Regulation of Histone Deacetylase 2 by Protein Kinase CK2
2002; Elsevier BV; Volume: 277; Issue: 35 Linguagem: Inglês
10.1074/jbc.m204149200
ISSN1083-351X
Autores Tópico(s)Epigenetics and DNA Methylation
ResumoHistone deacetylase 2 (HDAC2) is a member of a large family of enzymes that alter gene expression by catalyzing the removal of acetyl groups from core histones. Originally isolated as a transcriptional co-repressor, HDAC2 possesses extensive amino acid sequence homology to HDAC1 (the founding member and most extensively studied HDAC enzyme). Because of this high degree of sequence similarity between HDAC1 and HDAC2, coupled with the fact that the two always co-exist in the same complexes, it is difficult to assess whether different properties exist between these two proteins. We report here that HDAC2 is a phosphoprotein similar to HDAC1. In addition, like HDAC1, the phospho-acceptor sites in HDAC2 are located in the C-terminal portion of the protein. However, unlike HDAC1, which can be phosphorylated by protein kinase CK2, cAMP-dependent protein kinase, and protein kinase G, HDAC2 is phosphorylated uniquely by protein kinase CK2 in vitro. Studies using unfractionated cell extracts with CK2 inhibitors suggest that protein kinase CK2 is the major source of HDAC2 kinase. Finally, and perhaps most interesting, HDAC2 phosphorylation promotes enzymatic activity, selectively regulates complex formation, but has no effect on transcriptional repression. Together, our data indicate that like many HDACs, HDAC2 is regulated by post-translational modification, particularly phosphorylation. Furthermore, we demonstrate for the first time that there are similarities and differences in the regulation of HDAC1 and HDAC2 by phosphorylation. Histone deacetylase 2 (HDAC2) is a member of a large family of enzymes that alter gene expression by catalyzing the removal of acetyl groups from core histones. Originally isolated as a transcriptional co-repressor, HDAC2 possesses extensive amino acid sequence homology to HDAC1 (the founding member and most extensively studied HDAC enzyme). Because of this high degree of sequence similarity between HDAC1 and HDAC2, coupled with the fact that the two always co-exist in the same complexes, it is difficult to assess whether different properties exist between these two proteins. We report here that HDAC2 is a phosphoprotein similar to HDAC1. In addition, like HDAC1, the phospho-acceptor sites in HDAC2 are located in the C-terminal portion of the protein. However, unlike HDAC1, which can be phosphorylated by protein kinase CK2, cAMP-dependent protein kinase, and protein kinase G, HDAC2 is phosphorylated uniquely by protein kinase CK2 in vitro. Studies using unfractionated cell extracts with CK2 inhibitors suggest that protein kinase CK2 is the major source of HDAC2 kinase. Finally, and perhaps most interesting, HDAC2 phosphorylation promotes enzymatic activity, selectively regulates complex formation, but has no effect on transcriptional repression. Together, our data indicate that like many HDACs, HDAC2 is regulated by post-translational modification, particularly phosphorylation. Furthermore, we demonstrate for the first time that there are similarities and differences in the regulation of HDAC1 and HDAC2 by phosphorylation. histone deacetylase glutathione S-transferase cAMP-dependent protein kinase protein kinase C protein kinase G Dulbecco's modified Eagle's medium In eukaryotes, DNA is tightly bound to histones, forming repeating units of DNA-protein particles called nucleosomes. Each nucleosome contains a nucleosomal core particle, consisting of 146 bp of supercoiled DNA wrapped twice around a complex of eight histone molecules. The histone core complex consists of two molecules each of histones H2A, H2B, H3, and H4. Linker DNA of variable length connects the core particles to one another. The four core histones can undergo different post-translational modifications as follows: acetylation, phosphorylation, methylation, ADP-ribosylation, and ubiquitination (1Isenberg I. Annu. Rev. Biochem. 1979; 48: 159-191Crossref PubMed Scopus (463) Google Scholar). Many thousands of different combinations of histone modification are possible, providing an abundance of regulatory potential. In fact, it was proposed that the combinatorial nature of histone modification may form a "histone code" that is read by other proteins to bring about distinct downstream events (2Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7709) Google Scholar, 3Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6679) Google Scholar). The existence of this code would considerably extend the information potential of the genetic code. All core histones undergo postsynthetic acetylation of one or more lysine residues in the N-terminal third of the molecule, and numerous studies suggest that acetylation of histones correlates with gene expression. Although supporting experimental evidence is still lacking, it is reasonable to assume that acetylation of core histones can weaken their interaction with DNA. Recent evidence is also accumulating in support of a model in which acetylation/deacetylation, in addition to affecting the intrinsic folding properties of nucleosomal arrays, generates specific docking surfaces for proteins that, in turn, regulate chromatin folding and/or transcription. For instance, unacetylated tails may perhaps provide interaction sites for transcriptional repressors, whereas the acetylated tails may provide interaction sites for activating complexes (4Dhalluin C. Carlson J.E. Zeng L., He, C. Aggarwal A.K. Zhou M.M. Nature. 1999; 399: 491-496Crossref PubMed Scopus (1320) Google Scholar, 5Jacobson R.H. Ladurner A.G. King D.S. Tjian R. Science. 2000; 288: 1422-1425Crossref PubMed Scopus (680) Google Scholar). In addition to its effect on transcription and chromatin assembly, acetylation/deacetylation of histones may have important roles in many cellular processes including DNA replication and repair, recombination, and chromosome segregation. Comprehensive knowledge of the mechanisms regulating histone acetylation and deacetylation is a definite prerequisite for elucidation of the potential histone code and eventually understanding the complex mechanisms of gene regulation in eukaryotic cells. The first histone acetyltransferase enzyme was identified inTetrahymena and was found to possess a high degree of amino acid sequence similarity to the yeast transcriptional adapter GCN5 (6Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1292) Google Scholar). Subsequently, over 20 proteins, including some very well characterized transcription activators and co-activators, were found to contain histone acetylating activities (reviewed in Refs. 7Shelley B.L. Grant P.A. Workman J.L. Allis C.D. Elgin S.C.R. Workman J.L. Chromatin Structure and Gene Expression. Oxford University Press, New York2000: 135-155Google Scholar, 8Sterner D.E. Berger S.L. Microbiol. Mol. Biol. Rev. 2000; 64: 435-459Crossref PubMed Scopus (1412) Google Scholar, 9Marmorstein R. Roth S.Y. Curr. Opin. Genet. & Dev. 2001; 11: 155-161Crossref PubMed Scopus (318) Google Scholar, 10Roth S.Y. Denu J.M. Allis C.D. Annu. Rev. Biochem. 2001; 70: 81-120Crossref PubMed Scopus (1624) Google Scholar). Each of these histone acetyltransferases may have a particular histone substrate specificity, and different histone acetyltransferases are specific with regard to which histone amino acids they will acetylate. Moreover, some "histone" acetyltransferases have a wide range of protein substrates in addition to histones. Equally swift and significant advances have been made in the last 6 years toward identification of histone deacetylase (HDAC)1 enzymes. The first HDAC, HDAC1, was purified and cloned by Schreiber and colleagues (11Taunton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1542) Google Scholar) using a trapoxin affinity matrix. Sequence analysis revealed that HDAC1 is related to the yeast protein RPD3. At the same time, a second human histone deacetylase protein, HDAC2, also with high homology to yeast RPD3, was identified in our laboratory based on a yeast two-hybrid screen with the YY1 transcription factor as bait (12Yang W.M. Inouye C. Zeng Y. Bearss D. Seto E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12845-12850Crossref PubMed Scopus (485) Google Scholar). Human HDAC1 is highly homologous to the human HDAC2 protein with 75% identity in DNA sequence and 85% identity in protein sequence. HDAC1 and HDAC2 exist together in at least three distinct multiprotein complexes called the Sin3, the NuRD/NRD/Mi2, and the CoREST complexes (13Ayer D.E. Trends Cell Biol. 1999; 9: 193-198Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 14Hassig C.A. Fleischer T.C. Billin A.N. Schreiber S.L. Ayer D.E. Cell. 1997; 89: 341-347Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar, 15Humphrey G.W. Wang Y. Russanova V.R. Hirai T. Qin J. Nakatani Y. Howard B.H. J. Biol. Chem. 2001; 276: 6817-6824Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 16Laherty C.D. Yang W.M. Sun J.M. Davie J.R. Seto E. Eisenman R.N. Cell. 1997; 89: 349-356Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar, 17Ng H.H. Bird A. Trends Biochem. Sci. 2000; 25: 121-126Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 18Tong J.K. Hassig C.A. Schnitzler G.R. Kingston R.E. Schreiber S.L. Nature. 1998; 395: 917-921Crossref PubMed Scopus (552) Google Scholar, 19You A. Tong J.K. Grozinger C.M. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1454-1458Crossref PubMed Scopus (396) Google Scholar, 20Zhang Y. Iratni R. Erdjument-Bromage H. Tempst P. Reinberg D. Cell. 1997; 89: 357-364Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, 21Zhang Y. LeRoy G. Seelig H.-P. Lane W.S. Reinberg D. Cell. 1998; 95: 279-289Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar). In addition, many transcription factors interact directly with HDAC1/2 and thus may target HDAC1/2 to specific promoters (17Ng H.H. Bird A. Trends Biochem. Sci. 2000; 25: 121-126Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 22Cress W.D. Seto E. J. Cell. Physiol. 2000; 184: 1-16Crossref PubMed Scopus (579) Google Scholar). Detailed analyses of the HDAC1/2 complexes revealed an unprecedented connection between deacetylation, DNA methylation, and chromatin remodeling. Since the initial discovery of HDAC1 and HDAC2, many additional HDACs have been identified in various species including human, mouse, chicken, Xenopus, Drosophila, Caenorhabditis elegans, yeast, and maize. In humans and in mice, HDACs are divided into three categories (22Cress W.D. Seto E. J. Cell. Physiol. 2000; 184: 1-16Crossref PubMed Scopus (579) Google Scholar, 23Gray S.G. Ekstrom T.J. Exp. Cell Res. 2001; 262: 75-83Crossref PubMed Scopus (497) Google Scholar, 24Khochbin S. Kao H. FEBS Lett. 2001; 494: 141-144Crossref PubMed Scopus (31) Google Scholar, 25Khochbin S. Verdel A. Lemercier C. Seigneurin-Berny D. Curr. Opin. Genet. & Dev. 2001; 11: 162-166Crossref PubMed Scopus (334) Google Scholar): the class I RPD3-like proteins (HDAC1, HDAC2, HDAC3, and HDAC8); the class II HDA1-like proteins (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10); and the class III SIR2-like proteins (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7). The class III proteins do not show any sequence resemblance to class I/II HDACs and are unique in that their activity requires the cofactor NAD. Many observations suggest that the function of the class II HDACs is tightly regulated by phosphorylation. For example, the 14-3-3 proteins negatively regulate the actions of HDAC4 by excluding it from the nucleus (26Grozinger C.M. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7835-7840Crossref PubMed Scopus (505) Google Scholar, 27Wang A.H. Kruhlak M.J., Wu, J. Bertos N.R. Vezmar M. Posner B.I. Bazett-Jones D.P. Yang X.J. Mol. Cell. Biol. 2000; 20: 6904-6912Crossref PubMed Scopus (230) Google Scholar), and calcium/calmodulin-dependent protein kinase signaling induces nuclear export of HDAC4 and HDAC5 by phosphorylating these proteins (28McKinsey T.A. Zhang C.L., Lu, J. Olson E.N. Nature. 2000; 408: 106-111Crossref PubMed Scopus (881) Google Scholar). In contrast, activation of the Ras-mitogen-activated protein kinase pathway by expression of Ras or constitutively active mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 increases the nuclear:cytoplasmic ratio of HDAC4 (29Zhou X. Richon V.M. Wang A.H. Yang X.J. Rifkind R.A. Marks P.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14329-14333Crossref PubMed Scopus (106) Google Scholar). Recently, it was reported that like many class II HDACs, HDAC1 (the prototype HDAC) is also a phosphoprotein (30Pflum M.K. Tong J.K. Lane W.S. Schreiber S.L. J. Biol. Chem. 2001; 276: 47733-47741Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar,31Cai R. Kwon P. Yan-Neale Y. Sambuccetti L. Fischer D. Cohen D. Biochem. Biophys. Res. Commun. 2001; 283: 445-453Crossref PubMed Scopus (56) Google Scholar). Because HDAC2 typically co-exists in the same protein complexes as HDAC1 and plays a crucial role in gene regulation, a complete picture of how phosphorylation may regulate HDACs requires that HDAC2 kinases be identified and the functional consequences of HDAC2 phosphorylation clearly determined. We report that HDAC2 is phosphorylated at the C-terminal portion of the protein exclusively at serine residues. Mutational analysis revealed that Ser394 and potentially Ser411, Ser422, and Ser424 of HDAC2 are phosphorylated in vivo. Furthermore, we found that the protein kinase, CK2, is responsible for phosphorylation of HDAC2. Our data also indicate that phosphorylation of HDAC2 is more critical for complex formation with mSin3 and Mi2 than with HDAC1. Finally, our results suggest that phosphorylation of HDAC2 is essential for enzymatic activity but not for transcriptional repression. 2 × 105 HeLa cells were cultured in 60-mm tissue culture dishes in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. pBJ5-HD1-F (11Taunton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1542) Google Scholar), pME18S-HDAC2 (32Yang W.M. Yao Y.L. Sun J.M. Davie J.R. Seto E. J. Biol. Chem. 1997; 272: 28001-28007Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar), pCS2-MT- mSin3(16), pGal4-HDAC2 (32Yang W.M. Yao Y.L. Sun J.M. Davie J.R. Seto E. J. Biol. Chem. 1997; 272: 28001-28007Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar), pGEX-HDAC3 (32Yang W.M. Yao Y.L. Sun J.M. Davie J.R. Seto E. J. Biol. Chem. 1997; 272: 28001-28007Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar), pGEX4T-3-HDAC8 (33Yang W.M. Tsai S.C. Wen Y.D. Fejer G. Seto E. J. Biol. Chem. 2002; 277: 9447-9454Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), pGST-HDAC1 (32Yang W.M. Yao Y.L. Sun J.M. Davie J.R. Seto E. J. Biol. Chem. 1997; 272: 28001-28007Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar), and pGST-H2B (34Verreault A. Kaufman P.D. Kobayashi R. Stillman B. Curr. Biol. 1998; 8: 96-108Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar) have been described. pGEX4T-1-HDAC2, pCEP4FLAG-HDAC8, pGal4E1B-luc, and plasmids that express different FLAG-tagged or Gal4-fused HDAC2 mutants were constructed by standard PCR and recombinant DNA methods. All constructs were verified by automated DNA sequencing analysis. Polyclonal anti-HDAC2, anti-HDAC1, and anti-mSin3 have been described (16Laherty C.D. Yang W.M. Sun J.M. Davie J.R. Seto E. Eisenman R.N. Cell. 1997; 89: 349-356Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar, 35Tsai S.C. Valkov N. Yang W.M. Gump J. Sullivan D. Seto E. Nat. Genet. 2000; 26: 349-353Crossref PubMed Scopus (134) Google Scholar). Anti-FLAG M2 and anti-MYC antibodies were obtained from Sigma. Anti-Mi2 antibody was obtained from Santa Cruz Biotechnology. For immunoprecipitations, cells were rinsed with ice-cold Tris-buffered saline and lysed in 1 ml of either a modified RIPA buffer containing protease and phosphatase inhibitors (1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 0.15 m NaCl, 0.05m Tris (pH 7.5), 2 mm EDTA, 0.05 mNaF, 0.2 mm Na3VO4, 0.5 mm phenylmethylsulfonyl fluoride) or in a low stringency buffer (phosphate-buffered saline containing 0.1% Nonidet P-40). After incubation for 20 min at 4 °C, the lysates were clarified by centrifugation at 4 °C for 10 min in a microcentrifuge at maximum speed (14,000 × g), and primary antibodies were added to the resultant supernatants. After incubation for 1 h at 4 °C, immunocomplexes were collected, washed four times with the buffer used for lysis, and applied to 8% SDS-polyacrylamide gels. HeLa cells were washed once with phosphate-free DMEM and incubated in phosphate-free DMEM containing 10% dialyzed fetal calf serum and 2 mCi of [32P]orthophosphate for 4 h. For experiments that involved the labeling of FLAG-HDAC2 proteins in vivo, expression plasmids were transfected into cells using a standard calcium phosphate co-precipitation method. Cells were harvested 48 h after transfection. Phosphoamino acid analysis was performed as described (36Blume-Jensen P. Hunter T. Methods Mol. Biol. 2001; 124: 49-65PubMed Google Scholar). Briefly, 32P-labeled HDAC2 was transferred onto a polyvinylidene difluoride membrane. The transferred product was visualized by autoradiography, excised from the membrane, and hydrolyzed in 200 μl of 6 n HCl at 110 °C for 60 min. After centrifugation for 1 min at 14,000 × g, the hydrolysate was dried under vacuum and resuspended in 5 μl of buffer I (0.58 m formic acid, 1.36 m glacial acetic acid (pH 1.9)) plus a mixture of phosphoamino acid standards (1 μl of a mixture of phosphoserine, phosphothreonine, and phosphotyrosine at 1 mg/ml each). Approximately 3,000 cpm of the hydrolysate was spotted on a 20-cm2, 100-μm-thick glass-backed cellulose thin layer chromatography plate (EM Science). The first dimension was resolved by electrophoresis at 1,500 V for 25 min in buffer I at 16 °C. Electrophoresis of the second dimension was carried out at 1,300 V for 20 min in buffer II (0.87 m glacial acetic acid, 0.5% pyridine, 0.5 mm EDTA (pH 3.5)). After being dried, plates were sprayed with 0.25% (w/v) ninhydrin in acetone and developed at 65 °C for 10 min to visualize the phosphoamino acid standards. Autoradiography was performed to visualize labeled HDAC2 fragments. Two μg of GST fusion proteins were incubated with either PKA (New England Biolab), PKC (Roche Molecular Biochemicals), PKG (Calbiochem), or CK2 (New England Biolab) in the presence of 5 μCi of [γ-32P]ATP, 100 μmATP, and manufacturer supplied kinase buffers in a total volume of 20 μl for 30 min at 30 °C. The reactions were terminated by the addition of 2× SDS loading buffer and boiled for 5 min. Proteins were separated on 8% SDS-polyacrylamide gels, and phosphorylated proteins were visualized by autoradiography. For analytical phosphorylation reactions, total cell extracts prepared from HeLa cells and 1 μg of purified FLAG-HDAC2 were incubated in a total volume of 20 μl at 30 °C for 30 min with recombinant CK2 and 5 μCi of [γ-32P]ATP (or [γ-32P]GTP) in CK2 buffer (25 mm Tris/HCl (pH 8.5), 10 mmMgCl2, 1 mm dithiothreitol). The kinase reactions were stopped by the addition of 2× sample loading buffer, boiled, centrifuged, and analyzed in SDS-polyacrylamide gels followed by autoradiography. CK2 inhibitors apigenin (chrysin) and 6-dichloro-1-d-ribofuranosylbenzimidazole were obtained from Sigma. Proteins were transferred from SDS-polyacrylamide gels onto polyvinylidene difluoride membranes. After blocking with 4% nonfat dried milk, the membranes were treated with diluted primary antibodies followed by 1:7500 diluted alkaline phosphatase-conjugated rabbit anti-mouse IgG (Promega). Subsequently, the blots were developed with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (Promega). 2 × 105 HeLa cells were seeded in 60-mm culture dishes 18 h before transfection. Plasmids directing the synthesis of various effector proteins and luciferase reporters were introduced into cells using a standard calcium phosphate co-precipitation method. Each transfection contained 5 μg each of effector and reporter DNAs, and all transfections were normalized to equal amounts of DNA with parental expression vectors. Forty eight hours after transfection, cells were collected, and luciferase activity was determined with the Dual Luciferase Reporter Assay System (Promega). [3H]Acetate-incorporated histones were isolated from butyrate-treated HeLa cells by acid extraction as described (37Carmen A.A. Rundlett S.E. Grunstein M. J. Biol. Chem. 1996; 271: 15837-15844Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Briefly, 5,000 cpm of purified core histones were incubated with immunoprecipitates in 150 μl of ice-cold HD buffer (20 mm Tris (pH 8.0), 150 mm NaCl, and 10% glycerol) at room temperature overnight with mild shaking. The reaction was terminated by the addition of an equal volume of stop solution (0.16 m acetic acid, 1.0 m HCl) and mixed well by vortexing. The released [3H]acetate was extracted with ethyl acetate and combined with scintillation mixture for analysis. Searches for protein kinase recognition motifs in HDAC1/2 were done using the PhosphoBase 2.0, a data base of phosphorylation sites at www.cbs.dtu.dk/databases/phosphobase. To determine whether HDAC2 is subject to phosphorylation modification, we prepared an extract from metabolically 32P-labeled HeLa cells, immunoprecipitated the endogenous HDAC2 protein, and resolved the product on an SDS-polyacrylamide gel. As a negative control, a mock immunoprecipitation was carried out side-by-side with the same labeled extract. As shown in Fig. 1A, HDAC2 is radioactively labeled by inorganic phosphate indicating that the protein is constitutively phosphorylated in vivo. By using partial acid hydrolysis in HCl followed by two-dimensional thin layer electrophoresis of the labeled phosphoamino acid, the presence of phosphoserine but not phosphothreonine or phosphotyrosine was unambiguously identified in HDAC2 (Fig. 1B). To identify the exact phosphorylation sites on HDAC2, we transfected HeLa cells with FLAG-tagged full-length wild-type HDAC2 or various C-terminal deletion mutants, repeated the in vivo labeling, and immunoprecipitated the FLAG fusion proteins with an anti-FLAG antibody for analysis on SDS-polyacrylamide gel. Fragments corresponding to residues 1–420 and 1–400 of HDAC2 were phosphorylated to similar levels compared with the full-length HDAC2-(1–488) (Fig. 2A, compare lane 1 to lanes 2 and 3; Fig.2B). A fragment containing residues 1–380 of HDAC2 (lane 4), however, clearly was not phosphorylated in vivo indicating that at least one phosphorylation site exists at 380–400 of HDAC2. Sequence analysis reveals that the only potential phosphorylation site located between 380 and 400 of HDAC2 is Ser394 (Fig.2C). To confirm that Ser394 is indeed a phosphorylation site, we tested the ability of the mutant 1–398 (S394A) to be phosphorylated. Our results showed that 1–398 (S394A) cannot be phosphorylated in vivo (Fig 2A, lanes 5 and 7; Fig. 2B), unequivocally demonstrating that Ser394 of HDAC2 is a phosphoacceptor site. To determine whether additional phosphoserines are present in HDAC2, we repeated the transfection, labeling, and immunoprecipitation with a full-length HDAC2 expression construct mutated in Ser394. As shown in Fig. 2A (lanes 8 and 12) and Fig. 2B, 1–488 (S394A) was phosphorylated in vivo, arguing for the existence of additional phosphorylation site(s). Besides Ser394, four additional conserved serine residues (Ser407, Ser411, Ser422, and Ser424) are present between 380 and 488 of HDAC2 (Fig.2C). Further analysis showed that 1–414 (S394A/S411A) was not labeled and 1–488 (S394A/S411A/S422A/S424A) was labeled very modestly in the presence of [γ-32P]ATP indicating that Ser407 is not a phosphorylation site in HDAC2, and any combination of Ser394 with Ser411, Ser422, or Ser424 could be phosphorylated (Fig.2A, lanes 9 and 13; Fig.2B). A careful inspection of the amino acid sequence of HDAC2 revealed that whereas Ser394, Ser422, and Ser424 all lie within protein kinase CK2 recognition motifs, Ser411could potentially be phosphorylated by PKA and PKC (Table I). To determine whether CK2, PKA, or PKC were indeed the kinases for HDAC2, we first tested the ability of purified CK2 to phosphorylate HDAC2 in vitro. As presented in Fig. 3A, GST-HDAC2 was readily phosphorylated by CK2 in vitro(lane 3). For comparison, we examined the other class I HDACs, and, interestingly, GST-HDAC1 and GST-HDAC3 but not GST-HDAC8 were phosphorylated in a similar fashion (Fig. 3A, lanes 2, 4, and 5).Table IPotential phosphorylation sites in HDAC1/2KinaseConsensusPotential sites in HDAC1Potential sites in HDAC2CK2X(S/T)XX(D/E)393421394423422445424460480465 PKARX1–2(S/T)X406407434411445 PKCX(S/T)X(R/K)410407411 PKG(R/K)2–3X(S/T)X406407434454 Open table in a new tab Intriguingly, we found that PKA does not phosphorylate GST-HDAC2 or GST-HDAC3 in vitro, although it efficiently phosphorylated GST-HDAC1 and GST-HDAC8 (Fig. 2B). Furthermore, PKC did not phosphorylate any class I HDACs under the same conditions (data not shown). In addition to consensus sequences for CK2, PKA, and PKC, residues 380–488 of HDAC2 contains recognition motifs for PKG in Ser407 and Thr454. As predicted, GST-HDAC2 was not phosphorylated by PKG in vitro (Fig.3C, lane 3) confirming that Ser407 is not a phosphorylation site in vivo (Fig. 2A) and that HDAC2 is phosphorylated exclusively at serine residues (Fig.1B). Like GST-HDAC2, GST-HDAC3 and GST-HDAC8 were not substrates for PKG (Fig. 3C, lanes 4 and 5). However, unlike GST-HDAC2, GST-HDAC1 is phosphorylated by PKG (lane 2). Further experiments using FLAG-HDAC2 and FLAG-(1–488) (S394A/S422A/S424A) expressed and purified from bacteria conclusively confirmed that HDAC2 is a substrate for CK2 in vitro (Fig. 3D). To confirm that CK2 is the major protein kinase and not just one of many kinases that phosphorylate HDAC2, we used a total HeLa cell lysate to phosphorylate purified HDAC2 in the presence or absence of specific CK2 inhibitors, apigenin, and 6-dichloro-1-d-ribofuranosylbenzimidazole. Results presented in Fig. 4, A and B, clearly showed that phosphorylation of HDAC2 was markedly reduced in the presence of CK2 inhibitors arguing that CK2 is the major and perhaps sole kinase responsible for phosphorylation of HDAC2. One of the unique features of CK2 among eukaryotic protein kinases is that CK2 can use both ATP and GTP as phosphoryl donors. Consistent with the observation that CK2 is the major kinase for HDAC2, we found that phosphorylation of HDAC2 by a total cell lysate was equally efficient using either ATP or GTP as phosphoryl donors (Fig.4B). HDAC2 was originally identified as a transcriptional co-repressor (12Yang W.M. Inouye C. Zeng Y. Bearss D. Seto E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12845-12850Crossref PubMed Scopus (485) Google Scholar). Early studies showed that a Gal4-HDAC2 fusion repressed transcription when targeted to promoters containing Gal4-binding sites (12Yang W.M. Inouye C. Zeng Y. Bearss D. Seto E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12845-12850Crossref PubMed Scopus (485) Google Scholar). Our findings that HDAC2 is modified by phosphorylation raise the important question of whether phosphorylation modulates the transcriptional activity of HDAC2 in the cell. To address this issue, we constructed plasmids that expressed Gal4-(1–488) (S394A) and Gal4-(1–488) (S394A/S411A/S422A/S424A), and we transfected them into cells together with a reporter containing Gal4-binding sites. Surprisingly, both of these Gal4 fusion mutant proteins repress transcription similar to wild-type Gal4-HDAC2 suggesting that phosphorylation of HDAC2 is not important for transcriptional repression (Fig.5A). Consistent with this observation, a Gal4-(1–380) fusion protein that can no longer be phosphorylated in vivo retained full repression activity (Fig. 5A). To determine whether phosphorylation influences HDAC2 enzymatic activity, we expressed various FLAG-HDAC2 mutants, immunoprecipitated the proteins with anti-FLAG antibody, and tested their abilities to deacetylate core histones. As shown in Fig. 5B, histone deacetylase activity of HDAC2 is strictly correlated with its ability to be phosphorylated in vivo. 1–488 (S394A), which is phosphorylated slightly less than wild-type HDAC2-(1–488), had slightly less enzymatic activity. 1–488 (S394A/S411A/S422A/S424A), which is severely impaired in phosphorylation in vivo, had ∼30% deacetylase activity compared with wild-type HDAC2. An HDAC2 mutant that was totally not phosphorylated in vivo, 1–380, did not possess any enzymatic activity. Taken together, our data suggest that although phosphorylation of HDAC2 has no role in transcriptional repression, it is absolutely critical for enzymatic activity. HDAC2, together with HDAC1, exists in two major complexes called the Sin3 complex and the NuRD/NRD/Mi2 complex. To determine whether phosphorylation of HDAC2 changes the ability of protein to interact with other proteins, we compared the amount of co-immunoprecipitated HDAC1, mSin3, and Mi2 from nuclear extracts prepared from cells that expressed wild-type or mutant HDAC2. In multiple experiments, we repeatedly found that compared with wild-type HDAC2, a significant (although somewhat smaller) fraction of endogenous HDAC1 coprecipitated with 1–488 (S394A) and 1–488 (S394A/S411A/S422A/S424A), as detected via Western blot analysis (Fig.6A, c
Referência(s)