MAPKAP Kinase 3pK Phosphorylates and Regulates Chromatin Association of the Polycomb Group Protein Bmi1
2004; Elsevier BV; Volume: 280; Issue: 7 Linguagem: Inglês
10.1074/jbc.m407155200
ISSN1083-351X
AutoresJan Willem Voncken, Hanneke E.C. Niessen, Bernd Neufeld, Ulrike Rennefahrt, Vivian E.H. Dahlmans, Nard Kubben, Barbara Holzer, Stephan Ludwig, Ulf R. Rapp,
Tópico(s)RNA Research and Splicing
ResumoPolycomb group (PcG) proteins form chromatin-associated, transcriptionally repressive complexes, which are critically involved in the control of cell proliferation and differentiation. Although the mechanisms involved in PcG-mediated repression are beginning to unravel, little is known about the regulation of PcG function. We showed previously that PcG complexes are phosphorylated in vivo, which regulates their association with chromatin. The nature of the responsible PcG kinases remained unknown. Here we present the novel finding that the PcG protein Bmi1 is phosphorylated by 3pK (MAPKAP kinase 3), a convergence point downstream of activated ERK and p38 signaling pathways and implicated in differentiation and developmental processes. We identified 3pK as an interaction partner of PcG proteins, in vitro and in vivo, by yeast two-hybrid interaction and co-immunoprecipitation, respectively. Activation or overexpression of 3pK resulted in phosphorylation of Bmi1 and other PcG members and their dissociation from chromatin. Phosphorylation and subsequent chromatin dissociation of PcG complexes were expected to result in de-repression of targets. One such reported Bmi1 target is the Cdkn2a/INK4A locus. Cells overexpressing 3pK showed PcG complex/chromatin dissociation and concomitant de-repression of p14ARF, which was encoded by the Cdkn2a/INK4A locus. Thus, 3pK is a candidate regulator of phosphorylation-dependent PcG/chromatin interaction. We speculate that phosphorylation may not only affect chromatin association but, in addition, the function of individual complex members. Our findings linked for the first time MAPK signaling pathways to the Polycomb transcriptional memory system. This suggests a novel mechanism by which a silenced gene status can be modulated and implicates PcG-mediated repression as a dynamically controlled process. Polycomb group (PcG) proteins form chromatin-associated, transcriptionally repressive complexes, which are critically involved in the control of cell proliferation and differentiation. Although the mechanisms involved in PcG-mediated repression are beginning to unravel, little is known about the regulation of PcG function. We showed previously that PcG complexes are phosphorylated in vivo, which regulates their association with chromatin. The nature of the responsible PcG kinases remained unknown. Here we present the novel finding that the PcG protein Bmi1 is phosphorylated by 3pK (MAPKAP kinase 3), a convergence point downstream of activated ERK and p38 signaling pathways and implicated in differentiation and developmental processes. We identified 3pK as an interaction partner of PcG proteins, in vitro and in vivo, by yeast two-hybrid interaction and co-immunoprecipitation, respectively. Activation or overexpression of 3pK resulted in phosphorylation of Bmi1 and other PcG members and their dissociation from chromatin. Phosphorylation and subsequent chromatin dissociation of PcG complexes were expected to result in de-repression of targets. One such reported Bmi1 target is the Cdkn2a/INK4A locus. Cells overexpressing 3pK showed PcG complex/chromatin dissociation and concomitant de-repression of p14ARF, which was encoded by the Cdkn2a/INK4A locus. Thus, 3pK is a candidate regulator of phosphorylation-dependent PcG/chromatin interaction. We speculate that phosphorylation may not only affect chromatin association but, in addition, the function of individual complex members. Our findings linked for the first time MAPK signaling pathways to the Polycomb transcriptional memory system. This suggests a novel mechanism by which a silenced gene status can be modulated and implicates PcG-mediated repression as a dynamically controlled process. Members of the mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; PcG, Polycomb group; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; SAPK, stress-activated protein kinase; 3pK, MAPK-activated protein kinase 3 (MAPKAPK3); aa, amino acid; GST, glutathione S-transferase; FCS, fetal calf serum; GFP, green fluorescent protein; mAb, monoclonal antibody; pAb, polyclonal antibody; TPA, tetradecanoyl phorbol acetate; HA, hemagglutinin; HDII/SPM/SAM, homology domain II/sex combs on midleg, polyhomeotic, l(3)mbt/self-association motif; IRES, internal ribosomal entry site. family (ERK, JNK/SAPK, and p38 kinases) are implicated in many biological processes, such as adaptation to environmental changes, differentiation, immune activation, inflammatory responses, cell cycle modulation, transformation, and apoptosis (1Tibbles L.A. Woodgett J.R. Cell. Mol. Life Sci. 1999; 55: 1230-1254Crossref PubMed Scopus (555) Google Scholar). These phosphorylation cascades may function as switching mechanisms that modulate gene activity (2Hazzalin C.A. Mahadevan L.C. Nat. Rev. Mol. Cell Biol. 2002; 3: 30-40Crossref PubMed Scopus (342) Google Scholar). 3pK, also known as MAPK-activated protein kinase 3 (MAPKAPK3), belongs to a growing family of kinases that are activated by one or more members of the MAPK family (3Ludwig S. Engel K. Hoffmeyer A. Sithanandam G. Neufeld B. Palm D. Gaestel M. Rapp U.R. Mol. Cell. Biol. 1996; 16: 6687-6697Crossref PubMed Scopus (154) Google Scholar). To better understand processes regulated by 3pK, we aimed to identify interactors and potential novel substrates of 3pK. Here, we report the finding of a group of novel interaction partners of 3pK, the Polycomb group (PcG) proteins, including the mammalian PcG proteins HPH2 and Bmi1 (4Gunster M.J. Satijn D.P. Hamer K.M. den Blaauwen J.L. de Bruijn D. Alkema M.J. van Lohuizen M. van Driel R. Otte A.P. Mol. Cell. Biol. 1997; 17: 2326-2335Crossref PubMed Scopus (149) Google Scholar, 5Alkema M.J. Bronk M. Verhoeven E. Otte A. van't Veer L.J. Berns A. van Lohuizen M. Genes Dev. 1997; 11: 226-240Crossref PubMed Scopus (228) Google Scholar). PcG·protein complexes maintain a transcriptionally repressed gene status in dividing and differentiating cells throughout developing eukaryotes (6Paro R. Trends Genet. 1990; 6: 416-421Abstract Full Text PDF PubMed Scopus (321) Google Scholar, 7van Lohuizen M. Cell. Mol. Life Sci. 1998; 54: 71-79Crossref PubMed Scopus (77) Google Scholar, 8Simon J.A. Tamkun J.W. Curr. Opin. Genet. Dev. 2002; 12: 210-218Crossref PubMed Scopus (291) Google Scholar). The mechanisms that contribute to PcG-mediated silencing are beginning to unravel. PcG proteins form large multimeric protein complexes that associate with chromatin (reviewed in Refs. 9Orlando V. Paro R. Curr. Opin. Genet. 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Cell Biol. 2004; 16: 239-246Crossref PubMed Scopus (253) Google Scholar, 21Levine S.S. King I.F. Kingston R.E. Trends Biochem. Sci. 2004; 29: 478-485Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Little is known about how chromatin association and, potentially, function of PcG complexes is controlled. In the present study, we identified Polycomb group (PcG) proteins as in vivo interaction partners of the MAPKAP kinase 3pK. We have provided evidence that MAPK signal transduction cascades target PcG·protein complex/chromatin interaction through phosphorylation. 3pK acts as a Bmi1 kinase in vitro and in vivo. Of relevance, 3pK overexpression causes PcG/protein dissociation from chromatin and de-repression of the Cdkn2a/INK4A locus. Our data support a model in which Polycomb-mediated repression is modulated by stress- and mitogen-activated protein kinase cascades. These findings point to a molecular mechanism by which a transcriptionally silenced gene status can be reprogrammed and implicates PcG-mediated repression as a dynamically controlled process. These observations are expected to have important implications in understanding epigenetic mechanisms in development and disease. Plasmid Construction—Eukaryotic expression vectors for untagged and tagged wild type or mutant 3pK have been described elsewhere (3Ludwig S. Engel K. Hoffmeyer A. Sithanandam G. Neufeld B. Palm D. Gaestel M. Rapp U.R. Mol. Cell. Biol. 1996; 16: 6687-6697Crossref PubMed Scopus (154) Google Scholar, 22Neufeld B. Grosse-Wilde A. Hoffmeyer A. Jordan B.W. Chen P. Dinev D. Ludwig S. Rapp U.R. J. Biol. Chem. 2000; 275: 20239-20242Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The nucleus-retained 3pK mutant 3pkΔC was created by deleting the C-terminal (aa 308–383) regulatory region from Gal4DB-3pK. pGAD10-HPH2 (C-298 aa) and pGAD10-HPH2 (432 aa) were described elsewhere (4Gunster M.J. Satijn D.P. Hamer K.M. den Blaauwen J.L. de Bruijn D. Alkema M.J. van Lohuizen M. van Driel R. Otte A.P. Mol. Cell. Biol. 1997; 17: 2326-2335Crossref PubMed Scopus (149) Google Scholar). HDII/SPM/SAM domain mutations within the C-198 HPH2 construct were generated by PCR-directed deletion of the 3′-most sequences or by site-directed mutagenesis (QuikChange; Stratagene Europe, Amsterdam, The Netherlands) within the fifth C-terminal-most α-helix. MT2SM-HA-Bmi1 (mouse) was kindly provided by Dr. M. van Lohuizen (5Alkema M.J. Bronk M. Verhoeven E. Otte A. van't Veer L.J. Berns A. van Lohuizen M. Genes Dev. 1997; 11: 226-240Crossref PubMed Scopus (228) Google Scholar). Glutathione S-transferase (GST)-tagged 3pK (3Ludwig S. Engel K. Hoffmeyer A. Sithanandam G. Neufeld B. Palm D. Gaestel M. Rapp U.R. Mol. Cell. Biol. 1996; 16: 6687-6697Crossref PubMed Scopus (154) Google Scholar, 22Neufeld B. Grosse-Wilde A. Hoffmeyer A. Jordan B.W. Chen P. Dinev D. Ludwig S. Rapp U.R. J. Biol. Chem. 2000; 275: 20239-20242Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) was recloned into pBABE- or LZRS-based retroviral expression vectors (23Kinsella T.M. Nolan G.P. Hum. Gene Ther. 1996; 7: 1405-1413Crossref PubMed Scopus (672) Google Scholar, 24Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1903) Google Scholar). The murine ecotropic receptor was recloned from an original construct by Dr. J. M. Cunningham (Harvard Medical School, Boston, MA) and shared by Dr. A. Lund (Netherlands Cancer Institute, Amsterdam, The Netherlands). RNA interfering 3pK sequences 5′-CGGCAAAGTGTCTGGAGTGC-3′, 5′-GCAGCTGATCCGCCTCCTG-3′, and 5′-GGAGGAGATGACCAGTGCC-3′ (Fig. 5C, respectively, lanes 1–3) were cloned into stable hsRNA vectors described elsewhere (25Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3971) Google Scholar). Yeast Two-hybrid System—A kinase-inactive 3pK mutant (K73M) was used as bait in two-hybrid screens against a human heart MATCH-MAKER cDNA library cloned into the pGAD10 vector (Clontech). Yeast strain CG-1945 (Clontech) was manipulated according the manufacturer's instructions. Positive clones were monitored by growth on SD/-Trp/-Leu/-His plates and activity of the lacZ reporter gene in filter assays. In direct two-hybrid assays, reselected SD/-Trp/-Leu/-His filters were incubated for 12–24 h at 30 °C before the β-Gal assay was performed. The liquid culture β-Gal assay with o-nitrophenyl-β-d-galactopyranoside (Sigma) as substrate was performed according to the manufacturer's instructions using the yeast strain Y190. Cell Culture and Cell Cycle Synchronization—Indicated cell lines were cultured at 37 °C, 5% CO2, 100% humidity in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS), antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin), 200 mm l-glutamine and 4.5 g/liter l-glucose. The TIG3-Bmi1.2PY has been described elsewhere (26Voncken J.W. Schweizer D. Aagaard L. Sattler L. Jantsch M.F. van Lohuizen M. J. Cell Sci. 1999; 112: 4627-4639Crossref PubMed Google Scholar). Human U2OS osteosarcoma cells and TIG3 primary human fibroblasts expressing the murine ecotropic receptor were provided by Dr. D. Shvarts (Utrecht Medical Center, Utrecht, The Netherlands) and Dr. D. Peeper (Netherlands Cancer Institute, Amsterdam, The Netherlands), respectively. Titers of LZRS·IRES·GFP (Bmi1, 3pK, or control) viral supernatants were sufficiently high to achieve near 100% infection; infection with pBABE-GST (3pK or control) virus was followed by selection with puromycin. G0/G1 arrest of cells was established by contact inhibition and/or a 48-h serum starvation period (in Dulbecco's modified Eagle's medium supplemented with 0.1% FCS). M phase enrichment was achieved by adding colcemid (0.01 μg/ml) to S phase-synchronized cells, acquired by a double thymidine block (2 × 2 mm; 15–15 h) essentially as published previously (27Ishida R. Sato M. Narita T. Utsumi K. Nishimoto T. Morita T. Nagata H. Andoh T. J. Cell Biol. 1994; 126: 1341-1351Crossref PubMed Scopus (155) Google Scholar). M phase enrichment was accomplished by mitotic shake-off. Immunofluorescence—Immunofluorescence on methanol-fixed U2OS cells was carried out as described previously (26Voncken J.W. Schweizer D. Aagaard L. Sattler L. Jantsch M.F. van Lohuizen M. J. Cell Sci. 1999; 112: 4627-4639Crossref PubMed Google Scholar). For immunocytochemical detection of cell cycle inhibitors, cells were infected on glass slides (Superfrost; Menzel GmbH & Co. KG); fixation was accomplished by a 3–5-min incubation in 100% methanol at –20°C, or a 20-min 3.7% formaldehyde fixation at ambient temperature, followed by a 7-min 100% acetone treatment at –20°C, and subsequent washing and staining were carried out 3–5 days following infection. Polyclonal rabbit antiserum against recombinant 3pK and GST antisera were obtained from rabbits immunized with purified GST. The anti-Bmi1 mAb F6 and anti-MPh 1 (HPH1) pAb 70092 were kindly provided by Dr. M. van Lohuizen. Other antisera used were anti-2Py mAb (MMS-115R; Babco, Richmond, CA); anti-Bmi1 mAb (6C9; Bmi1O), anti-HPc2 mAb (M9), anti-Ring1A mAb (M3) kindly provided by Dr. A. Otte (28Hamer K.M. Sewalt R.G. den Blaauwen J.L. Hendrix T. Satijn D.P. Otte A.P. Hybrid. Hybridomics. 2002; 21: 245-252Crossref PubMed Scopus (29) Google Scholar); anti-Rnf2 (Ring1B) mAb (29Atsuta T. Fujimura S. Moriya H. Vidal M. Akasaka T. Koseki H. Hybridoma. 2001; 20: 43-46Crossref PubMed Scopus (54) Google Scholar); anti-dinG (Ring1B) pAb (Dr. M. Dyer, University of Leicester, Leicester, UK); anti-HPH1 mAb (Dr. H. Koseki, RIKEN Yokohama Institute, Yokohama, Japan), anti-Gsk3β pAb (catalog number 9332; Cell Signaling), anti-AKT pAb (catalog number 9272; Cell Signaling), anti-p14ARF/p16β pAb (Ab-1; LabVision Corp., Fremont, CA), anti-pSer10 pAb (Upstate Biotechnology, Lake Placid, NY), and anti-pSer28 pAb (rat) kindly provided by Dr. M. Inagaki (30Goto H. Tomono Y. Ajiro K. Kosako H. Fujita M. Sakurai M. Okawa K. Iwamatsu A. Okigaki T. Takahashi T. Inagaki M. J. Biol. Chem. 1999; 274: 25543-25549Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). Monoclonal primary antibodies were detected with goat-anti-mouse Texas Red polyclonal primary antisera with goat-anti-rabbit fluorescein isothiocyanate. Transfections and 3pK Activation—HEK293 and U2OS cells were transfected at 10–30% confluency. Transfections were done by standard calcium phosphate co-precipitation. Production of infectious viral particles was carried out as described previously (31Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3994) Google Scholar). Cells were starved in Dulbecco's modified Eagle's medium with 0.1% (U2OS) or 0.3% (HEK293) FCS for 48 h. For activation of 3pK, the cells were stimulated with 0.2–0.5 mm sodium-meta-arsenite (Sigma) or with 5 or 10% FCS with or without 100 ng/ml tetradecanoyl phorbol acetate (TPA) for 1 h before harvesting, unless indicated otherwise. Preincubation with kinase inhibitor UO126 (32Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2754) Google Scholar) or SB202190 (33Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Keys J.R. Land Vatter S.W. Strickler J.E. Mclaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3147) Google Scholar) was 20–30 min before activation of the cells. Immunoprecipitation and Differential Extraction—Cell lysates were prepared as described previously (3Ludwig S. Engel K. Hoffmeyer A. Sithanandam G. Neufeld B. Palm D. Gaestel M. Rapp U.R. Mol. Cell. Biol. 1996; 16: 6687-6697Crossref PubMed Scopus (154) Google Scholar, 5Alkema M.J. Bronk M. Verhoeven E. Otte A. van't Veer L.J. Berns A. van Lohuizen M. Genes Dev. 1997; 11: 226-240Crossref PubMed Scopus (228) Google Scholar). For immunoprecipitation, cells were extracted with ELB buffer (5Alkema M.J. Bronk M. Verhoeven E. Otte A. van't Veer L.J. Berns A. van Lohuizen M. Genes Dev. 1997; 11: 226-240Crossref PubMed Scopus (228) Google Scholar, 26Voncken J.W. Schweizer D. Aagaard L. Sattler L. Jantsch M.F. van Lohuizen M. J. Cell Sci. 1999; 112: 4627-4639Crossref PubMed Google Scholar); immune complexes were precipitated with protein A- or G-agarose. Differential extraction was carried out as described previously (34Muchardt C. Reyes J.C. Bourachot B. Leguoy E. Yaniv M. EMBO J. 1996; 15: 3394-3402Crossref PubMed Scopus (194) Google Scholar). Briefly, the cells were washed twice in phosphate-buffered saline and carefully suspended in 200 μlof phosphate-buffered saline supplemented with 1 mm NaVO4 and 50 mm NaF and extracted in IP0.1 buffer (60 mm KCl, 15 mm NaCl, 5 mm MgCl2, 0.1 mm EGTA, 15 mm Tris·HCl, pH 7.4, 300 mm sucrose, 1 mm NaVO4 and 50 mm NaF) supplemented with 0.3% non-ionic detergent Igepal (Nonidet P-40). The cells were incubated for 3 min at ambient temperature, gently pelleted, and the supernatant was collected (soluble fraction). The pellet was carefully washed once in nuclear buffer and extracted in IP0.1 buffer (20 mm HEPES, pH 7.6, 10% glycerol, 25 mm MgCl2, 0.1 mm EDTA, 0.2% Nonidet P-40, 0.1 m Potassium acetate, 1 mm NaVO4, 50 mm NaF; chromatin-bound fraction) as described previously (34Muchardt C. Reyes J.C. Bourachot B. Leguoy E. Yaniv M. EMBO J. 1996; 15: 3394-3402Crossref PubMed Scopus (194) Google Scholar). Protein extracts were analyzed by standard Western blot technology. Immune Complex Kinase Assay—GST·3pK and HA·Bmi1 immune complexes were immunoprecipitated separately and combined and washed twice in high salt radioimmune precipitation assay buffer (500 mm NaCl) and kinase buffer (10 mm MgCl2, 25mm β-glycerophosphate, 25 mm HEPES, pH 7.5, 5 mm benzamidine, 0.5 mm dithiothreitol, 1 mm NaVO4; (3Ludwig S. Engel K. Hoffmeyer A. Sithanandam G. Neufeld B. Palm D. Gaestel M. Rapp U.R. Mol. Cell. Biol. 1996; 16: 6687-6697Crossref PubMed Scopus (154) Google Scholar)). In control experiments, GST·3pK-containing immune complexes were incubated with Hsp27 as a substrate. The kinase assays were performed at 30 °C for 30–120 min in kinase buffer supplemented with 3–5 μCi [γ-32P]ATP and 0.02–0.1 mm cold ATP. In vitro kinase assays with recombinant protein utilized 1 μg of kinase (3pK-EE) and 5 μg of substrate (His- or GST-tagged Bmi1). 3pK Binds the C Terminus of HPH2 in Vitro—In two independent yeast two-hybrid screens using an inactive 3pK (K73M) variant as bait, 20 overlapping clones containing cDNA of the human Polyhomeotic protein (HPH2) were isolated (Fig. 1A). These clones represented 73, 145, 198, or 209 C-terminal amino acid fragments of HPH2 (Fig. 1A). In a direct yeast two-hybrid test, 3pK also interacted with a larger HPH2 C-terminal fragment of 298 aa and the putative full-length HPH2 (432 aa) (Fig. 1, A and B). The C-terminal 3pK interaction domain of HPH2 (Fig. 1A) overlaps with the α-helical HDII/SPM/SAM domain involved in hetero- and homotypic interactions between several Polycomb proteins (4Gunster M.J. Satijn D.P. Hamer K.M. den Blaauwen J.L. de Bruijn D. Alkema M.J. van Lohuizen M. van Driel R. Otte A.P. Mol. Cell. Biol. 1997; 17: 2326-2335Crossref PubMed Scopus (149) Google Scholar, 5Alkema M.J. Bronk M. Verhoeven E. Otte A. van't Veer L.J. Berns A. van Lohuizen M. Genes Dev. 1997; 11: 226-240Crossref PubMed Scopus (228) Google Scholar, 35Peterson A.J. Kyba M. Bornemann D. Morgan K. Brock H.W. Simon J. Mol. Cell. Biol. 1997; 17: 6683-6692Crossref PubMed Scopus (123) Google Scholar, 36Kyba M. Brock H.W. Dev. Genet. 1998; 22: 74-84Crossref PubMed Scopus (64) Google Scholar). Mutational analysis of the C-198 fragment was used to further investigate the role of the HDII domain in the HPH2/3pK interaction. The HDII domain contains at least five distinct α-helical (α1–α5) structures (37Kim C.A. Gingery M. Pilpa R.M. Bowie J.U. Nat. Struct. Biol. 2002; 9: 453-457PubMed Google Scholar). C-terminal deletions removing these α-helices prevent HPH2/3pK interaction (Fig. 1A, lower inset). Removal of the C-terminal-most helix (α5) is sufficient to completely abolish interaction. The requirement of an intact α5 was confirmed by the introduction of selected point mutations in α5 (Fig. 1A, asterisks on lower panel), which are predicted to be present on the surface of the protein and therefore may be required for the interaction with 3pK. Removing the basic side chain through exchange of K422A prevented interaction between HPH2 C-198 and 3pK completely, whereas exchange of L417A led to a reduction of protein association. 3pK (K73M) displays high affinity for the shorter HPH2 fragments, in particular for the C-terminal 73 fragment (Fig. 1A). This may relate to binding-site masking in full-length HPH2. Interestingly, mutation of known phosphorylation sites on 3pK (e.g. T313E, T313A, T201E/T313E) does not alter binding affinity as compared with the wild type kinase (not shown), suggesting that HPH2 binding is independent of Thr201/Thr313 phosphorylation. Thus, the HDII/SPM/SAM domain plays an important role in HPH2/3pK interaction. 3pK Complexes with PcG Proteins in Vivo—Interaction between 3pK and the individual HPH2 fragments in mammalian cells was confirmed by co-immunoprecipitation experiments (data not shown) with results consistent with the quantitative yeast two-hybrid data (Fig. 1A). The finding that 3pK is a binding partner of HPH2 suggests that the kinase may be part of a PcG complex and may also be associated with other PcG proteins. Biochemical analysis indeed confirms that HA-tagged Bmi1, a direct binding partner of HPH1/2 (4Gunster M.J. Satijn D.P. Hamer K.M. den Blaauwen J.L. de Bruijn D. Alkema M.J. van Lohuizen M. van Driel R. Otte A.P. Mol. Cell. Biol. 1997; 17: 2326-2335Crossref PubMed Scopus (149) Google Scholar, 5Alkema M.J. Bronk M. Verhoeven E. Otte A. van't Veer L.J. Berns A. van Lohuizen M. Genes Dev. 1997; 11: 226-240Crossref PubMed Scopus (228) Google Scholar), is specifically co-immunoprecipitated with GST-3pK and vice versa (Fig. 1B). In vivo, GST-3pK co-precipitates with endogenous PcG proteins, such as (h)Rnf2 (Fig. 1C), which is part of the hPRC1 complex (38Hemenway C.S. Halligan B.W. Levy L.S. Oncogene. 1998; 16: 2541-2547Crossref PubMed Scopus (58) Google Scholar, 39Suzuki M. Mizutani-Koseki Y. Fujimura Y. Miyagishima H. Kaneko T. Takada Y. Akasaka T. Tanzawa H. Takihara Y. Nakano M. Masumoto H. Vidal M. Isono K. Koseki H. Development (Camb.). 2002; 29: 4171-4183Google Scholar). Exposure of cells to a 3pK-activating agent, the stress-inducer arsenite (3Ludwig S. Engel K. Hoffmeyer A. Sithanandam G. Neufeld B. Palm D. Gaestel M. Rapp U.R. Mol. Cell. Biol. 1996; 16: 6687-6697Crossref PubMed Scopus (154) Google Scholar), does not alter the interaction quantitatively in these cells (not shown), again suggesting that phosphorylation of 3pK does not affect PcG association. This is in line with the comparable interaction of 3pK phosphorylation-site mutants with HPH2 described above. In a reciprocal experiment, Bmi1 was immunoprecipitated with an anti-3pK antiserum in both U2OS cell extracts (Fig. 1C) and HeLa cell extracts with high Bmi1 levels (Fig. 1D). Finally, we showed that endogenous proteins co-precipitate (Fig. 1E); this interaction was specific, because two unrelated kinases, AKT and Gsk3β, did not co-precipitate hPRC1 core members (Fig. 1E). These biochemical data demonstrated association of 3pK with PcG proteins in vivo, without the need for overexpressed binding partners. Mitogen- and Stress-induced Phosphorylation of Bmi1 in Vitro and in Vivo—3pK is a kinase activated by both stress stimuli and mitogenic signals. Activation by mitogens, such as serum and TPA, occurs almost exclusively via the ERK pathway, whereas stress stimulation with arsenite recruits the MAPKAPK to the p38 MAPK cascade (3Ludwig S. Engel K. Hoffmeyer A. Sithanandam G. Neufeld B. Palm D. Gaestel M. Rapp U.R. Mol. Cell. Biol. 1996; 16: 6687-6697Crossref PubMed Scopus (154) Google Scholar). Thus, we next examined whether PcG proteins may represent downstream targets of these MAP kinase cascades by applying the respective stimuli. We focused on Bmi1, because chromatin association of Bmi1 correlates with its phosphorylation status (26Voncken J.W. Schweizer D. Aagaard L. Sattler L. Jantsch M.F. van Lohuizen M. J. Cell Sci. 1999; 112: 4627-4639Crossref PubMed Google Scholar). ERK activation, via mitogenic stimulation of serum-starved cells, leads to a rapid and readily detectable phosphorylation of Bmi1 in vivo; activation of p38, via arsenite, induces a much stronger hyperphosphorylation of Bmi1 as compared with mitogenic stimulation (Fig. 2A). Importantly, serum starvation prior to activation in these experiments precludes cell cycle-dependent phosphorylation (26Voncken J.W. Schweizer D. Aagaard L. Sattler L. Jantsch M.F. van Lohuizen M. J. Cell Sci. 1999; 112: 4627-4639Crossref PubMed Google Scholar) from interfering with these assays. Phosphatase treatment of PcG proteins extracted from arsenite-exposed cells establishes phosphorylation as the main post-translational modification on PcG proteins in response to p38 activation (Fig. 2B). We then studied the effect of protein phosphorylation on PcG core complex/protein interaction. As indicated in Fig. 2C, interaction of known PcG core complex partners, such as HPH1 and HPc2 (14Levine S.S. Weiss A. Erdjument-Bromage H. Shao Z. Tempst P. Kingston R.E. Mol. Cell. Biol. 2002; 22: 6070-6078Crossref PubMed Scopus (318) Google Scholar), is at least in part preserved, as they still co-immunoprecipitate with Bmi1. Stress (Fig. 2D) or mitogenic (data not shown) stimulation result in Bmi1 phosphorylation in HeLa and 293T cells and, importantly, also in primary human TIG3 fibroblasts, showing that the observation is not cell-type restricted or linked to a specific cellular phenotype. Ground-state phosphorylation comparison in the different cell types also reveals that Bmi1 is already phosphorylated in some serum-starved established cell lines (Fig. 2, A and D). This would be consistent with a constitutive activation of MAP kinase signaling pathways in some cancer cell lines. Arsenite-induced Bmi1 pho
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