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Timothy A Mckinsey - One of the best experts on this subject based on the ideXlab platform.
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HDAC5 catalytic activity suppresses cardiomyocyte oxidative stress and NRF2 target gene expression.
The Journal of biological chemistry, 2019Co-Authors: Friederike C. Schreiter, Rushita A. Bagchi, Philip D. Tatman, Mark Hannink, Timothy A MckinseyAbstract:Histone Deacetylase 5 (HDAC5) and HDAC9 are class IIa HDACs that function as signal-responsive repressors of the epigenetic program for pathological cardiomyocyte hypertrophy. The conserved Deacetylase domains of HDAC5 and HDAC9 are not required for inhibition of cardiac hypertrophy. Thus, the biological function of class IIa HDAC catalytic activity in the heart remains unknown. Here we demonstrate that catalytic activity of HDAC5, but not HDAC9, suppresses mitochondrial reactive oxygen species generation and subsequent induction of NF-E2-related factor 2 (NRF2)-dependent antioxidant gene expression in cardiomyocytes. Treatment of cardiomyocytes with TMP195 or TMP269, which are selective class IIa HDAC inhibitors, or shRNA-mediated knockdown of HDAC5 but not HDAC9 leads to stimulation of NRF2-mediated transcription in a reactive oxygen species-dependent manner. Conversely, ectopic expression of catalytically active HDAC5 decreases cardiomyocyte oxidative stress and represses NRF2 activation. These findings establish a role of the catalytic domain of HDAC5 in the control of cardiomyocyte redox homeostasis and define TMP195 and TMP269 as a novel class of NRF2 activators that function by suppressing the enzymatic activity of an epigenetic regulator.
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β adrenergic receptor stimulation and activation of protein kinase a protect against α1 adrenergic mediated phosphorylation of protein kinase d and Histone Deacetylase 5
Journal of Cardiac Failure, 2011Co-Authors: Carmen C. Sucharov, Timothy A Mckinsey, Karen Dockstader, Karin Nunley, Michael R. BristowAbstract:Abstract Introduction Chronic activation of β 1 -adrenergic receptor (β 1 -AR) signaling can have deleterious effects on the heart, and animal models overexpressing β 1 -ARs develop a dilated cardiomyopathy and heart failure. In the classic β-AR pathway, receptor occupancy by an agonist results in increased cyclic adenosine monophosphate (cAMP) levels and activation of protein kinase A (PKA). However, the role of PKA-dependent signaling in the development and progression of cardiomyopathies and heart failure is controversial, because β-AR signal transduction is generally desensitized in the failing heart and PKA activity is not increased. Methods and Results Neonatal rat ventricular myocytes were acutely (15 minutes) or chronically (48 hours) treated with isoproterenol, and phosphorylation of protein kinase D (PKD) and Histone Deacetylase 5 (HDAC5) was measured. Acute β 1 -AR stimulation or expression of constitutively active (CA) PKA reduced α 1 -adrenergic-mediated phosphorylation of HDAC5 and PKD by activation of a phosphatase. Overexpression of CA-PKA also reduced α 1 -adrenergic–mediated increased expression of contractile protein fetal isoforms and promoted repression of adult isoforms, but had no effect on α 1 -adrenergic–mediated cellular hypertrophy. Conclusions These data indicate that the PKA-dependent arm of β-AR signaling can be antihypertrophic and presumably beneficial, through dephosphorylation of PKD and HDAC5 and reduction of hypertrophic fetal isoform gene expression.
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β-Adrenergic Receptor Stimulation and Activation of Protein Kinase A Protect Against α1-Adrenergic–Mediated Phosphorylation of Protein Kinase D and Histone Deacetylase 5
Journal of cardiac failure, 2011Co-Authors: Carmen C. Sucharov, Timothy A Mckinsey, Karen Dockstader, Karin Nunley, Michael R. BristowAbstract:Abstract Introduction Chronic activation of β 1 -adrenergic receptor (β 1 -AR) signaling can have deleterious effects on the heart, and animal models overexpressing β 1 -ARs develop a dilated cardiomyopathy and heart failure. In the classic β-AR pathway, receptor occupancy by an agonist results in increased cyclic adenosine monophosphate (cAMP) levels and activation of protein kinase A (PKA). However, the role of PKA-dependent signaling in the development and progression of cardiomyopathies and heart failure is controversial, because β-AR signal transduction is generally desensitized in the failing heart and PKA activity is not increased. Methods and Results Neonatal rat ventricular myocytes were acutely (15 minutes) or chronically (48 hours) treated with isoproterenol, and phosphorylation of protein kinase D (PKD) and Histone Deacetylase 5 (HDAC5) was measured. Acute β 1 -AR stimulation or expression of constitutively active (CA) PKA reduced α 1 -adrenergic-mediated phosphorylation of HDAC5 and PKD by activation of a phosphatase. Overexpression of CA-PKA also reduced α 1 -adrenergic–mediated increased expression of contractile protein fetal isoforms and promoted repression of adult isoforms, but had no effect on α 1 -adrenergic–mediated cellular hypertrophy. Conclusions These data indicate that the PKA-dependent arm of β-AR signaling can be antihypertrophic and presumably beneficial, through dephosphorylation of PKD and HDAC5 and reduction of hypertrophic fetal isoform gene expression.
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protein kinase d dependent phosphorylation and nuclear export of Histone Deacetylase 5 mediates vascular endothelial growth factor induced gene expression and angiogenesis
Journal of Biological Chemistry, 2008Co-Authors: Chang Hoon Ha, Timothy A Mckinsey, Weiye Wang, Bong Sook Jhun, Chelsea Wong, Angelika Hausser, Klaus Pfizenmaier, Eric N OlsonAbstract:Vascular endothelial growth factor (VEGF) is essential for normal and pathological angiogenesis. However, the signaling pathways linked to gene regulation in VEGF-induced angiogenesis are not fully understood. Here we demonstrate a critical role of protein kinase D (PKD) and Histone Deacetylase 5 (HDAC5) in VEGF-induced gene expression and angiogenesis. We found that VEGF stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a VEGF receptor 2-phospholipase Cγ-protein kinase C-PKD-dependent pathway. We further showed that the PKD-HDAC5 pathway mediated myocyte enhancer factor-2 transcriptional activation and a specific subset of gene expression in response to VEGF, including NR4A1, an orphan nuclear receptor involved in angiogenesis. Specifically, inhibition of PKD by overexpression of the PKD kinase-negative mutant prevents VEGF-induced HDAC5 phosphorylation and nuclear export as well as NR4A1 induction. Moreover, a mutant of HDAC5 specifically deficient in PKD-dependent phosphorylation inhibited VEGF-mediated NR4A1 expression, endothelial cell migration, and in vitro angiogenesis. These findings suggest that the PKD-HDAC5 pathway plays an important role in VEGF regulation of gene transcription and angiogenesis.
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Histone Deacetylase 5 acquires calcium calmodulin dependent kinase ii responsiveness by oligomerization with Histone Deacetylase 4
Molecular and Cellular Biology, 2008Co-Authors: Johannes Backs, Thea Backs, Timothy A Mckinsey, Svetlana Bezprozvannaya, Eric N OlsonAbstract:Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Histone Deacetylase 4 (HDAC4), a class IIa HDAC, resulting in the cytosolic accumulation of HDAC4 and the derepression of the transcription factor myocyte enhancer factor 2. Phosphorylation by CaMKII requires docking of the kinase to a specific domain of HDAC4 not present in other HDACs. Paradoxically, however, CaMKII signaling can also promote the nuclear export of other class IIa HDACs, such as HDAC5. Here, we show that HDAC4 and HDAC5 form homo- and hetero-oligomers via a conserved coiled-coil domain near their amino termini. Whereas HDAC5 alone is unresponsive to CaMKII, it becomes responsive to CaMKII in the presence of HDAC4. The acquisition of CaMKII responsiveness by HDAC5 is mediated by HDAC5's direct association with HDAC4 and can occur by phosphorylation of HDAC4 or by transphosphorylation by CaMKII bound to HDAC4. Thus, HDAC4 integrates upstream Ca2+-dependent signals via its association with CaMKII and transmits these signals to HDAC5 by protein-protein interactions. We conclude that HDAC4 represents a point of convergence for CaMKII signaling to downstream HDAC-regulated genes, and we suggest that modulation of the interaction of CaMKII and HDAC4 represents a means of regulating CaMKII-dependent gene programs.
Q. Khai Huynh - One of the best experts on this subject based on the ideXlab platform.
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Evidence for the phosphorylation of serine259 of Histone Deacetylase 5 by protein kinase Cδ.
Archives of biochemistry and biophysics, 2010Co-Authors: Q. Khai HuynhAbstract:Abstract Signaling via pro-growth G protein coupled receptors triggers phosphorylation of HDAC5 on two serine residues (Ser 259 and Ser 498 ), resulting in nuclear export of HDAC5 and de-repression of downstream target genes. In the previous paper we reported the important role of PKD isozymes in the regulation of HDAC5 by phosphorylating Ser 498 of HDAC5 [Q.K. Huynh, T.A. Mckinsey, Arch. Biochem. Biophys. 450 (2006) 141–148]. In the present paper, we provide evidence that PKCδ can directly phosphorylate Ser 259 of HDAC5. The evidence is based on the following facts (a) isolated kinase fraction from human failing heart tissues contained PKCδ that phosphorylated HDAC5 Ser 259 peptide and no significant activity was found for the unbound fraction after they were immunoprecipitated with PKCδ specific antibody; (b) specific inhibitors for PKCδ inhibited kinase activity from isolated fraction and recombinant human PKCδ with similar IC 50 values; (c) recombinant human PKCδ can directly phosphorylate full length Ser 259 HDAC5 protein and HDAC5 Ser 259 peptide. The results suggest that in addition to activation of protein kinase D isozymes by phosphorylating Ser 744 and Ser 748 at their activation sites, PKCδ may also play a role in the regulation of HDAC5 by phosphorylation of Ser 259 .
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Protein kinase D directly phosphorylates Histone Deacetylase 5 via a random sequential kinetic mechanism.
Archives of biochemistry and biophysics, 2006Co-Authors: Q. Khai Huynh, Timothy A MckinseyAbstract:Abstract Class II Histone Deacetylases (HDACs) are signal-responsive repressors of gene transcription. In the heart, class II HDAC5 suppresses expression of genes that govern stress-induced cardiomyocyte growth. Signaling via pro-growth G protein coupled receptors triggers phosphorylation of HDAC5 on two serine residues (Ser 259 and Ser 498 ), resulting in nuclear export of HDAC5 and de-repression downstream target genes. Although prior studies established a role for protein kinase D (PKD) in the regulation of HDAC5 phosphorylation, it remained unclear whether PKD functions directly or indirectly to control the phosphorylation status of this transcriptional repressor. Here, we demonstrate that PKD catalyzes direct phosphoryl-group transfer to Ser 498 of HDAC5. Each of the three PKD family members, PKD1, PKD2, and PKD3, is capable of phosphorylating HDAC5 ( K m for substrate = 2.07, 3.12, and 1.43 μM, respectively), although PKD2 exhibits highest catalytic efficiency ( k cat / K m = 6.77 min −1 μM −1 ). Kinetic studies revealed that the three PKD isozymes phosphorylate HDAC5 through a random sequential mechanism, and that ATP has no effect on association of kinase with peptide substrate. In addition, we demonstrate that ADP competitively inhibits phosphorylation of HDAC5 ( K i = 8.50, 17.54, and 11.98 μM for PKD1, PKD2, and PKD3, respectively). These findings define PKD as an HDAC kinase and thus suggest key roles for PKD family members in the control of chromatin structure and gene expression.
Ashok K. Srivastava - One of the best experts on this subject based on the ideXlab platform.
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Angiotensin II-Induced Histone Deacetylase 5 Phosphorylation, Nuclear Export and Egr-1 Expression is mediated by Akt Pathway in A10 Vascular Smooth Muscle Cells.
American journal of physiology. Heart and circulatory physiology, 2021Co-Authors: Vanessa Truong, Ashish Jain, Madhu B. Anand-srivastava, Ashok K. SrivastavaAbstract:Angiotensin II (Ang II) regulates an array of physiological and pathological responses in vascular smooth muscle cells (VSMCs) by activating ERK1/2 and PI3K/Akt signaling pathways. We have demonstrated that Ang II and insulin-like growth factor-1 (IGF-1) induces the expression of early growth response protein-1 (Egr-1), a zinc finger transcription factor which regulates the transcription of cell cycle regulatory genes network in VSMCs. We have reported that IGF-1 induces the phosphorylation of Histone Deacetylase 5 (HDAC5) which has been implicated in the expression of genes linked to VSMC growth and hypertrophy, via a PI3K/Akt-dependent pathway in VSMCs. However, the involvement of PI3K/Akt pathways in Ang II-induced HDAC5 phosphorylation and the contribution of HDAC5 in Egr-1 expression and hypertrophy in VSMCs remains unexplored. Here, we show that pharmacological blockade of the PI3K/Akt pathway either by wortmannin/SC66 or siRNA-induced silencing of Akt attenuated Ang II-induced HDAC5 phosphorylation and its nuclear export. Moreover, SC66 or Akt knockdown also suppressed Ang II-induced Egr-1 expression. Further, pharmacological inhibition of HDAC5 by MC1568 or TMP-195, or knockdown of HDAC5 and the blockade of the nuclear export of HDAC5 by leptomycin B or KPT-330 significantly reduced Ang II-induced Egr-1 expression. In addition, depletion of either HDAC5 or Egr-1 by siRNA attenuated VSMC hypertrophy in response to Ang II. In summary, our results demonstrate that Ang II-induced HDAC5 phosphorylation and its nuclear exclusion is mediated by PI3K/Akt pathway, and HDAC5 is an upstream regulator of Egr-1 expression and hypertrophy in VSMCs.
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angiotensin ii induced Histone Deacetylase 5 phosphorylation nuclear export and egr 1 expression is mediated by akt pathway in a10 vascular smooth muscle cells
American Journal of Physiology-heart and Circulatory Physiology, 2021Co-Authors: Vanessa Truong, Ashish Jain, Madhu B Anandsrivastava, Ashok K. SrivastavaAbstract:Angiotensin II (Ang II) regulates an array of physiological and pathological responses in vascular smooth muscle cells (VSMCs) by activating ERK1/2 and PI3K/Akt signaling pathways. We have demonstr...
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Reactive Oxygen Species‐Dependent PKB Activation Contributes to Insulin‐Like Growth Factor 1‐Induced Phosphorylation of Histone Deacetylase 5 in Vascular Smooth Muscle Cells
The FASEB Journal, 2016Co-Authors: Paulina Pietruczuk, Ashok K. SrivastavaAbstract:Insulin-like growth factor 1 (IGF-1), a potent mitogenic and vasoactive factor, has been shown to play an important role in the development of cardiovascular diseases. This occurs through the gener...
Eric N Olson - One of the best experts on this subject based on the ideXlab platform.
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angiotensin ii induces skeletal muscle atrophy by activating tfeb mediated murf1 expression
Circulation Research, 2015Co-Authors: Philipp Du Bois, Eric N Olson, Cristina Pablo Tortola, Doerte Lodka, Franziska Schmidt, Kunhua Song, Sibylle Schmidt, Rhonda Basselduby, Jens FielitzAbstract:Rationale:Skeletal muscle wasting with accompanying cachexia is a life threatening complication in congestive heart failure. The molecular mechanisms are imperfectly understood, although an activated renin–angiotensin aldosterone system has been implicated. Angiotensin (Ang) II induces skeletal muscle atrophy in part by increased muscle-enriched E3 ubiquitin ligase muscle RING-finger-1 (MuRF1) expression, which may involve protein kinase D1 (PKD1). Objective:To elucidate the molecular mechanism of Ang II–induced skeletal muscle wasting. Methods and Results:A cDNA expression screen identified the lysosomal hydrolase-coordinating transcription factor EB (TFEB) as novel regulator of the human MuRF1 promoter. TFEB played a key role in regulating Ang II–induced skeletal muscle atrophy by transcriptional control of MuRF1 via conserved E-box elements. Inhibiting TFEB with small interfering RNA prevented Ang II–induced MuRF1 expression and atrophy. The Histone Deacetylase-5 (HDAC5), which was directly bound to an...
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protein kinase d dependent phosphorylation and nuclear export of Histone Deacetylase 5 mediates vascular endothelial growth factor induced gene expression and angiogenesis
Journal of Biological Chemistry, 2008Co-Authors: Chang Hoon Ha, Timothy A Mckinsey, Weiye Wang, Bong Sook Jhun, Chelsea Wong, Angelika Hausser, Klaus Pfizenmaier, Eric N OlsonAbstract:Vascular endothelial growth factor (VEGF) is essential for normal and pathological angiogenesis. However, the signaling pathways linked to gene regulation in VEGF-induced angiogenesis are not fully understood. Here we demonstrate a critical role of protein kinase D (PKD) and Histone Deacetylase 5 (HDAC5) in VEGF-induced gene expression and angiogenesis. We found that VEGF stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a VEGF receptor 2-phospholipase Cγ-protein kinase C-PKD-dependent pathway. We further showed that the PKD-HDAC5 pathway mediated myocyte enhancer factor-2 transcriptional activation and a specific subset of gene expression in response to VEGF, including NR4A1, an orphan nuclear receptor involved in angiogenesis. Specifically, inhibition of PKD by overexpression of the PKD kinase-negative mutant prevents VEGF-induced HDAC5 phosphorylation and nuclear export as well as NR4A1 induction. Moreover, a mutant of HDAC5 specifically deficient in PKD-dependent phosphorylation inhibited VEGF-mediated NR4A1 expression, endothelial cell migration, and in vitro angiogenesis. These findings suggest that the PKD-HDAC5 pathway plays an important role in VEGF regulation of gene transcription and angiogenesis.
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Histone Deacetylase 5 acquires calcium calmodulin dependent kinase ii responsiveness by oligomerization with Histone Deacetylase 4
Molecular and Cellular Biology, 2008Co-Authors: Johannes Backs, Thea Backs, Timothy A Mckinsey, Svetlana Bezprozvannaya, Eric N OlsonAbstract:Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Histone Deacetylase 4 (HDAC4), a class IIa HDAC, resulting in the cytosolic accumulation of HDAC4 and the derepression of the transcription factor myocyte enhancer factor 2. Phosphorylation by CaMKII requires docking of the kinase to a specific domain of HDAC4 not present in other HDACs. Paradoxically, however, CaMKII signaling can also promote the nuclear export of other class IIa HDACs, such as HDAC5. Here, we show that HDAC4 and HDAC5 form homo- and hetero-oligomers via a conserved coiled-coil domain near their amino termini. Whereas HDAC5 alone is unresponsive to CaMKII, it becomes responsive to CaMKII in the presence of HDAC4. The acquisition of CaMKII responsiveness by HDAC5 is mediated by HDAC5's direct association with HDAC4 and can occur by phosphorylation of HDAC4 or by transphosphorylation by CaMKII bound to HDAC4. Thus, HDAC4 integrates upstream Ca2+-dependent signals via its association with CaMKII and transmits these signals to HDAC5 by protein-protein interactions. We conclude that HDAC4 represents a point of convergence for CaMKII signaling to downstream HDAC-regulated genes, and we suggest that modulation of the interaction of CaMKII and HDAC4 represents a means of regulating CaMKII-dependent gene programs.
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Histone Deacetylase 5 Acquires Calcium/Calmodulin-Dependent Kinase II Responsiveness by Oligomerization with Histone Deacetylase 4
Molecular and Cellular Biology, 2008Co-Authors: Johannes Backs, Thea Backs, Timothy A Mckinsey, Svetlana Bezprozvannaya, Eric N OlsonAbstract:Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Histone Deacetylase 4 (HDAC4), a class IIa HDAC, resulting in the cytosolic accumulation of HDAC4 and the derepression of the transcription factor myocyte enhancer factor 2. Phosphorylation by CaMKII requires docking of the kinase to a specific domain of HDAC4 not present in other HDACs. Paradoxically, however, CaMKII signaling can also promote the nuclear export of other class IIa HDACs, such as HDAC5. Here, we show that HDAC4 and HDAC5 form homo- and hetero-oligomers via a conserved coiled-coil domain near their amino termini. Whereas HDAC5 alone is unresponsive to CaMKII, it becomes responsive to CaMKII in the presence of HDAC4. The acquisition of CaMKII responsiveness by HDAC5 is mediated by HDAC5's direct association with HDAC4 and can occur by phosphorylation of HDAC4 or by transphosphorylation by CaMKII bound to HDAC4. Thus, HDAC4 integrates upstream Ca2+-dependent signals via its association with CaMKII and transmits these signals to HDAC5 by protein-protein interactions. We conclude that HDAC4 represents a point of convergence for CaMKII signaling to downstream HDAC-regulated genes, and we suggest that modulation of the interaction of CaMKII and HDAC4 represents a means of regulating CaMKII-dependent gene programs.
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Angiotensin II Stimulates Protein Kinase D–Dependent Histone Deacetylase 5 Phosphorylation and Nuclear Export Leading to Vascular Smooth Muscle Cell Hypertrophy
Arteriosclerosis thrombosis and vascular biology, 2007Co-Authors: Chelsea Wong, Timothy A Mckinsey, Eric N Olson, Weiye Wang, Angelika Hausser, Klaus Pfizenmaier, Zheng Gen JinAbstract:Background— Angiotensin II (Ang II) induces the phenotypic modulation and hypertrophy of vascular smooth muscle cells (VSMCs), which is implicated in the pathogenesis of hypertension, atherosclerosis, and diabetes. In this study, we tested the hypothesis that Histone Deacetylases 5 (HDAC5) and its signal pathway play a role in Ang II-induced VSMC hypertrophy. Methods and Results— VSMCs were isolated from the thoracic aortas of male Sprague-Dawley rats and treated with Ang II. We found that Ang II rapidly stimulated phosphorylation of HDAC5 at Serine259/498 residues in a time- and dose- dependent manner. Ang II receptor-1, protein kinase C, and protein kinase D1 (PKD1) mediated HDAC5 phosphorylation. Furthermore, we observed that Ang II stimulated HDAC5 nuclear export, which was dependent on its PKD1-dependent phosphorylation. Consequently, both inhibiting PKD1 and HDAC5 Serine259/498 to Alanine mutant significantly attenuated Ang II-induced myocyte enhancer factor-2 (MEF2) transcriptional activity and protein synthesis in VSMCs. Conclusion— These findings demonstrate for the first time that PKD1-dependent HDAC5 phosphorylation and nuclear export mediates Ang II-induced MEF2 activation and VSMC hypertrophy, and suggest that PKD1 and HDAC5 may emerge as potential targets for the treatment of pathological vascular hypertrophy.
Timothy J. Richmond - One of the best experts on this subject based on the ideXlab platform.
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direct interaction of ca2 calmodulin inhibits Histone Deacetylase 5 repressor core binding to myocyte enhancer factor 2
Journal of Biological Chemistry, 2003Co-Authors: Imre Berger, Christoph Bieniossek, Christiane Schaffitzel, Markus Hassler, Eugenio Santelli, Timothy J. RichmondAbstract:Myocyte enhancer factor 2 (MEF2) proteins play a pivotal role in the differentiation of cardiac and skeletal muscle cells. MEF2 factors are regulated by Histone Deacetylase enzymes such as Histone Deacetylase 5 (HDAC5). HDAC5 in turn is responsive to Ca(2+) signaling mediated by the intracellular calcium sensor calmodulin. Here a combination of proteolytic fragmentation, matrix-assisted laser desorption ionization mass spectrometry, Edman degradation, circular dichroism, gel filtration, and surface plasmon resonance studies is utilized to define and characterize a stable core domain of HDAC5 and to examine its interactions with MEF2a and calmodulin. Results from real time binding experiments provide evidence for direct interaction of Ca(2+)/calmodulin with HDAC5 inhibiting MEF2a association with this enzyme.
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Direct Interaction of Ca2+/Calmodulin Inhibits Histone Deacetylase 5 Repressor Core Binding to Myocyte Enhancer Factor 2 *
The Journal of biological chemistry, 2003Co-Authors: Imre Berger, Christoph Bieniossek, Christiane Schaffitzel, Markus Hassler, Eugenio Santelli, Timothy J. RichmondAbstract:Myocyte enhancer factor 2 (MEF2) proteins play a pivotal role in the differentiation of cardiac and skeletal muscle cells. MEF2 factors are regulated by Histone Deacetylase enzymes such as Histone Deacetylase 5 (HDAC5). HDAC5 in turn is responsive to Ca(2+) signaling mediated by the intracellular calcium sensor calmodulin. Here a combination of proteolytic fragmentation, matrix-assisted laser desorption ionization mass spectrometry, Edman degradation, circular dichroism, gel filtration, and surface plasmon resonance studies is utilized to define and characterize a stable core domain of HDAC5 and to examine its interactions with MEF2a and calmodulin. Results from real time binding experiments provide evidence for direct interaction of Ca(2+)/calmodulin with HDAC5 inhibiting MEF2a association with this enzyme.