Four-and-a-half LIM domains proteins are novel regulators of the protein kinase D pathway in cardiac myocytes.
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.
Konstantina
K
Stathopoulou
Friederike
F
Cuello
*Cardiovascular Division, King's College London British Heart Foundation Centre, London SE1 7EH, U.K.
Alexandra J
AJ
Candasamy
*Cardiovascular Division, King's College London British Heart Foundation Centre, London SE1 7EH, U.K.
Elizabeth M
EM
Kemp
*Cardiovascular Division, King's College London British Heart Foundation Centre, London SE1 7EH, U.K.
Elisabeth
E
Ehler
*Cardiovascular Division, King's College London British Heart Foundation Centre, London SE1 7EH, U.K.
Robert S
RS
Haworth
*Cardiovascular Division, King's College London British Heart Foundation Centre, London SE1 7EH, U.K.
Metin
M
Avkiran
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000378:metabolism; D004789:Enzyme Activation; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D060588:LIM Domain Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D060850:LIM-Homeodomain Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D064326:MEF2 Transcription Factors / Q000378:metabolism; D051379:Mice; D009124:Muscle Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D019332:Endothelin-1; C105987:FHL2 protein, human; C486306:Fhl1 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D060588:LIM Domain Proteins; D060850:LIM-Homeodomain Proteins; D064326:MEF2 Transcription Factors; D009124:Muscle Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C089212:protein kinase D; D011493:Protein Kinase C; C498619:Hdac5 protein, rat; D006655:Histone Deacetylases