Protein Kinase D Activation Research Articles

Pancreatic oncogenic signaling cascades converge at Protein Kinase D1

Pancreatic oncogenic signaling cascades converge at Protein Kinase D1

Group B streptococci induce proinflammatory responses via a protein kinase D1-dependent pathway (INM7P.351)

Abstract Group B streptococci (GBS) are one of the leading causes of life-threatening sepsis in neonates. Despite intervention with antibiotic therapy, the incidence of lethality is high among GBS-infected infants. The clinical symptoms of sepsis are related to the host-pathogen interaction and exaggerated proinflammatory cytokine production during the process via pathways dependent on Toll-like receptors (TLRs). Over the last few years, it has become evident that protein kinase D1 (PKD1) plays a significant role in the inflammatory process mediated by TLRs. However, it is currently unknown whether PKD1 is activated, and contributes to proinflammatory responses induced by live or antibiotic-killed GBS. In the present study, we found that both live and antibiotic-killed GBS induce activation of PKD1 that is dependent on the TLR signaling adaptor MyD88 and its downstream kinase IRAK1, but not TRAF6. Inhibition of PKD using a pharmacological inhibitor revealed that PKD1 is indispensable for GBS-mediated expression of various cytokines/chemokines in vitro and in vivo. Furthermore, systemic administration of PKD inhibitor protects D-galactosamine-sensitized mice from shock-mediated death caused by antibiotic-killed GBS. Our findings imply that PKD1 is one of the critical factors that play a regulatory role in GBS-induced proinflammatory reactions, and inhibition of PKD1 activation together with antibiotic treatment in GBS-infected neonates might be an effective way to control GBS diseases.

Targeting vascular endothelial growth factor receptor 2 and protein kinase D1 related pathways by a multiple kinase inhibitor in angiogenesis and inflammation related processes in vitro.

Emerging evidence suggests that the vascular endothelial growth factor receptor 2 (VEGFR2) and protein kinase D1 (PKD1) signaling axis plays a critical role in normal and pathological angiogenesis and inflammation related processes. Despite all efforts, the currently available therapeutic interventions are limited. Prior studies have also proved that a multiple target inhibitor can be more efficient compared to a single target one. Therefore, development of novel inflammatory pathway-specific inhibitors would be of great value. To test this possibility, we screened our molecular library using recombinant kinase assays and identified the previously described compound VCC251801 with strong inhibitory effect on both VEGFR2 and PKD1. We further analyzed the effect of VCC251801 in the endothelium-derived EA.hy926 cell line and in different inflammatory cell types. In EA.hy926 cells, VCC251801 potently inhibited the intracellular activation and signaling of VEGFR2 and PKD1 which inhibition eventually resulted in diminished cell proliferation. In this model, our compound was also an efficient inhibitor of in vitro angiogenesis by interfering with endothelial cell migration and tube formation processes. Our results from functional assays in inflammatory cellular models such as neutrophils and mast cells suggested an anti-inflammatory effect of VCC251801. The neutrophil study showed that VCC251801 specifically blocked the immobilized immune-complex and the adhesion dependent TNF-α -fibrinogen stimulated neutrophil activation. Furthermore, similar results were found in mast cell degranulation assay where VCC251801 caused significant reduction of mast cell response. In summary, we described a novel function of a multiple kinase inhibitor which strongly inhibits the VEGFR2-PKD1 signaling and might be a novel inhibitor of pathological inflammatory pathways.

PKD Regulates Mitochondrial Morphology and Function via Phosphorylation of DLP1 in Cardiac Myocytes.

Regulation of mitochondrial morphology and dynamics is essential for maintaining cardiomyocyte function. Abnormal mitochondrial morphology and mitochondrial dysfunction are frequently observed in both human heart failure (HF) and animal HF models. However, it is still unclear which cardiac signaling pathway(s) regulate(s) mitochondrial morphology/function under pathophysiological conditions. Since Gq‐protein coupled receptor (GqPCR) signaling is critical for the development and progression of HF, we tested the hypothesis that GqPCR stimulation induces pathological changes in mitochondrial morphology/function in cardiomyocytes. We found that protein kinase D (PKD) was activated and translocated to the outer mitochondrial membrane by GqPCR stimulation. GqPCR‐mediated PKD activation induced mitochondrial fragmentation, increased reactive oxygen species generation and mitochondrial permeability transition pore opening followed by caspase‐3 activation. These morphological and functional changes in cardiac mitochondria were mediated via PKD‐dependent phosphorylation of Dynamin‐Like Protein 1 (DLP1) at S637. Importantly, PKD‐dependent DLP1 phosphorylation at S637 concurrent with abnormal mitochondrial morphology and apoptotic signaling were also observed in ventricular tissue from transgenic mice with cardiac‐specific overexpression of constitutively active Gq. In conclusion, we demonstrate that GqPCR stimulation induces mitochondrial fragmentation and dysfunction via PKD‐dependent phosphorylation of DLP1 at S637, which likely contributes to cardiomyocyte dysfunction during HF.

The PKD inhibitor CID755673 enhances cardiac function in diabetic db/db mice.

The development of diabetic cardiomyopathy is a key contributor to heart failure and mortality in obesity and type 2 diabetes (T2D). Current therapeutic interventions for T2D have limited impact on the development of diabetic cardiomyopathy. Clearly, new therapies are urgently needed. A potential therapeutic target is protein kinase D (PKD), which is activated by metabolic insults and implicated in the regulation of cardiac metabolism, contractility and hypertrophy. We therefore hypothesised that PKD inhibition would enhance cardiac function in T2D mice. We first validated the obese and T2D db/db mouse as a model of early stage diabetic cardiomyopathy, which was characterised by both diastolic and systolic dysfunction, without overt alterations in left ventricular morphology. These functional characteristics were also associated with increased PKD2 phosphorylation in the fed state and a gene expression signature characteristic of PKD activation. Acute administration of the PKD inhibitor CID755673 to normal mice reduced both PKD1 and 2 phosphorylation in a time and dose-dependent manner. Chronic CID755673 administration to T2D db/db mice for two weeks reduced expression of the gene expression signature of PKD activation, enhanced indices of both diastolic and systolic left ventricular function and was associated with reduced heart weight. These alterations in cardiac function were independent of changes in glucose homeostasis, insulin action and body composition. These findings suggest that PKD inhibition could be an effective strategy to enhance heart function in obese and diabetic patients and provide an impetus for further mechanistic investigations into the role of PKD in diabetic cardiomyopathy.

SD-208, a novel protein kinase D inhibitor, blocks prostate cancer cell proliferation and tumor growth in vivo by inducing G2/M cell cycle arrest.

Protein kinase D (PKD) has been implicated in many aspects of tumorigenesis and progression, and is an emerging molecular target for the development of anticancer therapy. Despite recent advancement in the development of potent and selective PKD small molecule inhibitors, the availability of in vivo active PKD inhibitors remains sparse. In this study, we describe the discovery of a novel PKD small molecule inhibitor, SD-208, from a targeted kinase inhibitor library screen, and the synthesis of a series of analogs to probe the structure-activity relationship (SAR) vs. PKD1. SD-208 displayed a narrow SAR profile, was an ATP-competitive pan-PKD inhibitor with low nanomolar potency and was cell active. Targeted inhibition of PKD by SD-208 resulted in potent inhibition of cell proliferation, an effect that could be reversed by overexpressed PKD1 or PKD3. SD-208 also blocked prostate cancer cell survival and invasion, and arrested cells in the G2/M phase of the cell cycle. Mechanistically, SD-208-induced G2/M arrest was accompanied by an increase in levels of p21 in DU145 and PC3 cells as well as elevated phosphorylation of Cdc2 and Cdc25C in DU145 cells. Most importantly, SD-208 given orally for 24 days significantly abrogated the growth of PC3 subcutaneous tumor xenografts in nude mice, which was accompanied by reduced proliferation and increased apoptosis and decreased expression of PKD biomarkers including survivin and Bcl-xL. Our study has identified SD-208 as a novel efficacious PKD small molecule inhibitor, demonstrating the therapeutic potential of targeted inhibition of PKD for prostate cancer treatment.

A computational model of PKD and CERT interactions at the trans-Golgi network of mammalian cells.

BackgroundIn mammalian cells protein-lipid interactions at the trans-Golgi network (TGN) determine the formation of vesicles, which transfer secretory proteins to the cellular membrane. This process is regulated by a complex molecular network including protein kinase D (PKD), which is directly involved in the fission of transport vesicles, and its interaction with the ceramide transfer protein CERT that transports ceramide from the endoplasmic reticulum to the TGN.ResultsHere we present a novel quantitative kinetic model for the interactions of the key players PKD, phosphatidylinositol 4-kinase III beta (PI4KIII β) and CERT at the TGN membranes. We use sampling-based Bayesian analysis and perturbation experiments for model calibration and validation.ConclusionsOur quantitative predictions of absolute molecular concentrations and reaction fluxes have major biological implications: Model comparison provides evidence that PKD and CERT interact in a cooperative manner to regulate ceramide transfer. Furthermore, we identify active PKD to be the dominant regulator of the network, especially of CERT-mediated ceramide transfer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-015-0147-1) contains supplementary material, which is available to authorized users.

Irbesartan ameliorates diabetic cardiomyopathy by regulating protein kinase D and ER stress activation in a type 2 diabetes rat model

Recent studies demonstrate an important role of protein kinase D (PKD) in the cardiovascular system. However, the potential role of PKD in the pathogenesis of diabetic cardiomyopathy (DCM) remains unclear. Irbesartan has beneficial effects against diabetes-induced heart damage, while the mechanisms were still poorly understood. Our present study was designed to investigate the effects of irbesartan in DCM and whether the cardioprotective effects of irbesartan were mediated by PKD and endoplasmic reticulum (ER) stress. We induced the type 2 diabetic rat model by high fat diet and low dose streptozotocin injection. The characteristics of type 2 DCM were evaluated by metabolic tests, echocardiography and histopathology. 8-weeks administration of irbesartan (15, 30 and 45mg/kg/day) was used to evaluate the effect irbesartan in DCM. Diabetic rats revealed severe metabolic abnormalities, left ventricular dysfunction, myocardial fibrosis and apoptosis. PKD and ER stress were excessive activated in the myocardium of diabetic rats. Furthermore, cardiac fibrosis, apoptosis, diastolic dysfunction and ER stress were all significantly related to PKD activation in diabetic rats. Irbesartan treatment attenuated the activation of PKD and ER stress, which paralleled its cardioprotective effects. Our study suggests that irbesartan could ameliorate cardiac remodeling and dysfunction in type 2 diabetes, and these beneficial effects were associated with its ability to suppress the activation of PKD and ER stress.

PKD1 is downregulated in non-small cell lung cancer and mediates the feedback inhibition of mTORC1-S6K1 axis in response to phorbol ester

Protein kinase D1 (PKD1) is increasingly implicated in multiple biological and molecular events that regulate the proliferation or invasiveness in several cancers. However, little is known about the expression and functions of PKD1 in non-small cell lung cancer (NSCLC). In the present study, 34 pairs of human NSCLC and matched normal bronchiolar epitheliums were enrolled and evaluated for PKD1 expression by quantitative real-time PCR. We showed that PKD1 was downregulated in 26 of 34 cancer tissues in comparison with matched normal epitheliums. Moreover, patients with venous invasion or lymph node metastasis showed significant lower expression of PKD1. Exposure of NSCLC A549 and H520 cells to the PKD family inhibitor kb NB 142-70(Kb), at concentrations that inhibited PKD1 activation, strikingly potentiated S6K1 phosphorylation at Thr389 and S6 phosphorylation at Ser235/236 in response to phorbol ester (PMA). Knockdown of PKD1 with siRNAs strikingly enhanced S6K1 phosphorylation whereas constitutively active PKD1 resulted in the S6K1 activity inhibition. Furthermore, the PI3K inhibitors LY294002, BKM120 and MEK inhibitors U0126, PD0325901 blocked the enhanced S6K1 activity induced by Kb. Collectively, our results identify decreased expression of the PKD1 as a marker for NSCLC and the loss of PKD1 expression increases the malignant potential of NSCLC cells. This may be due to the function of PKD1 as a negative regulator of mTORC1-S6K1. Our results suggest that re-expression or activation of PKD1 might serve as a potential therapeutic target for NSCLC treatment.

Small-Molecule PKD Inhibitor Prevents Mitochondrial Fragmentation and Dysfunction during Gq-Protein Coupled Receptor Stimulation in Cardiac Cells

Regulation of mitochondrial morphology and dynamics is crucial for the maintenance of various cellular functions in cardiomyocytes. Abnormal mitochondrial morphologies concomitant with mitochondrial dysfunction are frequently observed both in human heart failure (HF) and in animal HF models. However, it is still unclear which cardiac signaling pathways regulate mitochondrial morphology and function under pathophysiological conditions. Recent reports suggest that Gq-protein coupled receptor (GqPCR) signaling pathways are critical for the development and progression of HF. Therefore, we hypothesize that GqPCR stimulation induces mitochondrial fragmentation and dysfunction, which initiates cardiomyocyte death. We found that protein kinase D (PKD) activated by GqPCR signaling was translocated to outer mitochondrial membrane (OMM) observed by Western blot analysis of cytosolic and mitochondria-enriched fractionated proteins and by live cell imaging of fluorescence resonance energy transfer (FRET). We also found that GqPCR-mediated PKD activation induced mitochondrial fragmentation, leading to increased reactive oxygen species (ROS) generation as well as increased mitochondrial permeability transition pore (mPTP) opening, which initiates apoptotic signaling activation and cardiomyocyte death. These morphological and functional changes in cardiac mitochondria were mediated via PKD-dependent phosphorylation of mitochondrial fission protein, Dynamin-Like Protein 1 (DLP1) at S637. Moreover, pretreatment with a novel potent PKD inhibitor CRT0066101 effectively inhibited GqPCR-mediated PKD translocation to OMM, DLP1 phosphorylation at S637, mitochondrial fragmentation, ROS generation and mPTP activation. In conclusion, we demonstrate that GqPCR stimulation induces mitochondrial fragmentation and dysfunction through PKD-dependent phosphorylation of DLP1 at S637, which likely contributes to cardiomyocyte injury. Thus, small-molecule PKD inhibitor may become a novel and potent therapeutic for preventing cardiac cell injury and death during HF.

Protein kinase-D1 overexpression prevents lipid-induced cardiac insulin resistance

In the insulin resistant heart, energy fuel selection shifts away from glucose utilization towards almost complete dependence on long-chain fatty acids (LCFA). This shift results in excessive cardiac lipid accumulation and eventually heart failure. Lipid-induced cardiomyopathy may be averted by strategies that increase glucose uptake without elevating LCFA uptake. Protein kinase-D1 (PKD1) is involved in contraction-induced glucose, but not LCFA, uptake allowing to hypothesize that this kinase is an attractive target to treat lipid-induced cardiomyopathy. For this, cardiospecific constitutively active PKD1 overexpression (caPKD1)-mice were subjected to an insulin resistance-inducing high fat-diet for 20-weeks. Substrate utilization was assessed by microPET and cardiac function by echocardiography. Cardiomyocytes were isolated for measurement of substrate uptake, lipid accumulation and insulin sensitivity. Wild-type mice on a high fat-diet displayed increased basal myocellular LCFA uptake, increased lipid deposition, greatly impaired insulin signaling, and loss of insulin-stimulated glucose and LCFA uptake, which was associated with concentric hypertrophic remodeling. The caPKD1 mice on high-fat diet showed none of these characteristics, whereas on low-fat diet a shift towards cardiac glucose utilization in combination with hypertrophy and ventricular dilation was observed. In conclusion, these data suggest that PKD pathway activation may be an attractive therapeutic strategy to mitigate lipid accumulation, insulin resistance and maladaptive remodeling in the lipid-overloaded heart, but this requires further investigation.

The protein kinase D1 COOH terminus: marker or regulator of enzyme activity?

Protein kinase D1 (PKD1) is a Ser/Thr kinase implicated in a wide variety of cellular responses. PKD1 activation is generally attributed to a PKC-dependent pathway that leads to phosphorylation of the activation loop at Ser(744)/Ser(748). This modification increases catalytic activity, including that toward an autophosphorylation site (Ser(916)) in a postsynaptic density-95/disks large/zonula occludens-1 (PDZ)-binding motif at the extreme COOH terminus. However, there is growing evidence that PKD1 activation can also result from a PKC-independent autocatalytic reaction at Ser(744)/Ser(748) and that certain stimuli increase in PKD1 phosphorylation at Ser(744)/S(748) without an increase in autophosphorylation at Ser(916). This study exposes a mechanism that results in a discrepancy between PKD1 COOH-terminal autocatalytic activity and activity toward other substrates. We show that PKD1 constructs harboring COOH-terminal epitope tags display high levels of in vitro activation loop autocatalytic activity and activity toward syntide-2 (a peptide substrate), but no Ser(916) autocatalytic activity. Cell-based studies show that the COOH-terminal tag, adjacent to PKD1's PDZ1-binding motif, does not grossly influence PKD1 partitioning between soluble and particulate fractions in resting cells or PKD1 translocation to the particulate fraction following treatment with PMA. However, a COOH-terminal tag that confers a high level of activation loop autocatalytic activity decreases the PKC requirement for agonist-dependent PKD1 activation in cells. The recognition that COOH-terminal tags alter PKD1's pharmacological profile is important from a technical standpoint. The altered dynamics and activation mechanisms for COOH-terminal-tagged PKD1 enzymes also could model the signaling properties of localized pools of enzyme anchored through the COOH terminus to PDZ domain-containing scaffolding proteins.

Lipin-1 Regulates Autophagy Clearance and Intersects with Statin Drug Effects in Skeletal Muscle

LPIN1 encodes lipin-1, a phosphatidic acid phosphatase (PAP) enzyme that catalyzes the dephosphorylation of phosphatidic acid to form diacylglycerol. Homozygous LPIN1 gene mutations cause severe rhabdomyolysis, and heterozygous LPIN1 missense mutations may promote statin-induced myopathy. We demonstrate that lipin-1-related myopathy in the mouse is associated with a blockade in autophagic flux and accumulation of aberrant mitochondria. Lipin-1 PAP activity is required for maturation of autolysosomes, through its activation of the protein kinase D (PKD)-Vps34 phosphatidylinositol 3-kinase signaling cascade. Statin treatment also reduces PKD activation and autophagic flux, which are compounded by diminished mammalian target of rapamycin (mTOR) abundance in lipin-1-haploinsufficent and -deficient muscle. Lipin-1 restoration in skeletal muscle prevents myonecrosis and statin toxicity invivo, and activated PKD rescues autophagic flux in lipin-1-deficient cells. Our findings identify lipin-1 PAP activity as a component of the macroautophagy pathway and define the basis for lipin-1-related myopathies.

Protein kinase C and Src family kinases mediate angiotensin II-induced protein kinase D activation and acute aldosterone production

Recent evidence has shown a role for the serine/threonine protein kinase D (PKD) in the regulation of acute aldosterone secretion upon angiotensin II (AngII) stimulation. However, the mechanism by which AngII activates PKD remains unclear. In this study, using both pharmacological and molecular approaches, we demonstrate that AngII-induced PKD activation is mediated by protein kinase C (PKC) and Src family kinases in primary bovine adrenal glomerulosa cells and leads to increased aldosterone production. The pan PKC inhibitor Ro 31-8220 and the Src family kinase inhibitors PP2 and Src-1 inhibited both PKD activation and acute aldosterone production. Additionally, like the dominant-negative serine-738/742-to-alanine PKD mutant that cannot be phosphorylated by PKC, the dominant-negative tyrosine-463-to-phenylalanine PKD mutant, which is not phosphorylatable by the Src/Abl pathway, inhibited acute AngII-induced aldosterone production. Taken together, our results demonstrate that AngII activates PKD via a mechanism involving Src family kinases and PKC, to underlie increased aldosterone production.

Protein kinase D1 (PKD1) phosphorylation promotes dopaminergic neuronal survival during 6-OHDA-induced oxidative stress.

Oxidative stress is a major pathophysiological mediator of degenerative processes in many neurodegenerative diseases including Parkinson’s disease (PD). Aberrant cell signaling governed by protein phosphorylation has been linked to oxidative damage of dopaminergic neurons in PD. Although several studies have associated activation of certain protein kinases with apoptotic cell death in PD, very little is known about protein kinase regulation of cell survival and protection against oxidative damage and degeneration in dopaminergic neurons. Here, we characterized the PKD1-mediated protective pathway against oxidative damage in cell culture models of PD. Dopaminergic neurotoxicant 6-hydroxy dopamine (6-OHDA) was used to induce oxidative stress in the N27 dopaminergic cell model and in primary mesencephalic neurons. Our results indicated that 6-OHDA induced the PKD1 activation loop (PKD1S744/S748) phosphorylation during early stages of oxidative stress and that PKD1 activation preceded cell death. We also found that 6-OHDA rapidly increased phosphorylation of the C-terminal S916 in PKD1, which is required for PKD1 activation loop (PKD1S744/748) phosphorylation. Interestingly, negative modulation of PKD1 activation by RNAi knockdown or by the pharmacological inhibition of PKD1 by kbNB-14270 augmented 6-OHDA-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 (PKD1WT) or constitutively active PKD1 (PKD1S744E/S748E) attenuated 6-OHDA-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury. Collectively, our results demonstrate that PKD1 signaling plays a cell survival role during early stages of oxidative stress in dopaminergic neurons and therefore, positive modulation of the PKD1-mediated signal transduction pathway can provide a novel neuroprotective strategy against PD.

β-Adrenergic Signaling Inhibits G q -Dependent Protein Kinase D Activation by Preventing Protein Kinase D Translocation

Rationale: Both β-adrenergic receptor (β-AR) and G q -coupled receptor (G q R) agonist–driven signaling play key roles in the events, leading up to and during cardiac dysfunction. How these stimuli interact at the level of protein kinase D (PKD), a nodal point in cardiac hypertrophic signaling, remains unclear. Objective: To assess the spatiotemporal dynamics of PKD activation in response to β-AR signaling alone and on coactivation with G q R-agonists. This will test our hypothesis that compartmentalized PKD signaling reconciles disparate findings of PKA facilitation and inhibition of PKD activation. Methods and Results: We report on the spatial and temporal profiles of PKD activation using green fluorescent protein-tagged PKD (wildtype or mutant S427E) and targeted fluorescence resonance energy transfer–based biosensors (D-kinase activity reporters) in adult cardiomyocytes. We find that β-AR/PKA signaling drives local nuclear activation of PKD, without preceding sarcolemmal translocation. We also discover pronounced interference of β-AR/cAMP/PKA signaling on G q R-induced translocation and activation of PKD throughout the cardiomyocyte. We attribute these effects to direct, PKA-dependent phosphorylation of PKD-S427. We also show that phosphomimetic substitution of S427 likewise impedes G q R-induced PKD translocation and activation. In neonatal myocytes, S427E inhibits G q R-evoked cell growth and expression of hypertrophic markers. Finally, we show altered S427 phosphorylation in transverse aortic constriction–induced hypertrophy. Conclusions: β-AR signaling triggers local nuclear signaling and inhibits G q R-mediated PKD activation by preventing its intracellular translocation. PKA-dependent phosphorylation of PKD-S427 fine-tunes the PKD responsiveness to G q R-agonists, serving as a key integration point for β-adrenergic and G q -coupled stimuli.

Compensatory regulation of HDAC5 in muscle maintains metabolic adaptive responses and metabolism in response to energetic stress.

Some gene deletions or mutations have little effect on metabolism and metabolic adaptation because of redundancy and/or compensation in metabolic pathways. The mechanisms for redundancy and/or compensation in metabolic adaptation in mammalian cells are unidentified. Here, we show that in mouse muscle and myogenic cells, compensatory regulation of the histone deacetylase (HDAC5) transcriptional repressor maintains metabolic integrity. HDAC5 phosphorylation regulated the expression of diverse metabolic genes and glucose metabolism in mouse C2C12 myogenic cells. However, loss of AMP-activated protein kinase (AMPK), a HDAC5 kinase, in muscle did not affect HDAC5 phosphorylation in mouse skeletal muscle during exercise, but resulted in a compensatory increase (32.6%) in the activation of protein kinase D (PKD), an alternate HDAC5 kinase. Constitutive PKD activation in mouse C2C12 myogenic cells regulated metabolic genes and glucose metabolism. Although aspects of this response were HDAC5 phosphorylation dependent, blocking HDAC5 phosphorylation when PKD was active engaged an alternative compensatory adaptive mechanism, which involved post-transcriptional reductions in HDAC5 mRNA (-93.1%) and protein. This enhanced the expression of a specific subset of metabolic genes and mitochondrial metabolism. These data show that compensatory regulation of HDAC5 maintains metabolic integrity in mammalian cells and reinforces the importance of preserving the cellular metabolic adaptive response.

Angiotensin II-Induced Protein Kinase D Activates the ATF/CREB Family of Transcription Factors and Promotes StAR mRNA Expression

Aldosterone synthesis is initiated upon the transport of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol is hydrolyzed to pregnenolone. This process is the rate-limiting step in acute aldosterone production and is mediated by the steroidogenic acute regulatory (StAR) protein. We have previously shown that angiotensin II (AngII) activation of the serine/threonine protein kinase D (PKD) promotes acute aldosterone production in bovine adrenal glomerulosa cells, but the mechanism remains unclear. Thus, the purpose of this study was to determine the downstream signaling effectors of AngII-stimulated PKD activity. Our results demonstrate that overexpression of the constitutively active serine-to-glutamate PKD mutant enhances, whereas the dominant-negative serine-to-alanine PKD mutant inhibits, AngII-induced StAR mRNA expression relative to the vector control. PKD has been shown to phosphorylate members of the activating transcription factor (ATF)/cAMP response element binding protein (CREB) family of leucine zipper transcription factors, which have been shown previously to bind the StAR proximal promoter and induce StAR mRNA expression. In primary glomerulosa cells, AngII induces ATF-2 and CREB phosphorylation in a time-dependent manner. Furthermore, overexpression of the constitutively active PKD mutant enhances the AngII-elicited phosphorylation of ATF-2 and CREB, and the dominant-negative mutant inhibits this response. Furthermore, the constitutively active PKD mutant increases the binding of phosphorylated CREB to the StAR promoter. Thus, these data provide insight into the previously reported role of PKD in AngII-induced acute aldosterone production, providing a mechanism by which PKD may be mediating steroidogenesis in primary bovine adrenal glomerulosa cells.

PKD inhibitor treatment results in increased PKD activation loop phosphorylation (1055.1)

Protein kinase D (PKD) transduces an abundance of signals downstream of diacylglycerol (DAG) production and plays a role in many cellular functions, including regulation of Golgi sorting, apoptosis, cell growth, and cell survival. PKD1, originally described as a protein kinase C (PKC) family member and named PKCμ, is now known to be a member of a distinct family of kinases that includes, PKD1, PKD2, and PKD3. The PKD family is related to members of the calcium‐calmodulin kinase (CAMK) superfamily of kinases rather than the PKCs, which belong to the AGC kinase group. PKD activity is acutely regulated via binding to membrane DAG via its C1 domains. Subsequent to DAG binding, PKD is activated via phosphorylation by the upstream kinases, the novel PKCs, at the activation loop followed by autophosphorylation. Thus, this phosphorylated species is often used as a measure of kinase activity. Here we describe that PKD inhibitors promote activation loop phosphorylation downstream of G protein‐coupled receptors. This is reminiscent of similar observations for PKC and Akt, wherein active site inhibitors trap phosphates on the kinase; a phenomenon we have attributed to stabilization of a phosphatase‐resistant conformation of the kinase domain. For PKD, this effect occurred in the presence of active site inhibitors as well as in the presence of non‐competitive inhibitors, a distinction from PKC. These findings underscore the importance of using caution when interpreting kinase activity from kinase phosphorylation status in the presence of small molecule inhibitors.Grant Funding Source: Supported by NIH P01 DK54441

G‐protein beta‐gamma regulation of Golgi to cell surface transport (802.11)

Non‐canonical G‐protein signaling has been shown to regulate intracellular transport and secretion. G‐protein beta‐gamma localizes to the trans‐Golgi network and activates Protein Kinase D (PKD), resulting in fission of vesicles destined for the plasma membrane. While basolaterally‐targeted (BL) or secretory vesicle release is known to be downstream of PKD activation, apically‐targeted (AP) vesicle release from the Golgi occurs via a PKD‐independent pathway. A similar cargo selectivity for beta‐gamma has not been tested. Here, we generate GRK2ct variants that inhibit beta‐gamma in a spatial or temporal manner. We test regulation of AP and BL cargo trafficking via a temperature shift immunofluorescence transport assay, as well as trafficking of other AP and BL candidate cargoes. We find that lipid‐association of the GRK2ct is critical for its inhibitory function on beta‐gamma. We also generate an inducible system of recruitment of GRK2ct variants to distinct subcellular locations, towards the goal of inhibiting solely Golgi‐localized beta‐gamma signaling. Our system can also be used to inhibit beta‐gamma signaling at other endomembrane locations. The results indicate that beta‐gamma is an upstream regulator of vesicle transport from the Golgi to the cell surface.Grant Funding Source: NIH GM056444