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First published online 3 May 2005
doi: 10.1242/jcs.02373

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MAP kinase-mediated phosphorylation of distinct pools of histone H3 at S10 or S28 via mitogen- and stress-activated kinase 1/2

¹ Nuclear Signalling Laboratory, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK
² Aichi Cancer Centre Research Instititute, 1-1 Kanokoden, Chikusa-ku, Nagoya, Aichi 46 8681, Japan
³ MRC Protein Phosphorylation Unit, School of Life Sciences, Dundee, DD1 5EH, UK
⁴ Chromatin and Gene Expression, Institute of Biomedical Research, University of Birmingham, Vincent Drive, Edgbaston, Birmingham, B15 2TT, UK
⁵ The Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, OX3 9DS, UK

View larger version (69K): [in a new window]   Fig. 1. Rapid induction and TSA-hypersensitivity of histone H3 phosphorylated at S28. (A) Quiescent C3H 10T fibroblasts were treated with 50 ng/ml EGF (lanes 2-6), 25 ng/ml anisomycin (sAn) (lanes 7-11), 100 nM TPA (lanes 12-16) for the times indicated. Acid-soluble proteins were run on 15% SDS-PAGE and transferred to PVDF membrane for western blotting with anti-phosphoS28-H3 (panel i), anti-phosphoacetyl-H3 (panel ii) or anti-phosphoS10-H3occ (panel iii) antibodies, or Coomassie-stained (panel iv). (B) Quiescent C3H 10T fibroblasts were treated with TSA (500 ng/ml, 1 hour, lanes 4-6) or not (lanes 1-3) before stimulating them with TPA (30 minutes, 100 nM, lanes 2, 5), sAn (45 minutes, 25 ng/ml, lanes 3, 6) or not at all (con, lanes 1, 4). Acid-soluble proteins were run on 15% acid-urea gels and transferred to PVDF membrane for western blotting with anti-phosphoS28-H3 (panel i), anti-phosphoacetyl-H3 (panel ii), anti-phosphoS10-H3occ. (panel iii) or anti-phosphoS10-H3 (panel iv), or Coomassie-stained (panel v). Positions of differentially-modified H3 are indicated by bars (and numbered where space permits) according to increasing modification by comparison with Coomassie-stained gels or Ponceau-S-stained PVDF membrane. (C) Quiescent C3H 10T fibroblasts were treated with TSA (500 ng/ml, 1 hour, lanes 5-8) or not (lanes 1-4) before stimulating them with with EGF (30 minutes, 50 ng/ml, lanes 2, 5), TPA (30 minutes, 100 nM, lanes 3, 6), sAn (45 minutes, 25 ng/ml, lanes 4, 8), or not at all (lanes 1, 5). Acid-soluble proteins were separated by 15% SDS-PAGE and transferred to PVDF membrane for western blotting with anti-phosphoS28-H3 (panel i), anti-phosphoacetyl-H3 (panel ii), anti-phosphoS10occ. (panel iii) or anti-phosphoS10-H3 (panel iv), or Coomassie-stained (panel v).

View larger version (33K): [in a new window]   Fig. 2. In vitro phosphorylation of histone H3 S10 and S28 by MSK1. (A) [32P]-ATP kinase assays were performed with acid extracts from quiescent C3H 10T cells (lanes 1, 2), recombinant Drosophila H3 (lanes 3, 4), recombinant reconstituted Drosophila octamers (lanes 5, 6) or without substrate (lane 7) in the presence (lanes 2, 4, 6, 7) or absence (lanes 1, 3, 5) of recombinant MSK1 as described in Materials and Methods. Proteins were separated on 15% SDS-PAGE and Coomassie-stained (panel ii). Phosphorylated protein bands were detected by autoradiography (panel i). (B) Kinase assays were performed with acid extracts from quiescent C3H 10T cells (lanes 1, 2), recombinant Drosophila H3 (lanes 3, 4), recombinant reconstituted Drosophila octamers (lanes 5, 6) or without substrate (lane 7) in the presence (lanes 2, 4, 6, 7) or absence (lanes 1, 3, 5) of recombinant MSK1 as described in Materials and Methods. Proteins were separated on 15% SDS-PAGE and transferred to a PVDF membrane for western blotting using anti-phosphoS10-H3 (panel i) or anti-phosphoS28-H3 (panel ii), or Coomassie-stained (panel iii). (C) Kinase assays were performed using either recombinant Drosophila H3 (lanes 1-4), or recombinant reconstituted Drosophila octamers (lanes 5-8). Aliquots were removed after 0, 15, 30 and 45 minutes. Proteins were run on 15% acid-urea gels. Gels were transferred to PVDF membrane for western blotting with anti-phosphoS28-H3 (panel i) or anti-phosphoS10-H3 (panel ii), or were Coomassie-stained (panel iii).

View larger version (46K): [in a new window]   Fig. 3. S10 and S28 phosphorylation are targeted to distinct pools of histone H3. (A) Crosslinked chromatin was prepared from quiescent C3H 10T fibroblasts which were stimulated with 25 ng/ml anisomycin for 30 minutes (even-numbered lanes) or left untreated (odd-numbered lanes). Chromatin was immunodepleted with anti-phosphoS10-H3occ (lanes 5, 6), anti-phosphoacetyl-H3 (lanes 7, 8) or anti-phosphoS28-H3 (lanes 11, 12), or was mock immunoprecipitated in the absence of antibody (lanes 3, 4). Immunodepleted material and corresponding undepleted (input) samples (lanes 1, 2 and 9, 10) were analysed on 15% SDS-PAGE gels and western blotted with anti-phosphoS10-H3occ (panel i), anti-phosphoacetyl-H3 (panel ii) or anti-phosphoS28-H3 (panel iii). Before blotting, membranes were stained with Ponceau-S to show that no overall loss of bulk histones occurred by immunoprecipitation (data not shown), consistent with phosphorylation being targeted to a small fraction of total H3. (B) Quiescent C3H 10T fibroblasts were stimulated with 25 ng/ml anisomycin for 45 minutes (panels iv-ix) or left untreated (panels i-iii). Cells were fixed and histone H3 phospho-epitopes were detected by immunofluorescence as described in Materials and Methods. Primary antibodies used were: anti-phosphoS10-H3 (panels i, iv, vii) and anti-phosphoS28-H3 (panels ii, v, viii). Anti-rabbit-IgG-Alexa-488 and anti-rat-IgG-Cy3 were used as secondary antibodies. Images were collected with a confocal laser scanning microscope. Alexa-488-(green) and Cy3-(red) fluorescence images are shown and merge images are presented (panels iii, vi, ix). Images from control and stimulated cells were acquired with identical image collection protocols. Enlarged regions from stimulated nuclei are presented (inset, panels iv-ix). Scale bar, 4 µm. (C) sAn-stimulated nuclei were prepared for immunofluorescence and labelled with anti-phosphoS10-H3 and anti-phosphoS28-H3 antibodies as in (B). Lines were drawn at random through the nucleoplasm profiles of intensity for both the green (anti-phosphoS10-H3; green line) and red (anti-phosphoS28-H3; red line); channels were plotted (y-axis; arbitrary units) against the position along the line (x-axis; voxels). (D) CCF analysis was performed for anti-phosphoS10-H3 and anti-phosphoS28-H3 co-stained cells (solid line), and for cells labelled with anti-phosphoS10-H3 (non-occ.) detected by red- and green-fluorescent antibodies (broken line), as described in Materials and Methods. Two nuclei were chosen from independent experiments and for each nucleus, three independent line-scans were taken as in (C) for CCF analysis (six line-scans in total). CCF analysis produces a graph showing the dependency of the correlation between the fluorescent profiles of the red and green channel (calculated as Pearson's coefficient, RP, y-axis) on the shifting of the intensity profiles with respect to one another (X, x-axis). Each CCF represents the mean of the six line-scans processed.

View larger version (39K): [in a new window]   Fig. 4. Stable overexpression and activation of GFP-MSK1 in C3H 10T cells. (A) Quiescent wild-type (panels i-vi) or GFP-MSK1-overexpressing (panels vii-xii) cells were stimulated with 25 ng/ml anisomycin (sAn) for 45 minutes (panels iv-vi, x-xii) or left untreated (panels i-iii, vii-ix). Cells were fixed, permeabilised and processed for immunofluorescence as described in Materials and Methods. Cells were stained with rabbit anti-GFP primary and anti-rabbit-IgG-Alexa-488 secondary antibodies. Nuclei were stained with DAPI. Cells were examined by conventional fluorescence microscopy for Alexa-488/GFP- (green) and DAPI- (blue) fluorescence. Merged images are presented in the right column of images. Scale bar, 20 µm. (B) Quiescent wild-type or GFP-MSK1-overexpressing cells were stimulated with 100 nM TPA for 30 minutes (lanes 2, 4) or left untreated (lanes 1, 3). Cells were lysed and a fraction containing soluble nuclear and cytoplasmic proteins was obtained (see Materials and Methods). Proteins were separated by 10% SDS-PAGE and western blotted with anti-GFP (panel i), anti-MSK1 (panel ii) and anti-ERK (panel iii) antibodies. (C) Quiescent wild-type (lanes 1-9) or GFP-MSK1-overexpressing (lanes 10-18) cells were stimulated with 50 ng/ml EGF (lanes 2, 3 and 11, 12), 25 ng/ml sAn (lanes 4-6, 13-15) or 100 nM TPA (lanes 7-9, 16-18) for the times indicated. Cells were lysed and high-salt-extracted (see Materials and Methods) proteins were separated either on standard 10% SDS-PAGE (panel ii) or in modified gels that enhance phosphorylation-dependent electrophoretic retardation (panel i) (see Materials and Methods). Western blots were carried out with anti-MSK1 (panel i) and anti-ERK (panel ii) antibodies. (D) Quiescent wild-type (columns 1-3) or GFP-MSK1-overexpressing (columns 4-6) fibroblasts were stimulated for 30 minutes with 25 ng/ml sAn (columns 3, 6), for 20 minutes with 100 nM TPA (columns 2, 5), or left untreated (columns 1, 4). Cell lysates were immunoprecipitated with anti-GFP antibody, and immunoprecipitates assayed for kinase activity towards Crosstide peptide substrate as described in Materials and Methods. Assays were performed in triplicate on independent extracts. Error bars represent the standard deviation of the mean.

View larger version (54K): [in a new window]   Fig. 5. Effect of GFP-MSK1 overexpression on substrate phosphorylation. (A) Quiescent wild-type (lanes 1-9) or GFP-MSK1-overexpressing (lanes 10-18) cells were stimulated with 50 ng/ml EGF (lanes 2, 3 and 11, 12), 25 ng/ml sAn (lanes 4-6, 13-15) or 100 nM TPA (lanes 7-9, 16-18), for the times indicated. Acid-extracted proteins were separated on 15% SDS-PAGE and Coomassie-stained (panel vi) or transferred to PVDF for western blotting. Blotting was carried out using anti-phosphoS10-H3 (panel i); anti-phosphoS10-H3occ (panel ii); anti-phosphoacetyl-H3 (panel iii); anti-phosphoS28-H3 (panel iv); or anti-phosphoHMG-14 (panel v). (B) Quiescent wild-type (lanes 1-4) or GFP-MSK1-overexpressing (lanes 5-8) cells were stimulated with EGF (50 ng/ml, 15 minutes; lanes 2, 6), sAn (25 ng/ml, 30 minutes; lanes 3, 7), or TPA (100 nM, 30 minutes; lanes 4, 8), or left untreated (lanes 1, 5). Acid-extracted proteins were separated on 15% SDS-PAGE (panel ii) or 20% acid-urea (panel i) gels and transferred to PVDF membranes for western blotting. Blotting was carried out with anti-HMG-14 antibody (panels i,ii). In i, the lower and upper arrows indicate the positions of the unphosphorylated and phosphorylated proteins, respectively. (C) Quiescent wild-type (lanes 1-4) or GFP-MSK1-overexpressing (lanes 5-8) cells were stimulated with EGF (50 ng/ml, 15 minutes; lanes 2, 6), sAn (25 ng/ml, 30 minutes; lanes 3, 7), or TPA (100 nM, 30 minutes; lanes 4, 8), or left untreated (lanes 1, 5). Cells were lysed in high-salt buffer (see Materials and Methods). Proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes. Western blotting was carried out with an antibody which recognises CREB phosphorylated at S133 and ATF1 phosphorylated at S63 (panel i) or anti-ERK antibody (panel ii). In panel i, the arrows indicate the positions of phospho-CREB and phospho-ATF1.

View larger version (63K): [in a new window]   Fig. 6. Confocal microscopy images of the intranuclear distribution of phosphorylated histone H3 and GFP-MSK1. (A, B) Quiescent wild-type (A) or GFP-MSK1-overexpressing (B) cells were treated with 100 nM TPA for 20 minutes (central panels), 25 ng/ml sAn for 45 minutes (right panels) or left untreated (left panels). Cells were processed for immunofluorescence as described in Materials and Methods. Coverslips were incubated with rabbit anti-GFP (panels i-iii) and rat anti-phosphoS28-H3 (panels iv-vi) primary antibodies. Secondary antibodies used were anti-rabbit-IgG-Alexa-488 and anti-rat-IgG-Cy3 conjugates. Nuclei were examined by laser scanning confocal microscopy for Alexa-488/GFP- (green) and Cy3- (red)s fluorescence. Merged images are presented (panels vii-ix). Scale bar, 4 µm. (C) Quiescent wild-type (panels i-iii) or GFP-MSK1-overexpressing cells (panels iv-ix) were treated with 100 nM TPA for 20 minutes (central panels), 25 ng/ml sAn for 45 minutes (right panels) or left untreated (left panels). Cells were processed for immunofluorescence as described in Materials and Methods. Primary antibodies used were anti-phosphoS10-H3 (panels i-vi) or anti-GFP (panels vii-ix). Secondary antibodies were either Cy3-(panels i-vi) or Alexa-488-(panels vii-ix) conjugated anti-rabbit-IgG. Nuclei were identified by the weak GFP-fluorescence and examined by laser scanning confocal microscopy for Cy3-fluorescence. Scale bar, 4 µm.

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