The Role of Diacylglycerol and Cysteine‐rich Domains in Spatiotemporal Regulation of Protein Kinase D1 in Cardiac Myocytes

Protein Kinase D (PKD) has emerged as a signaling nodal point in the heart affecting contractility, gene transcription, cell survival and energy substrate use. PKD contains two cysteine‐rich domains (C1A and C1B) that bind diacylglycerol (DAG) and phorbol esters. We previously found distinct spatiotemporal activation of PKD in response to Gα q/11 ‐protein coupled receptor agonists Phenylephrine (PE) and Endothelin‐1 (ET): transient and minimal PKD translocation and activation at the sarcolemma followed by rapid nuclear translocation for PE, and prominent and sustained PKD recruitment to the sarcolemma for ET. Here we test the hypotheses that the C1 translocation modules are the primary spatiotemporal regulator of PKD in cardiomyocytes, and that differences in DAG production elicited by PE and ET account for the disparities in PKD response. Given the unique pattern of ligand recognition for the individual domains, the contributions of the individual C1 domains to the spatial regulation of PKD were assessed using GFP‐tagged PKD C1 domain mutants. Translocation to the membrane and nucleus was assessed using Total Internal Reflection Fluorescence (TIRF) and confocal microscopy respectively, while DAG production was monitored using a YFP‐tagged C1B PKCβII domain. Surprisingly, sarcolemmal recruitment of PKD1 was faster than that of C1B‐YFP in response to both ET and phorbol ester (PDBu) treatment. However, in the nucleus, C1B‐YFP accumulated faster than PKD1 in response to ET. The sustained membrane recruitment and nuclear export of C1B‐YFP in response to PDBu, was mirrored by PKD1. Both PE and ET treatment triggered nuclear import of PKD1 (+6.96±1.9% and +7.1 ±1.5% after 90 min), however only ET caused a significant increase in nuclear DAG accumulation (+20.8 ±2.3 %). These results indicate that local DAG signals are not the sole dictator of PKD translocation. We further speculate that PKD is basally anchored near the membrane and is poised to be recruited. PKD1 C1 mutants had membrane and nuclear translocation profiles in line with expectations based on their previously established properties, however, a few unexpected findings emerged. Chief among these were the first cases of nuclear export, rather than import, upon agonist stimulation; as well as evidence that access to the NLS motif is sterically regulated by interactions between the C1 domains. Unexpectedly, lack of the C1B domain did not lower nuclear targeting of PKD at baseline. However, two mutants that lacked the C1A domain (C1BC1B and ΔC1A) and one mutant with the order of the domains switched (C1BC1A) had greater nuclear levels (2.4, 1.8 and 1.8 times > WT), suggesting that the C1A domain inhibits or masks the NLS of the C1B domain through a direct interaction. Furthermore, these same mutants all showed nuclear export (−18.4 ±3.6, −14.5 ±1.3, and −11.2 ±2.3%) after PE treatment. ET‐1 treatment resulted in small nuclear export of C1BC1B and ΔC1A (−2.9 ±4.8 and −5.5 ±1.3%), but a large nuclear import of C1BC1A (peak of +24.8 ±9.8% at 60 min). These results indicate that PKD translocation, though influenced by DAG production, is a complex process which can be regulated by physical interaction between the C1 domains and influenced by kinase activation status. Support or Funding Information RO1 HL103933 to JB and 14PRE19920005 to BW