Abstract
PKD (protein kinase D) is a serine/threonine kinase implicated in multiple cardiac roles, including the phosphorylation of the class II HDAC5 (histone deacetylase isoform 5) and thereby de-repression of MEF2 (myocyte enhancer factor 2) transcription factor activity. In the present study we identify FHL1 (four-and-a-half LIM domains protein 1) and FHL2 as novel binding partners for PKD in cardiac myocytes. This was confirmed by pull-down assays using recombinant GST-fused proteins and heterologously or endogenously expressed PKD in adult rat ventricular myocytes or NRVMs (neonatal rat ventricular myocytes) respectively, and by co-immunoprecipitation of FHL1 and FHL2 with GFP-PKD1 fusion protein expressed in NRVMs. In vitro kinase assays showed that neither FHL1 nor FHL2 is a PKD1 substrate. Selective knockdown of FHL1 expression in NRVMs significantly inhibited PKD activation and HDAC5 phosphorylation in response to endothelin 1, but not to the α₁-adrenoceptor agonist phenylephrine. In contrast, selective knockdown of FHL2 expression caused a significant reduction in PKD activation and HDAC5 phosphorylation in response to both stimuli. Interestingly, neither intervention affected MEF2 activation by endothelin 1 or phenylephrine. We conclude that FHL1 and FHL2 are novel cardiac PKD partners, which differentially facilitate PKD activation and HDAC5 phosphorylation by distinct neurohormonal stimuli, but are unlikely to regulate MEF2-driven transcriptional reprogramming.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Animals, Newborn
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Cells, Cultured
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Endothelin-1 / metabolism
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Enzyme Activation
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Heart Ventricles / cytology
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Heart Ventricles / metabolism
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Histone Deacetylases / metabolism
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Humans
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Intracellular Signaling Peptides and Proteins / antagonists & inhibitors
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Intracellular Signaling Peptides and Proteins / chemistry
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Intracellular Signaling Peptides and Proteins / genetics
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Intracellular Signaling Peptides and Proteins / metabolism*
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Isoenzymes / genetics
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Isoenzymes / metabolism
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LIM Domain Proteins / antagonists & inhibitors
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LIM Domain Proteins / chemistry
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LIM Domain Proteins / genetics
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LIM Domain Proteins / metabolism*
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LIM-Homeodomain Proteins / antagonists & inhibitors
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LIM-Homeodomain Proteins / chemistry
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LIM-Homeodomain Proteins / genetics
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LIM-Homeodomain Proteins / metabolism*
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MEF2 Transcription Factors / metabolism
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Mice
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Muscle Proteins / antagonists & inhibitors
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Muscle Proteins / chemistry
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Muscle Proteins / genetics
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Muscle Proteins / metabolism*
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Myocytes, Cardiac / cytology
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Myocytes, Cardiac / metabolism*
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Peptide Fragments / antagonists & inhibitors
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Peptide Fragments / chemistry
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Peptide Fragments / genetics
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Peptide Fragments / metabolism
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Phosphorylation
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Protein Kinase C / genetics
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Protein Kinase C / metabolism*
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Protein Processing, Post-Translational
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Rats
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / metabolism
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Transcription Factors / antagonists & inhibitors
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Transcription Factors / chemistry
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Transcription Factors / genetics
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Transcription Factors / metabolism*
Substances
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Endothelin-1
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FHL2 protein, human
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Fhl1 protein, mouse
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Intracellular Signaling Peptides and Proteins
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Isoenzymes
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LIM Domain Proteins
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LIM-Homeodomain Proteins
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MEF2 Transcription Factors
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Muscle Proteins
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Peptide Fragments
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Recombinant Fusion Proteins
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Transcription Factors
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protein kinase D
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Protein Kinase C
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Hdac5 protein, rat
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Histone Deacetylases