View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

FEBS Letters 584 (2010) 631–637

journal homepage: www.FEBSLetters.org

A novel inhibitor establishes a predominant role for D as a cardiac class IIa histone deacetylase kinase

Lauren Monovich a,*, Richard B. Vega a, Erik Meredith a, Karl Miranda a, Chang Rao a, Michael Capparelli a, Douglas D. Lemon b, Dillon Phan b, Keith A. Koch b, Joseph A. Chapo b, David B. Hood b, Timothy A. McKinsey b,*

a Novartis Institutes for Biomedical Research, 3333 Walnut Street, Boulder, CO 80301, United States b Gilead Colorado, Inc., 3333 Walnut Street, Boulder, CO 80301, United States

article info abstract

Article history: Class IIa histone deacetylases (HDACs) repress involved in pathological cardiac hypertrophy. Received 13 November 2009 The anti-hypertrophic action of class IIa HDACs is overcome by signals that promote their phosphor- Revised 8 December 2009 ylation-dependent nuclear export. Several have been shown to phosphorylate class IIa Accepted 11 December 2009 HDACs, including calcium/calmodulin-dependent protein kinase (CaMK), protein kinase D (PKD) Available online 14 December 2009 and G protein-coupled receptor kinase (GRK). However, the identity of the kinase(s) responsible Edited by Ivan Sadowski for phosphorylating class IIa HDACs during cardiac hypertrophy has remained controversial. We describe a novel and selective small molecule inhibitor of PKD, bipyridyl PKD inhibitor (BPKDi). BPKDi blocks signal-dependent phosphorylation and nuclear export of class IIa HDACs in cardio- Keywords: Kinase myocytes and concomitantly suppresses hypertrophy of these cells. These studies define PKD as a Histone deacetylase principal cardiac class IIa HDAC kinase. Transcription Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Signaling Cardiomyocyte

1. Introduction (SIK) [7], which is also a member of the CaMK superfamily, and G protein- coupled receptor kinase 5 (GRK5) [8], a member of Pathological cardiac hypertrophy is accompanied by diverse the the unrelated AGC kinase superfamily, have been described alterations in myocyte expression. For example, stress signal- as class IIa HDAC kinases. ing in the adult heart results in up-regulation of dormant ‘‘fetal Using high throughput screening and medicinal chemistry, we cardiac genes” encoding embryonic isoforms of proteins that gov- have identified a series of selective small molecule inhibitors of ern growth, contractility, calcium handling and energetics, with PKD. We describe the in vitro and cellular activity of one of these concomitant down-regulation of the adult isoforms [1,2]. This gene compounds, which we refer to here as bipyridyl PKD inhibitor reprogramming is contingent on phosphorylation-dependent nu- (BPKDi). BPKDi inhibits the three members of the PKD family,

clear export of class IIa histone deacetylases (HDACs), which func- PKD1, PKD2 and PKD3, with IC50 values of 1 nM, 9 nM and 1 nM, tion as endogenous repressors of cardiac [3]. respectively. In contrast, the compound fails to significantly inhibit Intense investigation has focused on identification of class IIa other putative class IIa HDAC kinases. In cultured cardiac HDAC kinases. Over 500 genes in the encode ki- myocytes, BPKDi blocks agonist-dependent PKD activation and nases, and this ‘‘kinome” is comprised of seven superfamilies [4]. phosphorylation-dependent nuclear export of class IIa HDACs -4 Molecular, biochemical and pharmacological investigations have and -5. Pharmacological inhibition of PKD activity is associated led to the identification of three families of class IIa HDAC kinases: with attenuation of myocyte hypertrophy. These studies establish Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein ki- a key role for PKD in cardiomyocyte signaling and gene regulation. nase D (PKD) and microtubule affinity-regulating kinase (MARK), all of which possess related catalytic domains and fall within the 2. Methods CaMK superfamily [5,6]. More recently, salt-inducible kinase 2.1. PKD1 enzymatic assay

* Corresponding authors. Fax: +1 720 887 8681 (T.A. McKinsey). E-mail addresses: [email protected] (L. Monovich), timothy.mck- The assay to measure (PKD1) activity was a [email protected] (T.A. McKinsey). time-resolved fluorescence resonance transfer (TR-FRET) assay

0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.12.014 632 L. Monovich et al. / FEBS Letters 584 (2010) 631–637 using Perkin–Elmer’s LANCE™ technology. Full-length, wild-type HDAC4 or HDAC5 [12] or control pre-immune serum and Exacta human PKD1 was expressed and purified from Sf9 insect cells. Bio- Cruz beads (Santa Cruz Biotechnology; 10 ll packed volume). tinylated syntide-2 peptide was used as the substrate. The reaction Immunoprecipitation was conducted overnight at 4 °C with rock- buffer consisted of Tris–HCl (35 mM; pH 7.5), MgCl2 (5 mM), ing and beads were subsequently washed five times in lysis buffer Tween-20 (0.02%), ATP (20 lM), DTT (1 mM) and PKD1 (0.2 lg/ and prepared for SDS–PAGE. ml) . Reactions were initiated by the addition of syntide- 2 peptide (2 lM) and carried out for 50 min at room temperature. 2.4. Hypertrophy and toxicity measurements Reactions were stopped by a stop/detection buffer consisting of EDTA (50 mM), rabbit polyclonal anti-phospho syntide-2 antibody Myomaxin, alpha-skeletal actin (a-Sk-actin), b-myosin heavy (0.18 mg/ml), europium (Eu)-labeled anti-rabbit IgG (0.5 nM) and chain (b-MyHC), and atrial natriuretic factor (ANF) mRNA streptavidin-conjugated allophycocyanin (APC; 10 nM). After 1 h expression was quantified from NRVM lysates using the branched- of incubation at room temperature, FRET signal was read using DNA-technology-based QuantiGene Reagent System (Panomics, an Envision 2100 Reader and the LANCE™ Eu/APC dual protocol Fremont CA), according to the manufacturer’s protocols. Three- (Perkin–Elmer). Additional details of the screen and medicinal dimensional measurements of NRVMs were conducted using a chemistry follow-up will be described elsewhere. Kinase selectiv- Coulter Model Z2 instrument (Beckman), as previously described ity testing was performed by Invitrogen (SelectscreenTM) using [9]. Briefly, cells in 6-well dishes (2.5 105 cells/well) were the indicated recombinant kinases. washed 1 with PBS and trypsinized (200 ll trypsin; 2 min at room temperature). DMEM containing 10% FBS (2 ml) was added 2.2. NRVM preparation, culture and stimulation to trypsinized cells and cells were dispersed by vigorous pipetting. Cells were washed 1 with PBS and transferred to cuvettes con- Hearts were dissected from 1 to 3 day-old Sprague–Dawley rats, taining Isoflow buffer (Coulter; 10 ml). Volume measurements minced, and digested with collagenase (Worthington; 600 lg/ml) were obtained for 1 104 cells with Coulter aperture diameter and pancreatin (Sigma; 1X activity equivalent) in 1 Ads buffer limits between 6 and 26 lm. Cell volumes are expressed as lm3. (NaCl [116 mM], HEPES [20 mM; pH 7.4], NaH2PO4 [4.8 mM], KCl Adenylate kinase (AK) was measured in culture supernatants [5 mM], MgSO4 [400 lM], and glucose [5.5 mM]). Cells were cen- employing the bioluminescent ToxiLight kit (Cambrex) according trifuged through a step gradient of Percoll (Pharmacia) to separate to the manufacturer’s instructions. myocytes from fibroblasts, and the myocyte pool was further en- riched by pre-plating for 2 h to remove adherent fibroblasts from the cell population. Cells were plated on gelatin-coated plates in 3. Results DMEM containing FBS (10%), penicillin–streptomycin and L-gluta- mine (DMEM complete). After 12 h, plating medium was replaced 3.1. Identification of BPKDi, a selective inhibitor of PKD with serum-free DMEM supplemented with Nutridoma-SP (0.1%; Roche Applied Sciences). For studies with adenoviruses, cells were High-throughput screening was performed to identify small mixed with virus (multiplicity of infection = 25) at the time of plat- molecule inhibitors of recombinant protein kinase D1 (PKD1). A li- ing and washed 12 h post-plating. Cells were fixed with 10% forma- brary of >650 000 compounds was screened using a time-resolved lin and washed with PBS prior to visualize of GFP-HDACs with an fluorescence resonance transfer (TR-FRET) assay (Perkin–Elmer’s inverted fluorescence microscope. Adenoviruses for GFP-HDAC4 LANCE™) to measure activity of full-length, human PKD1 overex- and GFP-HDAC5 have been previously described [9,10]. Phenyleph- pressed in insect cells, as described in Section 2. The substrate rine (PE) and phorbol-12-myristate-13-acetate (PMA) were pur- for PKD1 was a biotinylated syntide-2 peptide. Medicinal chemis- chased from Sigma–Aldrich and endothelin-1 (ET-1) from try and structure–activity-relationship (SAR) studies yielded an Phoenix Pharmaceuticals. amido bipyridyl analog of a hit compound, termed BPKDi (Fig. 1A), which potently inhibited PKD1, as well as PKD2 and 2.3. Immunoblotting and immunoprecipitation PKD3 (Table 1). Further details of the discovery of this novel PKD inhibitor will be described elsewhere. NRVMs were lysed in Tris buffer (50 mM, pH 7.5) containing BPKDi was tested for its ability to inhibit other putative class IIa EDTA (5 mM), Triton-X100 (1%), protease and phosphatase inhibi- HDAC kinases in vitro using the Invitrogen Selectscreen service. As tor cocktails (Thermo Scientific). Homogenates were briefly soni- shown in Table 1, the compound failed to inhibit members of the cated and cleared by centrifugation. Total protein was resolved CaMKII and MARK families, and also lacked the ability to signifi- by SDS–polyacrylamide gel electrophoresis (PAGE) with gradient cantly inhibit SIK1 and GRK5 at the 1 lM screening concentration. gels (4–20% polyacrylamide; Invitrogen). Proteins were transferred In contrast, dose–response studies revealed that BPKDi inhibited to nitrocellulose membranes (Bio-Rad) and probed with one of the recombinant forms of each of the three PKD isoforms with low following antibodies: anti-phospho-PKD serine-916 (Cell Signaling nanomolar potency. The activity of BPKDi against the entire Invit- Technology #2051), anti-phospho-PKD serines-744/748 (Cell Sig- rogen panel of over 250 kinases will be described elsewhere. naling Technology #2054), anti-total PKD (Cell Signaling Technol- ogy #2052), anti-phospho-HDAC5 serine-259 (cross-reacts with 3.2. BPKDi selectively blocks cellular PKD activity phospho-HDAC4 serine-246) [11], anti-phospho-HDAC5 serine- 498 (cross-reacts with phospho-HDAC4 serine-467) (Abcam (PKC)-mediated phosphorylation of activation #47283). All antibody incubations were performed with 1:500 loop serines on PKD1 results in PKD1 activation, as evidenced by dilutions of antibody in TBS-T containing 3% BSA, overnight at auto-phosphorylation of PKD1 [13,14]. To determine whether 4 °C with rocking. Secondary HRP-conjugated anti-rabbit antibody BPKDi blocks endogenous, cellular PKD1 activity, neonatal rat ven- was purchased from Southern Biotechnology. Proteins were visual- tricular myocytes (NRVMs) were pre-treated with compound and ized using a chemiluminescence system (Thermo Scientific, Super- then stimulated with the PKC activator, phorbol myristate acetate Signal West Pico system). For immunoprecipitation experiments, (PMA). As shown in Fig. 1B, BPKDi significantly attenuated PMA-in- five 10-cm plates of NRVMs (1.8 106 cells/plate) were used per duced auto-phosphorylation of PKD1 on serine-916. Importantly, experimental condition. Lysates were prepared as described for BPKDi had no effect on PKC-mediated phosphorylation of the immunoblotting and subjected to rabbit anti-serum against PKD1 activation loop serines 744/748, illustrating the cellular L. Monovich et al. / FEBS Letters 584 (2010) 631–637 633

Fig. 1. BPKDi inhibits endogenous PKD1 signaling in cardiac myocytes. (A) Structure of BPKDi. (B) Neonatal rat ventricular myocytes (NRVMs; 2 106 cells/10-cm plate) were cultured in serum-free medium and incubated in the absence or presence of BPKDi (1 lM) for 30 min prior to exposure to PMA (25 nM) for 1 h, as indicated. Total protein was prepared and analyzed by immunoblotting with an antibody that recognizes PKD1 when auto-phosphorylated on serine-916 (upper panel), an antibody that recognizes PKD1 when phosphorylated by PKC on serines-744 and 748 (middle panel), or an antibody to total PKD1 (lower panel) to control for protein loading. (C) NRVMs were treated as described in (B) and stimulated for 1 h with ET-1 (25 nM), PGF2a (10 lM) or PE (10 lM) prior to protein lysate preparation and immunoblotting. Each experiment shown in this figure was performed with NRVMs prepared on separate days.

tivity [15], and thus the immunoblots shown here reflect effects of Table 1 BPKDi specifically inhibits PKD family members. BPKDi (1 lM) was tested for its BPKDi specifically on PKD1 auto-phosphorylation. ability to block activity the indicated recombinant kinases. IC50 values were determined for PKD1, PKD2 and PKD3. Kinase assays were performed by Invitrogen. n.d., not determined. 3.3. Endogenous PKD regulates phosphorylation of class IIa HDACs

Kinase % Inhibition at 1 lM BPKDi IC (lM) 50 Serines-259 and -498 are the primary phospho-acceptors that PKD1 102 0.001 control nuclear export of human HDAC5 [12,16]. The equivalent PKD2 102 0.009 sites in human HDAC4 are serines-246 and -467. Upon phosphor- PKD3 109 0.001 CaMKId 6 n.d. ylation, these sites are bound by the 14-3-3 chaperone protein CaMKIIa 0 n.d. [16–18], which exposes a cryptic nuclear export sequence in class CaMKIIb 3 n.d. IIa HDACs. BPKDi was employed to determine the role of PKD in CaMKIId 26 n.d. the regulation of HDAC4/5 phosphorylation in cardiac myocytes. CaMKIV 1 n.d. Initial studies were performed by overexpressing GFP-tagged MARK1 24 n.d. MARK2 27 n.d. HDAC4 or HDAC5 in NRVMs using adenoviruses. Cells were pre- SIK1 7 n.d. treated with vehicle or BPKDi and subsequently stimulated with GRK5 2 n.d. either PE or ET-1. As shown in Fig. 2A, agonist-dependent phos- PKCd 22 n.d. phorylation of HDAC5 was significantly attenuated by BPKDi. Sim- PKCe 3 n.d. ilar results were obtained with HDAC4 (Fig. 2B). Sequential immunoprecipitation and immunoblotting was per- formed with lysates from uninfected NRVMs to specifically evalu- selectivity of BPKDi for PKD. In addition, BPKDi efficiently blocked ate the phosphorylation state of endogenous HDAC4 and HDAC5 in PKD1 auto-phosphorylation in response to three independent G response to PE in the absence and presence of BPKDi. As shown in protein-coupled receptor (GPCR) agonists, endothelin-1 (ET-1), Fig. 2C, PE-mediated phosphorylation of both HDAC4 and HDAC5 prostaglandin-F2a (PGF2a) and phenylephrine (PE) (Fig. 1C). The was attenuated by BPKDi. Since the compound does not affect antibody against phospho-serine-916 cross-reacts with PKD2, but the activity of other putative class IIa HDAC kinases, these results PKD3 lacks an equivalent site. Prior siRNA studies demonstrated support a role for PKD as a dominant class IIa HDAC kinase in car- that PKD1-specfic knockdown in NRVMs abolished antibody reac- diac myocytes. 634 L. Monovich et al. / FEBS Letters 584 (2010) 631–637

Fig. 2. BPKDi blocks agonist-dependent phosphorylation of HDAC4 and HDAC5. (A) NRVMs (2 106/10-cm plate) were infected with adenovirus encoding GFP-tagged HDAC5. After 14 h of infection, cells were washed and cultured in serum-free medium. Cells were exposed to BPKDi (1 lM) for 30 min prior to stimulation for 1 h with either PE (10 lM) or ET-1 (25 nM), as indicated. Total protein was prepared and analyzed by immunoblotting with an antibody that recognizes HDAC5 when phosphorylatedon serine-259 (upper panel), an antibody that recognizes HDAC5 when phosphorylated on serine-498 (middle panel), or an antibody to GFP (lower panel) to control for protein loading. (B) NRVMs (2 106 cells/10-cm plate) were infected with adenovirus encoding GFP-tagged HDAC4. Cells were treated with BPKDi (1 lM) and agonists as described in (A), and immunoblotting was performed to assess HDAC4 phosphorylation. Note that HDAC4 is basally phosphorylated. The reduced phospho-signals seen in lane two (upper and middle panels) appear to be a consequence of lower total HDAC4 loading (bottom panel). (C) Uninfected NRVMs were cultured in serum-free medium in the absence or presence of BPKDi (1 lM) and stimulated for 1 h with PE (20 lM), as indicated. Total protein lysates were immunoprecipitated (IP) with antibody to either HDAC5 (left-hand panels) or HDAC4 (middle panels). Immunoprecipitates were split in two and run on separate gels for immunoblotting (IB) with either anti-phospho-HDAC antibody (upper panels) or total HDAC antibody (lower panels). Separate immunoprecipitations were performed with normal rabbit serum as a negative control for non- specific pulldown (right-hand panels).

3.4. PKD regulates nuclear export of class IIa HDACs in cardiac tainty, the results support a role for PKD in the control of agonist- myocytes dependent phosphorylation of HDACs 4 and 5 in cardiac myocytes.

Fluorescence microscopy was used to determine if PKD regu- 3.5. BPKDi blunts pathologic cardiac gene expression and myocyte lates phosphorylation-dependent nuclear export of class IIa HDACs hypertrophy in NRVMs. Cells were infected with adenoviruses encoding GFP- tagged forms on HDAC4 or HDAC5. HDAC5 was predominantly nu- Class IIa HDACs associate with MEF2 transcription factors and clear in unstimulated NRVMs but rapidly relocated to the cyto- thereby repress target genes harboring MEF2 binding sites. BPKDi plasm in response to PMA, PE or ET-1 treatment (Fig. 3A, upper was used to determine the role of endogenous PKD in the control panels). BPKDi completely blocked agonist-dependent nuclear ex- of the cardiac-specific MEF2 target gene, myomaxin [19]. NRVMs port of HDAC5 (Fig. 3A, lower panels). were exposed to the hypertrophic agonist PE in the absence or GFP-HDAC4 was primarily localized to nuclei of infected NRVMs, presence of BPKDi for 72 h. As shown in Fig. 4A, BPKDi completely although some cytoplasmic and pan-cellular distribution was blocked agonist-dependent induction of myomaxin mRNA expres- noted. In response to PMA, PE and ET-1, HDAC4 efficiently redistrib- sion, supporting the notion that PKD derepresses MEF2 target uted to the cytoplasm (Fig. 3B, upper panels). However, HDAC4 re- genes via class IIa HDACs. mained nuclear in agonist-stimulated cells pre-treated with BPKDi BPKDi was employed to examine the contribution of PKD sig- (Fig. 3B, lower panels). BPKDi had only marginal effects on basal nu- naling to activation of additional hypertrophic marker genes, clear export of HDAC4 (data not shown), suggesting that basal phos- including those encoding atrial natriuretic factor (ANF), b-myosin phorylation of HDAC4 is regulated by a kinase distinct from PKD. heavy chain (b-MyHC) and a-skeletal actin (a-Sk-actin). As shown Alternatively, the 1 h duration of compound treatment was not suf- in Fig. 4B and C, BPKDi completely blocked PE-mediated induction ficient to allow de-phosphorylation of HDAC4. Despite this uncer- a-Sk-actin and b-MyHC mRNA expression. In contrast, ANF expres- L. Monovich et al. / FEBS Letters 584 (2010) 631–637 635

Fig. 3. BPKDi blocks agonist-dependent nuclear export of HDAC4 and HDAC5. NRVMs were cultured in 6-well plates (2 105 cells/well) and infected with adenovirus encoding GFP-tagged HDAC5 (A) or GFP-tagged HDAC4 (B). After 14 h of infection, cells were washed and cultured in serum-free medium. Cells were exposed to BPKDi (1 lM) for 30 min prior to stimulation with PMA (25 nM), PE (10 lM) or ET-1 (25 nM) for 1 h. GFP-HDAC was visualized using a fluorescence microscope. Scale bar = 10 lm. sion was not repressed, but rather was further enhanced by the phy. Here, using a novel and highly selective kinase inhibitor, we combination of BPKDi and PE (Fig. 4D). These results suggest that provide evidence that PKD plays a critical role in this process by di- PKD signaling does not serve a general role in cardiac gene regula- rectly phosphorylating class IIa HDACs in response to GPCR tion, but instead selectively functions to control a subset of genes agonists. that are induced during cardiac hypertrophy. Prior genetic studies have implicated PKD in the control of class To more directly examine the effect of PKD inhibition on cardiac IIa HDAC phosphorylation and cardiac hypertrophy. Overexpres- hypertrophy, the ability of BPKDi to alter NRVM growth was as- sion of constitutively active forms of PKD triggers nuclear export sessed. Seventy two hours of stimulation with PE caused a 21% in- of class IIa HDACs [11,20,21]. Conversely, siRNA knockdown of crease in NRVM cell volume that was blunted by 40% by BPKDi PKD1 in NRVMs blunts hypertrophy and nuclear export of HDAC5 (Fig. 4E). Adenylate kinase (AK), which is released from cells when [15]. Transgenic overexpression of active PKD1 in mouse heart plasma membrane integrity is compromised, was measured in the causes pathological cardiac remodeling [11], while cardiac-specific culture supernatants to access possible cytotoxic effects of BPKDi knockout of the gene encoding PKD1 blunts hypertrophy in re- on NRVMs. Importantly, the addition of 1 lM BPKDi to NRVMs for sponse to pressure overload [22]. Our studies with BPKDi further 72 h did not promote AK release from cells (data not shown), and implicate PKD in the control of cardiac hypertrophy via class IIa thus the results reflect authentic anti-hypertrophic activity of the HDACs. Future in vivo studies with this compound or analogs compound rather than non-specific toxicity. Additional studies of should establish the relative importance of PKD signaling to path- AK release demonstrated that 10 lM BPKDi was well tolerated by ological remodeling of the heart under various conditions. NRVMs exposed to the compound for 72 h. BPKDi was also non- Another small molecule inhibitor of PKD, Gö-6976 [23], has toxic to cultured adult rat ventricular myocytes (data not shown). been widely used to assess the contribution of PKD to various processes, including class IIa HDAC nuclear export [11]. However, 4. Discussion Gö-6976 has been shown to inhibit a variety of kinases in addition to PKD [24], including PKC, p70 , checkpoint Signal-dependent nuclear export of class IIa HDACs is required kinase-2 and 30-phosphoinositide-dependent protein kinase-1, for derepression of genes that promote cardiomyocyte hypertro- and thus results obtained with this compound are equivocal. 636 L. Monovich et al. / FEBS Letters 584 (2010) 631–637

Fig. 4. Effect of BPKDi on cultured cardiac myocyte gene expression and hypertrophy. (A–D) NRVMs (2 106 cells/10-cm plate) were cultured in serum-free medium. Cells were stimulated for 72 h with PE (20 lM) in the absence or presence of BPKDi (1 lM), as indicated. Total RNA was prepared and mRNA transcripts for the indicated genes were quantified as described in Section 2. Values represent averages from three independent plates of cells ± standard deviation. Statistical significance was assessed using 1- way ANOVA with Newman–Keuls post-test. (E) NRVMs were cultured in 6-wells dishes (2 105 cells/well) and treated for 72 h as described above. Cell volume (lm3) was quantified using a Coulter Model Z2 instrument. Values represent averages from six independent wells of cells ± standard deviation. Statistical significance was assessed using 1-way ANOVA with Newman–Keuls post-test.

Preliminary characterization of a benzoxoloazepinolone PKD knockdown of PKD1 in NRVMs only partially prevented HDAC5 nu- inhibitor, CID755673, was described [25]. Unlike BPKDi, which is clear export [15], further suggesting that multiple PKD family an ATP-competitive PKD inhibitor (data not shown), CID755673- members control class IIa HDAC phosphorylation. The profound ef- mediated inhibition of PKD is non-competitive with respect to fect of BPKDi on class IIa HDAC phosphorylation and nuclear export ATP. The degree of selectivity of CID755673 for PKD, and thus is likely due to its ability to inhibit all PKD family members. How- the specificity of the compound, has recently been questioned [26]. ever, it is important to note that the immunoblotting results shown BPKDi does not distinguish between PKD1, PKD2 and PKD3. in Fig. 1 only capture the activation state of PKD1. Prior gene targeting studies in chicken B lymphocytes demon- CaMKII has been strongly associated with HDAC4 regulation in strated that PKD1 and PKD3 function redundantly to control the heart [28]. HDAC4 harbors a unique CaMKII docking domain HDAC5 phosphorylation in these cells [27]. Additionally, siRNA not found in other class IIa HDACs. BPKDi does not inhibit CaMKII, L. Monovich et al. / FEBS Letters 584 (2010) 631–637 637 yet it efficiently prevented phosphorylation and nuclear export of [11] Vega, R.B., Harrison, B.C., Meadows, E., Roberts, C.R., Papst, P.J., Olson, E.N., HDAC4 in NRVMs treated with PE and ET-1. It is possible that et al. (2004) Protein kinases C and D mediate agonist-dependent cardiac hypertrophy through nuclear export of histone deacetylase 5. Mol. Cell Biol. HDAC4 is regulated by multiple kinases in cardiac myocytes, and 24, 8374–8385. that the operative kinase is dependent on the nature of activating [12] McKinsey, T.A., Zhang, C.L., Lu, J. and Olson, E.N. (2000) Signal-dependent stimulus. The same possibility exists for HDAC5, which is capable nuclear export of a histone deacetylase regulates muscle differentiation. Nature 408, 106–111. of being phosphorylated by GRK5 in cardiac myocytes [8]. None- [13] Iglesias, T., Waldron, R.T. and Rozengurt, E. (1998) Identification of in vivo theless, the results presented here demonstrate that in NRVMs phosphorylation sites required for protein kinase D activation. J. Biol. Chem. treated with agonists that stimulate Gaq-coupled receptors, PKD 273, 27662–27667. [14] Matthews, S.A., Rozengurt, E. and Cantrell, D. (1999) Characterization of serine is likely the principal kinase for HDAC4 and HDAC5. 916 as an in vivo autophosphorylation site for protein kinase D/protein kinase Our findings solidify a role for PKD in cardiomyocyte signal C mu. J. Biol. Chem. 274, 26543–26549. transduction through effects on class IIa HDACs. Prior genetic stud- [15] Harrison, B.C., Kim, M.S., Van, R.E., Plato, C.F., Papst, P.J., Vega, R.B., et al. (2006) Regulation of cardiac stress signaling by protein kinase D1. Mol. Cell Biol. 26, ies have also suggested that PKD regulates cardiac contractility 3875–3888. through phosphorylation of troponin I [29], and PKD isoforms have [16] Grozinger, C.M. and Schreiber, S.L. (2000) Regulation of histone deacetylase 4 been implicated in a wide variety of additional biochemical and and 5 and transcriptional activity by 14-3-3-dependent cellular localization. biological pathways, including regulation of NF-jB and MAP kinase Proc. Natl. Acad. Sci. USA 97, 7835–7840. [17] McKinsey, T.A., Zhang, C.L. and Olson, E.N. (2000) Activation of the myocyte signaling, cell proliferation, apoptosis, motility and Golgi transport enhancer factor-2 transcription factor by calcium/calmodulin-dependent [30]. BPKDi is a powerful chemical tool that will enable confirma- protein kinase-stimulated binding of 14-3-3 to histone deacetylase 5. Proc. tion or refinement of the role(s) of PKD signaling in these pro- Natl. Acad. Sci. USA 97, 14400–14405. [18] Wang, A.H., Kruhlak, M.J., Wu, J., Bertos, N.R., Vezmar, M., Posner, B.I., et al. cesses, and will undoubtedly uncover previously unrecognized (2000) Regulation of histone deacetylase 4 by binding of 14 3–3 proteins. Mol. functions for the PKD family. Cell Biol. 20, 6904–6912. [19] Huang, H.T., Brand, O.M., Mathew, M., Ignatiou, C., Ewen, E.P., McCalmon, S.A., et al. (2006) Myomaxin is a novel transcriptional target of MEF2A that encodes Acknowledgements a Xin related alpha actinin-interacting protein. J. Biol. Chem. 281, 39370– 39379. [20] Dequiedt, F., Van, L.J., Lecomte, E., Van, D., Seufferlein, V.T., Vandenheede, J.R., We thank P. Papst for adenovirus production and L. Cable, T. et al. (2005) Phosphorylation of histone deacetylase 7 by protein kinase D Horn, D. Koditek, G. Lundgaard, J. Loeb and Y. Peng for NRVMs. mediates T cell receptor-induced Nur77 expression and apoptosis. J. Exp. Med. 201, 793–804. [21] Parra, M., Kasler, H., McKinsey, T.A., Olson, E.N. and Verdin, E. (2005) Protein References kinase D1 phosphorylates HDAC7 and induces its nuclear export after T-cell receptor activation. J. Biol. Chem. 280, 13762–13770. [1] Frey, N. and Olson, E.N. (2003) Cardiac hypertrophy: the good, the bad, and the [22] Fielitz, J., Kim, M.S., Shelton, J.M., Qi, X., Hill, J.A., Richardson, J.A., et al. (2008) ugly. Annu. Rev. Physiol. 65, 45–79. Requirement of protein kinase D1 for pathological cardiac remodeling. Proc. [2] McKinsey, T.A. and Kass, D.A. (2007) Small-molecule therapies for cardiac Natl. Acad. Sci. USA 105, 3059–3063. hypertrophy: moving beneath the cell surface. Nat. Rev. Drug Disc. 6, 617–635. [23] Gschwendt, M., Dieterich, S., Rennecke, J., Kittstein, W., Mueller, H.J. and [3] Zhang, C.L., McKinsey, T.A., Chang, S., Antos, C.L., Hill, J.A. and Olson, E.N. Johannes, F.J. (1996) Inhibition of protein kinase C mu by various inhibitors. (2002) Class II histone deacetylases act as signal-responsive repressors of Differentiation from protein kinase c isoenzymes. FEBS Lett. 392, 77–80. cardiac hypertrophy. Cell 110, 479–488. [24] Davies, S.P., Reddy, H., Caivano, M. and Cohen, P. (2000) Specificity and [4] Manning, G., Whyte, D.B., Martinez, R., Hunter, T. and Sudarsanam, S. (2002) mechanism of action of some commonly used protein kinase inhibitors. The protein kinase complement of the human genome. Science 298, 1912– Biochem. J. 351, 95–105. 1934. [25] Sharlow, E.R., Giridhar, K.V., LaValle, C.R., Chen, J., Leimgruber, S., Barrett, R., [5] Avkiran, M., Rowland, A.J., Cuello, F. and Haworth, R.S. (2008) Protein kinase D et al. (2008) Potent and selective disruption of protein kinase D functionality in the cardiovascular system: emerging roles in health and disease. Circ. Res. by a benzoxoloazepinolone. J. Biol. Chem. 283, 33516–33526. 102, 157–163. [26] Torres-Marquez, E., Sinnett-Smith, J., Guha, S., Kui, R., Waldron, R.T., Rey, O. [6] McKinsey, T.A. (2007) Derepression of pathological cardiac genes by members and Rozengurt, E. (2009) CID755673 enhances mitogenic signaling by phorbol of the CaM kinase superfamily. Cardiovasc. Res. 73, 667–677. esters, bombesin and EGF through a protein kinase D-independent pathway. [7] Berdeaux, R., Goebel, N., Banaszynski, L., Takemori, H., Wandless, T., Shelton, Biochem. Biophys. Res. Commun., in press. G.D., et al. (2007) SIK1 is a class II HDAC kinase that promotes survival of [27] Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A., skeletal myocytes. Nat. Med. 13, 597–603. et al. (2006) Essential role for protein kinase D family kinases in the regulation [8] Martini, J.S., Raake, P., Vinge, L.E., DeGeorge, B.R., Chuprun, J.K., Harris, D.M., of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569– et al. (2008) Uncovering G protein-coupled receptor kinase-5 as a histone 1577. deacetylase kinase in the nucleus of cardiomyocytes. Proc. Natl. Acad. Sci. USA [28] Backs, J., Song, K., Bezprozvannaya, S., Chang, S. and Olson, E.N. (2006) CaM 105, 12457–12462. kinase II selectively signals to histone deacetylase 4 during cardiomyocyte [9] Harrison, B.C., Roberts, C.R., Hood, D.B., Sweeney, M., Gould, J.M., Bush, E.W., hypertrophy. J. Clin. Invest. 116, 1853–1864. et al. (2004) The CRM1 nuclear export receptor controls pathological cardiac [29] Cuello, F., Bardswell, S.C., Haworth, R.S., Yin, X., Lutz, S., Wieland, T., et al. gene expression. Mol. Cell Biol. 24, 10636–10649. (2007) Protein kinase D selectively targets cardiac troponin I and regulates 2+ [10] Monovich, L., Koch, K.A., Burgis, R., Osimboni, E., Mann, T., Wall, D., et al. (2009) myofilament Ca sensitivity in ventricular myocytes. Circ. Res. 100, 864–873. Suppression of HDAC nuclear export and cardiomyocyte hypertrophy by novel [30] Rozengurt, E., Rey, O. and Waldron, R.T. (2005) Protein kinase D signaling. J. irreversible inhibitors of CRM1. Biochim. Biophys. Acta. 1789, 422–431. Biol. Chem. 280, 13205–13208.