Phosphorylation and nuclear export of HDAC7 is transient after PMA treatment or TCR activation and is reversed by a cellular phosphatase. (A) Western blot showing phosphorylation of Ser155, Ser318, and Ser448 in HDAC7. DO11.10-HDAC7-Flag cells were treated or not with PMA for 30 min. HDAC7 phosphorylation levels were determined by immunoprecipitation of HDAC7-Flag followed by immunoblotting with antisera specific for phosphorylated Ser155, Ser318, and Ser448. The immunoprecipitated material was incubated with CIP before Western blotting where indicated. An anti-Flag Western blot shows equal amounts of HDAC7-Flag were immunoprecipitated. (B) DO11.10-HDAC7-Flag cells were treated with PMA alone (left panel), or with PMA + okadaic acid (right panel) for the times indicated. Equal amounts of HDAC7-Flag were immunoblotted with anti-phospho-HDAC7 antisera. (C) Immunofluorescence was performed in DO11.10 cells nucleofected with an HDAC7-GFP expression vector followed 24 h later by PMA treatment for the indicated times. HDAC7 subcellular distribution was analyzed by immunofluorescence microscopy (representative fields are shown). (D) Quantitation of immunofluorescence microscopy in C. The percentage of cells showing nuclear exclusion of HDAC7 is indicated at each time point. One-hundred cells were counted for each point. Error bars represent SEM for four independent experiments. (E) Western blot showing total cell lysates prepared from DO11.10 cells, treated as in B, and analyzed by Western blotting with antisera against Nur77 and actin. (F) DO11.10-HDAC7-Flag cells were treated or not with anti-CD3 antibody for the indicated times. HDAC7 phosphorylation was analyzed as in B. (G) Total cell lysates were prepared from DO11.10 cells treated as in E and analyzed by Western blotting with antisera against Nur77 and actin.

Myosin phosphatase dephosphorylates HDAC7 and induces its nuclear reimport after TCR activation. (A) DO11.10-HDAC7-Flag cells were either left untreated or treated with PMA for 30 min. HDAC7 phosphorylation was determined as in Figure 1A. A mixture of recombinant PP1 isoforms was added to the immunoprecipitated material before Western blotting with phospho-specific antisera for HDAC7. (B) DO11.10-HDAC7-Flag cells nucleofected with either siCo or siPP1β + MYPT1 were treated with PMA for the indicated times. HDAC7 phosphorylation was determined as in Figure 1A. HDAC7-Flag, PP1β, MYPT1, and actin protein levels are shown. (C) Depletion of PP1β and MYPT1 in DO11.10 cells by siRNA-mediated knockdown. Western blot analysis of PP1β and MYPT1 protein levels 48 h after nucleofection of control (siCo) or PP1β and MYPT1 (siPP1β + MYPT1) siRNA oligonucleotides. (D) DO11.10 cells were nucleofected with either siCo or siPP1β + MYPT1 and with an HDAC7-GFP expression vector. Cells were treated with PMA 24 h for the indicated periods of time. HDAC7 was localized by immunofluorescence microscopy. The percentage of cells showing nuclear exclusion of HDAC7 is indicated at each time point after PMA addition. One-hundred cells were counted for each point. Error bars represent SEM for four independent experiments. (*) p < 0.05.

Myosin phosphatase subunits PP1β and MYPT1 coimmunoprecipitate with HDAC7. (A) Coomassie gel of HDAC7-Flag-tagged-containing complexes immunoprecipitated from DO11.10 cells with anti-M2 agarose beads. The HDAC7-interacting proteins are indicated by arrows and were identified by mass spectrometry (see Supplemental Material for details). (B) Cell lysates from DO11.10-Empty and DO11.10-HDAC7-Flag cells were immunoprecipitated with anti-M2 agarose beads and analyzed by Western blotting with the indicated antibodies. (C,D) Total cell lysates from mouse primary thymocytes were immunoprecipitated with antibodies against endogenous PP1β, PP1γ, MYPT1, and 14–3–3ε, followed by immunoblotting with anti-HDAC7 antibody. “No Ab” indicates no antibody.

Myosin phosphatase regulates Nur77 activation and thymocyte apoptosis after TCR activation. (A) Depletion of PP1β, MYPT1, or both in DO11.10 cells by siRNA-mediated knockdown. PP1β and MYPT1 protein levels were analyzed 48 h after nucleofection of the different siRNAs. (B) DO11.10-Empty or DO11.10-HDAC7ΔP-Flag cells were nucleofected with either siCo, siPP1β, siMYPT1, or siPP1β + MYPT1. After 24 h, cells were treated with anti-CD3 antibody and analyzed by Western blotting with antisera against Nur77 and actin. (C) Depletion of PP1α, PP1β, or PP1γ in DO11.10 cells by siRNA-mediated knockdown. PP1α, PP1β, or PP1γ protein levels were analyzed 48 h after nucleofection of the different siRNAs. (D) DO11.10-Empty or DO11.10-HDAC7ΔP-Flag cells were nucleofected with siRNAs for the different PP1 isoforms. Cells were treated and analyzed as in Figure 1A. (E) Primary thymocytes were nucleofected with the indicated siRNAs. Cells were treated with anti-CD3 antibody for 16 h, followed by staining with AnnexinV-APC, anti-CD4-PE, and anti-CD8-FITC. A representative FACS plot is shown that quantifies Annexin V binding (X-axis) in CD4⁺CD8⁺ cells. Data are represented as mean ± SEM of three independent experiments. (*) p < 0.001. In the bottom panel, flow histograms illustrate the percentage of apoptotic cells in a representative experiment. (F) Signaling pathways responsible for HDAC7 nucleocytoplasmic shuttling after TCR activation. The nucleocytoplasmic location of HDAC7 is under the competing influences of a kinase (PKD1) and a phosphatase (myosin phosphatase).