HDAC4 interacts with 14-3-3. (A) Expression plasmids for Flag-HDAC4 and HA-14-3-3β were cotransfected into 293 cells as indicated. At 48 h after transfection, cell extracts were prepared for affinity purification (AP) on M2 agarose beads (lanes 1 to 4) or immunoprecipitation (IP) with the anti-HA monoclonal antibody (lanes 5 to 8). Bound proteins, eluted with Flag peptide (lanes 1 to 4) or the SDS sample buffer (lanes 5 to 8), were subjected to Western analyses with the anti-Flag (top) or anti-HA antibody (bottom). H, IgG heavy chain; L, light chain. Note that in lanes 1 to 4, no heavy- and light-chain bands are visible because the bound antigens were eluted with Flag peptide from M2 agarose beads, on which the anti-Flag antibody is covalently cross-linked. Whether the bands at the light-chain position in lanes 3 and 4 (bottom) are due to light chains is unclear. (B) NIH 3T3 extracts (lane 1) were subjected to immunoprecipitation with a rabbit preimmune IgG (lane 2) or the rabbit anti-HDAC4 antibody (lane 3) and subsequent Western analysis with the rabbit anti-HDAC4 antibody (top) or a mouse anti-14-3-3 monoclonal antibody (bottom).

Effects of point mutations of S246, S467, and S632 of HDAC4 on its subcellular localization. (A) Representative images of green fluorescence of NIH 3T3 cells expressing HDAC4 and its mutants fused to GFP. (B) Quantitative representation of NIH 3T3 cells expressing HDAC4 or its mutants fused to GFP. Blank bar (C>N), more green fluorescence in the cytoplasm; shaded bar (C=N), fluorescence equally in the cytoplasm and the nucleus; solid bar (N>C), more fluorescence in the nucleus. Average values of three independent experiments are shown with standard deviation indicated by error bars. (C) Representative images of green fluorescence of 293 cells expressing GFP-S246/467/632A.

Repression ability of HDAC4 and its mutant S246/467/632A. (A and B) The reporter (200 ng), MEF2-E4-Luc (A) or pJLuc (B), was transfected into NIH 3T3 cells with a MEF2C expression plasmid (100 ng), an internal control plasmid (CMV-β-Gal; 50 ng), and the expression plasmid for Flag-tagged HDAC4 or S246/467/632A at the indicated amount. The normalized luciferase activity from the transfection without any effector plasmid was arbitrarily set to 1.0. Average values of at least three independent experiments are shown with standard deviation indicated by error bars. (C) A 200-ng sample of the Gal4-tk-Luc reporter was transfected into NIH 3T3 cells with a Gal4-VP16 expression plasmid (5 ng), the internal control plasmid CMV-β-Gal (50 ng), and the expression plasmid for Flag-tagged HDAC4 or S246/467/632A at the indicated amount. The reporter activities were measured as for panels A and B. (D) The Gal4-tk-Luc reporter was transfected into NIH 3T3 cells along with an expression plasmid for Gal4-HDAC4 or Gal4-S246/467/632A. Normalized luciferase activities from transfection with effector plasmids at the indicated amounts were compared with that from the reporter alone to calculate the relative repression. Average values of four independent experiments are shown with standard deviation indicated by error bars.

Cytoplasmic localization of HDAC4. (A) Affinity-purified Flag-HDAC4 (lane 1) and cytoplasmic (lanes 2 and 5) and nuclear (lanes 3 and 6) extracts of NIH 3T3 cells were subjected to immunoblotting with the anti-HDAC4 (lanes 1 to 3), anti-14-3-3 (lanes 5 and 6, top), or anti-MEF2D (lanes 5 and 6, bottom) antibody. The amount of extracts was normalized according to cell numbers. The 55-kDa band in lane 2 may not be specific, since it was not reproducibly detected by different bleeds of the anti-HDAC4 antibody. (B) Representative green fluorescence images of NIH 3T3 and 293 cells expressing GFP-HDAC4. (C) Green fluorescence images of two SKN cells (cells a and b) expressing GFP-HDAC4. After initial examination for green fluorescence, LMB (10 ng/ml) was added to the medium and cell b was then analyzed for redistribution of green fluorescence at the indicated times. Under similar conditions, LMB had minimal effects on the pancellular localization of GFP itself (data not shown).

Mapping of 14-3-3 binding sites. (A) Expression plasmids for HDAC4 and its deletion mutants (all Flag tagged) were transfected into 293 cells, and cell extracts were prepared for affinity purification on M2 agarose. Bound proteins were eluted with the Flag peptide and subjected to Western analyses with the anti-Flag (top) or anti-14-3-3 (bottom) antibody. C (lane 1), control affinity purification using nontransfected cells. For HDAC4 proteins, bands with expected molecular masses are indicated by asterisks. (B) Schematic representation of HDAC4 and its mutants, with their 14-3-3 binding ability indicated at the right. (C) Expression plasmids for HA-tagged hm9 and hm10 were transfected into 293 cells, and cell extracts were prepared for immunoprecipitation with the anti-HA antibody. Immunocomplexes were subjected to immunoblotting with the anti-HA (lanes 1 to 3) or anti-14-3-3 antibody (lanes 4 to 6). H, IgG heavy chain; L, light chain. (D and E) Interaction of Flag-tagged deletion mutants hm11 to hm15 (D) and full-length point mutants (E) with 14-3-3 proteins. The migration difference between hm11 and hm12 may be due to differential phosphorylation. The Flag-tagged HDAC4 proteins were expressed, affinity purified, and analyzed as in panel A.

Effects of point mutations of S246, S467, and S632 of HDAC4 on its deacetylase activity. (A) Deacetylase activity of HDAC4 and its mutant S246/467/632A. Expression plasmids for Flag-tagged fusion proteins were transfected into 293 cells, and cell extracts were prepared for affinity purification on M2 agarose. Activities of eluted proteins (left) were determined by measuring the release of [³H]acetate from [³H]acetyl-histones. (B) The amount of the eluted proteins was analyzed by immunoblotting with the anti-Flag antibody. The migration position of full-length proteins is indicated by an asterisk.

Model depicting possible modes of regulation of HDAC4 by 14-3-3 proteins. HDAC4 is actively shuttled between the cytoplasm (C) and the nucleus (N), and the relative rate of nuclear import and export may determine the subcellular localization. 14-3-3 binding may shift the distribution equilibrium of HDAC4 toward cytoplasmic accumulation by hindering its nuclear import (A) and/or facilitating its nuclear export (B). 14-3-3 proteins have been shown to be subject to active nuclear export (40), so they can interact with HDAC4 in the nucleus (B). Association of HDAC4 with other proteins may also affect its localization. In this study, we have investigated how 14-3-3 proteins regulate the functions of HDAC4. Theoretically, it is also possible that HDAC4 regulates the functions of 14-3-3 proteins such as their ability to regulate the function of their binding partners (1, 4, 19, 38, 39) and to bind to cruciform DNA molecules (19, 50).