Abstract
In cardiomyocytes, protein kinase D1 (PKD1) plays a central role in the response to stress signals. From a yeast two-hybrid assay, we have identified Enigma Homolog 1 (ENH1) as a new binding partner of PKD1. Since in neurons, ENH1, associated with protein kinase Cepsilon, was shown to modulate the activity of N-type calcium channels, and the pore-forming subunit of the cardiac L-type voltage-gated calcium channel, alpha1C, possesses a potential phosphorylation site for PKD1, we studied here a possible role of ENH1 and PKD1 in the regulation of the cardiac L-type voltage-gated calcium channel. PKD1-interacting proteins were searched by yeast two-hybrid screening. In vivo protein interactions in cardiomyocytes isolated from heart ventricles of newborn rats were tested by co-immunoprecipitation. Small interfering RNA and a dominant negative mutant of PKD1 were delivered into cardiomyocytes by use of an adenovirus. Calcium currents were measured by the patch-clamp technique. Both ENH1 and PKD1 interact with alpha1C in cardiomyocytes. This interaction is increased upon stimulation. Silencing of ENH1 prevented the binding of PKD1 to alpha1C. Moreover, a dominant negative mutant of PKD1 or the silencing of ENH1 inhibited the alpha-adrenergic-induced increase of L-type calcium currents. We found a new binding partner, ENH1, and a new target, alpha1C, for PKD1 in neonatal rat cardiomyocytes. We propose a model where ENH1 scaffolds PKD1 to alpha1C in order to form a signalling complex that regulates the activity of cardiac L-type voltage-gated Ca(2+) channels.
Highlights
In heart, neurohumoral factors or mechanical stress activate intracellular signalling pathways that induce increases in contraction frequency and strength and lead to hypertrophy and heart failure.[1,2] The effectors of signalling pathways modulate the activities of many proteins residing at different subcellular localizations, simultaneously or sequentially, to achieve the cardiomyocyte response to the stimuli
We show that Enigma Homolog 1 (ENH1) and protein kinase D1 (PKD1) interact with a1C, the pore subunit of L-type voltage-gated calcium channels (L-VCC) in neonatal rat cardiomyocytes
Our results strongly suggest that ENH1, PKD1, and a1C form a signalling complex that regulates the activity of L-VCC in cardiomyocytes
Summary
Neurohumoral factors or mechanical stress activate intracellular signalling pathways that induce increases in contraction frequency and strength and lead to hypertrophy and heart failure.[1,2] The effectors of signalling pathways modulate the activities of many proteins residing at different subcellular localizations, simultaneously or sequentially, to achieve the cardiomyocyte response to the stimuli. Effectors such as protein kinases have to be selectively translocated to various locations: e.g. protein kinase C1 (PKC1) is localized at transverse tubules. PKD1 phosphorylates different proteins of the myofibrils, such as cardiac troponin I, telethonin, myosin-binding protein C, and myomesin.[15]
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