Four-and-a-half LIM domains proteins are novel regulators of the protein kinase D pathway in cardiac myocytes
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Four-and-a-half LIM domains proteins are novel regulators of the protein kinase D pathway in cardiac myocytes. / Stathopoulou, Konstantina; Cuello, Friederike; Candasamy, Alexandra J; Kemp, Elizabeth M; Ehler, Elisabeth; Haworth, Robert S; Avkiran, Metin.
in: BIOCHEM J, Jahrgang 457, Nr. 3, 01.02.2014, S. 451-461.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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T1 - Four-and-a-half LIM domains proteins are novel regulators of the protein kinase D pathway in cardiac myocytes
AU - Stathopoulou, Konstantina
AU - Cuello, Friederike
AU - Candasamy, Alexandra J
AU - Kemp, Elizabeth M
AU - Ehler, Elisabeth
AU - Haworth, Robert S
AU - Avkiran, Metin
PY - 2014/2/1
Y1 - 2014/2/1
N2 - 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.
AB - 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.
KW - Animals
KW - Animals, Newborn
KW - Cells, Cultured
KW - Endothelin-1
KW - Enzyme Activation
KW - Heart Ventricles
KW - Histone Deacetylases
KW - Humans
KW - Intracellular Signaling Peptides and Proteins
KW - Isoenzymes
KW - LIM Domain Proteins
KW - LIM-Homeodomain Proteins
KW - MEF2 Transcription Factors
KW - Mice
KW - Muscle Proteins
KW - Myocytes, Cardiac
KW - Peptide Fragments
KW - Phosphorylation
KW - Protein Kinase C
KW - Protein Processing, Post-Translational
KW - Rats
KW - Recombinant Fusion Proteins
KW - Transcription Factors
U2 - 10.1042/BJ20131026
DO - 10.1042/BJ20131026
M3 - SCORING: Journal article
C2 - 24219103
VL - 457
SP - 451
EP - 461
JO - BIOCHEM J
JF - BIOCHEM J
SN - 0264-6021
IS - 3
ER -