At the sarcolemma, enigma homolog 1 scaffolds PKD1 to increase Ca²⁺ current of voltage-gated Ca²⁺ channels in response to α1-AR but not β-AR stimulation (Maturana et al., 2008). PKD phosphorylation of Serine 1884 in the L-type Ca²⁺ channel (LTCC) results in increased open probability of the channel (Aita et al., 2011). PKD1 also regulates LTCC trafficking and function indirectly via phosphorylation of the GTP-binding protein Rem1 (for Rad and Gem-related) at S18. This relieves Rem1 inhibition of LTCCs, resulting in greater T-tubule expression and a corresponding increase in Ca²⁺ current density (Jhun et al., 2012).

Protein Kinase D1 has also been implicated in the phosphorylation and regulation of constitutive nitric oxide synthases (endothelial and neuronal NOS; Aicart-Ramos et al., 2014; Sanchez-Ruiloba et al., 2014). In endothelial cells, VEGF activation of PKD promotes vasodilation and angiogenesis via S1179 phosphorylation, and activation, of eNOS. Numerous signaling cascades and kinases target this regulatory site to boost NO production, including Akt. The importance of PKD as a regulatory kinase of eNOS and vascular tone was confirmed in vivo, where inhibition of PKD dramatically reduced VEGF-induced dilation of the carotid artery. Given its role in control of cell proliferation, migration and angiogenic gene expression (Liu et al., 2014; Ren, 2016), PKD signaling is seen as a key driver of angiogenesis with interesting therapeutic possibilities. Several studies have already shown pan-KD inhibition is beneficial in cancer models [where both the tumor cells and angiogenesis are targeted (Lavalle et al., 2010; Borges et al., 2015; Tandon et al., 2015)]. However, given the plethora of signaling cues that modulate angiogenesis in physiological and pathological conditions, the precise role of PKD isoforms in these processes should be worked out.

The role of PKD in transcriptional regulation is well-documented. PKD is involved in the regulation of several transcription factors such as the activating enhancer binding protein-2 (AP2; Kondo et al., 2011), T-cell factor (TCF; via beta catenin; Jaggi et al., 2008), Snail1 (Eiseler et al., 2012), cAMP-response element binding protein (CREB; Johannessen et al., 2007; Ozgen et al., 2008), myocyte enhancer factor2 (MEF2; Vega et al., 2004) and NFκB (nuclear factor κ-light-chain-enhancer of activated B-cells; Holden et al., 2008). However, only CREB and MEF2 regulation have been confirmed in the heart. Here activation of PKD by Gq leads to CREB serine 133 phosphorylation and the induction of CRE-responsive genes such as Bcl-2 contributing to cell survival (Ozgen et al., 2008). Low levels of oxidative stress also promote CREB phosphorylation, but this is associated with decreased CREB abundance and no change CREB target gene transcription (Ozgen et al., 2009). The importance of PKD-dependent CREB phosphorylation in cardiac remodeling processes is still unclear.

Protein Kinase D also influences gene expression by modulating the epigenetic machinery, specifically the transcriptional repressors class II HDACs. The Olson and McKinsey labs firmly established PKD isoforms as HDAC kinases (McKinsey and Olson, 2005). Following activation and translocation to the nucleus, PKD associates with and phosphorylates the 14-3-3 binding sites on the HDAC protein. This unmasks a nuclear export sequence on HDAC, culminating in crm1-dependent nuclear export of the HDAC-chaperone protein complex (McKinsey et al., 2001). The release of class II HDACs from MEF2 allows for histone acetyltransferases (HATs) to associate with MEF2 inducing chromatin relaxation and transcriptional activation of fetal cardiac genes. PKD regulation of HDACs exemplifies an emerging theme in PKD regulation of its substrates: PKD target phosphorylation alters association with 14-3-3 chaperone proteins and consequently intracellular location of its substrates.

In contrast to CaMKII, each PKD isoforms can phosphorylate all of the class IIa HDACs (HDAC4, 5, 7, and 9) but their functional redundancy is not fully addressed and may be signal-dependent (Ellwanger and Hausser, 2013). In B lymphocytes, disruption of both PKD1 and 3 was required to block HDAC7 phosphorylation in antigen receptor signaling (Matthews et al., 2006). In the PKD1 cKO mice, fetal gene activation and stress-induced hypertrophy is blunted, suggesting that neither PKD2 and PKD3, nor CaMKII can fully compensate for the loss of PKD1 (Fielitz et al., 2008).

A number of regulatory interactions for PKD-HDAC signaling axis have also been identified. Disruption of the small heat shock protein 20 (hsp20)-PKD interaction, which chaperones nuclear translocation of PKD1, inhibited nuclear import of PKD and β-AR induced hypertrophy (Sin et al., 2015). In microvascular endothelial cells, PKD1/HDAC7 signaling to FoxO1 initiates proangiogenic and proarteriogenic transcription by repression of CD36 transcription (Ren et al., 2016). Four-and-a-half LIM domain proteins (FHL) 1 and 2, while not actual PKD targets, were identified as novel PKD binding partners that differentially facilitate neurohormonal activation of PKD (FHL1 for ET, and FHL2 for both ET and α1-AR agonists). Curiously, PKD regulation by FHL proteins affected HDAC5 phosphorylation but not MEF2 activation (Stathopoulou et al., 2014). Graeme Carnegie demonstrated that the AKAP-Lbc (AKAP13) scaffold protein facilitates hypertrophic PKD-HDAC signaling (Carnegie et al., 2008). This scaffolding protein, located at the nuclear envelope and cytoskeleton, nucleates PKCα, PKCη, PKA, and PKD, and promotes Rho activation (Carnegie et al., 2004). Gene-trap mice expressing an AKAP-Lbc variant that abolishes the PKD interaction (AKAP-Lbc-ΔPKD) exhibited blunted cardiac hypertrophy in response to Ang/PE treatment or pressure overload (Taglieri et al., 2014; Johnson et al., 2015), in agreement with the PKD1cKO mice. Unlike the PKD1cKO mice, the AKAP-Lbc-ΔPKD mice had greater collagen deposition and displayed an accelerated progression to cardiac dysfunction. This study further highlights the compartmentalization aspect of PKD signaling and suggests a critical in vivo role for AKAP-Lbc-anchored PKD signaling in compensatory hypertrophy development. In contrast, the Smrcka group proposes that the perinuclear mAKAP-PLC𝜀 complex, which generates DAG from PI4P in the Golgi apparatus in close proximity to the nuclear envelope, is the crucial scaffold complex to regulate activation of nuclear PKD and hypertrophic signaling pathways (Zhang et al., 2013). The discordant result might be explained by signal-specific pathways but both groups included ET as stimulus in their study.

Besides the long term nuclear effects, PKD signaling may also contribute to excitation-contraction coupling regulation via effects on sarcomeric proteins. A number of PKD substrates have been identified thus far: the titin-cap protein telethonin (Candasamy et al., 2014), troponin I (TnI; Cuello et al., 2007) and Myosin Binding Protein c (MyBPc; Bardswell et al., 2010; Dirkx et al., 2012a). Upon phosphorylation of TnI at Serine 22 and 23, the sites also targeted by PKA, myofilament Ca²⁺ sensitivity is reduced. Phosphorylation of MyBPc at Serine 302 and Serine 315 accelerates crossbridge cycling kinetics and increases maximal Ca²⁺-activated contraction tension.

Force generation was not modulated by the coordinated phosphorylation of TnI and MyBPc. Instead, active PKD can affect force generation by increasing Ca²⁺ current via increased surface expression of LTCCs. Paradoxically, α1-AR stimulation with PE, elicits a triphasic response in both the left ventricle (LV) and right ventricle (RV), but an overall positive inotropic response in LV trabeculae and an overall negative inotropic response in RV trabeculae (Wang et al., 2006). The overall response of each ventricle does not correspond to the response of individual myocytes, however, as α1-AR signaling causes both a positive and negative inotropic effect in individual myocytes of both ventricles that is independent of which α1-AR subtype is stimulated (Chu et al., 2013). In what is likely a compensatory mechanism, heart failure shifts the overall negative response to PE in the RV to a positive one (Wang et al., 2010). Although PKD is a key target of α1-AR signaling it is unknown whether α1-adrenergic-PKD signaling is involved in either the heterogeneous inotropic response of individual myocytes or the switch of inotropy during HF.

Despite a well-established nuclear translocation effect from the membrane to the nucleus, the spatiotemporal dynamics of PKD at its sarcomeric targets has not been assessed. It is unclear whether the active PKD that phosphorylates TnI and MyBPc originates from the same pool that is destined for the nucleus, or whether there is a separate scaffolded pool of PKD available to respond locally within the sarcomeres.

In endothelial cells, PKD1 was found to be a critical mediator of H₂O₂- but not TNF-induced apoptosis signal-regulating kinase 1 (ASK1) activation of JNK (Waldron et al., 2004; Zhang et al., 2005). Here H₂O₂ triggered PKD activation and translocation from the membrane to the perinuclear region, where it associated with ASK1, before progressing to the nucleus. The ASK1-PKD1 interaction (via the PKD PH domain) was critically dependent on 14-3-3 binding to PKD at two pairs of phosphorylated serines (205/208 and 219/223). The H₂O₂ –induced ASK-JNK activation and endothelial cell apoptosis was blocked with PKD inhibition or siRNA knockdown. Whether ASK1 is also modulated by PKD in cardiomyocytes or whether PKD1 regulation of ASK1 activity is via direct phosphorylation (as shown for CaMKII) is still unknown.

The Storz group identified a pathway where PKD activation by mitochondrial ROS led to induction of antioxidative genes through activation of NFκB. In HeLa cells, mitochondrial oxidative stress promoted local DAG production by phospholipase D1 evoking PKD translocation to the mitochondria (Cowell et al., 2009). PKD activation at the mitochondria was dependent upon the tyrosine kinases c-Abl and Src (Storz et al., 2003). c-Abl phosphorylation of Tyrosine 463 within the PKD PH domain led to loss of autoinhibition and Src phosphorylation of PKD Tyrosine 95. These phosphotyrosines create a docking site for PKCδ via its C2 domain and subsequently phosphorylation of Serine 744/748 in the activation loop. PKCδ recognizes this phosphorylated tyrosine and associates with PKD resulting in phosphorylation at Serines 744 and 748 (Storz et al., 2004). Active PKD then phosphorylates inhibitor of NFκB kinase β (IKKβ) at Serine 181, which allows for IκBα degradation and NFκB activation (Storz and Toker, 2003b). In the nucleus NFκB promotes SOD2 gene transcription which encodes the mitochondrial protein manganese depend superoxide dismutase (MnSOD; Storz et al., 2005). The subsequent detoxification of ROS resulted in increased cell survival but perturbation of any components in this pathway led to increased cell death. It is not clear whether this pathway operates in cardiac myocytes. It should also be noted that growth factor-dependent PKD1 signaling cascades do not activate NFκB nor induce MnSOD, underscoring the impact of contextual cues and specific post-translational modifications of PKD1 on achieving unique cellular outcomes.

Protein Kinase D1-mediated protection from oxidative stress may also be mediated via RhoA signaling (Xiang et al., 2013a). During ischemia-reperfusion injury cardiomyocytes release the cardioprotectant sphingosine 1-phosphate (S1P; Vessey et al., 2009). The signaling cascade initiated by S1P via its Gα_12/13-coupled receptor, produce guanine nucleotide exchange factors for RhoA activation which in turn activates PLC𝜀 (Wing et al., 2003; Xiang et al., 2013b). The surge in DAG, activates PKD1 which then phosphorylates and inhibits Slingshot 1L (SSH1L), a phosphatase/activator of cofilin (actin depolymerization factor). S1P thus attenuates cofilin translocation to mitochondria and association with the pro-apoptotic BAX (bcl-2-associated X protein), preserving mitochondrial integrity and cell survival in the face of oxidative stress (Klamt et al., 2009). In mice with genetic deletion of PKD1 or PLC𝜀, S1P-mediated cardioprotection against ischemia/reperfusion injury was reduced (Xiang et al., 2013b). The PKD-slinghot-cofilin signaling axis is one of many PKD-mediated pathways regulating actin dynamics described thus far (Olayioye et al., 2013). Determining if PKD also fulfills this role in cardiac cell types may shed light on cytoskeletal and actin rearrangement seen with heart failure.

In pancreatic β-cells, PKD1 is essential for glucose-stimulated insulin secretion signaling downstream of the long-chain fatty acid (LCFA) receptor GPR40 (Ferdaoussi et al., 2012). In these cells PKD activity is negatively controlled by MAPKp38δ phosphorylation (at Serines 397/401). Accordingly, p38δ KO mice displayed high constitutive PKD activity and were protected against high-fat-feeding induced insulin resistance and oxidative stress–induced βcell failure (Sumara et al., 2009).

In the heart, PKD is crucial for contraction-induced translocation of the glucose transporter type 4 (GLUT4), an essential step to stimulate cardiac glucose uptake during increased energy demand (Luiken et al., 2008). The underlying mechanism is not a direct effect of ATP/AMP levels, as with AMP-activated kinase (AMPK), but rather a result of contraction-induced ROS production, association of death-activated protein kinase (DAPK) and subsequent PKD activation (Dirkx et al., 2012b). AMPK represents the other obligatory signaling branch regulating GLUT4 translocation in cardiomyocytes, but unlike PKD, AMPK also mediates increased CD36 membrane translocation, allowing for LCFA to enter the cell (Dirkx et al., 2012b). Indeed, PKD1 cKO myocytes, despite having intact AMPK levels, do not increase their glucose uptake with pacing and their LCFA uptake is enhanced (Dirkx et al., 2014). Conversely, mice expressing constitutively active PKD1 (caPKD1) have 1.5 times the level of basal glucose uptake and a more pronounced increase in uptake in response to pacing than WT myocytes (Dirkx et al., 2014). Distinct roles for PKD and AMPK are also seen in rat cardiomyocytes with low glucose uptake induced by high insulin or high palmitate culture conditions. Either PKD or AMPK overexpression prevents loss of insulin-stimulated glucose uptake. But while overexpression of PKD in cardiomyocytes prevents lipid loading, AMPK overexpression promotes retention of insulin-stimulated Akt signaling (Steinbusch et al., 2013). Given its independence from the insulin signaling axis, the PKD signaling pathway represents a strategic option to increase glucose uptake in the insulin-resistant diabetic heart.

Conversely, PKD also regulates cardiomyocyte lipoprotein lipase (LPL) secretion, which hydrolyzes lipoproteins at the vascular lumen (Kim et al., 2008). In diabetes, the observed increase in LPL facilitates the switch to the disproportionate use of fatty acids (FA) which ultimately leads to excessive cardiac lipid accumulation and dysfunction (Wang and Rodrigues, 2015). The action of PKD1 to regulate LPL-mediated triglyceride accumulation was discovered in mice treated with diazoxide to decrease insulin secretion and cause hyperglycemia. The mechanism relied on heat shock protein 25 dissociation from PKCδ, permitting PKCδ association with and activation of PKD. Active PKD then promoted vesicular trafficking and release of LPL. A subsequent study recapitulated these findings in rats using low dose streptozotocin (STZ) to induce moderate hypoinsulinemia (Kim et al., 2009). Whereas in rats with severe hypoinsulinemia induced by a higher dose of STZ, LPL activity at the vascular lumen was diminished. The loss of LPL stimulation was attributed to caspase-dependent cleavage of PKD (resulting in a modest increase in basal activity of the kinase, but severe limitation of its maximal activation). These observations contrast with the finding that cardiac lipid overload and insulin resistance are largely prevented in caPKD mice fed a high fed diet (Dirkx et al., 2014). Clearly further investigation is needed to clarify the beneficial and pathogenic roles of PKD in regulating energy substrate utilization.

Several groups have performed proof-of-concept studies targeting PKD in diabetic models. Some groups have targeted PKD-mediated cardiac remodeling in diabetic cardiomyopathy, while others have attempted to capitalize on PKD modulation of fuel selection. Using the early stage type 2 diabetes model db/db mice, which have a point mutation in one of the leptin receptor genes, administration of the pan-PKD inhibitor CID755673 for 2 weeks effectively inhibited PKD isoforms, suppressed the gene expression signature of PKD activation and enhanced diastolic and systolic function as well as reduced heart weight. The improvement in cardiac indices was independent of effects on glucose homeostasis, insulin action and body composition (Venardos et al., 2015). Liu et al. likewise found that the observed cardiac fibrosis, apoptosis, diastolic dysfunction and ER stress were all related to PKD activation in a rat diabetic model achieved by a combined low dose STZ and high fat diet (4 weeks). Irbertesan treatment ameliorated cardiac remodeling and function in this model, which was attributed to inhibition of PKD activation and ER stress function (Liu et al., 2015).

A third model, mice on a chronic (8 weeks) high fat diet, displayed the characteristic insulin resistance, cardiac switch to LCFA and lipid deposition, and LV concentric hypertrophy but also reduced PKD expression and activity levels (Dirkx et al., 2014). Interestingly, applying the high fat diet to caPKD1 mice rescued their baseline dilated cardiomyopathy phenotype (without inducing hypertrophy) and also prevented cardiac lipid overload and insulin resistance. The only two mutual responses to high fat diet of WT and caPKD mice were reduced protein levels of PKD and decreased levels of HDAC5 phosphorylation. Of note, despite the decreased PKD expression, PKD activity levels were unchanged, suggesting that not just the expression but also the location and/or substrate of PKD is altered by metabolic stress. Some of these conflicting reports likely reflect variations in the animal models and our poor understanding of the temporal and complex role of PKD signaling in metabolism. There is a tendency to oversimplify the mechanistic basis of PKD effects to the context of single downstream targets (e.g., cardiac remodeling to the HDAC5-Mef2 axis), which is clearly not the only mode of action. Much remains to be learned about the precise regulation and effectors of PKD signaling in response to metabolic stress.