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.
Protein kinase D has multiple roles in cardiac myocytes, where its regulatory mechanisms remain incompletely defined. In the present study we identify four-and-a-half LIM domains proteins 1 and 2 as novel binding partners and regulators of protein kinase D in this cell type.
cardiac myocyte; four-and-a-half LIM (FHL); histone deacetylase; neurohormonal stimulation; protein kinase; signal transduction; ARVM, adult rat ventricular myocyte; BPKDi, bipyridyl PKD inhibitor; CaMK, Ca2+/calmodulin-dependent protein kinase; caPKD, constitutively active catalytic domain of PKD; cMyBP-C, cardiac myosin-binding protein C; CRM1, chromosome region maintenance 1; cTnI, inhibitory subunit of cardiac troponin; ERK, extracellular-signal-regulated kinase; ET1, endothelin 1; FHL, four-and-a-half LIM domains; HDAC, histone deacetylase; IVK, in vitro kinase; MEF2, myocyte enhancer factor 2; MOI, multiplicity of infection; MuRF, muscle RING finger; NRVM, neonatal rat ventricular myocyte; PE, phenylephrine; pfu, plaque-forming unit; PKC, protein kinase C; PKD, protein kinase D; TAC, transverse aortic constriction