Nuclear Rho Kinase, ROCK2, Targets p300 Acetyltransferase
2006; Elsevier BV; Volume: 281; Issue: 22 Linguagem: Inglês
10.1074/jbc.m510954200
ISSN1083-351X
AutoresToru Tanaka, D. Nishimura, Ray‐Chang Wu, Mutsuki Amano, Tatsuya Iso, Larry Kedes, Hiroshi Nishida, Kozo Kaibuchi, Yasuo Hamamori,
Tópico(s)Cellular Mechanics and Interactions
ResumoRho-associated coiled-coil protein kinase (ROCK) is an effector for the small GTPase Rho and plays a pivotal role in diverse cellular activities, including cell adhesion, cytokinesis, and gene expression, primarily through an alteration of actin cytoskeleton dynamics. Here, we show that ROCK2 is localized in the nucleus and associates with p300 acetyltransferase both in vitro and in cells. Nuclear ROCK2 is present in a large protein complex and partially cofractionates with p300 by gel filtration analysis. By immunofluorescence, ROCK2 partially colocalizes with p300 in distinct insoluble nuclear structures. ROCK2 phosphorylates p300 in vitro, and nuclear-restricted expression of constitutively active ROCK2 induces p300 phosphorylation in cells. p300 acetyltransferase activity is dependent on its phosphorylation status in cells, and p300 phosphorylation by ROCK2 results in an increase in its acetyltransferase activity in vitro. These observations suggest that nucleus-localized ROCK2 targets p300 for phosphorylation to regulate its acetyltransferase activity. Rho-associated coiled-coil protein kinase (ROCK) is an effector for the small GTPase Rho and plays a pivotal role in diverse cellular activities, including cell adhesion, cytokinesis, and gene expression, primarily through an alteration of actin cytoskeleton dynamics. Here, we show that ROCK2 is localized in the nucleus and associates with p300 acetyltransferase both in vitro and in cells. Nuclear ROCK2 is present in a large protein complex and partially cofractionates with p300 by gel filtration analysis. By immunofluorescence, ROCK2 partially colocalizes with p300 in distinct insoluble nuclear structures. ROCK2 phosphorylates p300 in vitro, and nuclear-restricted expression of constitutively active ROCK2 induces p300 phosphorylation in cells. p300 acetyltransferase activity is dependent on its phosphorylation status in cells, and p300 phosphorylation by ROCK2 results in an increase in its acetyltransferase activity in vitro. These observations suggest that nucleus-localized ROCK2 targets p300 for phosphorylation to regulate its acetyltransferase activity. The Rho family of small GTPases, which includes Rho, Rac, and Cdc42, is ubiquitously expressed and plays a pivotal role in the regulation of a wide variety of cellular processes, including cell adhesion and migration, cytokinesis, cell cycle progression, and gene expression (1Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2101) Google Scholar, 2Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar, 3Kaibuchi K. Kuroda S. Amano M. Annu. Rev. 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ROCK2 phosphorylates p300 in vitro, and nucleus-restricted expression of a constitutively active ROCK2 mutant induces p300 phosphorylation in cells. We find that acetyltransferase activity of p300 is dependent on its phosphorylation status in cells, and p300 phosphorylation by ROCK2 causes an increased acetyltransferase activity in vitro. These observations suggest that nuclear ROCK2 associates with p300, phosphorylates p300, and enhances its acetyltransferase activity through a phosphorylation-dependent mechanism. Plasmids—The plasmids for GST-p300 fragments were kindly provided by Dr. Hottiger (University of Zurich, Zurich, Switzerland) (52Hasan S. Hassa P.O. Imhof R. Hottiger M.O. Nature. 2001; 410: 387-391Crossref PubMed Scopus (148) Google Scholar). The bovine ROCK2 (Rho kinase) mutants (14Amano M. Chihara K. Nakamura N. Fukata Y. Yano T. Shibata M. Ikebe M. Kaibuchi K. Genes Cells. 1998; 3: 177-188Crossref PubMed Scopus (220) Google Scholar) were subcloned into pcDNA3 vector for in vitro translation and mammalian expression. GST-p300 (CH3 domain), GST-ROCK2 (H-85) (bovine Rho kinase amino acids 775–860), and GST-ROCK1 (H-85) (human ROCK1 amino acids 755–840) plasmids were constructed by subcloning PCR products into pGEX vector (Amersham Biosciences). The GST-MBS construct was previously described (53Kawano Y. Fukata Y. Oshiro N. Amano M. Nakamura T. Ito M. Matsumura F. Inagaki M. Kaibuchi K. J. Cell Biol. 1999; 147: 1023-1038Crossref PubMed Scopus (477) Google Scholar). For nucleus-restricted expression of the ROCK2 catalytic domain, CMV-CAT-nls and CMV-CAT-KD-nls were constructed by subcloning bovine ROCK2 CAT (amino acids 6–553) and its kinase-deficient mutant CAT-KD (Lys121 → Gly mutation) (54Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (951) Google Scholar) into pCMV-Myc-nuc vector (Invitrogen), respectively. pCAG-Myc-ROCK1 and Gal-p300 vectors were generously provided by Dr. Narumiya (Kyoto University, Kyoto, Japan) (55Fujisawa K. Fujita A. Ishizaki T. Saito Y. Narumiya S. J. Biol. Chem. 1996; 271: 23022-23028Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) and Dr. Giordano (Temple University) (43Yuan W. Condorelli G. Caruso M. Felsani A. Giordano A. J. Biol. Chem. 1996; 271: 9009-9013Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar), respectively. Yeast Two-hybrid Screening—The yeast two-hybrid screening was performed using the Matchmaker two-hybrid system (Clontech) according to the manufacturer's protocol. The cDNA encoding the CH3 domain of human p300 (amino acids 1540–1930) was cloned in frame into the EcoRI-BamHI site of pGBT9. The cDNA libraries of whole mouse embryo (embryonic day 9.5 and 10.5) cloned into pVP16 vector are generous gifts from Dr. Stanley Hollenberg (Oregon Health Sciences University). Antibodies—Antibodies against ROCK2 (ROCK2 H-85), ROCK1 (H-85), p300 (N-15), Rho-GDI (A-20), histone H3 (FL136), lamin B (M-20), and Myc (9E10) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal p300 antibody (NM11) was from BD Biosciences. Normal rabbit IgG and β-tublin were from Sigma. Tissue Culture and Transient Transfection—HeLa, 293T, and U2OS cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Transfection was performed by the BES-buffered saline protocol as described (47Hamamori Y. Sartorelli V. Ogryzko V. Puri P.L. Wu H.Y. Wang J.Y. Nakatani Y. Kedes L. Cell. 1999; 96: 405-413Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Luciferase reporter gene assay was performed as described (47Hamamori Y. Sartorelli V. Ogryzko V. Puri P.L. Wu H.Y. Wang J.Y. Nakatani Y. Kedes L. Cell. 1999; 96: 405-413Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Preparation of Recombinant Protein and Protein-Protein Interaction Assays—GST fusion proteins were prepared in Escherichia coli (BL21-CodonPlus (DE3)-RP; Stratagene) by the method described (47Hamamori Y. Sartorelli V. Ogryzko V. Puri P.L. Wu H.Y. Wang J.Y. Nakatani Y. Kedes L. Cell. 1999; 96: 405-413Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). In vitro translated [35S]methionine-labeled proteins were prepared using the TNT coupled transcription-translation system (Promega). For GST-p300 pull-down experiments with endogenous ROCK2, whole cell extracts were prepared by incubating cells in the NETN buffer (20 mm Tris-HCl, pH 8, 100 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40) supplemented with freshly prepared protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 2 μg/ml pepstatin A, and aprotinin). Five hundred-microgram extracts were incubated with ∼100 ng of GST fusion proteins. For co-immunoprecipitation studies, 1 mg of whole cell extracts or nuclear extracts were incubated with 4 μgof either anti-p300 (N-15) or control normal mouse IgG overnight, followed by incubation of protein A-agarose (Sigma) for 2 h at 4 °C. After washing four times, bound proteins were detected by Western analysis with anti-ROCK2 antibody (H-85). Immunofluorescence Analysis—Cells were fixed with methanol or 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min and were permeabilized for 10 min in HBS-PBS (5% horse serum, 1% bovine serum albumin and 0.1% saponin in PBS). After three washings with PBS, the cells were incubated with primary antibodies for 1 h at room temperature and then with Cy3- or fluorescein isothiocyanate-conjugated secondary antibodies (Sigma) in HBS-PBS for 1 h at room temperature with vigorous washings after incubation with each antibody. DAPI staining was performed to show chromatin DNA. To test the specificity of ROCK2 (H-85) and ROCK1 (H-85) antibodies, an excess of GST-ROCK2-H85 or GST-ROCK1-H85 protein (40-fold excess over the antibodies in molar ratio) was preincubated with the primary antibodies. Subcellular fractionation for immunofluorescence studies was performed as described (56Stenoien D.L. Patel K. Mancini M.G. Dutertre M. Smith C.L. O'Malley B.W. Mancini M.A. Nat. Cell Biol. 2001; 3: 15-23Crossref PubMed Scopus (341) Google Scholar). Briefly, cells were incubated with CSK buffer (10 mm PIPES, pH 6.8, 300 mm sucrose, 100 mm NaCl, 3 mm MgCl2, 1 mm EGTA, 0.5% Triton X-100, and the proteinase inhibitors) for 3 min on ice to give insoluble chromatin plus nuclear matrix fractions. Chromatin was removed by digesting genomic DNA with RNase-free DNase I (400 units/ml; Roche Applied Science) in the digestion buffer (CSK plus 50 mm NaCl and proteinase inhibitors) for 1 h at 37 °C. Cells were further treated with ammonium sulfate to a final concentration of 0.25 m for 5 min at room temperature and incubated with 2 m NaCl for 5 min at room temperature, with washings with the digestion buffer after each treatment, to give nuclear matrix. Cells were stained after each treatment, using polyclonal rabbit anti-ROCK2 (H-85) and mouse monoclonal p300 (NM11) antibodies. Cells were examined using an Axioplan2 microscope (Carl Zeiss Inc.). Subcellular Fractionation—Subcellular fractionation was performed as described (53Kawano Y. Fukata Y. Oshiro N. Amano M. Nakamura T. Ito M. Matsumura F. Inagaki M. Kaibuchi K. J. Cell Biol. 1999; 147: 1023-1038Crossref PubMed Scopus (477) Google Scholar, 57Reyes J.C. Muchardt C. Yaniv M. J. Cell Biol. 1997; 137: 263-274Crossref PubMed Scopus (200) Google Scholar) with modifications. Cells were homogenized in buffer A (10 mm Tris-HCl, pH 7.4, 1.5 mm MgCl2, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 0.1% Triton X-100, and the proteinase inhibitors) with a Dounce homogenizer. The homogenates were loaded onto 1 m sucrose (final concentration at 270 mm) and centrifuged at 2,000 × g for 10 min. The precipitates contained nuclei. The supernatant fraction was further centrifuged at 20,000 × g for 20 min, and the resulting supernatant was used as cytoplasmic fraction. The precipitates containing nuclei were resuspended in buffer A and centrifuged at 2,000 × g for 5 min. The pellet that contains almost only nuclei under phase-contrast microscopy was resuspended in CSK buffer, and the soluble nucleoplasmic proteins were extracted by centrifugation at 20,000 × g for 20 min. The pellet that contained a mixture of insoluble chromatin and nuclear matrix was incubated in digestion buffer containing RNase-free DNase I and the proteinase inhibitors for 1 h at 37 °C. Then 1 m ammonium sulfate was added to the sample to a final concentration of 0.25 m for 5 min on ice. After centrifugation at 2,000 × g for 10 min, the supernatant was used as a chromatin fraction. The pellet was further incubated with 2 m NaCl in digestion buffer for 10 min on ice and then centrifuged. The resulting pellet was solubilized in the urea buffer (10 mm Tris-HCl, pH 8.0, 8 m urea, 100 mm sodium phosphate, and the proteinase inhibitors) and used as the nuclear matrix fraction. Protein concentration was determined by the Bradford method (Bio-Rad). Phosphorylation Assay—Phosphorylation assay was performed as described (54Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (951) Google Scholar) with some modifications. For the immunocomplex kinase assay, endogenous ROCK2 was immunoprecipitated with 2 μg of anti-ROCK2 antibody (H-85) or control IgG from 1 mg of the cytoplasmic or nucleoplasmic fraction of HeLa cells. After washing twice in NETN buffer and twice in 1× kinase buffer (50 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 1 mm EDTA, 1 mm EGTA), the immunoprecipitates were preincubated with or without 10 μm Y27632 for 10 min at room temperature. A kinase reaction was performed in the presence of 500 ng of GST-MBS-CT (C terminus of MBS) (53Kawano Y. Fukata Y. Oshiro N. Amano M. Nakamura T. Ito M. Matsumura F. Inagaki M. Kaibuchi K. J. Cell Biol. 1999; 147: 1023-1038Crossref PubMed Scopus (477) Google Scholar) as a substrate and 1 μCi of [γ-32P]ATP (6000 Ci/mmol; Amersham Biosciences) for the indicated time at 30 °C. Reaction mixtures were resolved by SDS-PAGE, dried, and exposed to x-ray film. To test kinase activity of exogenously expressed CAT-nls, 293T cells were transiently transfected with expression vectors for Myc-tagged CAT-nls or empty vector (pCMV-Myc-nuc), and whole cell extracts were subjected to immunoprecipitation with anti-Myc antibody, followed by the kinase reaction as described above. Gel Filtration Chromatography—Gel filtration of HeLa nuclear extracts was performed using Superose 6, HR 10/30 column (Amersham Biosciences) as described (58Wu R.C. Qin J. Hashimoto Y. Wong J. Xu J. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. Biol. 2002; 22: 3549-3561Crossref PubMed Scopus (238) Google Scholar). Trichloroacetic acid protein precipitation was performed following fractionation, and the protein samples were separated by SDS-PAGE and analyzed by Western blot with anti-ROCK2 (H-85) and -p300 (N-15) antibodies by reprobing the same membrane. Molecular weight was estimated using the molecular weight marker kit for gel chromatography (Sigma) as well as reprobing the membrane with other reference antibodies, including the N-CoR (1.5–2 MDa) (59Yoon H.G. Chan D.W. Reynolds A.B. Qin J. Wong J. Mol. Cell. 2003; 12: 723-734Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar) and SRC-1 (∼600 kDa) (58Wu R.C. Qin J. Hashimoto Y. Wong J. Xu J. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. Biol. 2002; 22: 3549-3561Crossref PubMed Scopus (238) Google Scholar) (not shown). The signals were quantified using Densitometer and ImageQuant version 5.2 (Amersham Biosciences). Nuclear extracts prepared by two different methods, one with CSK buffer (57Reyes J.C. Muchardt C. Yaniv M. J. Cell Biol. 1997; 137: 263-274Crossr
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