Abstract
BackgroundNuclear import of protein kinase D1 (PKD1) is an important event in the transcriptional regulation of cardiac gene reprogramming leading to the hypertrophic growth response, however, little is known about the molecular events that govern this event. We have identified a novel complex between PKD1 and a heat shock protein (Hsp), Hsp20, which has been implicated as cardioprotective. This study aims to characterize the role of the complex in PKD1-mediated myocardial regulatory mechanisms that depend on PKD1 nuclear translocation.ResultsIn mapping the Hsp20 binding sites on PKD1 within its catalytic unit using peptide array analysis, we were able to develop a cell-permeable peptide that disrupts the Hsp20-PKD1 complex. We use this peptide to show that formation of the Hsp20-PKD1 complex is essential for PKD1 nuclear translocation, signaling mechanisms leading to hypertrophy, activation of the fetal gene programme and pathological cardiac remodeling leading to cardiac fibrosis.ConclusionsThese results identify a new signaling complex that is pivotal to pathological remodelling of the heart that could be targeted therapeutically.
Highlights
Cardiac myocytes respond to pathological stress via activation of the fetal gene programme
heat shock protein 20 (Hsp20) and protein kinase D1 (PKD1) form a complex in cardiac myocytes The protective functions of Hsp20 in a cardiac setting have received much attention and in an attempt to elucidate the molecular mechanisms involved, we endeavored to characterise putative interactors of Hsp20 using highdensity ProtoArray Human Protein Microarray (Invitrogen, UK) analysis
Co-localisation immunostaining (Figure 1F) demonstrated positive correlation in HEK293 cells overexpressing Hsp20 and PKD1 (Pearson’s 0.62 ± 0.03, Manders’ 0.81 ± 0.02, M1 0.96 ± 0.01, M2 0.95 ± 0.01) and in myocytes expressing endogenous Hsp20 and PKD1 (Pearson’s 0.81 ± 0.02, Manders’ 0.88 ± 0.01, M1 0.86 ± 0.03, M2 0.93 ± 0.01). This data suggests that Hsp20 and PKD1 locate to the same region of cells
Summary
Cardiac myocytes respond to pathological stress via activation of the fetal gene programme (reviewed in [1]). Essential to this process, is the activation of cardiac protein kinase D (PKD). Phosphorylation and activation of PKD1 occurs initially via binding to diacylglycerol [3] at its cystein-rich domain with kinase activation/translocation being triggered by PKC-phosphorylation and the release of pleckstrin homology (PH)-mediated PKD1 inhibition [4]. Nuclear import of protein kinase D1 (PKD1) is an important event in the transcriptional regulation of cardiac gene reprogramming leading to the hypertrophic growth response, little is known about the molecular events that govern this event. This study aims to characterize the role of the complex in PKD1-mediated myocardial regulatory mechanisms that depend on PKD1 nuclear translocation
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