Chronic neurohormonal stimulation can have direct adverse effects on the structure and function of the heart. Heart failure develops and progresses as a result of the deleterious changes. It is well established that phosphorylation of class II HDAC5 (histone deacetylase 5) is an important event in the transcriptional regulation of cardiac gene reprogramming that results in the hypertrophic growth response. To date, experimentation on phosphorylation-mediated translocation of HDAC5 has focused on the regulatory properties of PKD (protein kinase D) within intact cells. With regard to the potential role of PKD in myocardium, recent observations raise the possibility that PKD-mediated myocardial regulatory mechanisms may represent promising therapeutic avenues for the treatment of heart failure. The present review summarizes the most recent and important insights into the role of PKD in hypertrophic signalling pathways.
Since its discovery in 1994, PKD (protein kinase D) [1,2] has received much attention because of its important role in the cardiac signalling field. The PKD family comprises three isoforms: PKD1 [formerly PKC (protein kinase C) μ], PKD2 and PKD3 (also named PKCν). All of these enzymes share similar architecture and exhibit a high degree of homology. PKD is a 918-amino-acid intracellular serine/threonine kinase that contains an N-terminal regulatory domain [containing two zinc-finger-like cysteine-rich motifs that bind both DAG (diacylglycerol) and phorbol esters with high affinity], a PH (pleckstrin homology) domain which negatively regulates enzyme activity and a C-terminal catalytic domain. PKD shows distinct structural and enzymatic properties, and does not phosphorylate a variety of substrates targeted by PKC [1–3]. On the basis of the sequence homology of catalytic domain and substrate specificity, PKD is now thought to be distinct from PKC, being classified into the CaMK (Ca2+/calmodulin-dependent kinase) group in the human kinome . Nevertheless, recent evidence has shown that PKD is activated in living cells through a PKC-dependent pathway, suggesting that PKD can function either downstream of, or in parallel with, PKC . Indeed, in many cases, PKD activation is known to be attenuated by PKC inhibition [6,7]. Specifically, activation of PKD requires the phosphorylation of Ser744 and Ser748 in the activation loop of the catalytic domain, along with an alleviation on the inhibitory effect of the PH domain via a PKC-dependent signalling pathway [8–10]. In addition to the two activation loop sites, PKD also contains an autophosphorylation site at Ser916 in the C-terminus, which is not trans-phosphorylated by other upstream kinases .
Role of PKD in cardiac hypertrophy
Several recent advances in PKD research have provided insights into its functions. Being a major PKD isoform in the heart, PKD1 has been the focus of interest in the cardiovascular research. Previous studies have reported that PKD is involved in the regulation of myocardial contraction, hypertrophy and pathological cardiac remodelling [12,13]. In fact, PKD has been implicated in the phosphorylation of several sarcomeric proteins, such as cardiac troponin I, cMyBP-C (cardiac myosin binding protein C) and telethonin , as well as class II HDAC (histone deacetylase) proteins . These putative PKD substrates commonly conform to the PKD phosphorylation motif LXRXXS/T, where an aliphatic amino acid (isoleucine/leucine/valine) and a basic amino acid (arginine) are located at the −5 and −3 positions relative to the serine target site respectively [15,16].
Cardiac hypertrophy often develops in response to long-term haemodynamic stress such as pressure and volume overload. Because cardiomyocytes are unable to divide, they tend to increase in cell volume in response to stress, and this can lead to thickening of the heart wall. Whereas cardiac hypertrophy may initially be an adaptive response that normalizes wall stress and compensates for increased workload, prolonged hypertrophy may eventually impair cardiac functions, resulting in reduced pump function and the progression of heart failure .
Previous studies have suggested key role for PKD as a regulator of cardiac signalling. It is reported that PKD and its upstream activator PKC, in particular PKCϵ, are implicated in the control of pathological remodelling of the heart . Both kinases have been shown to phosphorylate HDAC proteins, in particular HDAC5, creating docking sites for 14-3-3 proteins which promote shuttling of HDAC5 from the nucleus to the cytoplasm in a CRM1 (chromosome region maintenance 1)-dependent manner [19,20]. Interestingly, PKD has been shown previously to translocate to the nucleus in response to GPCR (G-protein-coupled receptor) stimulation. Notably, PKD is bound by DAG at its cysteine-rich domain, followed by phosphorylation on serine residues in the activation loop by PKC, thereby promoting PKD activation and redistribution . These observations pinpoint a pertinent role for PKD as a cardiac signalling mediator that has two major sites of action. First, PKD resides mainly in the cytosol under basal conditions and then moves into the nucleus upon receptor activation. However, the mechanism underlying PKD1 nuclear import is still not well defined. Regardless, subcellular localization of PKD is likely to be associated with its activation status, resulting in different biological responses. Further work is required to elucidate the precise mechanisms which control PKD localization and translocation. Another facet of PKD's important role in cardiac signalling is in regulating HDACs anti-hypertrophic properties through inhibition of MEF2 (myocyte enhancer factor 2) transcriptional activity. When phosphorylated, Class II HDACs bind to 14-3-3 proteins and subsequently dissociate from MEF2, thereby promoting transcriptional derepression . Consequently, the activation of MEF2 transcription factors evokes a reprogramming event of the cardiac gene expression that promotes fetal gene response, resulting in a hypertrophic phenotype .
Another kinase that impinges on PKD's actions is PKA (protein kinase A). PKA activity can impose a block on α1-adrenergic stimulation and suppress PKD phosphorylation . In this context, PKA may act as a counter-regulator to modulate PKD activation. This notion has been supported by groundbreaking work showing that acute β-adrenergic stimulation and activation of PKA inhibits phosphorylation of not only PKD, but also HDAC5, highlighting its pertinent role in the induction of hypertrophy-associated genes . PKD activity has also been shown to undergo stimulation by GPCRs in response to PKC activation, suggesting a causal relationship between PKC and PKD in the regulation of myocardial responses . Interestingly, it has recently been reported that cardiac AKAP (A-kinase-anchoring protein)–Lbc functions as a scaffolding protein for PKA and PKC to mediate activation of PKD [25,26]. Upon neurohormonal stimuli such as ET (endothelin)-1 or phenylephrine, the up-regulation of AKAP–Lbc facilitates PKD/HDAC5/MEF2 signalling and enhances the transduction of hypertrophic response. Conversely, reduced AKAP–Lbc expression prevents the translocation of HDAC5, thereby hindering the progression of hypertrophy. Since PKA and PKC are components of the AKAP–Lbc complex, this may suggest the involvement of both cAMP and phospholipid signalling pathways in the regulation of PKD activation. Indeed, recent findings have revealed the roles of compartmentalized PDEs (phosphodiesterases) in cAMP-dependent PKA-mediated PKD activation. It was reported that the combined inhibition of PDE3 and PDE4 is required to achieve substantial attenuation of ET-1-induced PKD activation through increased PKA activity . On the basis of these findings, it is tempting to postulate that AKAP–Lbc serves as a connector, providing a locus for signalling cross-talk between the kinases in a stimulus-dependent manner within the hypertrophy signalling cascades (Figure 1).
Schematic diagram depicting the hypertrophic signalling cascades
Conclusions and future perspectives
In conclusion, it is increasingly apparent that PKD1 is a key player in the regulation of cardiac hypertrophy, most likely through its effect on the transcriptional regulation of fetal gene programming via the phosphorylation of HDAC5. Recent research suggests that the function of PKD is dependent upon (i) its phosphorylation state, either activated directly by neurohormonal stimuli or by an upstream regulator, and (ii) its localization within the cell. These findings open new routes for the investigation of the role of PKD as a potential target for novel therapeutic intervention in the treatment of cardiac hypertrophy. Nevertheless, the detailed intracellular mechanism(s) underlying fetal gene reprogramming remains to be determined. Novel insights may be gleaned from further exploration of the signalling network involving AKAP–Lbc and its scaffold partners. Further work is also required to clarify the mechanistic details underpinning the cross-talk between different signal transduction pathways which contribute to the induction of the hypertrophic response, with a particular emphasis on understanding the upstream regulators and downstream functional consequences of PKD activation.
Signalling 2011: a Biochemical Society Centenary Celebration: A Biochemical Society Focused Meeting held at the University of Edinburgh, U.K., 8–10 June 2011. Organized and Edited by Nicholas Brindle (Leicester, U.K.), Simon Cook (The Babraham Institute, U.K.), Jeff McIlhinney (Oxford, U.K.), Simon Morley (University of Sussex, U.K.), Sandip Patel (University College London, U.K.), Susan Pyne (University of Strathclyde, U.K.), Colin Taylor (Cambridge, U.K.), Alan Wallace (AstraZeneca, U.K.) and Stephen Yarwood (Glasgow, U.K.).
Y.Y.S. is a recipient of a Wellcome Trust postgraduate scholarship and Overseas Research Students Awards Scheme (ORSAS).