abstract "By presenting antigenic peptides on major histocompatibility complex class (MHC) II determinants to CD4+ T cells, macrophages help to direct the establishment of adaptive immunity. We found that in these cells, lipopolysaccharide stimulates the expression of MHC II genes via the activation of Erk1/2, which is mediated by Toll-like receptor 4. Erk1/2 then phosphorylates the serine at position 357, which is located in a degron of CIITA isoform 1 that leads to its monoubiquitylation. Thus modified, CIITA isoform 1 binds P-TEFb, which mediates the elongation of RNA polymerase II and co-transcriptional processing of nascent transcripts. This induction leads to the expression of MHC II genes. Subsequent polyubiquitylation results in the degradation of CIITA isoform 1. Thus, the signaling cascade from Toll-like receptor 4 to CIITA isoform 1 represents one connection between innate and adaptive immunity in macrophages. By presenting antigenic peptides on major histocompatibility complex class (MHC) II determinants to CD4+ T cells, macrophages help to direct the establishment of adaptive immunity. We found that in these cells, lipopolysaccharide stimulates the expression of MHC II genes via the activation of Erk1/2, which is mediated by Toll-like receptor 4. Erk1/2 then phosphorylates the serine at position 357, which is located in a degron of CIITA isoform 1 that leads to its monoubiquitylation. Thus modified, CIITA isoform 1 binds P-TEFb, which mediates the elongation of RNA polymerase II and co-transcriptional processing of nascent transcripts. This induction leads to the expression of MHC II genes. Subsequent polyubiquitylation results in the degradation of CIITA isoform 1. Thus, the signaling cascade from Toll-like receptor 4 to CIITA isoform 1 represents one connection between innate and adaptive immunity in macrophages. The immune system is composed of innate immunity, which performs the function of immune surveillance, and adaptive immunity, which eliminates non-self-antigens and creates the immune memory. Constituents of the former are antigen-presenting cells and of the latter are B and T cells. Because the establishment of adaptive immunity is dependent on innate immunity, appropriate interactions between them are indispensable for the normal function of the immune system. Macrophages are antigen-presenting cells that remove and digest invading pathogens as well as present antigenic peptides, thus directing the immune response against them. They sense the presence of these pathogens via their pathogen-associated molecular patterns, such as lipopolysaccharide (LPS), 2The abbreviations used are: LPS, lipopolysaccharide; MHC, major histocompatibility complex class; Erk, extracellular signal-regulated kinase; TLR, Toll-like receptor; MAPK, mitogen-activated protein kinase; JNK, Jun N-terminal kinase; CIITA, class II transactivator; IF, isoform; AAD, acidic activation domain; P-TEFb, positive transcription elongation factor b; CycT1, cyclin T1; MEK, MAPK/Erk kinase; CAT, chloramphenicol acetyltransferase; ChIP, chromatin immunoprecipitation; RT, reverse transcription; ALLN, N-acetyl-leucyl-leucyl-l-norleucinal. flagellin, etc., which is mediated by Toll-like receptors (TLRs) (1Iwasaki A. Medzhitov R. Nat. Immunol. 2004; 5: 987-995Crossref PubMed Scopus (3346) Google Scholar). For example, the binding between LPS and TLR4 triggers a signaling cascade that results in the activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular regulated kinase 1/2 (Erk1/2), and Jun kinases (JNK), as well as of nuclear factor κB (NF-κB) (2Barton G.M. Medzhitov R. Science. 2003; 300: 1524-1525Crossref PubMed Scopus (1057) Google Scholar). This signaling leads to the presentation of antigenic peptides in the groove of major histocompatibility complex class II (MHC II) molecules to CD4+ T cells and subsequently to the establishment of adaptive immunity (1Iwasaki A. Medzhitov R. Nat. Immunol. 2004; 5: 987-995Crossref PubMed Scopus (3346) Google Scholar). Because MHC II determinants are involved directly in the establishment of adaptive immunity, it is not surprising that their expression is tightly regulated. One of the levels of this regulation is at transcription and is mediated by the class II transactivator (CIITA). Transcription of CIITA can be initiated from three distinct promoters called PI, PIII, and PIV, which direct the synthesis of three isoforms (IF) of CIITA: IF1, IF3 and IF4 (3Muhlethaler-Mottet A. Otten L.A. Steimle V. Mach B. EMBO J. 1997; 16: 2851-2860Crossref PubMed Scopus (435) Google Scholar). Different isoforms are expressed following distinct stimuli in different cells. Whereas CIITA IF1 is expressed in macrophages and myeloid dendritic cells, CIITA IF3 is expressed in B cells and plasmacytoid dendritic cells (4LeibundGut-Landmann S. Waldburger J.M. Reis e Sousa C. Acha-Orbea H. Reith W. Nat. Immunol. 2004; 5: 899-908Crossref PubMed Scopus (114) Google Scholar). Only one study has been performed on CIITA IF1 (5Nickerson K. Sisk T.J. Inohara N. Yee C.S. Kennell J. Cho M.C. Yannie II, P.J. Nunez G. Chang C.H. J. Biol. Chem. 2001; 276: 19089-19093Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In contrast, CIITA IF3 has been studied in great detail (6Greer S.F. Harton J.A. Linhoff M.W. Janczak C.A. Ting J.P. Cressman D.E. J. Immunol. 2004; 173: 376-383Crossref PubMed Scopus (27) Google Scholar, 7Greer S.F. Zika E. Conti B. Zhu X.S. Ting J.P. Nat. Immunol. 2003; 4: 1074-1082Crossref PubMed Scopus (83) Google Scholar, 8Li G. Harton J.A. Zhu X. Ting J.P. Mol. Cell. Biol. 2001; 21: 4626-4635Crossref PubMed Scopus (56) Google Scholar, 9Schnappauf F. Hake S.B. Camacho Carvajal M.M. Bontron S. Lisowska-Grospierre B. Steimle V. Eur. J. Immunol. 2003; 33: 2337-2347Crossref PubMed Scopus (36) Google Scholar, 10Sisk T.J. Nickerson K. Kwok R.P. Chang C.H. Int. Immunol. 2003; 15: 1195-1205Crossref PubMed Scopus (37) Google Scholar, 11Spilianakis C. Papamatheakis J. Kretsovali A. Mol. Cell. Biol. 2000; 20: 8489-8498Crossref PubMed Scopus (134) Google Scholar, 12Tosi G. Jabrane-Ferrat N. Peterlin B.M. EMBO J. 2002; 21: 5467-5476Crossref PubMed Scopus (55) Google Scholar). CIITA isoforms do not bind to DNA. Rather they bind to the preformed enhanceosome on MHC II promoters, where they recruit general transcription factors (13Fontes J.D. Jabrane-Ferrat N. Toth C.R. Peterlin B.M. J. Exp. Med. 1996; 183: 2517-2521Crossref PubMed Scopus (41) Google Scholar, 14Fontes J.D. Jiang B. Peterlin B.M. Nucleic Acids Res. 1997; 25: 2522-2528Crossref PubMed Scopus (116) Google Scholar, 15Kanazawa S. Okamoto T. Peterlin B.M. Immunity. 2000; 12: 61-70Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 16Mahanta S.K. Scholl T. Yang F.C. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6324-6329Crossref PubMed Scopus (108) Google Scholar, 17Kohoutek J. Blazek D. Peterlin B.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 17349-17354Crossref PubMed Scopus (13) Google Scholar) as well as coactivators (11Spilianakis C. Papamatheakis J. Kretsovali A. Mol. Cell. Biol. 2000; 20: 8489-8498Crossref PubMed Scopus (134) Google Scholar, 18Fontes J.D. Kanazawa S. Jean D. Peterlin B.M. Mol. Cell. Biol. 1999; 19: 941-947Crossref PubMed Scopus (137) Google Scholar, 19Kretsovali A. Agalioti T. Spilianakis C. Tzortzakaki E. Merika M. Papamatheakis J. Mol. Cell. Biol. 1998; 18: 6777-6783Crossref PubMed Scopus (154) Google Scholar, 20Mudhasani R. Fontes J.D. Mol. Cell. Biol. 2002; 22: 5019-5026Crossref PubMed Scopus (47) Google Scholar, 21Zika E. Greer S.F. Zhu X.S. Ting J.P. Mol. Cell. Biol. 2003; 23: 3091-3102Crossref PubMed Scopus (84) Google Scholar) and function as transcriptional integrators. Transcription of eukaryotic genes is a highly coordinated process and is often regulated by post-translational modifications of activators. The most studied are post-translational modifications of proteins containing class II B or acidic activation domains (AADs). The phosphorylation of proteolytic signaling elements, called degrons, which are located in or near these AADs, leads to their monoubiquitylation and higher transcriptional activity. Subsequent polyubiquitylation finally results in their degradation (22Muratani M. Tansey W.P. Nat. Rev. Mol. Cell. Biol. 2003; 4: 192-201Crossref PubMed Scopus (676) Google Scholar). Indeed, by binding the positive transcription elongation factor b (P-TEFb), which is composed of cyclin T1 (CycT1) and cyclin-dependent kinase 9 (Cdk9), the monoubiquitylated VP16 protein increases the rates of elongation rather than initiation of transcription (23Kurosu T. Peterlin B.M. Curr. Biol. 2004; 14: 1112-1116Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Of note, cyclin-dependent kinase 9 phosphorylates the C-terminal domain of RNA polymerase II and the negative transcription elongation factor, which contains 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole-sensitivity inducing factor and negative elongation factor (24Sims III, R.J. Belotserkovskaya R. Reinberg D. Genes Dev. 2004; 18: 2437-2468Crossref PubMed Scopus (569) Google Scholar). These changes lead additionally to co-transcriptional processing of nascent mRNA species. LPS increases the levels of MHC II molecules on macrophages via an unknown mechanism (25Figueiredo F. Koerner T.J. Adams D.O. J. Immunol. 1989; 143: 3781-3786PubMed Google Scholar, 26Wentworth P.A. Ziegler H.K. J. Immunol. 1987; 138: 3167-3173PubMed Google Scholar). In this study, we found that TLR4, which binds LPS, directs several post-translational modifications of CIITA IF1. These modifications include its phosphorylation and monoubiquitylation. They lead to the binding between CIITA IF1 and P-TEFb, which increases the transcriptional activity of CIITA IF1 and thus the expression of MHC II genes. Subsequent polyubiquitylation results in a rapid degradation of CIITA IF1. Thus, these findings connect the innate and adaptive arms of the immune response. Animals, Cells, and Cell Culture—C57BL/10ScN mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and bred in our colony. HeLa, COS, and RAW 264.7 cells were maintained in Dulbecco's modified Eagle's medium, 5% fetal calf serum, and antibiotics. Bone marrow-derived macrophages were prepared and maintained as described previously (27Nishiya T. DeFranco A.L. J. Biol. Chem. 2004; 279: 19008-19017Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). LPS Re595 was obtained from Sigma. MEK1/2 inhibitor UO 126 was obtained from Promega (Madison, WI), and proteasome inhibitor ALLN was purchased from Calbiochem (La Jolla, CA). λ-Phosphatase was obtained from Sigma. Plasmid DNAs—Reporter plasmid pDRASCAT was described previously (12Tosi G. Jabrane-Ferrat N. Peterlin B.M. EMBO J. 2002; 21: 5467-5476Crossref PubMed Scopus (55) Google Scholar). Plasmid m:Ub was described previously (23Kurosu T. Peterlin B.M. Curr. Biol. 2004; 14: 1112-1116Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). To construct a plasmid m:CycT1, the coding sequence of CycT1 was cloned into the expression vector pEF-Myc between EcoRI and XbaI restriction sites. Plasmids f:CIITA1 and h:CIITA1 were gift from Dr. Ting (University of North Carolina) and Dr. Chang (Indiana University School of Medicine). To construct a plasmid coding for f:CIITA1(S357A), the f:CIITA1 plasmid was subjected to site-directed mutagenesis with the QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. To construct plasmids coding for the h:Ub.CIITA1 and h:Ub(K48,63R).CIITA1, the Ub and Ub(K48,63R) plasmids were digested with EcoRI and cloned into h:CIITA1. Murine TLR4 was cloned into the pMX-pie bicistronic retroviral vector, as described previously (Onishi 1996). All of the plasmids were verified by DNA sequencing. Immunoreagents—The monoclonal anti-CIITA (sc-13556), anti-Ub (sc-8017), and anti-Myc (9E10) antibodies, and the polyclonal anti-CycT1 (sc-8127), anti-CIITA (sc-9870 and sc-9869), and anti-Erk1/2 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal anti-phospho-Erk1/2 antibody was purchased from Cell Signaling Technology (Beverly, MA). The monoclonal anti-I-Aα (KH 118) antibody was obtained from Becton Dickinson (San Diego, CA). The monoclonal anti-FLAG M2 antibody and the anti-FLAG M2 beads were purchased from Sigma. Viral Production and Infection—Retroviruses were produced by triple transfection of HEK293T cells with retroviral constructs along with gag-pol and vesicular stomatitis virus G glycoprotein expression constructs (28Yee J.K. Miyanohara A. LaPorte P. Bouic K. Burns J.C. Friedmann T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9564-9568Crossref PubMed Scopus (444) Google Scholar). Bone marrow-derived macrophages were infected as described previously (27Nishiya T. DeFranco A.L. J. Biol. Chem. 2004; 279: 19008-19017Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Transient Transfection and CAT Assay—The cells were seeded into 100-mm-diameter Petri dishes ∼12 h prior to transfection. The cells were transfected with FuGene 6 reagent according to the manufacturer's instructions (Invitrogen). CAT enzymatic assays were performed as described (29Fujinaga K. Cujec T.P. Peng J. Garriga J. Price D.H. Grana X. Peterlin B.M. J. Virol. 1998; 72: 7154-7159Crossref PubMed Google Scholar). Fold transactivation represents the ratio between the CIITA-activated transcription and the activity of the reporter plasmid alone. Immunoprecipitation Assay and Western Blot Analysis—The cells were transfected with 2 μg of indicated plasmid vectors. About 18 h after transfection, the immunoprecipitations were performed as described previously (12Tosi G. Jabrane-Ferrat N. Peterlin B.M. EMBO J. 2002; 21: 5467-5476Crossref PubMed Scopus (55) Google Scholar). Precipitated proteins were resolved on SDS-PAGE and analyzed by immunoblotting with the appropriate antibody followed by horseradish peroxidase-conjugated secondary antibody. The blots were developed by chemiluminiscence assay from PerkinElmer Life Sciences. Chromatin Immunoprecipitation (ChIP) Assays, RT-PCR, and Quantitative Real-time PCR—ChIP assays were performed as described previously (30Jiang H. Zhang F. Kurosu T. Peterlin B.M. Mol. Cell. Biol. 2005; 25: 10675-10683Crossref PubMed Scopus (40) Google Scholar). RNA was extracted from RAW 264.7 cells using the TRIzol protocol from Invitrogen. RT-PCR was described previously (31Lin X. Irwin D. Kanazawa S. Huang L. Romeo J. Yen T.S. Peterlin B.M. J. Virol. 2003; 77: 8227-8236Crossref PubMed Scopus (77) Google Scholar). cDNAs were then amplified by primers described previously (32Takeuchi O. Sims T.N. Takei Y. Ramassar V. Famulski K.S. Halloran P.F. J. Am. Soc. Nephrol. 2003; 14: 2823-2832Crossref PubMed Scopus (11) Google Scholar). Quantitative PCR was performed by Stratagene Mx3005P quantitative real-time PCR system, according to the manufacturer's protocol. In Vitro Transcription and Translation and in Vitro Kinase Assay—f:CIITA1 and f:CIITA1(S357A) were expressed in vitro by using coupled rabbit reticulocyte lysate transcription and translation system from Promega (Madison, WI). In vitro kinase assay with Erk1/2 was performed according to the manufacturer's instructions (Upstate Biotechnology Inc., Lake Placid, NY). Pulse-Chase Analysis—18 h after transfection, COS cells were starved for 2 h in medium without cysteine and methionine. Radioactive labeling with [S35]cysteine and [S35]methionine was performed for 40 min. After the labeling, the cells were washed three times in phosphate-buffered saline and maintained in Dulbecco's modified Eagle's medium for indicated time periods. The cells were lysed for 45 min at 4 °C. The cell lysates were then subjected to immunoprecipitation. Precipitated proteins were resolved on SDS-PAGE and analyzed by radiography. LPS Increases the Expression of MHC II Determinants via TLR4 in Mouse Macrophages—Upon stimulation with LPS, macrophages increase the surface levels of MHC II molecules (1Iwasaki A. Medzhitov R. Nat. Immunol. 2004; 5: 987-995Crossref PubMed Scopus (3346) Google Scholar). Via TLR4, LPS also activates Erk1/2, JNK, and p38 MAPKs (2Barton G.M. Medzhitov R. Science. 2003; 300: 1524-1525Crossref PubMed Scopus (1057) Google Scholar). Thus, we asked two questions. First, does the expression of MHC II determinants depend on TLR4? Second, does this induction depend on any of these kinases? To answer the first question, we infected primary bone marrow-derived macrophages from TLR4–/– mice with TLR4 (Fig. 1A, lanes 3 and 4) or an empty vector as the control (Fig. 1A, lanes 1 and 2). Two days later, we stimulated these macrophages with LPS for 2 h (Fig. 1A, lanes 2 and 3) or left them untreated (Fig. 1A, lanes 1 and 4). To exclude a possible translocation of MHC II molecules from the cytoplasm to the cell surface, we examined their levels by Western blotting rather than by fluorescence-activated cell sorter. Indeed, LPS induced the expression of MHC II determinants only in macrophages that expressed TLR4 (12 h; Fig. 1A, top panel, compare lane 3 with lanes 1, 2, and 4), which correlated with the activation of Erk1/2 by 20 min (Fig. 1A, middle panel, compare lanes 1–4). Levels of Erk1/2 were comparable in all samples (Fig. 1A, bottom panel). These results indicate that LPS activates Erk1/2 and increases the expression of MHC II determinants in primary macrophages via TLR4. To answer the second question, we duplicated these experiments in RAW 264.7 cells, which are a mouse macrophage cell line. At identical time points, we determined levels of MHC II determinants (Fig. 1B, top panel, lanes 1 and 2). Indeed, LPS induced the expression of MHC II genes in RAW 264.7 cells equivalently to primary macrophages (Fig. 1B, top panel, compare lanes 1 and 2). In contrast, when these cells were treated with the MEK inhibitor UO 126 prior to the addition of LPS, the expression of MHC II determinants was abolished (Fig. 1B, top panel, compare lanes 2 and 3). These experiments were repeated with inhibitors of JNK and p38 MAPKs, but they had no effect on the induction of MHC II determinants by LPS (data not presented). UO 126 is a specific MEK1/2 inhibitor that prevents the phosphorylation and subsequent activation of Erk1/2. These findings were confirmed by Western blotting with anti-phospho-Erk1/2 antibodies (20 min; Fig. 1B, bottom panel, compare lanes 2 and 3). Moreover, as presented in supplemental Fig. S1 (top panel, lanes 1–6), the activation of Erk1/2 was detectable by 10 min and disappeared by 2 h upon the addition of LPS in primary cells. We conclude that the activation of Erk1/2 precedes and is required for the induction of MHC II determinants by LPS. To determine effects of LPS on MHC II transcription, we stimulated RAW 264.7 cells with LPS and measured levels of I-Aα transcripts starting at the zero time point. After 2 h, the cells were washed, and the medium was changed. We performed RT-PCR at the indicated time points (Fig. 1C, top panel). Initially, we did not detect MHC II transcripts in unstimulated cells (Fig. 1C, top panel, lane 1). They appeared 1 h and peaked 2 h after the addition of LPS (Fig. 1C, top panel, compare lanes 3 and 4). 4 h later, MHC II transcripts disappeared almost completely (Fig. 1C, top panel, lane 5). Importantly, the levels of actin mRNA, which we used as the internal control, were equivalent in all samples (Fig. 1C, bottom panel). Thus, LPS activates transiently Erk1/2, which is followed by the induction of MHC II transcription. Next, we wanted to determine whether the expression of MHC II genes depended on the de novo synthesis of CIITA. Because different isoforms of CIITA had been described in RAW 264.7 cells (33Giroux M. Schmidt M. Descoteaux A. J. Immunol. 2003; 171: 4187-4194Crossref PubMed Scopus (73) Google Scholar, 34Nikcevich K.M. Piskurich J.F. Hellendall R.P. Wang Y. Ting J.P. J. Neuroimmunol. 1999; 99: 195-204Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), we investigated first CIITA IF1, IF3, and IF4 transcripts by RT-PCR in the presence and absence of LPS in these cells. However, as presented in supplemental Fig. S2, only CIITA IF1 mRNA encodes a functional CIITA protein in RAW 264.7 cells. Moreover, CIITA IF1 was the only isoform that we detected by immunoprecipitation and subsequent Western blotting in these cells (see Fig. 3D, bottom panel). Next, we performed quantitative real time RT-PCR to quantify mRNA levels of CIITA IF1 in stimulated and unstimulated cells (Fig. 1D). Time points 0, 2, and 4 h correspond to samples from Fig. 1C (lanes 1, 4, and 5). Surprisingly, the stimulation with LPS for 2 h decreased levels of CIITA IF1 mRNA up to 7-fold, compared with those in unstimulated cells. Thus, we conclude that LPS induces the transcription of MHC II genes without a concomitant increase in CIITA IF1 transcripts in macrophages. Moreover, this stimulation depends on TLR4 and the activation of Erk1/2 and leads to a transient induction of transcription of MHC II genes. Erk1/2 Phosphorylates CIITA IF1 on the Serine at Position 357—Effects of LPS on levels of MHC class II determinants depend on the activation of Erk1/2. Moreover, CIITA IF1 directs the transcription of MHC II genes in macrophages. However, because the amounts of CIITA IF1 mRNA actually decreased (Fig. 1D), we hypothesized that Erk1/2 modifies the preformed CIITA IF1 for its increased transcriptional activity. When analyzed by gel electrophoresis, CIITA IF3 migrates as a double band, which has been attributed to its phosphorylation (6Greer S.F. Harton J.A. Linhoff M.W. Janczak C.A. Ting J.P. Cressman D.E. J. Immunol. 2004; 173: 376-383Crossref PubMed Scopus (27) Google Scholar, 10Sisk T.J. Nickerson K. Kwok R.P. Chang C.H. Int. Immunol. 2003; 15: 1195-1205Crossref PubMed Scopus (37) Google Scholar, 12Tosi G. Jabrane-Ferrat N. Peterlin B.M. EMBO J. 2002; 21: 5467-5476Crossref PubMed Scopus (55) Google Scholar). Moreover, treatment with λ-phosphatase and direct labeling revealed that the upper band represents the phosphorylated form of CIITA IF3 (12Tosi G. Jabrane-Ferrat N. Peterlin B.M. EMBO J. 2002; 21: 5467-5476Crossref PubMed Scopus (55) Google Scholar). Because it also appears as a double band, we wanted to determine whether CIITA IF1 is also modified by phosphorylation. Thus, we expressed the FLAG epitope-tagged CIITA IF1 protein (f:CIITA1) (Fig. 2A, lane 1) in COS cells. In these cells, Erk1/2 is active constitutively. Thus, they do not need the stimulation by LPS. An aliquot of cell lysates was incubated with λ-phosphatase, which removes phosphates from serines and threonines (Fig. 2A, lane 2). As expected, the upper f:CIITA1 band disappeared completely (Fig. 2A, compare lanes 1 and 2). We conclude that CIITA IF1 is also phosphorylated in cells. Because in Fig. 1 we showed that the increased expression of MHC II determinants after LPS stimulation depended on the activation of Erk1/2, we searched for putative Erk1/2 phosphorylation sites in CIITA IF1. Erk1/2 is a proline-directed kinase with the consensus phosphorylation sequence PX(S/T)P, where the serine or threonine is phosphorylated. Indeed, there is only one consensus Erk1/2 phosphorylation site in CIITA IF1, which contains a serine at position 357 (S357) (Fig. 2B). To determine whether this site is phosphorylated, we created the mutant f:CIITA1 protein, where the serine at position 357 was changed to alanine (f:CIITA1(S357A)). Next, we expressed both the wild type f:CIITA1 and the mutant f:CIITA1(S357A) proteins in COS cells (Fig. 2C). We made two observations. First, expression levels of the mutant f:CIITA1(S357A) protein were 4-fold higher than those of the f:CIITA1 protein (Fig. 2C, compare lanes 1 and 2). Second, the mutant f:CIITA1(S357A) protein migrated as a single lower band (Fig. 2C, compare lanes 1 and 2), which represents the nonphosphorylated form of CIITA IF1. Thus, we conclude that CIITA IF1 is phosphorylated on Ser357 in cells. Next, we wanted to determine whether Erk1/2 is involved in the phosphorylation of CIITA IF1 and whether the upper band of CIITA IF1 would diminish under the influence of UO 126. Again we expressed f:CIITA1 (Fig. 2D, lanes 1 and 2) and treated COS cells with UO 126 (Fig. 2D, lane 2) or the solvent as the control (Fig. 2D, lane 1). As expected, in the presence of UO 126, the upper band of f:CIITA1 disappeared (Fig. 2D, top panel, compare lanes 1 and 2), which correlated with the absence of phosphorylated Erk1/2 (Fig. 2D, bottom panel, compare lanes 1 and 2). This finding suggests that Erk1/2 is involved in the phosphorylation of CIITA IF1. To extend our findings, we determined whether Erk1/2 could phosphorylate CIITA IF1 in vitro and whether this phosphorylation depended on Ser357. Thus, we performed in vitro kinase assays with Erk1/2. Wild type f:CIITA1 and mutant f:CIITA1(S357A) proteins were transcribed and translated using the rabbit reticulocyte lysate in vitro (Fig. 2E). Aliquots of the reaction were incubated with Erk1/2 (Fig. 2E, lanes 2 and 4) or with the reaction buffer as the control (Fig. 2E, lanes 1 and 3). Indeed, as indicated by the appearance of the upper band, Erk1/2 phosphorylated the wild type f:CIITA1 protein (Fig. 2E, compare lanes 1 and 2). In contrast, Erk1/2 did not phosphorylate the mutant f:CIITA1(S357A) protein, which was confirmed by the absence of the upper band (Fig. 2E, compare lanes 3 and 4 with lane 2). Overall, we conclude that Erk1/2 phosphorylates Ser357 in CIITA IF1 in vitro and in vivo. CIITA IF1 Is Ubiquitylated in Cells—Thus far, we determined not only that Erk1/2 phosphorylates Ser357 in f:CIITA1, but we also observed that this phosphorylation affects the stability of f:CIITA1. Namely, expression levels of the mutant f:CIITA1(S357A) protein were higher than those of f:CIITA1 (Fig. 2C, compare lanes 1 and 2). These findings led us to hypothesize that the phosphorylation and degradation of CIITA IF1 could be connected via ubiquitin. To address this notion, we determined first whether different expression levels of f:CIITA1 and mutant f:CIITA1(S357A) proteins were due to differences in their rates of degradation. We expressed f:CIITA1 (Fig. 3A, top panel) and the mutant f:CIITA1(S357A) proteins (Fig. 3A, bottom panel) and performed pulse-chase analyses in COS cells. Indeed, the mutant f:CIITA1(S357A) protein had a half-life of ∼2 h, which was four times longer than that of f:CIITA1 (Fig. 3A, compare top and bottom panels). We conclude that the phosphorylation of CIITA IF1 leads to its degradation. Next, we wanted to determine whether CIITA IF1 was degraded via the proteasome. We expressed f:CIITA1 (Fig. 3B, lanes 1 and 2) and the mutant f:CIITA1(S357A) (Fig. 3B, lanes 3 and 4) proteins in COS cells. Transfected cells were treated with the proteasomal inhibitor ALLN for 6 h (Fig. 3B, lanes 2 and 4) and the solvent as the control (Fig. 3B, lanes 1 and 3). ALLN increased levels of only the phosphorylated form of f:CIITA1 ∼4-fold (Fig. 3B, lanes 1 and 2, compare top and bottom bands). It had almost no effect on levels of the mutant f:CIITA1(S357A) protein (Fig. 3B, compare lanes 3 and 4). Thus, we conclude that the phosphorylated CIITA IF1 is also degraded via the proteasome. To determine whether CIITA IF1 is ubiquitylated, we performed ubiquitylation assays in vivo. In COS cells, we co-expressed f:CIITA1 with the Myc epitope-tagged ubiquitin (m:Ub) (Fig. 3C, lane 2) or an empty plasmid vector as the control (Fig. 3C, lane 1). Indeed, in the presence of f:CIITA1 and m:Ub, there was a strong ubiquitylation ladder (Fig. 3C, lane 2). Because the mutant f:CIITA1(S357A) protein was more stable than f:CIITA1, we assumed that the ubiquitylation of f:CIITA1 was dependent on the phosphorylation of Ser357. Thus, we performed the ubiquitylation assay in vivo with the mutant f:CIITA1(S357A) protein (Fig. 3C, lanes 3 and 4). Indeed, by densitometry we determined that the mutant f:CIITA1(S357A) protein was ubiquitylated 4-fold less than f:CIITA1 (Fig. 3C, compare lanes 2 and 4). We conclude that CIITA IF1 is ubiquitylated, which is promoted by the phosphorylation of Ser357 in CIITA IF1. Finally, we asked whether the endogenous CIITA IF1 is also ubiquitylated in mouse macrophages. To address this question, we repeated ubiquitylation assays in RAW 264.7 cells (Fig. 3D). Because of low levels of expression or accelerated degradation, we were not able to detect CIITA IF1 or its ubiquitylation in untreated cells (Fig. 3D, lane 1) or cells treated with LPS and UO 126, respectively (data not presented). However, we were able to observe the heavily ubiquitylated CIITA IF1 only after treatment with LPS and ALLN (Fig. 3D, lane 2). We treated cells with ALLN for 2 h prior to 40 min of treatment with LPS. Afterward, the cells were incubated with ALLN for an additional 2 h. Interestingly, CIITA IF1 was present in phosphorylated and unphosphorylated forms (Fig. 3D, bottom panel, lane 2), which can be explained by transient activation of Erk1/2 by LPS (Fig. 1D). Thus, we conclude that the endogenous CIITA IF1 is phosphorylated and ubiquitylated in RAW 264.7 cells. In CIITA IF1, there is a predicted PEST sequence from positions 360 to 385 (Fig. 3E). The phosphorylation of these sequences decreases the stability of proteins (22Muratani M. Tansey W.P. Nat. Rev. Mol. Cell. Biol. 2003; 4: 192-201Crossref PubMed Scopus (676) Google Scholar, 35Yeh E. Cunningham M. Arnold H. Chasse D. Monteith T. Ivaldi G. Hahn W.C. Stukenberg P.T. Shenolikar S. Uchida T. Counter C.M. Nevins J.R. Means A.R. Sears R. Nat. Cell Biol. 2004; 6: 308-318Crossref PubMed Scopus (621) Google Scholar). Because the phosphorylation of Ser357, which is flanked by a proline, a serine, and an acidic residue, led to the degradation of CIITA IF1, we surmise that the actual PEST seq" @default.
• W2090757676 created "2016-06-24" @default.
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• W2090757676 title "Sequential Modifications in Class II Transactivator Isoform 1 Induced by Lipopolysaccharide Stimulate Major Histocompatibility Complex Class II Transcription in Macrophages" @default.
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