Four-and-a-half LIM domains proteins are novel regulators of the protein kinase D pathway in cardiac myocytes

Protein kinase D (PKD) is a Ser/Thr kinase implicated in multiple cardiac roles, including the phosphorylation of the class II histone deacetylase HDAC5 and thereby de-repression of myocyte enhancer factor 2 (MEF2) transcription factor activity. Here we identify four-and-a-half LIM domains proteins 1 and 2 (FHL1 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 or neonatal rat ventricular myocytes (NRVM), respectively, and by co-immunoprecipitation of FHL1 and FHL2 with GFP-PKD1 fusion protein expressed in NRVM. In vitro kinase assays showed that neither FHL1 nor FHL2 is a PKD1 substrate. Selective knock-down of FHL1 expression in NRVM significantly inhibited PKD activation and HDAC5 phosphorylation in response to endothelin 1, but not to the α1-adrenoceptor agonist phenylephrine. In contrast, selective knock-down 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.
Source: BJ Cell - Category: Biochemistry Authors: Tags: BJ Signal Source Type: research