Dominant Negative Protein Kinase D Research Articles

Reducing hepatic PKD activity lowers circulating VLDL cholesterol.

Protein kinase D (PKD) is emerging as an important kinase regulating energy balance and glucose metabolism; however, whether hepatic PKD activity can be targeted to regulate these processes is currently unclear. In this study, hepatic PKD activity was reduced using adeno-associated virus vectors to express a dominant-negative (DN) version of PKD1, which impairs the action of all three PKD isoforms. In chow-fed mice, hepatic DN PKD expression increased whole-body glucose oxidation, but had only mild effects on glucose and insulin tolerance and no effects on glucose homeostasis following fasting and refeeding. However, circulating VLDL cholesterol was reduced under these conditions and was associated with hepatic fatty acid accumulation, but not lipids involved in lipoprotein synthesis. The limited effects on glucose homeostasis in DN PKD mice was despite reduced expression of gluconeogenic genes under both fasted and refed conditions, and enhanced pyruvate tolerance. The requirement for PKD for gluconeogenic capacity was supported by in vitro studies in cultured FAO hepatoma cells expressing DN PKD, which produced less glucose under basal conditions. Although these pathways are increased in obesity, the expression of DN PKD in the liver of mice fed a high-fat diet had no impact on glucose tolerance, insulin action, pyruvate tolerance or plasma VLDL. Together, these data suggest that PKD signalling in the liver regulates metabolic pathways involved in substrate redistribution under conditions of normal nutrient availability, but not under conditions of overnutrition such as in obesity.

Protein Kinase D Regulates L-type Ca2+ Current In Cardiac Ventricular Myocytes

Cardiac excitation-contraction (EC) coupling is dynamically regulated. through an integration of kinase-phosphatase activities that alter the phosphorylation state of key Ca2+ cycling proteins, such as the L-type Ca2+ Channel (LTCC). This dynamic balance allows for active modulation of Ca2+ influx (ICa) through the LTCC. This is crucial for normal myocyte function and contractility, and is dysregulated in the failing human heart. Previous studies from our group show that treatment of ventricular myocytes with the phosphatase inhibitor calyculin A (caly A) increased contractility by augmenting ICa in the absence of humoral stimulation (steady-state). These results provided preliminary evidence that endogenous, LTCC-directed protein kinase activity is responsible for whole-cell ICa regulation in the steady-state. We explore the hypothesis that protein kinase D (PKD) plays a role in modulating the LTCC in the steady-state. This investigation focuses on the effect of the expression of genetically modified PKD on contractility, and ICa in cultured adult rat ventricular myocytes. Whole-cell ICa was recorded from myocytes infected with control adenovirus, constitutive active PKD, and dominant negative PKD. In addition, we studied the effect of caly A on these three different groups. In the constitutively active PKD myocytes, there is an increase in the peak ICa prior to the administration of caly A in comparison to the control adenovirus myocytes. The increase in peak ICa in the constitutively active group provide increasing evidence that PKD is required for modulating steady-state ICa.