Nuclear import of histone deacetylase 5 by requisite nuclear localization signal phosphorylation

Abstract

Histone deacetylase 5 (HDAC5), a class IIa deacetylase, is a prominent regulator of cellular and epigenetic processes that underlie the progression of human disease, ranging from cardiac hypertrophy to cancer. Although it is established that phosphorylation mediates 14-3-3 protein binding and provides the essential link between HDAC5 nucleo-cytoplasmic shuttling and transcriptional repression, thus far only four phospho-acceptor sites have been functionally characterized. Here, using a combinatorial proteomics approach and phosphomutant screening, we present the first evidence that HDAC5 has at least 17 in vivo phosphorylation sites within functional domains, including Ser278 and Ser279 within the nuclear localization signal (NLS), Ser1108 within the nuclear export signal, and Ser755 in deacetylase domain. Global and targeted MS/MS analyses of NLS peptides demonstrated the presence of single (Ser278 and Ser279) and double (Ser278/Ser279) phosphorylations. The double S278/279A mutation showed reduced association with HDAC3, slightly decreased deacetylation activity, and significantly increased cytoplasmic localization compared with wild type HDAC5, whereas the S278A and S1108A phosphomutants were not altered. Live cell imaging revealed a deficiency in nuclear import of S278/279A HDAC5. Phosphomutant stable cell lines confirmed the cellular redistribution of NLS mutants and revealed a more pronounced cytoplasmic localization for the single S279A mutant. Proteomic analysis of immunoisolated S278/279A, S279A, and S259/498A mutants linked altered cellular localization to changes in protein interactions. S278/279A and S279A HDAC5 showed reduced association with the NCoR-HDAC3 nuclear corepressor complex as well as protein kinase D enzymes, which were potentiated in the S259/498A mutant. These results provide the first link between phosphorylation outside the known 14-3-3 sites and downstream changes in protein interactions. Together these studies identify Ser279 as a critical phosphorylation within the NLS involved in the nuclear import of HDAC5, providing a regulatory point in nucleo-cytoplasmic shuttling that may be conserved in other class IIa HDACs-HDAC4 and HDAC9.

Fig. 1.

HDAC5-EGFP has deacetylation activity and associates in vivo with the N-CoR-HDAC3 complex and heterotrimeric PP2A enzyme. A, Schematic of EGFP-FLAG-tagged HDAC5 with functional domains: MEF2, myocyte enhancer factor 2 binding domain; NLS, nuclear localization signal; Deacetylase, deacetylase activity domain; NES, nuclear export signal. B, The HDAC5-EGFP localizes to both the nucleus and cytoplasm in the HEK293 cell line; HDAC5 (green, EGFP), nuclear periphery (red, Mab414 against nuclear pore complex proteins), nucleus (blue, DAPI); 100× oil-immersion lens; Bar, 5 μm. C, HDAC5-EGFP-FLAG has deacetylation activity. Deacetylation activity of HDAC5-EGFP and EGFP, isolated from HEK293 cell lines, was measured (n = 6, AFU±S.D.) using Fluor-de-Lys assay in the absence (-) or presence (+) of trichostatin A. D, HDAC5-EGFP associates with known interacting partners. Immunoaffinity purifications were performed via the EGFP tag, co-isolated proteins were resolved by one-dimensional SDS-PAGE, stained with Coomassie Blue, and analyzed by mass spectrometry. Red, known members of the N-CoR-HDAC3 corepressor complex; Blue, the three subunits of protein phosphatase 2A.

Fig. 2.

Combinatorial mass spectrometric approach allows a comprehensive view of in vivo phosphorylation on HDAC5. A, Workflow for phosphopeptide characterization of HDAC5 using immunoaffinity isolation, in-gel digestion, and complementary mass spectrometric approaches. Proteomics strategies employed include (i) MALDI-IMAC analysis, (ii) ESI-LC CID analysis, and (iii) ESI-LC HCD/ETD analysis. B, 87% sequence coverage of HDAC5, including unmodified and phosphorylated peptides, was obtained. C, MALDI CID MS/MS of tryptic phosphopeptide. Prominent neutral losses of phosphoric acid (H₃PO₄) and water were observed. MS³ fragmentation of the neutral loss species (M+H⁺- H₃PO₄) localized the phosphorylation site (Ser611) as a dehydroalanine, marked with *. (D and E) MS/MS ETD spectra corresponding to multiply charged Lys-N phosphopeptides. Phosphosites (Ser755 and Ser1108) were unambiguously localized, with nearly full sequence coverage of both c and z ion series. F, MS/MS HCD spectrum of tryptic phosphopeptide. Neutral losses of H₃PO₄ from the precursor and y₆ and y₇ fragment ions supported the phosphosite at Ser259. G, MS/MS ETD spectrum of a doubly phosphorylated peptide (Ser278 and Ser279) within the nuclear localization signal. For all mass spectra, the precursor m/z, charge state, mass error (Δm), and cross correlation score (X_c) are indicated. H, Venn diagram comparison of phosphopeptides identified from Lys-N and trypsin-digested HDAC5, and each as a function of fragmentation technique (ETD versus HCD). I, Venn diagram of the number of phosphosites unambiguously localized by HCD and ETD. Representative annotated, mass labeled phosphopeptide spectra are included in Supplemental Fig. S2.

Fig. 3.

In vivo HDAC5 phosphorylation reveals site conservation among class IIa HDACs. A, Location of phosphorylated sites along the HDAC5-EGFP-FLAG sequence. B, Conservation of phosphosites among HDAC5 vertebrate species. NCBI gi numbers used were: Homo sapiens 62750347, Pongo abelii 55730067, Pan troglodytes 114666888, Macaca mulatta 109116100, Bos taurus 194676212, Equus caballus 194216834, Canis familiaris 73965560 (isoform 1), Cricetulus griseus 28627825, Mus musculus 148702151, Rattus norvegicus 109491954, Danio rerio 189517029. The cladogram of vertebrate HDAC5 sequence divergence was generated from NCBI BLASTp by the TreeView program (43).

Fig. 4.

HDAC5 phosphorylation influences its deacetylation activity and association to HDAC3. A, HEK293 cells were transiently transfected with EGFP, HDAC5-EGFP (wild type), or Ser to Ala HDAC5-EGFP mutants. Following isolations via EGFP, the deacetylation activity was measured using the Fluor-de-Lys assay, and expressed as fold change ± S.D. versus wild type (n ≥ 3). Statistical significance was performed by student's t test comparing wild type to mutant HDAC5; * and ** indicate p = 0.03 and p = 0.0001, respectively. B, Isolated HDAC5 mutants were assessed for their association with HDAC3 and 14–3-3 ε by Western blotting; a quantitative assessment of the relative intensities for the illustrated images was performed by densitometry and shown in Supplemental Fig. S3. C, Crystal structure of the HDAC4 deacetylation domain (PDB 2VQJ) highlighting Asn726, located between α-helix 5 and 6, and which aligns to Ser755 in HDAC5.

Fig. 5.

Phosphorylation within the NLS affects HDAC5 localization. A, Cellular localization of transiently transfected wild type HDAC5, and S278/279A, S278A, and S1108A mutants was assessed by direct immunofluorescence microscopy in U2OS cells (bars, 10 μm); green (EGFP), blue (DAPI). B, wild type and mutant HDAC5-EGFP localization was classified as cytoplasmic, nuclear, or both (pan-cellular) (% ± S.D., n ≥ 220 cells from triplicate experiments). C, WT and S278/279A mutants were transiently transfected in U2OS cells, treated with leptomycin B, and visualized by live cell imaging for up to 4 h after treatment. Images are representative of three independent experiments.

Fig. 6.

Mutation of 14–3-3-binding sites and NLS phosphorylations leads to changes in HDAC5 interactions. Wild type, S259/498A and S278/279A HDAC5-EGFP were immunoaffinity purified from HEK293 stable cell lines with associating proteins, resolved by 1-D SDS-PAGE (Fig. S5) and analyzed by mass spectrometry. Changes in protein interactions were assessed using spectral counting (n = 2 biological replicates). A, The relative size of the circles associated with the mutant and WT (wild type) HDAC5 reflect increased or decreased relative abundance as assessed by the fold change in spectral counts for mutant versus WT. Color-coding illustrates functional clustering according to protein families or complexes: yellow, 14-3-3 protein isoforms, blue, PP2A subunits, purple, corepressor complex, orange, PKD enzymes. B, The average fold changes in spectral counts for each interaction illustrated in (A) are shown for the isolated mutant versus the wild type HDAC5.* indicates fold change for 14-3-3 isoforms representing the average of six isoforms (S.D. < ± 0.1).

Fig. 7.

Preclusion of serine 279 phosphorylation alters HDAC5 localization. A, Cellular localization of stably transfected wild type (WT), S278A/S279A, and S279A HDAC5-EGFP was assessed by direct immunofluorescence microscopy in U2OS cells (bar, 20 μm). Increased cytoplasmic accumulation of HDAC5 (EGFP) was observed because of Ser-to-Ala mutations. Nuclear and HDAC5 (EGFP) boundaries used for the quantification of HDAC5 cellular localization are shown. B, Distribution plot of the percent of HDAC5-EGFP-expressing U2OS cells as a function of the fraction of total HDAC5-EGFP within the cytoplasm (cytoplasmic proportion). Cytoplasmic proportion of HDAC5 was calculated by dividing the integrated intensity of the EGFP signal within the cytoplasm by the total integrated intensity of EGFP within each cell (see Experimental Procedures). WT HDAC5 (red bars; n = 848), S278A/S279A (black bars; n = 868), and S279A (green bars; n = 970) cells showed a median cytoplasmic proportion of 0.20, 0.36, and 0.45, respectively.

Fig. 8.

Targeted CID-MS² analysis of NLS region demonstrated the independence of Ser278 phosphorylation from Ser279 phosphorylation. A, Neutral loss extracted ion chromatogram (NL-XIC) (top) and CID MS/MS spectrum (bottom) of the tryptic phosphopeptide pS₂₇₈APLLR from the NLS region demonstrated the presence of Ser278 phosphorylation in S279A HDAC5-EGFP. This peptide ion (m/z = 368.69) was absent in the equivalent NL-XIC from S278/279A HDAC5-EGFP. B, Phosphopeptide CID spectra corresponding to doubly charged tryptic peptides from the NLS region of wild-type HDAC5 localized a single phosphorylation to Ser279 (top) and confirmed the previous identification of Ser278 phosphorylation by ETD (bottom). y and b ion fragments are indicated above each spectra, with site determining ions (b₂ and y₅) highlighted in bold.