MARK/Par-1 kinases, C-TAK1 and EMK, associate with the N terminus of HDAC7. (A) GST fusion proteins corresponding to Ser¹⁵⁵ of HDAC7 (GST-S155), to the serine-to-alanine mutant (GST-A155), or to GST alone were incubated with HEK293 cell extracts in a pull-down assay. Reactions were resolved by SDS-PAGE and analyzed for the presence of endogenous PKC family members (α-PKC), RSK1 and RSK2 (α-RSK), C-TAK1 (α-C-TAK1), and MSK1 and MSK2 (α-MSK) by Western blotting. (B) Binding of endogenous C-TAK1 to GST fusion proteins corresponding to Ser²⁴⁶ of HDAC4, Ser²⁵⁹ of HDAC5, and Ser¹⁵⁵ of HDAC7 (GST-HDAC4, GST-HDAC5, and GST-HDAC7, respectively) after a pull-down assay was analyzed by Western blotting or Coomassie staining for loading control. (C) Pull-down reactions described in panel A were analyzed by SDS-PAGE, followed by Western blotting for detection of associated endogenous EMK or by Coomassie staining for control loading. Input equals 10% of the cell extract used in each reaction. The arrow indicates the band corresponding to EMK in the pull-down reactions. Other bands result from cross-reactivity of the anti-EMK antiserum with bacterial proteins copurifying with the GST-fusion proteins. WB, Western blotting.

Class IIa HDACs are subjected to phosphorylation-dependent nucleocytoplasmic efflux. (A) Recombinant GFP-HDAC7 was transfected into Cos7 cells. Forty-eight hours posttransfection, cells were left untreated (No Treatment) or treated with staurosporine (Stauro) for 1 h before the localization of HDAC7 was examined by confocal microscopy. (B) Cos7 cells were transfected with constructs expressing GFP fusion proteins corresponding to HDAC7 (HDAC7wt) or a mutant of HDAC7 in which the residues Ser¹⁵⁵, Ser¹⁸¹, Ser³²¹, and Ser⁴⁴⁹ were mutated to alanines (HDAC7ΔP). Forty-eight hours after transfection, cells were left untreated or treated with staurosporine (Stauro) or leptomycin B (LMB) as indicated. The cytoplasm of transfected cells was repeatedly bleached, and the loss of fluorescence in the nuclear region was assessed by confocal microscopy. (C) Relative loss of fluorescence in the nuclear region was determined as described in Materials and Methods. FLIP data are represented as nonlinear fit curves according to the legend at right of the graph. HDACwt +LMB, HDAC7 wt with leptomycin B treatment; HDAC7wt+Stauro, HDAC7 wt with staurosporine treatment.

Class IIa HDACs are constitutively phosphorylated, bound to 14-3-3 proteins, and subjected to phosphorylation-dependent nucleo-cytoplasmic shuttling. (A) Do11.10 cells were labeled with [³²P]orthophosphate and subsequently treated with staurosporine (+) or left untreated (−). Endogenous HDAC7 was immunoprecipitated and analyzed by SDS-PAGE followed by Western blotting (α-HDAC7) or autoradiography (³²P). (B) Do11.10 cells, transduced with FLAG-tagged HDAC7 (right) or not (left), were left untreated or treated with staurosporine for 1 h. Endogenous (left) or ectopically expressed HDAC7 (right) was immunoprecipitated from cell lysates using the indicated antibodies. Immunoprecipitated material was then subjected to Western blot analysis with antibodies directed against endogenous HDAC7 (α-HDAC7), the FLAG epitope (α-FLAG), or 14-3-3 proteins (α-14-3-3). (C) HeLa cells expressing GFP-HDAC4 or GFP-HDAC7 were either left untreated (No Treatment) or treated with staurosporine. After 1 h of treatment, the subcellular distribution of GFP-HDACs was determined using confocal microscopy. Bar histograms represent the mean percentages of cells showing predominant cytoplasmic localization of GFP-HDACs in each condition. Stauro, staurosporine; IP, immunoprecipitation.

HDAC7 is phosphorylated on Ser¹⁵⁵ in vivo. (A) Total cell lysates were prepared from Do11.10 cells which were first treated with staurosporine (+) for 1 h or left untreated (−). Phosphorylation of endogenous HDAC7 was detected by Western blotting with the antibody specific for phosphorylated Ser¹⁵⁵ (α-pS155). As a loading control, the same membrane was stripped and immunoblotted with an antibody against endogenous HDAC7 (α-HDAC7). (B) HEK293 cells were transiently transfected with expression vectors encoding FLAG-tagged versions of wild-type HDAC4, HDAC5, and HDAC7 or the empty vector (Empty). Total cellular extracts were analyzed by SDS-PAGE and subjected to immunoblotting using antibodies directed either against the FLAG epitope (α-FLAG) or phosphorylated Ser¹⁵⁵ (α-pS155). (C) Nuclear (Nuc) and cytoplasmic (Cyto) extracts were prepared from HEK293 cells transiently expressing FLAG-tagged HDAC7 or HDAC4. Both extracts were analyzed by Western blotting with antibodies against the FLAG epitope (α-FLAG) and phosphorylated Ser¹⁵⁵ (α-pHDACs). As a control, extracts were also analyzed by Western blotting with antisera against the cytoplasmic protein tubulin (α-tubulin) and the nuclear protein HDAC1 (α-HDAC1). (D) Nuclear (Nuc) and cytoplasmic (Cyto) extracts were prepared from HeLa or Cos7 cells transiently expressing FLAG-tagged HDAC7. Equal protein amounts from each extract were analyzed by Western blotting with antisera against the FLAG epitope (α-FLAG) and phosphorylated Ser¹⁵⁵ (α-pS155). As a control, extracts were also analyzed by Western blotting with antibody against the cytoplasmic protein tubulin (α-tubulin) and the nuclear protein HDAC1 (α-HDAC1). Stauro, staurosporine.

The 14-3-3 binding sites in HDAC7 are hierarchically phosphorylated. (A) Cos7 cells were transfected with constructs expressing GFP fusion proteins corresponding to wild-type HDAC7 (HDAC7wt), a mutant of HDAC7 in which the residues Ser¹⁵⁵ was mutated to alanine alone [HDAC7(S155A)], or together with Ser¹⁸¹, Ser³²¹, and Ser⁴⁴⁹ (HDAC7ΔP). Forty-eight hours posttransfection, the cytoplasm of fluorescent cells was bleached, and relative loss of fluorescence in the nuclear region was measured, as described in Materials and Methods. FLIP data are represented as nonlinear fit curves according to the legend at the right of the graph. (B) HEK293 cells expressing wild-type or S155A HDAC7 were labeled in vivo with [³²P]orthophosphate. The labeled proteins were purified by immunoaffinity, digested with trypsin, and examined by HPLC analysis as described in Materials and Methods. (C) Expression constructs for FLAG-tagged wild-type HDAC7 (HDAC7wt) or mutant of HDAC7 in which the residues Ser¹⁵⁵, Ser¹⁸¹, Ser³²¹, or Ser⁴⁴⁹ were mutated to alanine independently [HDAC7(S155A), HDAC7(S181A), HDAC7(S321A), and HDAC7(S449A), respectively] or together (HDAC7ΔP) were transiently expressed in HEK293 cells. Cell extracts were examined by Western blotting with antibodies directed against the FLAG epitope (α-FLAG) or the phosphorylated forms of Ser¹⁵⁵ (α-pS155) or Ser¹⁸¹ (α-pS181). (D) HEK293 cells were transiently transfected with expression vectors encoding FLAG-tagged versions of wild-type HDAC7 (HDAC7wt) or the L150A mutant [HDAC7(L150A)]. Total cellular extracts were analyzed by SDS-PAGE and subjected to immunoblotting using antibodies directed against either the FLAG epitope (α-FLAG) or HDAC7 phosphorylated at Ser¹⁵⁵ (α-pS155) or Ser¹⁸¹ (α-pS181). (E) HEK293 cells expressing the L150A HDAC7 mutant were labeled in vivo with [³²P]orthophosphate. The labeled protein was purified by immunoaffinity, digested with trypsin, and examined by HPLC analysis as described in Materials and Methods.

C-TAK1 and EMK specifically phosphorylates Ser¹⁵⁵ of HDAC7 in vitro. (A) The C- and N-terminal domains of HDAC7 and wild-type or S216A mutant Cdc25C were produced as GST fusion proteins [GST-HDAC7Cter, GST-HDAC7Nter, GST-Cdc25Cwt, and GST-Cdc25C(S216A), respectively]. Equal amounts of purified recombinant proteins were used in IVK assays with recombinant active C-TAK1. IVK reactions were analyzed by SDS-PAGE and Coomassie staining (bottom) prior to autoradiography (top). The arrow indicates the signal resulting from autophosphorylated C-TAK1 (rC-TAK1). (B) Sequences around the four phosphorylatable serine residues in HDAC7 match with the canonical C-TAK1 phosphorylation motif. (C) GST-fusion proteins corresponding to the sequences surrounding Ser¹⁵⁵, Ser¹⁸¹, Ser³²¹, and Ser⁴⁴⁹ of HDAC7 were used as substrates in independent IVK assays with active recombinant C-TAK1. Reactions were resolved by SDS-PAGE, and the gel was stained with Coomassie (bottom), dried, and analyzed by autoradiography (top). (D) The N terminus of HDAC7 was expressed as a GST fusion protein and used as a substrate for recombinant PKD or C-TAK1 in IVK assays. Labeled proteins were resolved by SDS-PAGE and digested with trypsin. The resulting peptides were then separated by HPLC. Positions of the peptides containing each phosphorylatable serine are indicated on the radioactivity profile (as confirmed by mass spectrometry). (E) GST fusion proteins corresponding to the N-terminal domain of HDAC7 (GST-Nter) or to the same region with a serine to alanine substitution at position Ser¹⁵⁵ (GST-NterS155A) were phosphorylated in vitro by EMK or C-TAK1. IVK reactions were analyzed by SDS-PAGE and Coomassie staining (bottom) prior to autoradiography (top). MW, molecular weight.

The N terminus of class IIa HDACs associates with an 85-kDa autophosphorylating kinase. (A) The N terminus of HDAC7 was expressed as a GST fusion protein (GST-Nter) and incubated with total cellular extracts from unstimulated, normally growing HEK293 cells. A control reaction was performed in parallel with the GST protein alone. Associated autophosphorylating kinase activities were revealed by IGK assay and autoradiography. The input lane corresponds to 10% of the lysate engaged in the pull-down. (B) GST fusion proteins corresponding to the sequences surrounding the four phosphorylatable serine residues in HDAC7 (GST-S155, GST-S181, GST-S321, and GST-S449) were used in independent pull-down reactions with lysates from HEK293 cells. Autophosphorylating protein kinase activities in the associated material were detected by IGK assay. The gel was stained with Coomassie, dried, and analyzed by autoradiography. The lane marked “input” equals 10% of the cell lysate used in the reaction. (C) A GST fusion protein corresponding to the sequences around Ser¹⁵⁵ of HDAC7 (GST-S155) was incubated with HEK293 cell extracts in a pull-down assay. A mutant fusion protein harboring a serine to alanine substitution (GST-A155) was used as control. Pull-down reactions were resolved by SDS-PAGE and analyzed by IGK assay. The gel was stained with Coomassie and dried, and autophosphorylating protein kinase activity was visualized by autoradiography. Input amounts to 10% of the cell lysate were used in each reaction. (D) GST fusion proteins corresponding to Ser²⁴⁶ of HDAC4, Ser²⁵⁹ of HDAC5, and Ser¹⁵⁵ of HDAC7 (GST-HDAC4, GST-HDAC5, and GST-HDAC7, respectively) were incubated with extracts from HEK293 cells. A control reaction was performed with the GST protein alone. Pull-down reactions were analyzed by IGK and autoradiography to visualize autophosphorylating kinase activities. The input lane corresponds to 10% of the material used in each reaction. IGKA, IGK assay.

EMK and C-TAK1 control nuclear export of class IIa HDACs and regulate their repressive activity. (A) Cos7 cells were transfected with expression vectors for GFP-HDAC7 and FLAG-tagged EMK or C-TAK1. The intracellular localization of HDAC7 was detected by direct immunofluorescence (GFP) while MARKs were revealed by indirect immunofluorescence using an AlexaFluor 568-labeled anti-FLAG antibody (FLAG). (B) Cos7 cells were transfected with expression vectors for GFP-HDAC4 or GFP-HDAC7 and constructs for EMK or C-TAK1. The subcellular distribution of the GFP-fused HDACs proteins was examined by confocal immunofluorescence microscopy. Bar histograms represent the mean percentages of cells showing predominant cytoplasmic staining. (C) A construct encoding GFP-HDAC7 was transfected into Cos7 cells, either with expressing vectors for EMK, C-TAK1, or a control plasmid (Empty). The cytoplasm of GFP-positive cells was repeatedly bleached, and FLIP measurements were performed as described in Materials and Methods. Analyses of the FLIP data from three independent experiments are shown as fit curves. (D) Do11.10 cells were transiently transfected with a luciferase reporter plasmid driven by the Nur77 promoter and the expression plasmids for the indicated proteins. Luciferase activities are presented relative to the basal luciferase activity of the reporter. Results are from five independent experiments, each performed in triplicate. (E) Total cellular lysates were prepared from Cos7 cells transiently expressing FLAG-tagged versions of EMK and C-TAK1. Cell lysates were examined by Western blotting with antisera for c-jun, FLAG, and actin.

EMK and c-TAK1 phosphorylate Ser155 of HDAC7 in vivo. (A) FLAG-tagged versions of wild-type HDAC7 (HDAC7wt) or the HDAC7(L150A) mutant were transiently expressed in HEK293 cells, immunoprecipitated, and examined by Western blot analysis with antibodies directed against the FLAG epitope (α-FLAG), 14-3-3 family members (α-14-3-3), or HDAC7 phosphorylated at Ser¹⁵⁵ (α-pS155). (B) HeLa cells were transfected with constructs encoding GFP fusion proteins of wild-type HDAC7 (HDAC7wt) or the HDAC7(L150A) mutant. Subcellular localization of the fluorescent proteins was observed by confocal microscopy. (C) Cos7 cells were transfected with constructs encoding GFP-tagged wild-type or L150A HDAC7. Cytoplamsic photobleaching and FLIP measurements were performed as described in Materials and Methods. Graphical analyses of the FLIP data from at least three independent experiments are shown as fit curves. (D) HeLa cells were transfected with FLAG-tagged HDAC7, along with pooled siRNA against EMK and C-TAK1 or with control siRNA. The next day, cells were transfected with a mix of siRNAs targeting an alternative sequence both in EMK and C-TAK1 or with control siRNA (see Materials and Methods). Seventy-two hours after the initial transfection, cells were harvested and lysed. Whole-cell lysates were analyzed by Western blotting with antibodies against EMK, C-TAK1, actin, the FLAG epitope or phosphorylated Ser155. (E) HeLa cells were transfected with an expression vector for GFP-HDAC7 along with pools of siRNAs directed against two different sequences of both EMK and C-TAK1 or control siRNA, as described in panel D. Sixty hours after the initial transfection, localization of GFP-HDAC7 was examined by confocal immunofluorescence microscopy. Bar histograms represent the mean percentages of cells showing predominant cytoplasmic staining.