FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1967355315> ?p ?o ?g. }

• W1967355315 endingPage "13770" @default.
• W1967355315 startingPage "13762" @default.
• W1967355315 abstract "HDAC7, a class II histone deacetylase that is highly expressed in thymocytes, inhibits both transcription of the orphan steroid nuclear receptor Nur77 and induction of apoptosis in response to activation of the T-cell receptor (TCR). Here, we report that HDAC7 is exported to the cytoplasm by a calcium-independent signaling pathway after TCR activation. Protein kinase D1 (PKD1) was activated after TCR engagement, interacted with HDAC7, and phosphorylated three serines (Ser155, Ser318, and Ser448) at its N terminus, leading to its export from the nucleus. Mutation of Ser155, Ser318, and Ser448 blocked the nucleocytoplasmic shuttling of HDAC7 in response to TCR activation, as did overexpression of a kinase-inactive form of PKD1. Consistent with the regulatory role of HDAC7 in Nur77 expression, PKD1 activation led to the transcriptional activation of Nur77 via myocyte enhancer factor 2-binding sites in its promoter. In a mouse model of negative selection, PKD1 was activated during thymocyte activation. These observations indicate that PKD1 regulates the expression of Nur77 during thymocyte activation at least in part by phosphorylating HDAC7. HDAC7, a class II histone deacetylase that is highly expressed in thymocytes, inhibits both transcription of the orphan steroid nuclear receptor Nur77 and induction of apoptosis in response to activation of the T-cell receptor (TCR). Here, we report that HDAC7 is exported to the cytoplasm by a calcium-independent signaling pathway after TCR activation. Protein kinase D1 (PKD1) was activated after TCR engagement, interacted with HDAC7, and phosphorylated three serines (Ser155, Ser318, and Ser448) at its N terminus, leading to its export from the nucleus. Mutation of Ser155, Ser318, and Ser448 blocked the nucleocytoplasmic shuttling of HDAC7 in response to TCR activation, as did overexpression of a kinase-inactive form of PKD1. Consistent with the regulatory role of HDAC7 in Nur77 expression, PKD1 activation led to the transcriptional activation of Nur77 via myocyte enhancer factor 2-binding sites in its promoter. In a mouse model of negative selection, PKD1 was activated during thymocyte activation. These observations indicate that PKD1 regulates the expression of Nur77 during thymocyte activation at least in part by phosphorylating HDAC7. Histone acetylation and deacetylation are important in modifying chromatin structure and in regulating gene expression in eukaryotes. Mammalian histone deacetylases (HDACs) 1The abbreviations used are: HDACs, histone deacetylases; MEF2, myocyte enhancer factor 2; CaMK, calcium/calmodulin-dependent kinase; TCR, T-cell receptor; DAG, diacylglycerol; PKC, protein kinase C; PKD, protein kinase D; GFP, green fluorescent protein; GST, glutathione S-transferase; PMA, phorbol 12-myristate 13-acetate; HA, hemagglutinin; CsA, cyclosporin A. are divided into three classes based on their homology to yeast proteins. Class I HDACs are homologous to Rpd3; class II HDACs are related to Hda1; and class III HDACs are homologous to Sir2. Class II HDACs are further subdivided into class IIa (HDAC4, HDAC5, HDAC7, and HDAC9 and the HDAC9 splice variant MITR) and class IIb (HDAC6 and HDAC10) (reviewed in Ref. 1.Verdin E. Dequiedt F. Kasler H.G. Trends Genet. 2003; 19: 286-293Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar). The class IIa HDACs possess a conserved C-terminal catalytic HDAC domain and interact with myocyte enhancer factor 2 (MEF2) transcription factors through an N-terminal 17-amino acid motif. This interaction leads to the recruitment of class IIa HDACs to select promoters, where MEF2 is bound, resulting in the repression of its transcriptional activity. The repressive activity of class IIa HDACs is tightly regulated by nucleocytoplasmic shuttling and by their phosphorylation-dependent association with the intracellular 14-3-3 proteins (2.Dressel U. Bailey P.J. Wang S.C. Downes M. Evans R.M. Muscat G.E. J. Biol. Chem. 2001; 276: 17007-17013Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 3.Grozinger C.M. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7835-7840Crossref PubMed Scopus (505) Google Scholar, 4.Kao H.Y. Verdel A. Tsai C.C. Simon C. Juguilon H. Khochbin S. J. Biol. Chem. 2001; 276: 47496-47507Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 5.Zhou X. Marks P.A. Rifkind R.A. Richon V.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10572-10577Crossref PubMed Scopus (208) Google Scholar). Phosphorylation of conserved residues in the N-terminal regions of HDAC4, HDAC5, HDAC7, and HDAC9 in response to cellular signals leads to interaction with 14-3-3 proteins, dissociation of the class IIa HDAC·MEF2 complexes, and a conformational change culminating in export from the nucleus (6.McKinsey T.A. Zhang C.L. Lu J. Olson E.N. Nature. 2000; 408: 106-111Crossref PubMed Scopus (881) Google Scholar, 7.McKinsey T.A. Zhang C.L. Olson E.N. Mol. Cell. Biol. 2001; 21: 6312-6321Crossref PubMed Scopus (239) Google Scholar). Given the central role of phosphorylation in the regulation of class IIa HDAC activity, there is considerable interest in the identification of the responsible kinase(s). It has been reported that the calcium/calmodulin-dependent kinase (CaMK) phosphorylates HDAC4, HDAC5, and HDAC9, leading to their export from the nucleus and to gene activation in skeletal and cardiac myocytes (6.McKinsey T.A. Zhang C.L. Lu J. Olson E.N. Nature. 2000; 408: 106-111Crossref PubMed Scopus (881) Google Scholar, 7.McKinsey T.A. Zhang C.L. Olson E.N. Mol. Cell. Biol. 2001; 21: 6312-6321Crossref PubMed Scopus (239) Google Scholar, 8.Passier R. Zeng H. Frey N. Naya F.J. Nicol R.L. McKinsey T.A. Overbeek P. Richardson J.A. Grant S.R. Olson E.N. J. Clin. Investig. 2000; 105: 1395-1406Crossref PubMed Scopus (422) Google Scholar, 9.Lu J. McKinsey T.A. Zhang C.L. Olson E.N. Mol. Cell. 2000; 6: 233-244Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 10.McKinsey T.A. Zhang C.L. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14400-14405Crossref PubMed Scopus (428) Google Scholar, 11.Lu J. McKinsey T.A. Nicol R.L. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4070-4075Crossref PubMed Scopus (424) Google Scholar). However, the endogenous HDAC kinase activity in cardiac myocytes is resistant to pharmacological inhibitors of CaMK, suggesting that other kinases may be responsible for HDAC phosphorylation (12.Zhang C.L. McKinsey T.A. Chang S. Antos C.L. Hill J.A. Olson E.N. Cell. 2002; 110: 479-488Abstract Full Text Full Text PDF PubMed Scopus (825) Google Scholar). We recently reported that HDAC7 is expressed at high levels in thymocytes during the CD4+ CD8+ double-positive stage (14.Dequiedt F. Kasler H. Fischle W. Kiermer V. Weinstein M. Herndier B.G. Verdin E. Immunity. 2003; 18: 687-698Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). In resting thymocytes, HDAC7 is localized in the cell nucleus and represses the expression of the orphan steroid nuclear receptor Nur77 by interacting with the transcription factor MEF2D, which binds constitutively to the nur77 promoter (13.Woronicz J.D. Lina A. Calnan B.J. Szychowski S. Cheng L. Winoto A. Mol. Cell. Biol. 1995; 15: 6364-6376Crossref PubMed Scopus (193) Google Scholar, 14.Dequiedt F. Kasler H. Fischle W. Kiermer V. Weinstein M. Herndier B.G. Verdin E. Immunity. 2003; 18: 687-698Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). nur77, an immediate-early gene, is up-regulated in response to T-cell receptor (TCR) activation and has been implicated in the negative selection (apoptosis) of T cells (15.Liu Z.G. Smith S.W. McLaughlin K.A. Schwartz L.M. Osborne B.A. Nature. 1994; 367: 281-284Crossref PubMed Scopus (505) Google Scholar, 16.Woronicz J.D. Calnan B. Ngo V. Winoto A. Nature. 1994; 367: 277-281Crossref PubMed Scopus (508) Google Scholar). After TCR activation, HDAC7 is exported to the cytoplasm, leading to the derepression of the nur77 promoter and the induction of apoptosis. Interestingly, mutation of three serine residues at the N terminus of HDAC7 inhibits its nucleocytoplasmic shuttling in response to TCR activation and also suppresses TCR-induced apoptosis (14.Dequiedt F. Kasler H. Fischle W. Kiermer V. Weinstein M. Herndier B.G. Verdin E. Immunity. 2003; 18: 687-698Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). These observations indicate that an intracellular signaling pathway, originating at the TCR, is involved in the phosphorylation and subcellular localization of HDAC7 in thymocytes and that this signal controls the apoptosis of thymocytes in response to TCR activation. T cells use a complex array of signal transduction pathways to control their proliferation, differentiation, survival, and apoptosis. TCR engagement leads to the activation of phospholipase Cγ1, which in turn initiates the activation of two well characterized major signaling pathways. Phospholipase Cγ1 induces the cleavage of phosphatidylinositol in the plasma membrane, producing inositol triphosphate and diacylglycerol. Inositol polyphosphates increase intracellular calcium levels by binding to specific receptors in the endoplasmic reticulum, resulting in the activation of the calcineurin/NF-AT cascade. Diacylglycerol (DAG) induces a calcium-independent signal transduction pathway, which comprises the activation of protein kinase C (PKC) and protein kinase D (PKD) signaling modules (reviewed in Ref. 17.Cantrell D.A. Curr. Opin. Immunol. 2003; 15: 294-298Crossref PubMed Scopus (22) Google Scholar). PKD1, also called PKCμ, is a serine/threonine kinase that belongs to a new family of protein kinases with two other members, PKD2 and PKD3/PKCν. PKD1, the main isoform expressed in T cells (18.Marklund U. Lightfoot K. Cantrell D. Immunity. 2003; 19: 491-501Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), is mainly activated by a phospholipase C-DAG-PKC signal transduction pathway (reviewed in Ref. 19.Rykx A. De Kimpe L. Mikhalap S. Vantus T. Seufferlein T. Vandenheede J.R. Van Lint J. FEBS Lett. 2003; 546: 81-86Crossref PubMed Scopus (196) Google Scholar). PKD1 activation is mediated by the PKC-dependent phosphorylation of Ser744 and Ser748 in the activation loop of its catalytic domain (20.Iglesias T. Waldron R.T. Rozengurt E. J. Biol. Chem. 1998; 273: 27662-27667Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 21.Waldron R.T. Rey O. Iglesias T. Tugal T. Cantrell D. Rozengurt E. J. Biol. Chem. 2001; 276: 32606-32615Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). PKD1 is activated in B- and T-cells after engagement of their respective receptors (18.Marklund U. Lightfoot K. Cantrell D. Immunity. 2003; 19: 491-501Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 22.Matthews S.A. Dayalu R. Thompson L.J. Scharenberg A.M. J. Biol. Chem. 2003; 278: 9086-9091Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 23.Yuan J. Bae D. Cantrell D. Nel A.E. Rozengurt E. Biochem. Biophys. Res. Commun. 2002; 291: 444-452Crossref PubMed Scopus (61) Google Scholar, 24.Sidorenko S.P. Law C.L. Klaus S.J. Chandran K.A. Takata M. Kurosaki T. Clark E.A. Immunity. 1996; 5: 353-363Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). However, the specific substrates for PKD and its exact function in lymphocytes remain unclear. Here, we show that a calcium-independent signaling pathway is responsible for the nucleocytoplasmic shuttling of HDAC7 in a thymocyte hybridoma cell line (DO11.10) after TCR activation. Moreover, PKD1, which is activated after TCR engagement, interacts with and phosphorylates HDAC7, leading to its nuclear export and to the activation of Nur77 transcription. Finally, we show that PKD1 is activated by TCR activation in vivo in a mouse model of negative selection. Plasmids—The pcDNA3.1-based expression vector for FLAG-tagged human HDAC7 has been described (25.Fischle W. Dequiedt F. Fillion M. Hendzel M.J. Voelter W. Verdin E. J. Biol. Chem. 2001; 276: 35826-35835Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). C-terminal green fluorescent protein (GFP) fusions were constructed in pEGFP-N1 (Clontech). Deletion constructs of HDAC7 were generated by PCR and the cloning procedures described previously (25.Fischle W. Dequiedt F. Fillion M. Hendzel M.J. Voelter W. Verdin E. J. Biol. Chem. 2001; 276: 35826-35835Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Site-directed mutagenesis was performed with a QuikChange kit (Stratagene, La Jolla, CA). All mutations were verified by DNA sequencing. The glutathione S-transferase (GST) fusion proteins containing the N or C terminus of HDAC7 have been described (25.Fischle W. Dequiedt F. Fillion M. Hendzel M.J. Voelter W. Verdin E. J. Biol. Chem. 2001; 276: 35826-35835Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The luciferase reporter plasmid driven by the nur77 promoter (pNur77-Luc) was generated by cloning the –3800 to +87 genomic sequences of the nur77 promoter (a kind gift from Astar Winoto, University of California, Berkeley, CA) (13.Woronicz J.D. Lina A. Calnan B.J. Szychowski S. Cheng L. Winoto A. Mol. Cell. Biol. 1995; 15: 6364-6376Crossref PubMed Scopus (193) Google Scholar) into pGL2-Basic (Promega). Minimal wild-type and mutant MEF2 reporter constructs (pMEF2wt-Luc and pMEF2mt-Luc) were as described (13.Woronicz J.D. Lina A. Calnan B.J. Szychowski S. Cheng L. Winoto A. Mol. Cell. Biol. 1995; 15: 6364-6376Crossref PubMed Scopus (193) Google Scholar). PKD1 expression vectors were provided by Dr. Alex Toker (Beth Israel Deaconess Medical Center, Boston, MA). Cell Culture, Transfections, and Reporter Assay—DO11.10 T-cell hybridomas and 293T cells were grown at 37 °C in RPMI 1640 medium and Dulbecco's modified Eagle's medium, respectively, supplemented with 10% fetal bovine serum, 2 mm glutamine, and 50 units/ml streptomycin/penicillin. DO11.10 cells were transfected by the DEAE-dextran/chloroquine method. The DNA concentration was kept constant in different samples by using the corresponding empty vector. In some cases, cells were treated with phorbol 12-myristate 13-acetate (PMA; 10 ng/ml) and ionomycin (0.5 μm) for 4 h, beginning 36 h after transfection, and harvested for reporter assays. All transfections represent the result of at least three independent experiments performed in triplicate. Luciferase reporter assays were performed with the Dual-Luciferase reporter assay system (Promega) using an elongation factor 1α promoter-driven Renilla luciferase expression vector as an internal control. 293T cells were transfected by the standard calcium phosphate precipitation method. For anti-CD3/CD28 antibody stimulation, monoclonal antibodies 500A2 and 37.51 were bound to the culture flask by incubating a 1:2000 dilution in phosphate-buffered saline overnight at 4 °C, followed by three rinses with phosphate-buffered saline. Immunoprecipitation—Total cellular extracts from DO11.10 cells or primary thymocytes were prepared in 20 mm Hepes (pH 7.5), 10 mm EGTA, 2.5 mm MgCl2, 1% Nonidet P-40, 2 mm orthovanadate, 1 mm dithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride supplemented with protease inhibitors (Complete, Roche Applied Science). Total cell lysates from transiently transfected 293T cells were prepared in IPLS buffer (50 mm Tris-HCl (pH 7.5), 0.5 mm EDTA, 0.5% Nonidet P-40, and 150 mm NaCl) supplemented with protease inhibitors (26.Fischle W. Emiliani S. Hendzel M.J. Nagase T. Nomura N. Voelter W. Verdin E. J. Biol. Chem. 1999; 274: 11713-11720Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Immunoprecipitations were carried out overnight at 4 °C. For FLAG-tagged proteins, antibody M2-agarose (Sigma) was used at 15 μl/ml. Immunoprecipitated material was washed three times with IPLS buffer (26.Fischle W. Emiliani S. Hendzel M.J. Nagase T. Nomura N. Voelter W. Verdin E. J. Biol. Chem. 1999; 274: 11713-11720Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). For immunoprecipitation of endogenous PKD1, anti-PKD1 antiserum was used at 2 μg/ml in combination with a 50% protein A-Sepharose slurry (Amersham Biosciences). Immunoprecipitated PKD1 was washed three times with phosphate-buffered saline containing 1% Nonidet P-40 and 2 mm orthovanadate and once with PKD1 kinase buffer (20 mm Hepes (pH 7.5), 20 mm MgCl2, 0.1 mm orthovanadate, and 2 mm dithiothreitol). Bound proteins were subjected to either SDS-PAGE and Western blotting analysis or protein kinase assays. Protein Kinase Assays—Immunoprecipitated PKD1 was incubated with myelin basic protein or purified GST-HDAC7 fusion proteins. Phosphorylation reactions were performed in 30 μl of PKD1 kinase buffer supplemented with 20 μm ATP and 5 μCi of [γ-32P]ATP at 30 °C for 30 min. Reactions were stopped by the addition of 4× Laemmli sample buffer and resolved by SDS-PAGE on 8% gels. Immunofluorescence—DO11.10 cells were transfected with HDAC7-GFP fusion constructs and, where indicated, with PKD1 expression vectors. After transfection, cells were stimulated for 1 h with PMA/ionomycin, PMA, ionomycin, or anti-CD3/CD28 antibodies. Proteins were localized by immunofluorescence microscopy with an Olympus BX60 confocal fluorescence microscope (Bio-Rad). SDS-PAGE and Western Blotting—SDS-PAGE and Western blot analysis were performed according to standard procedures (27.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Western blots were developed with an ECL detection kit (Amersham Biosciences). Anti-PKD1 and anti-hemagglutinin (HA) antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-tubulin antibody was from Sigma. Anti-mouse Nur77 antibody was from Pharmingen (San Diego, CA). The antibody specific for PKD1 phosphorylated at Ser744 and Ser748 was from Cell Signaling (Beverly, MA). GST Fusion Proteins and Pull-down Assays—These assays were performed as described (28.Fischle W. Dequiedt F. Hendzel M.J. Guenther M.G. Lazar M.A. Voelter W. Verdin E. Mol. Cell. 2002; 9: 45-57Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). Peptide Injection of Mice—DO11.10 transgenic mice have been described previously (29.Murphy K.M. Heimberger A.B. Loh D.Y. Science. 1990; 250: 1720-1723Crossref PubMed Scopus (1653) Google Scholar). Balb/c wild-type mice and DO11.10 transgenic mice were injected intraperitoneally with 250 μl of a sterile 100 μm solution of ovalbumin peptide (ISQAVHAHAEINEAGR). Mice were sacrificed at the indicated times after injection. A Calcium-independent Pathway Regulates Nucleocytoplasmic Shuttling of HDAC7—Our previous work (14.Dequiedt F. Kasler H. Fischle W. Kiermer V. Weinstein M. Herndier B.G. Verdin E. Immunity. 2003; 18: 687-698Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) has documented that signals emanating from the TCR lead to the phosphorylation of HDAC7 and to its export from the nucleus. Accordingly, treatment of the thymocyte hybridoma cell line DO11.10 with phorbol esters and calcium ionophores (PMA/ionomycin), a treatment that mimics the two main pathways activated by the TCR, led to the nuclear export of an HDAC7-GFP fusion protein. In contrast, an HDAC7 mutant in which phosphorylation was prevented by substituting Ser155, Ser318, and Ser448 with alanines (HDAC7ΔP-GFP) remained in the nucleus (Fig. 1). Surprisingly, treatment with PMA alone (but not ionomycin) led to the nuclear export of HDAC7 (Fig. 1), suggesting that a calcium-independent pathway is responsible for the nucleocytoplasmic shuttling of HDAC7 after TCR activation. The HDAC7 mutant was also unresponsive to PMA treatment, indicating that the conserved serines in HDAC7 are potential phosphorylation targets for this intracellular signaling cascade. A PKC/PKD-dependent Pathway Regulates Nucleocytoplasmic Shuttling of HDAC7 after TCR Activation—To further define the factors involved in the nucleocytoplasmic shuttling of HDAC7, we used a number of specific inhibitors of distinct signaling pathways. Before PMA was added, DO11.10 cells expressing HDAC7-GFP were treated for 30 min with Gö6976, an inhibitor that targets both calcium-dependent PKC isoforms and PKD; with Gö6983 and GF109203X, two general inhibitors of PKCs; with KN62, a specific inhibitor of CaMK; or with cyclosporin A (CsA), a specific inhibitor of calcineurin. Pretreatment with Gö6976, Gö6983, and GF109203X completely inhibited HDAC7 nuclear export mediated by PMA (Fig. 2). In contrast, KN62 and CsA, which inhibit calcium-dependent pathways, had no effect on the cellular distribution of HDAC7 in response to PMA. Importantly, similar results were obtained after cross-linking the T-cell antigen receptor with anti-CD3/CD28 antibodies, a more physiologically relevant stimulus (Fig. 2). These observations indicate that CaMK and calcineurin are not involved in the nucleocytoplasmic shuttling of HDAC7 after TCR activation and also that a PKC/PKD-dependent mechanism is responsible for the nucleocytoplasmic shuttling of HDAC7 after TCR activation. A PKC/PKD-dependent Pathway Controls Nur77 Expression and Transcriptional Activation after TCR Engagement—In view of the key role of HDAC7 in regulating Nur77 expression after TCR activation, we tested the role of a PKC/PKD signaling pathway in Nur77 induction after TCR activation. In agreement with the results shown in Fig. 2, pretreatment with the inhibitors Gö6976, Gö6983, and GF109203X suppressed the induction of Nur77 protein expression in response to PMA, whereas KN62 and CsA surprisingly potentiated the effect of PMA (Fig. 3A). Similar results were obtained in cells transfected with a construct containing the nur77 promoter driving a luciferase reporter. The PMA-mediated transcriptional activation of the nur77 promoter was inhibited by the PKC inhibitors and potentiated by KN62 and CsA (Fig. 3B). The effects of the same inhibitors were tested after the induction of Nur77 expression by anti-CD3/CD28 antibodies. Pretreatment of DO11.10 cells with the PKC/PKD inhibitor (Gö6976) abolished the induction of Nur77 at the level of both protein expression and promoter induction (Fig. 3, C and D). The other inhibitors (Gö6983, GF109203X, and CsA) partially suppressed Nur77 expression and its promoter (Fig. 3, C and D). The calmodulin inhibitor (KN62) had no effect on the induction of Nur77 after TCR activation, further confirming that CaMKs are not involved in the HDAC7-dependent regulation of Nur77 by PMA or the TCR (Fig. 3, C and D). PKD1 Is Activated in Thymocyte Hybridoma Cells after TCR Engagement—Next, we analyzed the amino acid sequences surrounding the conserved serines in HDAC7 for homology to known consensus protein kinase sites. The three conserved serines in HDAC7 (Ser155, Ser318, and Ser448) and other class IIa HDACs are consensus sites for CaMK ((R/K)XX(S/T)). However, as shown above, chemical blockage of CaMK did not suppress HDAC7 nuclear export after TCR activation. Interestingly, leucine was present at position –5 relative to each serine (Fig. 4A), a requirement for substrates of PKD1 (30.Nishikawa K. Toker A. Johannes F.J. Songyang Z. Cantley L.C. J. Biol. Chem. 1997; 272: 952-960Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar). To determine whether PKD1 is activated by antigen receptor signals, we used an antibody specific for the active/phosphorylated form of PKD1 (Ser744 and Ser748). Treatment of DO11.10 cells with PMA/ionomycin induced PKD1 phosphorylation in a time-dependent manner (Fig. 4B). PMA alone (but not ionomycin) had a similar effect (Fig. 4B), demonstrating that a calcium-independent mechanism is involved. Cell treatment with anti-CD3/CD28 antibody also activated PKD1 (Fig. 4B). To further confirm the activation of PKD1 in DO11.10 cells, we performed an in vitro kinase assay. The cells were treated with PMA/ionomycin, and endogenous PKD1 was immunoprecipitated with anti-PKD1 antibody, followed by a kinase assay using myelin basic protein as substrate. Endogenous PKD1 was activated after TCR activation, as indicated by the PKD1 autophosphorylation band and the phosphorylation of myelin basic protein (Fig. 4C). These results demonstrate that PKD1 is activated in DO11.10 cells by TCR activation independently of calcium signaling. PKD1 Interacts with and Phosphorylates HDAC7—To test whether HDAC7 interacts with PKD1, we incubated GST fusion proteins containing the N terminus (GST-HDAC7(N-ter)) or the C terminus (GST-HDAC7(C-ter)) of HDAC7 after expression in bacteria with extracts of DO11.10 cells, either untreated or treated with PMA/ionomycin. Immunoblotting with an antibody specific for PKD1 revealed that endogenous PKD1 bound to GST-HDAC7(N-ter), but not to GST-HDAC7(C-ter) (Fig. 5A). HDAC7 bound to PKD1 present in resting or activated extracts (PMA/ionomycin) (Fig. 5A), suggesting that enzymatic activation of PKD1 is not necessary for HDAC7 binding. To further confirm the interaction of HDAC7 with PKD1, FLAG-HDAC7 and HA-PKD1 expression plasmids were coexpressed in 293T cells. FLAG-HDAC7 was immunoprecipitated, and the immunoprecipitate was probed for the presence of PKD1. Both proteins interacted, regardless of the order of immunoprecipitation (Fig. 5B). To investigate whether PKD1 phosphorylates HDAC7 directly, we immunoprecipitated endogenous PKD1 from DO11.10 cells, untreated or treated with PMA/ionomycin, and used it for kinase activity assay with GST-HDAC7(N-ter) or GST-HDAC7(C-ter) as substrate. PKD1 was activated after PMA/ionomycin treatment, as indicated by the PKD1 autophosphorylation band (Fig. 5C). As predicted, only the N-terminal region of HDAC7, which bears the conserved residues Ser155, Ser318, and Ser448, was phosphorylated by PKD1. To confirm that the predicted PKD1 phosphorylation sites were indeed phosphorylated, we performed an in vitro kinase assay using a GST-HDAC7 construct in which the conserved residues Ser155, Ser318, and Ser448 were mutated to alanines (GST-HDAC7ΔP) as substrate. The HDAC7 triple phosphorylation mutant was not phosphorylated by PKD1 after cell treatment with PMA/ionomycin (Fig. 5D). These observations demonstrate the ability of PKD1 to bind and phosphorylate HDAC7 and the requirement for the conserved HDAC7 residues Ser155, Ser318, and Ser448. PKD1 Activity Modulates HDAC7 Nucleocytoplasmic Shuttling—Because HDAC7 Ser155, Ser318, and Ser448 were required for nuclear export after TCR activation, we tested whether PKD1 could regulate the subcellular distribution of HDAC7. DO11.10 cells were cotransfected with HDAC7-GFP or the phosphorylation mutant HDAC7ΔP-GFP fusion construct as well as an expression vector for wild-type, constitutively active (PKD1 SS/EE), or kinase-inactive (PKD1 SS/AA) PKD1. HDAC7 was exported to the cytoplasm after PMA treatment in the absence or presence of wild-type PKD1 (Fig. 6). In contrast, overexpression of the constitutively active form of PKD1 (PKD1 SS/EE) led to export of HDAC7 from the nucleus to the cytoplasm, even in untreated cells (Fig. 6). Importantly, the kinase-inactive form of PKD1 (PKD1 SS/AA) inhibited HDAC7 nuclear export induced by PMA (Fig. 6). The subcellular localization of HDAC7ΔP-GFP was not affected by any stimulus, and the protein was observed in the nucleus under all experimental conditions (Fig. 6). These results demonstrate that PKD1 activity is both necessary and sufficient for the regulation of the nucleocytoplasmic shuttling of HDAC7. PKD1 Overexpression Activates Nur77 Expression through MEF2—To determine whether PKD1-induced HDAC7 nuclear export is sufficient to activate the nur77 promoter, we cotransfected DO11.10 cells with a nur77 promoter-reporter construct and a vector encoding constitutively active PKD1 (PKD1 SS/EE). PKD1 SS/EE increased the basal transcriptional activity of the nur77 promoter by ∼5-fold; activation was more modest in the presence of PMA/ionomycin (Fig. 7A). We reported previously that HDAC7 overexpression can block the TCR-mediated activation of the nur77 promoter (14.Dequiedt F. Kasler H. Fischle W. Kiermer V. Weinstein M. Herndier B.G. Verdin E. Immunity. 2003; 18: 687-698Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). To test whether PKD1 overexpression can block the repressive activity of HDAC7, we transfected DO11.10 cells with the HDAC7 or HDAC7ΔP expression vector. As expected, overexpression of HDAC7 inhibited the transcriptional activation of Nur77 (75%) induced by PMA/ionomycin (Fig. 7B). Overexpression of constitutively active PKD1 reversed the repressive effect of wild-type HDAC7 (Fig. 7B). Importantly, PKD1 SS/EE had no effect on the transcriptional repression mediated by the HDAC7ΔP phosphorylation mutant (Fig. 7B). To confirm that MEF2 is a target of the PKD1-dependent activation, we cotransfected DO11.10 cells with a reporter construct containing four MEF2D-binding sites upstream of a minimal promoter (pMEF2wt-Luc) and the PKD1 SS/EE expression vector. A construct containing mutant MEF2D-binding sites served as a control (pMEF2mt-Luc). PMA/ionomycin increased the activity of the pMEF2wt-Luc reporter construct by 18-fold, but had no effect on the pMEF2mt-Luc construct (Fig. 7C). Overexpression of PKD1 SS/EE caused a 12-fold activation of pMEF2wt-Luc under basal conditions and further increased its activity in response to PMA/ionomycin (Fig. 7C). As predicted, active PKD1 had no effect on pMEF2mt-Luc (Fig. 7C). PKD1 Is Activated during Negative Selection of T cells—Our observations indicated that PKD1 regulated gene expression in thymocytes during T-cell activation in an MEF2- and HDAC7-dependent manner. To test the relevance of these observations in vivo, we examined the status of PKD1 in a mouse model of negative selection" @default.
• W1967355315 created "2016-06-24" @default.
• W1967355315 creator A5025168089 @default.
• W1967355315 creator A5069989737 @default.
• W1967355315 creator A5075358012 @default.
• W1967355315 creator A5081693620 @default.
• W1967355315 creator A5084182062 @default.
• W1967355315 date "2005-04-01" @default.
• W1967355315 modified "2023-10-16" @default.
• W1967355315 title "Protein Kinase D1 Phosphorylates HDAC7 and Induces Its Nuclear Export after T-cell Receptor Activation" @default.
• W1967355315 cites W1543101698 @default.
• W1967355315 cites W1900565974 @default.
• W1967355315 cites W1923015860 @default.
• W1967355315 cites W1980655244 @default.
• W1967355315 cites W1984695968 @default.
• W1967355315 cites W1992263554 @default.
• W1967355315 cites W1992725701 @default.
• W1967355315 cites W2005471059 @default.
• W1967355315 cites W2008408686 @default.
• W1967355315 cites W2019097955 @default.
• W1967355315 cites W2020912062 @default.
• W1967355315 cites W2021714562 @default.
• W1967355315 cites W2030945682 @default.
• W1967355315 cites W2045008512 @default.
• W1967355315 cites W2048943247 @default.
• W1967355315 cites W2051933489 @default.
• W1967355315 cites W2052006994 @default.
• W1967355315 cites W2052373759 @default.
• W1967355315 cites W2056108005 @default.
• W1967355315 cites W2061763306 @default.
• W1967355315 cites W2072387160 @default.
• W1967355315 cites W2076614498 @default.
• W1967355315 cites W2080115585 @default.
• W1967355315 cites W2081509331 @default.
• W1967355315 cites W2093456806 @default.
• W1967355315 cites W2099093659 @default.
• W1967355315 cites W2099792852 @default.
• W1967355315 cites W2099959808 @default.
• W1967355315 cites W2100503396 @default.
• W1967355315 cites W2105499046 @default.
• W1967355315 cites W2117346996 @default.
• W1967355315 cites W2117813626 @default.
• W1967355315 cites W2121296685 @default.
• W1967355315 cites W2133446638 @default.
• W1967355315 cites W2154075930 @default.
• W1967355315 cites W2154572495 @default.
• W1967355315 cites W2171347864 @default.
• W1967355315 doi "https://doi.org/10.1074/jbc.m413396200" @default.
• W1967355315 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15623513" @default.
• W1967355315 hasPublicationYear "2005" @default.
• W1967355315 type Work @default.
• W1967355315 sameAs 1967355315 @default.
• W1967355315 citedByCount "132" @default.
• W1967355315 countsByYear W19673553152012 @default.
• W1967355315 countsByYear W19673553152013 @default.
• W1967355315 countsByYear W19673553152014 @default.
• W1967355315 countsByYear W19673553152015 @default.
• W1967355315 countsByYear W19673553152016 @default.
• W1967355315 countsByYear W19673553152017 @default.
• W1967355315 countsByYear W19673553152018 @default.
• W1967355315 countsByYear W19673553152019 @default.
• W1967355315 countsByYear W19673553152020 @default.
• W1967355315 countsByYear W19673553152021 @default.
• W1967355315 countsByYear W19673553152022 @default.
• W1967355315 crossrefType "journal-article" @default.
• W1967355315 hasAuthorship W1967355315A5025168089 @default.
• W1967355315 hasAuthorship W1967355315A5069989737 @default.
• W1967355315 hasAuthorship W1967355315A5075358012 @default.
• W1967355315 hasAuthorship W1967355315A5081693620 @default.
• W1967355315 hasAuthorship W1967355315A5084182062 @default.
• W1967355315 hasBestOaLocation W19673553151 @default.
• W1967355315 hasConcept C11960822 @default.
• W1967355315 hasConcept C184235292 @default.
• W1967355315 hasConcept C185592680 @default.
• W1967355315 hasConcept C190062978 @default.
• W1967355315 hasConcept C2780114586 @default.
• W1967355315 hasConcept C54757728 @default.
• W1967355315 hasConcept C86803240 @default.
• W1967355315 hasConcept C95444343 @default.
• W1967355315 hasConcept C97029542 @default.
• W1967355315 hasConceptScore W1967355315C11960822 @default.
• W1967355315 hasConceptScore W1967355315C184235292 @default.
• W1967355315 hasConceptScore W1967355315C185592680 @default.
• W1967355315 hasConceptScore W1967355315C190062978 @default.
• W1967355315 hasConceptScore W1967355315C2780114586 @default.
• W1967355315 hasConceptScore W1967355315C54757728 @default.
• W1967355315 hasConceptScore W1967355315C86803240 @default.
• W1967355315 hasConceptScore W1967355315C95444343 @default.
• W1967355315 hasConceptScore W1967355315C97029542 @default.
• W1967355315 hasIssue "14" @default.
• W1967355315 hasLocation W19673553151 @default.
• W1967355315 hasOpenAccess W1967355315 @default.
• W1967355315 hasPrimaryLocation W19673553151 @default.
• W1967355315 hasRelatedWork W1964515703 @default.
• W1967355315 hasRelatedWork W1978425204 @default.
• W1967355315 hasRelatedWork W1983994417 @default.
• W1967355315 hasRelatedWork W2051108283 @default.
• W1967355315 hasRelatedWork W2158951696 @default.

• next