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Trends Pharmacol Sci. Jan 2010; 31(1): 8–14.
PMCID: PMC2809215
Protein kinase Cα: disease regulator and therapeutic target
Olga Konopatskaya and Alastair W. Poole
Department of Physiology & Pharmacology, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK
Alastair W. Poole: a.poole/at/bris.ac.uk
This document was posted here by permission of the publisher. At the time of the deposit, it included all changes made during peer review, copy editing, and publishing. The U. S. National Library of Medicine is responsible for all links within the document and for incorporating any publisher-supplied amendments or retractions issued subsequently. The published journal article, guaranteed to be such by Elsevier, is available for free, on ScienceDirect, at: http://dx.crossref.org/10.1016/j.tips.2009.10.006
Protein kinase Cα (PKCα) is a member of the AGC (which includes PKD, PKG and PKC) family of serine/threonine protein kinases that is widely expressed in mammalian tissues. It is closely related in structure, function and regulation to other members of the protein kinase C family, but has specific functions within the tissues in which it is expressed. There is substantial recent evidence, from gene knockout studies in particular, that PKCα activity regulates cardiac contractility, atherogenesis, cancer and arterial thrombosis. Selective targeting of PKCα therefore has potential therapeutic value in a wide variety of disease states, although will be technically complicated by the ubiquitous expression and multiple functions of the molecule.
Protein kinase C (PKC) is a family of serine/threonine protein kinases that regulate various cellular functions, including adhesion, secretion, proliferation, differentiation and apoptosis. The family is classified into three groups on the basis of the arrangement of their regulatory domains w2[1]. Conventional isoforms (cPKC; α, β, γ) contain a diacylglycerol (DAG)/phorbol ester-binding C1 domain and a Ca2+-binding C2 domain. Novel isoforms (nPKC; δ, epsilon, η, θ) also contain C1 domains, but their C2 domains are unable to bind Ca2+. Atypical isoforms (aPKC; ζ and ι/λ) lack a C2 domain and have an atypical C1 domain, and are therefore regulated independently of Ca2+ or DAG.
Here we focus on PKCα because of recent evidence, particularly derived from genetic studies, implicating this kinase in several major disease processes [2–4], and because of the multiple approaches that may be adopted to target PKCα pharmacologically [5–7]. PKCα is widely expressed and being a conventional PKC isoform, it may be regulated downstream of the multitude of receptors that couple to activation of phospholipase C, including Gq-coupled GPCRs (G-protein-coupled receptors), growth factor receptors and adhesion receptors. Pharmacological studies have shown PKCα to regulate multiple biological processes, including cell proliferation, apoptosis, differentiation, migration and adhesion. These roles are described and discussed elsewhere [1,8–10] and are outside of the scope of this article. The more recent introduction of PKCα gene knockout (Prkca−/−) mice has provided definitive evidence for the function of this kinase in physiological processes including insulin coupling to PI3kinase [11], and immune cell functions such as IgG class switching [12] and down-regulation of the T cell receptor [13], such as prostate and breast cancer, heart failure and pulmonary disease.
PKCα gene knockout mice are now also revealing critical roles for this gene in disease processes. In this opinion, we concentrate on the role of PKCα in the three best studied of these: cardiovascular dysfunction, arterial thrombosis and cancer (Fig. 1). We suggest that, through a variety of pharmacological strategies (Box 1), PKCα may emerge as a key target in the future treatment and management of these diseases.
Figure 1
Figure 1
PKCα plays key physiological and pathological roles. The ubiquitously expressed protein kinase PKCα plays roles in multiple cellular processes, including many not detailed in the Figure. Depicted are the three areas discussed principally (more ...)
Box 1. Approaches to targeting PKCα
Several approaches are used to target PKCα therapeutically, as summarized in Figure I.
  • 1.
    ATP (adenosine triphosphate) binding site
The ATP binding site is attractive for drug targeting, although selectivity may be difficult to achieve due to conservation of sequence and structure for this domain within the PKC family. The high concentration of ATP competes also with inhibitors at this site, decreasing their apparent activity. However, this site is still highly attractive for drug development, particularly by structure-based approaches, which are proving useful for PKCθ inhibitors [71]. A publicly available crystal structure for PKCα is still absent, and is clearly a requirement for future development.
Some bisinolylmaleimides, derivatives of staurosporine, show good selectivity within the PKC family. Ruboxistaurin (LY333531) and enzostaurin (LY317615) are designed to selectively target PKCβ, although a recent report suggests that ruboxistaurin may inhibit PKCα equipotently [72]. Both enzastaurin and ruboxistaurin are well tolerated [73,74]. Enzastaurin shows promise in phase I trials for the treatment of advanced cancer [75]. Ruboxistaurin has completed phase III trials for the management of diabetic retinopathy [70,74], principally through targeting PKCβ.
  • 2.
    Peptide substrate mimetics
Peptide substrate mimetics are short peptides that resemble the preferred substrate sequence for PKCα, often mimicking the pseudosubstrate domain, and compete for substrate binding. In addition, a small-molecule inhibitor, chelerythrine, competitively binds to the substrate-binding pocket, although its specificity for PKCα is poor [76].
  • 3.
    C1 domain
The C1 domain binds diacylglycerol (DAG) and phorbol esters. Bryostatin is a macrocyclic lactone that competes with DAG for binding [77], and simplified analogues have been designed with improved activity [78]. Calphostin C also binds PKCα, and other PKC isoforms, at the C1 domain. The inhibition is irreversible, and requires prior light activation of the molecule [79].
  • 4.
    C2 domain
The C2 domain binds Ca2+ and mediates interaction with proteins [80], in particular Receptors-for-Activated-C-Kinase (RACKs) [81]. RACK peptides therefore disrupt the localization of PKCs to their substrates, thereby effectively inhibiting their activity. This inhibition has been shown most extensively for PKCδ, where KAI-9803 is undergoing clinical trial for cardiac ST segment elevation of cardiogram in patients with myocardial infarction [82]. PKCα interacts with fascin and lamin A, and inhibition of these interactions might disrupt cytoskeleton- and nucleus-dependent signalling [83,84].
  • 5.
    Gene therapy
Although not shown in Figure I, an antisense drug, aprinocarsen (ISIS 3521, LY900003), has been used to reduce expression of PKCα. This drug has undergone phase III clinical trials for the treatment of various human tumors [61] but has not progressed through to the clinic, due to non-achievement of efficacy endpoints and associated toxicity.
Heart failure
It has been established that various PKC isoforms, including PKCα, contribute to impaired left ventricular filling and ejection in heart failure [6]. PKCα is both necessary and sufficient to induce cardiomyocyte hypertrophy - an adaptive mechanism triggered by decreased cardiac output and, if sustained, leading to heart failure and death - by an increase in protein synthesis, protein-DNA ratio, and cell surface area [14]. Further, PKCα antisense treatment reduces phenylephrine-induced increases in α-actin mRNA and atrial natriuretic peptide secretion [15], thereby causing a loss of some markers of pathological hypertrophy. Overexpression of PKCα also increases cardiomyocyte surface area and atrial natriuretic peptide expression, indicating that PKCα activation induces cardiomyocyte hypertrophy [16].
This regulation of cardiac hypertrophy translates into end-stage heart failure, and in animal models of this disease, expression and activity of PKCα are up-regulated [17]. Ablation of myocardial PKCα by targeted gene knockout leads to increased myocardial contractility, whereas transgenic overexpression of PKCα results in ventricular dysfunction and alterations in Ca2+ homeostasis [3]. The mechanism of PKCα-dependent depression of myofilament contractility involves phosphorylation of the cardiac troponins cTnI and/or cTnT functionally important in controlling maximum tension, ATPase activity, and Ca2+ sensitivity of the myofilaments [18].
Atherosclerosis
Atherosclerosis is a disease of large and medium-sized vessels that involves multiple cells and processes including endothelial dysfunction, inflammation and proliferation. In the arterial wall the disease is characterized by the emergence in the intima of atherosclerotic fatty streaks, later transforming into plaques, consisting of connective tissue and some smooth muscle cells (SMCs). It is accompanied by thickening and hardening (sclerosis) of the arterial wall, loss of elasticity due to infiltration of the intima with mononuclear cells, SMCs, and increased endothelial cell depositions. Atherosclerosis designates a special form of arteriosclerosis that is additionally characterized by the occurrence of foam cells, that is, lipid-laden macrophages and SMCs that may rupture and release their contents into the lesional areas. There is substantial evidence that PKCα plays key roles in these processes.
Endothelial cell proliferation
Pathological angiogenesis has been implicated in a number of vascular diseases, including unstable atherosclerotic plaque development [19]. Although there are some reports to the contrary [20,21], PKCα has generally been shown to be a positive regulator of angiogenesis [22], involving the processes of endothelial cell migration, adhesion and tube formation mediated by vascular endothelial growth factor (VEGF).
In vivo, knockdown of PKCα prevents myocardial vessel formation in a murine model of myocardial infarction [23]. There seems to be positive feedback between the two signalling components, PKCα and VEGF, because VEGF expression in endothelial cells depends on PKCα, but in turn VEGF leads to activation of PKCα [24]. Overexpression of a PKCα pseudosubstrate inhibitory peptide also slows the rate of endothelial cell migration [25]. Additionally, PKCα mediates interleukin-1β-induced expression of matrix metalloproteinase-2, a molecule implicated in angiogenesis [26].
Endothelial barrier function
Endothelial permeability is tightly regulated by a balance of contractile forces generated by the cytoskeleton and adhesive forces generated by cell–cell and cell–matrix contact [27]. Early studies suggested that PKC mediates the acute rise in endothelial permeability in vitro, via receptor-mediated (e.g. through thrombin, histamine or bradykinin receptors), or receptor-independent (by oxidants or shear stress) increases in phospholipase C activity [28]. Subsequently, several studies have narrowed this effect specifically to PKCα and its role in endothelial cell contraction and disassembly of VE-cadherin junctions [29,30].
At the molecular level, the mechanism might involve PKCα-dependent regulation of transient receptor potential canonical-1 (TRPC-1) channels [31] and Rho GTPases [32,33]. The increase in pulmonary endothelial cell permeability caused by TNFα is also associated with an increase in PKCα activity [34], and protein phosphatase type 2B (PP2B), which might regulate PKCα activity, is also implicated in regulating the permeability of pulmonary endothelial cells [35].
Lastly, endothelial permeability is altered in diabetes mellitus, and PKCα has been shown to mediate glucose-induced increases in endothelial cell permeability [36]. Therefore, there is high consensus among data supporting the idea that PKCα positively regulates endothelial permeability through various molecular mechanisms.
Oxidative stress and monocyte adhesion
Endothelial cell dysfunction elicited by oxidative stress plays a critical role in the pathogenesis of atherosclerosis [37]. Low-density lipoproteins (LDLs) may be oxidized when excessive free radicals react with the lipoproteins. In turn, these oxidized LDLs (Ox-LDLs) become deposited in the arterial walls and can lead to plaque development by stimulating macrophage uptake and foam cell formation.
A detrimental feedback loop operates in endothelial cells, where superoxides lead to generation of Ox-LDLs, which in turn stimulate further superoxide generation. Fleming et al. [38] have shown that Ox-LDL stimulation of endothelial cells leads to uncoupling of endothelial nitric oxide synthase (eNOS) from its generation of nitric oxide, and a greater accumulation of superoxide free radicals. The signalling pathway in endothelial cells from Ox-LDL to eNOS has been attributed to inactivation and down-regulation of PKCα, which results in diminished phosphorylation of eNOS on Thr-495 [38].
Additionally, PKCα in inflammatory cells might mediate their adhesion to and interaction with endothelial cells. The adhesion of circulating monocytes to endothelial cells contributes importantly to the inflammatory aspects of atherogenesis, which describes the development of atherosclerotic plaques. Pre-incubation of monocytes with Gö6976, the inhibitor of classical PKC isoforms, significantly reduces monocyte adhesion to HUVECs (human umbilical vein endothelial cells) [39]. Another inflammatory mediator, apolipoprotein CIII, a constituent of apolipoprotein B, enhances the adhesion of monocytes to endothelial cells via PKCα-mediated β1-integrin activation [40]. Lastly, endothelial cell PKCα is important in also regulating inflammatory cell adhesion, through expression of the adhesion molecule PECAM-1 [41], and through regulating exocytosis and surface expression of P-selectin, which mediates neutrophil-endothelial cell interaction [42].
In summary, although there is a mixed picture in terms of the role of PKCα in atherogenesis and cardiomyocyte hypertrophy, there is reasonable consensus that, on balance, inhibitors of PKCα are likely to be useful in the management of these diseases.
Platelets play a central role in mediating atherothrombosis, characterized by an unpredictable atherosclerotic plaque disruption, and are therefore the target of numerous therapies aimed at reducing their activity, particularly in the prevention of coronary artery thrombosis in heart attacks [43]. PKC has been established, largely by pharmacological studies, as a major regulator of multiple platelet activities [44], and it is increasingly clear that the different isozymes of PKC expressed in platelets perform distinct functions.
On activation, platelets release a multitude of active substances from secretory granules, essential for platelet pro-aggregatory responses and development of a stable thrombus in arteries. Yoshioka et al. [45] have analyzed the mechanisms governing Ca2+-induced secretion of α-granules and dense-core granules in permeabilized human platelets, and identified PKCα as an essential component of the mechanism. Using similar biochemical approaches, the same group demonstrated that PKCα is also involved in the regulation of Ca2+-induced platelet aggregation [46]. Pula et al. [47] have shown that two tyrosine kinases, Syk and Src, physically interact with PKCα, leading to distinct functional consequences. Although they found that PKCα activity was dependent on Syk, Syk activity was not regulated by PKCα; however, Src activity was negatively regulated by PKCα. These results suggest that PKCα is an important factor in the complex interaction with tyrosine kinases for regulation of functional activities in platelets. Additionally, in a recent elegant study reconstructing the signalling pathway regulating platelet integrin αIIbβ3 in a heterologous cell system, it was shown that PKCα expression is required for activation of the integrin through the Rap1 pathway [48]. Key molecular functions of platelet activation are therefore mediated by PKCα.
On the basis of pharmacological data, the specificity of the role played by PKCα has been a contentious issue, due to the lack of selectivity of the reagents available. We have therefore developed a genetic approach using PKCα knockout (Prkca−/−) mice to determine the role of PKCα in regulating platelet function and thrombus formation [4]. With regard to the suggested role of PKCα in regulating secretion of dense- and α-granules [45], we were able to confirm definitively that secretion of these granules was substantially diminished in Prkca−/− platelets. Prkca−/− platelets showed significantly reduced functionally relevant phosphorylation on Ser95 of synaptosomal associated protein SNAP-23, an essential component of the membrane fusion machinery and an important regulator of vesicle docking and fusion [4]. This observation therefore may potentially explain the observed secretion defect in PKCα-deficient platelets.
In addition, Prkca−/− platelets show a marked knockdown in activation of integrin αIIbβ3 in response either to thrombin or collagen-related peptide, paralleling a deficit in their ability to undergo aggregation at submaximal concentrations of agonist. This is also consistent with the impaired ability of Prkca−/− platelets to form a thrombus in vitro in blood flowing over a collagen-coated surface and in an in vivo laser-induced model of thrombus formation (Fig. 2). The defect in secretion was shown, however, to be the central significant event, because addition of exogenous ADP (adenosine diphosphate), the major constituent released from platelet dense granules, rescued the deficits in responses seen in Prkca−/− platelets, confirming that granule secretion is likely to be the primary function for PKCα.
Figure 2
Figure 2
PKCα plays a critical role regulating platelet function and thrombosis in arteries. Platelets are the smallest cellular component of the blood, and flow close to the endothelial lining of arteries continually surveying the vessel for breaches (more ...)
These findings, together with the fact that Prkca−/− mice do not demonstrate any evidence of overt bleeding, have revealed that PKCα is a potential drug target for antithrombotic therapy, because selective inhibitors would be expected to exert a major effect on thrombus formation while sparing primary platelet adhesive functions.
PKCα has long been recognized to have a role in regulating aspects of tumor growth and development [10], although this role is clearly complex and highly tissue-dependent because in some cases it acts as a tumor promoter, and in others it functions as a tumor suppressor. Nonetheless, PKC in general has, for many years, been a drug target for the treatment of cancer. Pharmacological approaches to control of cancer by PKC inhibitors have been discussed elsewhere [7,49] and some of these approaches for PKCα are discussed in Box 1.
Its most consistent role is probably regulation of cell motility, and certainly PKCα activation can result in increased cell motility in several in vivo and in vitro cancer models, the effect of which may be reversed on PKCα inhibition[50,51]. However, the predominant overall picture is of a lack of consistency, which can be illustrated by its differing expression patterns in different tumors. Overexpression of PKCα has been demonstrated in tissue samples of prostate, endometrial, high-grade urinary bladder and hepatocellular cancers[52–55], while for haematological malignancies up- or down-regulation of PKCα has been described [56], and in basal cell carcinoma and colon cancers, down-regulation of PKCα has been observed [2,57]. A mixed picture also pertains to breast cancer cells, where it has been studied extensively. Whereas activation or over-expression of PKCα has been shown in breast cancer cells and in breast tumor samples by some [58,59], down-regulation has been demonstrated by others [60].
In terms of cancer therapeutics, PKCα has been a target for the drug aprinocarsen (ISIS 3521; see also Box 1), a phosphorothioate antisense oligonucleotide (ASO) that targets the 3’-untranslated region of human PKCα mRNA, leading to decreased expression of the protein [61]. Aprinocarsen has been studied as a single agent, as well as in combination with standard chemotherapeutics, in cancer patients in over 20 trials from phase I to phase III (reviewed in [61,62]). The studies include individuals with non-small cell lung cancer [63], colon [64], ovarian [65], breast [66] and prostate cancers [67] and non-Hodgkin's lymphoma [68]. Although encouraging results have been observed in phase II trials, disappointingly aprinocarsen has failed to meet efficacy endpoints in phase III trials when used in combination with conventional chemotherapy for late-stage lung cancer [69,70]. Reasons for this lack of efficacy could be manifold, including lack of prior screening of patients for expression levels of PKCα, dose reduction due to toxicity during the trial and driving of cell proliferation by other related PKC isoforms [69].
PKCα plays important roles in several cellular processes and pathologies, involving cancer, cardiovascular disorders, atherogenesis and thrombosis. Its ubiquity and multiple roles, however, present a potential difficulty in its use as a drug target, although the mutual functional redundancy of various PKC isoforms might help to overcome possible unwanted effects of specific pharmacological intervention. It is clear, for instance, that thrombus formation is markedly diminished in the Prkca−/− mouse, whereas haemostasis is normal [4]. This functional redundancy with related PKC isoforms may also obscure other potential roles for PKCα, determined in particular from gene knockout strategies. There are clearly many strategies that can be adopted to target this kinase therapeutically (Box 1), and in this regard a key advance will be the determination of the crystal structure of the catalytic domain of PKCα. It will be important to continue developing strategies for PKCα targeting in the future, because specific drug targeting could have great value in the treatment of multiple disease states.
Conflict of interest declaration
The authors have declared that no conflict of interest exists.
Figure I
Figure I
PKCα domain structure mapped against pharmacological inhibitors. The structure comprises an N-terminal regulatory domain, consisting of the diacylglycerol (DAG)-binding C1 domain and the Ca2+-binding C2 domain, and a C-terminal catalytic domain, (more ...)
Acknowledgments
The authors thank Drs Matthew Jones and Matthew Harper for their advice and critical reading of the manuscript. Work in the authors’ laboratory is supported by grants from the British Heart Foundation (grant nos. RG/05/015 and PG/07/118/24152).
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