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1.  Protein kinase C signaling and cell cycle regulation 
A link between T cell proliferation and the protein kinase C (PKC) family of serine/threonine kinases has been recognized for about 30 years. However, despite the wealth of information on PKC-mediated control of, T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. The outcome of PKC activation is highly context-dependent, with the precise cell cycle target(s) and overall effects determined by the specific isozyme involved, the timing of PKC activation, the cell type, and the signaling environment. Although PKCs can regulate all stages of the cell cycle, they appear to predominantly affect G0/G1 and G2. PKCs can modulate multiple cell cycle regulatory molecules, including cyclins, cyclin-dependent kinases (cdks), cdk inhibitors and cdc25 phosphatases; however, evidence points to Cip/Kip cdk inhibitors and D-type cyclins as key mediators of PKC-regulated cell cycle-specific effects. Several PKC isozymes can target Cip/Kip proteins to control G0/G1 → S and/or G2 → M transit, while effects on D-type cyclins regulate entry into and progression through G1. Analysis of PKC signaling in T cells has largely focused on its roles in T cell activation; thus, observed cell cycle effects are mainly positive. A prominent role is emerging for PKCθ, with non-redundant functions of other isozymes also described. Additional evidence points to PKCδ as a negative regulator of the cell cycle in these cells. As in other cell types, context-dependent effects of individual isozymes have been noted in T cells, and Cip/Kip cdk inhibitors and D-type cyclins appear to be major PKC targets. Future studies are anticipated to take advantage of the similarities between these various systems to enhance understanding of PKC-mediated cell cycle regulation in T cells.
doi:10.3389/fimmu.2012.00423
PMCID: PMC3547298  PMID: 23335926
protein kinase C; signal transduction; T cell activation; cell cycle; cyclin; cyclin-dependent kinase; cyclin-dependent kinase inhibitor
2.  Protein kinase C-theta isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes. 
Molecular and Cellular Biology  1996;16(4):1842-1850.
T-lymphocyte stimulation requires activation of several protein kinases, including the major phorbol ester receptor protein kinase C (PKC), ultimately leading to induction of lymphokines, such as interleukin-2 (IL-2). The revelant PKC isoforms which are involved in the activation cascades of nuclear transcription factors involved in IL-2 production have not yet been clearly defined. We have examined the potential role of two representative PKC isoforms in the induction of the IL-2 gene, i.e., PKC-alpha and PKC-theta, the latter being expressed predominantly in hematopoietic cell lines, particularly T cells. Similar to that of PKC-alpha, PKC-theta overexpression in murine EL4 thymoma cells caused a significant increase in phorbol 12-myristate 13-acetate (PMA)-induced transcriptional activation of full-length IL-2-chloramphenicol acetyltransferase (CAT) and NF-AT-CAT but not of NF-IL2A-CAT or NF-kappaB promoter-CAT reporter gene constructs. Importantly, the critical AP-1 enhancer element was differentially modulated by these two distinct PKC isoenzymes, since only PKC-theta but not PKC-alpha overexpression resulted in an approximately 2.8-fold increase in AP-1-collagenase promoter CAT expression in comparison with the vector control. Deletion of the AP-1 enhancer site in the collagenase promoter rendered it unresponsive to PKC-theta. Expression of a constitutively active mutant PKC-theta A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-theta K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-RasS17N completely inhibited the PKC-O A148E-induced signal, PKC-O. Expression of a constitutively active mutant PKC-O A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-O K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-enRasS17N completely inhibited in the PKC-O A148E-induced signal, identifying PKC-theta as a specific constituent upstream of or parallel to Ras in the signaling cascade leading to AP transcriptional activation.
PMCID: PMC231171  PMID: 8657160
3.  Proteomic technologies in the study of kinases: Novel tools for the investigation of PKC in the heart 
Recent developments in the field of protein separation allows for the analysis of qualitative and quantitative global protein changes in a particular state of a biological system. Due to the enormous number of proteins potentially present in a cell, sub-fractionation and the enrichment of specific organelles are emerging as a necessary step to allow a more comprehensive representation of the protein content. The proteomic studies demonstrate that a key to understand the mechanisms underlying physiological or pathological phenotypes lies, at least in part, in post-translational modifications (PTMs), including phosphorylation of proteins. Rapid improvements in proteomic characterization of amino acid modifications are further expanding our comprehension of the importance of these mechanisms.
The present review will provide an overview of technologies available for the study of a proteome, including tools to asses changes in protein quantity (abundance) as well as in quality (PTM forms). Examples of the recent application of these technologies and strategies in the field of kinase signalling will be provided with particular attention on the role of PKC in the heart. Studies of PKC mediated phosphorylation of cytoskeletal, myofilament and mitochondrial proteins in the heart have provided great insight into the phenotypes of heart failure, hypertrophy and cardioprotection. Proteomics studies of the mitochondria have provided novel evidences for kinase signalling cascades localized to the mitochondria, some of which are known to involve various isoforms of PKC. Proteomics technologies allow for the identification of the different PTM forms of specific proteins and this information is likely to provide insight into the determinants of morphological as well as metabolic mal-adaptations, both in the heart and other tissues.
doi:10.1016/j.phrs.2007.04.012
PMCID: PMC2693016  PMID: 17548206
4.  Lambda-interacting protein, a novel protein that specifically interacts with the zinc finger domain of the atypical protein kinase C isotype lambda/iota and stimulates its kinase activity in vitro and in vivo. 
Molecular and Cellular Biology  1996;16(1):105-114.
The members of the atypical subfamily of protein kinase C (PKC) show dramatic structural and functional differences from other PKC isotypes. Thus, in contrast to the classical or novel PKCs, they are not activated by diacylglycerol or phorbol esters. However, the atypical PKCs are the target of important lipid second messengers such as ceramide, phosphatidic acid, and 3'-phosphoinositides. The catalytic and pseudosubstrate sequences in the two atypical PKCs (lambda/iota PKC and zeta PKC) are identical but are significantly different from those of conventional or novel PKCs. It has been shown that microinjection of a peptide with the sequence of the pseudosubstrate of the atypical PKC isotypes but not of alpha PKC or epsilon PKC dramatically inhibited maturation and NF-kappa B activation in Xenopus oocytes, as well as reinitiation of DNA synthesis in quiescent mouse fibroblasts. This indicates that either or both atypical isoforms are important in cell signalling. Besides the pseudosubstrate, the major differences in the sequence between lambda/iota PKC and zeta PKC are located in the regulatory domain. Therefore, any functional divergence between the two types of atypical PKCs will presumably reside in that region. We report here the molecular characterization of lambda-interacting protein (LIP), a novel protein that specifically interacts with the zinc finger of lambda/iota PKC but not zeta PKC. We show in this paper that this interaction is detected not only in vitro but also in vivo, that LIP activates lambda/iota PKC but not zeta PKC in vitro and in vivo, and that this interaction is functionally relevant. Thus, expression of LIP leads to the transactivation of a kappa B-dependent promoter in a manner that is dependent on lambda/iota PKC. To our knowledge, this is the first report on the cloning and characterization of a protein activator of a PKC that binds to the zinc finger domain, which has so far been considered a site for binding of lipid modulators. The fact that LIP binds to lambda/iota PKC but not to the highly related zeta PKC isoform suggests that the specificity of the activation of the members of the different PKC subfamilies will most probably be accounted for by proteins like LIP rather than by lipid activators.
PMCID: PMC230983  PMID: 8524286
5.  Protein Kinase Cα as a Heart Failure Therapeutic Target 
Heart failure afflicts ~5 million people and causes ~300,000 deaths a year in the United States alone. Heart failure is defined as a deficiency in the ability of the heart to pump sufficient blood in response to systemic demands, which results in fatigue, dyspnea, and/or edema. Identifying new therapeutic targets is a major focus of current research in the field. We and others have identified critical roles for protein kinase C (PKC) family members in programming aspects of heart failure pathogenesis. More specifically, mechanistic data have emerged over the past 6–7 years that directly implicate PKCα, a conventional PKC family member, as a nodal regulator of heart failure propensity. Indeed, deletion of the PKCα gene in mice, or its inhibition in rodents with drugs or a dominant negative mutant and/or inhibitory peptide, have shown dramatic protective effects that antagonize the development of heart failure. This review will weigh all the evidence implicating PKCα as a novel therapeutic target to consider for the treatment of heart failure.
doi:10.1016/j.yjmcc.2010.10.004
PMCID: PMC3204459  PMID: 20937286
6.  Molecular Analysis Reveals Localization of Saccharomyces cerevisiae Protein Kinase C to Sites of Polarized Growth and Pkc1p Targeting to the Nucleus and Mitotic Spindle 
Eukaryotic Cell  2005;4(1):36-45.
The catalytic activity and intracellular localization of protein kinase C (PKC) are both highly regulated in vivo. This family of kinases contains conserved regulatory motifs, i.e., the C1, C2, and HR1 domains, which target PKC isoforms to specific subcellular compartments and restrict their activity spatially. Saccharomyces cerevisiae contains a single PKC isozyme, Pkc1p, which contains all of the regulatory motifs found in mammalian PKCs. Pkc1p localizes to sites of polarized growth, consistent with its main function in maintaining cell integrity. We dissected the molecular basis of Pkc1p localization by expressing each of its domains individually and in combinations as green fluorescent protein fusions. We find that the Rho1p-binding domains, HR1 and C1, are responsible for targeting Pkc1p to the bud tip and cell periphery, respectively. We demonstrate that Pkc1p activity is required for its normal localization to the bud neck, which also depends on the integrity of the septin ring. In addition, we show for the first time that yeast protein kinase C can accumulate in the nucleus, and we identify a nuclear exit signal as well as nuclear localization signals within the Pkc1p sequence. Thus, we propose that Pkc1p shuttles in and out of the nucleus and consequently has access to nuclear substrates. Surprisingly, we find that deletion of the HR1 domain results in Pkc1p localization to the mitotic spindle and that the C2 domain is responsible for this targeting. This novel nuclear and spindle localization of Pkc1p may provide a molecular explanation for previous observations that suggest a role for Pkc1p in regulating microtubule function.
doi:10.1128/EC.4.1.36-45.2005
PMCID: PMC544167  PMID: 15643058
7.  Hyperglycaemia promotes human brain microvascular endothelial cell apoptosis via induction of protein kinase C-ßI and prooxidant enzyme NADPH oxidase 
Redox Biology  2014;2:694-701.
Blood–brain barrier disruption represents a key feature in hyperglycaemia-aggravated cerebral damage after an ischaemic stroke. Although the underlying mechanisms remain largely unknown, activation of protein kinase C (PKC) is thought to play a critical role. This study examined whether apoptosis of human brain microvascular endothelial cells (HBMEC) might contribute to hyperglycaemia-evoked barrier damage and assessed the specific role of PKC in this phenomenon. Treatments with hyperglycaemia (25 mM) or phorbol myristate acetate (PMA, a protein kinase C activator, 100 nM) significantly increased NADPH oxidase activity, O2•- generation, proapoptotic protein Bax expression, TUNEL-positive staining and caspase-3/7 activities. Pharmacological inhibition of NADPH oxidase, PKC-a, PKC-ß or PKC-ßI via their specific inhibitors and neutralisation of O2•- by a cell-permeable superoxide dismutase mimetic, MnTBAP normalised all the aforementioned increases induced by hyperglycaemia. Suppression of these PKC isoforms also negated the stimulatory effects of hyperglycaemia on the protein expression of NADPH oxidase membrane-bound components, Nox2 and p22-phox which determine the overall enzymatic activity. Silencing of PKC-ßI gene through use of specific siRNAs abolished the effects of both hyperglycaemia and PMA on endothelial cell NADPH oxidase activity, O2•- production and apoptosis and consequently improved the integrity and function of an in vitro model of human cerebral barrier comprising HBMEC, astrocytes and pericytes. Hyperglycaemia-mediated apoptosis of HBMEC contributes to cerebral barrier dysfunction and is modulated by sequential activations of PKC-ßI and NADPH oxidase.
Highlights
•Hyperglycemia disrupts cerebral barrier via protein kinase C-ßI-evoked apoptosis.•Protein kinase C-ßI promotes oxidative stress through NADPH oxidase activation.•NADPH oxidase regulates apoptotic pathway by elevating caspase-3/7 activity.•Like hyperglycemia, induction of protein kinase C perse perturbs barrier function.•Silencing of protein kinase C-ßI protects cerebral barrier from both pathologies.
Graphical abstract
doi:10.1016/j.redox.2014.05.005
PMCID: PMC4052534  PMID: 24936444
Apoptosis; Endothelial cell; Hyperglycaemia; Protein kinase C; NADPH oxidase
8.  Protein kinase C depresses cardiac myocyte power output and attenuates myofilament responses induced by protein kinase A 
Following activation by G-protein-coupled receptor agonists, protein kinase C (PKC) modulates cardiac myocyte function by phosphorylation of intracellular targets including myofilament proteins cardiac troponin I (cTnI) and cardiac myosin binding protein C (cMyBP-C). Since PKC phosphorylation has been shown to decrease myofibril ATPase activity, we hypothesized that PKC phosphorylation of cTnI and cMyBP-C will lower myocyte power output and, in addition, attenuate the elevation in power in response to protein kinase A (PKA)-mediated phosphorylation. We compared isometric force and power generating capacity of rat skinned cardiac myocytes before and after treatment with the catalytic subunit of PKC. PKC increased phosphorylation levels of cMyBP-C and cTnI and decreased both maximal Ca2+ activated force and Ca2+ sensitivity of force. Moreover, during submaximal Ca2+ activations PKC decreased power output by 62 %, which arose from both the fall in force and slower loaded shortening velocities since depressed power persisted even when force levels were matched before and after PKC. In addition, PKC blunted the phosphorylation of cTnI by PKA, reduced PKA-induced spontaneous oscillatory contractions, and diminished PKA-mediated elevations in myocyte power. To test whether altered thin filament function plays an essential role in these contractile changes we investigated the effects of chronic cTnI pseudo-phosphorylation on myofilament function using myocyte preparations from transgenic animals in which either only PKA phosphorylation sites (Ser-23/Ser-24) (PP) or both PKA and PKC phosphorylation sites (Ser-23/Ser-24/Ser-43/Ser-45/T-144) (All-P) were replaced with aspartic acid. Cardiac myocytes from All-P transgenic mice exhibited reductions in maximal force, Ca2+ sensitivity of force, and power. Similarly diminished power generating capacity was observed in hearts from All-P mice as determined by in situ pressure–volume measurements. These results imply that PKC-mediated phosphorylation of cTnI plays a dominant role in depressing contractility, and, thus, increased PKC isozyme activity may contribute to maladaptive behavior exhibited during the progression to heart failure.
doi:10.1007/s10974-012-9294-9
PMCID: PMC3568763  PMID: 22527640
PKC; PKA; Cardiac myocyte; Cardiac troponin I; Power output
9.  Protein Kinase C Isoforms as Specific Targets for Modulation of Vascular Smooth Muscle Function in Hypertension 
Biochemical pharmacology  2005;70(11):1537-1547.
Vascular contraction is an important determinant of the peripheral vascular resistance and blood pressure. The mechanisms underlying vascular smooth muscle (VSM) contraction and the pathological changes that occur in hypertension have been the subject of numerous studies and interpretations. Activation of VSM by vasoconstrictor stimuli at the cell surface causes an increase in [Ca2+]i, Ca2+-dependent activation of myosin light chain (MLC) kinase, MLC phosphorylation, actin-myosin interaction and VSM contraction. Additional signaling pathways involving Rho-kinase and protein kinase C (PKC) may increase the myofilament force sensitivity to [Ca2+]i and MLC phosphorylation, and thereby maintain vascular contraction. PKC is a particularly intriguing protein kinase as it comprises a family of Ca2+-dependent and Ca2+-independent isoforms, which have different tissue and subcellular distribution, and undergo differential translocation during cell activation. PKC translocation to the cell surface may trigger a cascade of protein kinases such as mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) that ultimately interact with the contractile myofilaments and cause VSM contraction. Also, PKC translocation to the nucleus may promote VSM growth and proliferation. Increased PKC expression and activity have been identified in several forms of hypertension. The subcellular location of PKC may determine the state of VSM activity, and may be useful in the diagnosis/prognosis of hypertension. Vascular PKC isoforms may represent specific targets for modulation of VSM hyperactivity, and isoform-specific PKC inhibitors may be useful in treatment of Ca2+ antagonist-resistant forms of hypertension.
doi:10.1016/j.bcp.2005.07.017
PMCID: PMC1343531  PMID: 16139252
signal transduction; vascular smooth muscle; calcium; blood pressure; AngII, angiotensin II; ATP, adenosine triphosphate; CPI-17, PKC-potentiated phosphatase inhibitor protein-17 kDa; CAM, calmodulin; DAG, diacylglycerol; ET-1, endothelin; IP3, inositol 1,4,5-trisphosphate; MAPK, mitogen-activated protein kinase; MARCKs, myristoylated alanine-rich C-kinase substrate; MEK, MAPK kinase; MLC, myosin light chain; NADP, nicotinamide adenine dinucleotide phosphate; O·2−, superoxide; PDBu, phorbol 12,13-dibutyrate; PIP2, phosphatidylinositol 4,5-bisphosphate; PLD, phospholipase D; PKC, protein kinase C; PMA, phorbol myristate acetate; RACKs, receptors for activated C-kinase; Rho-kinase, Rho-associated kinase; ROS, reactive oxygen species; SHR, spontaneously hypertensive rat; TPA, 12-o-tetradecanoylphorbol-13-acetate; VSM, vascular smooth muscle; WKY, Wistar-Kyoto
10.  An aPKC-Exocyst Complex Controls Paxillin Phosphorylation and Migration through Localised JNK1 Activation 
PLoS Biology  2009;7(11):e1000235.
The exocyst/aPKC complex controls the spatiotemporal activation of the kinases JNK and ERK at the leading edge of migrating cells and thereby controls the dynamic behaviour of the adhesion protein paxillin during cell migration.
Atypical protein kinase C (aPKC) isoforms have been implicated in cell polarisation and migration through association with Cdc42 and Par6. In distinct migratory models, the Exocyst complex has been shown to be involved in secretory events and migration. By RNA interference (RNAi) we show that the polarised delivery of the Exocyst to the leading edge of migrating NRK cells is dependent upon aPKCs. Reciprocally we demonstrate that aPKC localisation at the leading edge is dependent upon the Exocyst. The basis of this inter-dependence derives from two-hybrid, mass spectrometry, and co-immunoprecipitation studies, which demonstrate the existence of an aPKC–Exocyst interaction mediated by Kibra. Using RNAi and small molecule inhibitors, the aPKCs, Kibra, and the Exocyst are shown to be required for NRK cell migration and it is further demonstrated that they are necessary for the localized activation of JNK at the leading edge. The migration associated control of JNK by aPKCs determines JNK phosphorylation of the plasma membrane substrate Paxillin, but not the phosphorylation of the nuclear JNK substrate, c-jun. This plasma membrane localized JNK cascade serves to control the stability of focal adhesion complexes, regulating migration. The study integrates the polarising behaviour of aPKCs with the pro-migratory properties of the Exocyst complex, defining a higher order complex associated with the localised activation of JNK at the leading edge of migrating cells that determines migration rate.
Author Summary
Cell migration is an essential process in multicellular organisms during such events as embryonic development, the immune response, and wound healing. Cell migration is also instrumental in the development of pathologies such as cancer cell invasion of healthy tissues. To make cells move, key molecules must be engaged in a coordinated manner; understanding which molecules, and how and when they work (for example, under physiological versus pathological conditions) will impact on new therapies designed to suppress abnormal migration. Migrating cells must coordinate two key processes: extension of the front or ‘leading’ edge of the cell and retraction of the back edge. Both processes require the turnover of protein assemblies known as focal adhesion complexes. In this paper we show that two different groups of regulators of migration – aPKC, a protein kinase, and exocyst, a complex of proteins also known to be required for exocytosis – interact physically via the scaffold protein kibra. All these components are required for efficient cell migration and all are enriched at the leading edge of moving cells, in a mutually dependent manner. At the leading edge, these components control the local activation of two additional protein kinases, ERK and JNK. The activation of ERK and JNK at the front of migrating cells in turn controls the phosphorylation of paxillin, a component of focal adhesions. Phosphorylation of paxillin is associated with the presence of more dynamic focal adhesions. Our data thus indicate that an aPKC-kibra-exocyst complex plays a crucial role in delivering local stimulatory signals to the leading edge of migrating cells.
doi:10.1371/journal.pbio.1000235
PMCID: PMC2762617  PMID: 19885391
11.  Detecting PKC phosphorylation as part of the Wnt/calcium pathway in cutaneous melanoma 
Signaling networks play crucial roles in the changes leading to malignancy. In melanoma, increased Wnt5A expression increases melanoma cell motility via activation of Protein Kinase C (PKC). PKC isoforms comprise a family of serine/threonine kinases that are involved in the transduction of signals for cell proliferation, differentiation and metastasis. The important role of PKC in processes leading to carcinogenesis and tumor cell invasion would render PKC a suitable target for cancer therapy, if not for its ubiquitous nature. Thus, targeting more tumor-specific pathways leading to PKC activation, such as the non-canonical Wnt pathway, may prove to be the key to targeting PKC in cancer. Here we summarize the current understanding of the Wnt/Calcium pathway and discuss methods of detecting activated/phosphorylated PKC as a result of Wnt signaling in malignant melanoma. We have shown that overexpression of Wnt5A results in the activation of PKC, while inhibition of Wnt5A via siRNA treatment results in its inactivation. In addition, the use of PKC activators and inhibitors has allowed us to study Wnt5A effects on downstream genes that may prove to be key targets for molecular therapy.
doi:10.1007/978-1-59745-249-6_12
PMCID: PMC2814177  PMID: 19099253
melanoma; Wnt5A; Protein Kinase C (PKC)
12.  Differential expression of protein kinase C isoforms in coronary arteries of diabetic mice lacking the G-protein Gα11 
Background
Diabetes mellitus counts as a major risk factor for developing atherosclerosis. The activation of protein kinase C (PKC) is commonly known to take a pivotal part in the pathogenesis of atherosclerosis, though the influence of specific PKC isozymes remains unclear. There is evidence from large clinical trials suggesting excessive neurohumoral stimulation, amongst other pathways leading to PKC activation, as a central mechanism in the pathogenesis of diabetic heart disease. The present study was therefore designed to determine the role of Gq-protein signalling via Gα11 in diabetes for the expression of PKC isozymes in the coronary vessels.
Methods
The role of Gα11 in diabetes was examined in knockout mice with global deletion of Gα11 compared to wildtype controls. An experimental type 1-diabetes was induced in both groups by injection of streptozotocin. Expression and localization of the PKC isozymes α, βII, δ, ε, and ζ was examined by quantitative immunohistochemistry.
Results
8 weeks after induction of diabetes a diminished expression of PKC ε was observed in wildtype animals. This alteration was not seen in Gα11 knockout animals, however, these mice showed a diminished expression of PKCζ. Direct comparison of wildtype and knockout control animals revealed a diminished expression of PKC δ and ε in Gα11 knockout animals.
Conclusion
The present study shows that expression of the nPKCs δ and ε in coronary vessels is under control of the g-protein Gα11. The reduced expression of PKC ζ that we observed in coronary arteries from Gα11-knockout mice compared to wildtype controls upon induction of diabetes could reduce apoptosis and promote plaque stability. These findings suggest a mechanism that may in part underlie the therapeutic benefit of RAS inhibition on cardiovascular endpoints in diabetic patients.
doi:10.1186/1475-2840-9-93
PMCID: PMC3024287  PMID: 21190563
13.  Protein kinase C, an elusive therapeutic target? 
Nature reviews. Drug discovery  2012;11(12):937-957.
Preface
Protein kinase C (PKC) has been a tantalizing target for drug discovery ever since it was first identified as the receptor for the tumor promoter phorbol ester in 19821. Although initial therapeutic efforts focused on cancer, additional diseases, including diabetic complications, heart failure, myocardial infarction, pain and bipolar disease were targeted as researchers developed a better understanding of the roles that PKC’s eight conventional and novel isozymes play in health and disease. Unfortunately, both academic and pharmaceutical efforts have yet to result in the approval of a single new drug that specifically targets PKC. Why does PKC remain an elusive drug target? This review will provide a short account of some of the efforts, challenges and opportunities in developing PKC modulators to address unmet clinical needs.
doi:10.1038/nrd3871
PMCID: PMC3760692  PMID: 23197040
14.  Hypoxia alters the subcellular distribution of protein kinase C isoforms in neonatal rat ventricular myocytes. 
Cardiac myocytes coexpress multiple protein kinase C (PKC) isoforms which likely play distinct roles in signaling pathways leading to changes in contractility, hypertrophy, and ischemic preconditioning. Although PKC has been reported to be activated during myocardial ischemia, the effect of ischemia/hypoxia on individual PKC isoforms has not been determined. This study examines the effect of hypoxia on the subcellular distribution of individual PKC isoforms in cultured neonatal rat ventricular myocytes. Hypoxia induces the redistribution of PKC alpha and PKC epsilon from the soluble to the particulate compartment. This effect (which is presumed to represent activation of PKC alpha and PKC epsilon) is detectable by 1 h, sustained for up to 24 h, and reversible within 1 h of reoxygenation. Inhibition of phospholipase C with tricyclodecan-9-yl-xanthogenate (D609) prevents the hypoxia-induced redistribution of PKC alpha and PKC epsilon, whereas chelation of intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) blocks the redistribution of PKC alpha, but not PKC epsilon; D609 and BAPTA do not influence the partitioning of PKC alpha and PKC epsilon in normoxic myocytes. Hypoxia, in contrast, decreases the membrane association of PKC delta via a mechanism that is distinct from the hypoxia-induced translocation/activation of PKC alpha/PKC epsilon, since the response is slower in onset, slowly reversible upon reoxygenation, and not blocked by D609 or BAPTA. The hypoxia-induced shift of PKC delta to the soluble compartment does not prevent subsequent 4-beta phorbol 12-myristate-13-acetate-dependent translocation/activation of PKC delta. Hypoxia does not alter the abundance of any PKC isoform nor does it alter the subcellular distribution of PKC lambda. The selective hypoxia-induced activation of PKC isoforms through a pathway involving phospholipase C (PKC alpha/PKC epsilon) and intracellular calcium (PKC alpha) may critically influence cardiac myocyte contractility, gene expression, and/or tolerance to ischemia.
PMCID: PMC507767  PMID: 9011576
15.  Kinase/phosphatase overexpression reveals pathways regulating hippocampal neuron morphology 
Kinases and phosphatases that regulate neurite number versus branching versus extension are weakly correlated.The kinase family that most strongly enhances neurite growth is a family of non-protein kinases; sugar kinases related to NADK.Pathway analysis revealed that genes in several cancer pathways were highly active in enhancing neurite growth.
In neural development, neuronal precursors differentiate, migrate, extend long axons and dendrites, and finally establish connections with their targets. Clinical conditions such as spinal cord injury, traumatic brain injury, stroke, multiple sclerosis, Parkinson's disease, Huntington's disease, and Alzheimer's disease are often associated with a loss of axon and/or dendrite connectivity and treatment strategies would be enhanced by new therapies targeting cell intrinsic mechanisms of axon elongation and regeneration.
Phosphorylation controls most cellular processes, including the cell cycle, proliferation, metabolism, and apoptosis. Neuronal differentiation, including axon formation and elongation, is also regulated by a wide range of kinases and phosphatases. For example, the non-receptor tyrosine kinase Src is required for cell adhesion molecule-dependent neurite outgrowth. In addition to individual kinases and phosphatases, signaling pathways like the MAPK, growth factor signaling, PIP3, cytoskeletal, and calcium-dependent pathways have been shown to impinge on or control neuronal process development. Recent results have implicated GSK3 and PTEN as therapeutically relevant targets in axonal regeneration after injury. However, these and other experiments have studied only a small fraction of the total kinases and phosphatases in the genome. Because of recent advances in genomic knowledge, large-scale cDNA production, and high-throughput phenotypic analysis, it is now possible to take a more comprehensive approach to understanding the functions of kinases and phosphatases in neurons.
We performed a large, unbiased set of experiments to answer the question ‘what effect does the overexpression of genes encoding kinases, phosphatases, and related proteins have on neuronal morphology?' We used ‘high-content analysis' to obtain detailed results about the specific phenotypes of neurons. We studied embryonic rat hippocampal neurons because of their stereotypical development in vitro (Dotti et al, 1988) and their widespread use in studies of neuronal differentiation and signaling. We transfected over 700 clones encoding kinases and phosphatases into hippocampal neurons and analyzed the resulting changes in neuronal morphology.
Many known genes, including PP1a, ERK1, ErbB2, atypical PKC, Calcineurin, CaMK2, IGF1R, FGFR, GSK3, and PIK3 were observed to have significant effects on neurite outgrowth in our system, consistent with earlier findings in the literature.
We obtained quantitative data for many cellular and neuronal morphological parameters from each neuron imaged. These included nuclear morphology (nuclear area and Hoechst dye intensity), soma morphology (tubulin intensity, area, and shape), and numerous parameters of neurite morphology (e.g. tubulin intensity along the neurites, number of primary neurites, neurite length, number of branches, distance from the cell body to the branches, number of crossing points, width and area of the neurites, and longest neurite; Supplementary Figure 1). Other parameters were reported on a ‘per well' basis, including the percentage of transfected neurons in a condition, as well as the percentage of neurons initiating neurite growth. Data for each treatment were normalized to a control (pSport CAT) within the same experiment, then aggregated across replicate experiments.
Correlations among the 19 normalized parameters were analyzed for neurons transfected with all kinase and phosphatase clones (Figure 2). On the basis of this analysis, the primary variables that define the neurite morphology are primary neurite count, neurite average length, and average branches. Interestingly, primary neurite count was not well correlated with neurite length or branching. The Pearson correlation coefficient (r2) between the number of primary neurites and the average length of the neurites was 0.3, and between the number of primary neurites and average branching was 0.2. In contrast, the correlation coefficient of average branching with neurite average length was 0.7. The most likely explanation is that signaling mechanisms underlying the neurite number determination are different than those controlling length/branching of the neurites.
Related proteins are often involved in similar neuronal functions. For example, families of receptor protein tyrosine phosphatases are involved in motor axon extension and guidance in both Drosophila and in vertebrates, and a large family of Eph receptor tyrosine kinases regulates guidance of retinotectal projections, motor axons, and axons in the corpus callosum. We therefore asked whether families of related genes produced similar phenotypes when overexpressed in hippocampal neurons. Our set of genes covered 40% of the known protein kinases, and many of the non-protein kinases and phosphatases.
Gene families commonly exhibit redundant function. Redundant gene function has often been identified when two or more knockouts are required to produce a phenotype. Our technique allowed us to measure whether different members of gene families had similar (potentially redundant) or distinct effects on neuronal phenotype.
To determine whether groups of related genes affect neuronal morphology in similar ways, we used sequence alignment information to construct gene clusters (Figure 6). Genes were clustered at nine different thresholds of similarity (called ‘tiers'). The functional effect for a particular parameter was then averaged within each cluster of a given tier, and statistics were performed to determine the significance of the effect. We analyzed the results for three key neurite parameters (average neurite length, primary neurite count, and average branching). Genes that perturbed each of these phenotypes are grouped in Figure 6. Eight families, most with only a few genes, produced significant changes for one or two parameters. A diverse family of non-protein kinases had a positive effect on neurite outgrowth in three of the four parameters analyzed. This family of kinases consisted of a variety of enzymes, mostly sugar and lipid kinases. A similar analysis was performed using pathway cluster analysis with pathways from the KEGG database, rather than sequence homology. Interestingly, pathways involved in cancer cell proliferation potentiated neurite extension and branching.
Our studies have identified a large number of kinases and phosphatases, as well as structurally and functionally defined families of these proteins, that affect neuronal process formation in specific ways. We have provided an analytical methodology and new tools to analyze functional data, and have implicated genes with novel functions in neuronal development. Our studies are an important step towards the goal of a molecular description of the intrinsic control of axodendritic growth.
Development and regeneration of the nervous system requires the precise formation of axons and dendrites. Kinases and phosphatases are pervasive regulators of cellular function and have been implicated in controlling axodendritic development and regeneration. We undertook a gain-of-function analysis to determine the functions of kinases and phosphatases in the regulation of neuron morphology. Over 300 kinases and 124 esterases and phosphatases were studied by high-content analysis of rat hippocampal neurons. Proteins previously implicated in neurite growth, such as ERK1, GSK3, EphA8, FGFR, PI3K, PKC, p38, and PP1a, were confirmed to have effects in our functional assays. We also identified novel positive and negative neurite growth regulators. These include neuronal-developmentally regulated kinases such as the activin receptor, interferon regulatory factor 6 (IRF6) and neural leucine-rich repeat 1 (LRRN1). The protein kinase N2 (PKN2) and choline kinase α (CHKA) kinases, and the phosphatases PPEF2 and SMPD1, have little or no established functions in neuronal function, but were sufficient to promote neurite growth. In addition, pathway analysis revealed that members of signaling pathways involved in cancer progression and axis formation enhanced neurite outgrowth, whereas cytokine-related pathways significantly inhibited neurite formation.
doi:10.1038/msb.2010.52
PMCID: PMC2925531  PMID: 20664637
bioinformatics; development; functional genomics; metabolic and regulatory networks; neuroscience
16.  Sevoflurane Stimulates MAP Kinase Signal transduction through the Activation of PKC α and βII in Fetal Rat Cerebral Cortex Cultured Neuron 
Acta Histochemica et Cytochemica  2006;39(6):163-172.
Protein kinase C (PKC) is a key enzyme that participates in various neuronal functions. PKC has also been identified as a target molecule for general anesthetic actions. Raf, mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK1/2) have been thought to be target effectors of PKC. In the present study, we attempted to evaluate the effect of sevoflurane on PKC/MAPK cascade signaling in cultured fetal rat cerebral ­cortex neurons, prepared from embryonic day 18 fetuses. The effects of sevoflurane on the translocation of 7 PKC isoforms (α, βI, βII, γ, δ, ɛ and ζ) were observed by immunoblotting using isoform-selective antibodies to PKCs. The treatment of neurons with sevoflurane induced the translocation of PKC α and PKC βII species from the cytosol to the membrane fraction, which indicated the activation of these PKC isoforms. In contrast, there was no clear change in the distribution of other PKC isoforms. We next examined whether the specific activation of PKC α and βII by sevoflurane could stimulate the MAP kinase signaling pathway in cultured neurons. Raf phosphorylation was increased by the administration of 0.25 mM sevoflurane. The phosphorylation of Raf proteins reached a maximum at 5–10 min. Subsequently, the phosphorylation of MEK proteins was increased at 10–15 min after sevoflurane treatments. That of ERK proteins was induced at 15–60 min. Moreover, the phosphorylation of ERK induced by sevoflurane was significantly decreased by the treatment of PKC inhibitor (staurosporine) and MEK inhibitor (PD98059). On the other hand, the contents of total Raf, MEK and ERK proteins were relatively constant at all times examined. To examine the ­localization of phosphorylated-ERK protein, immunohistochemical staining of sevoflurane-treated cultured neurons was performed. The phosphorylated-ERK proteins were markedly accumulated in both the cytosol of the cell body and the neurites in the neuronal cells with time after 0.25 mM sevoflurane-treatment. These results demonstrated that sevoflurane induced the phosphorylation of the MAP kinase cascade through the activation of the PKC α and PKC βII species.
doi:10.1267/ahc.06022
PMCID: PMC1779947  PMID: 17327903
sevoflurane; protein kinase C (PKC); mitogen-activated protein (MAP); MAP kinase; neuron
17.  Distinct mechanisms of the inhibition of vasculogenesis by different species of ionizing particles 
Journal of Radiation Research  2014;55(Suppl 1):i44-i45.
The human vasculature includes a vast network of microcapillaries networking the body and is a major target for non-carcinogenic effects of space radiation in the body. The brain microvascular system is crucial to healthy functioning of the brain and its pathology is not only a primary event in a range of neurodegenerative diseases but also an important influencing factor in many others. The vasculature is maintained by angiogenesis regenerating vessels as they are needed, this is particularly relevant if the blood–brain barrier is damaged by agents such as space radiation, thereby creating the need for angiogenic regeneration. The resulting lack of vasculature due to the inhibition of re-growth of vessels can, in turn, lead to a negative feedback loop and further pathologies.
Using three-dimensional human vessel cultures with human umbilical vein and brain microvascular endothelial cells, we have developed assays that determine at what stage angiogenesis is inhibited by ionizing radiation. The relative biological effect of high linear energy transfer (LET) 1 GeV Fe ions compared with low LET 1 GeV protons is only one for developing vessels but greater than four for mature vessels. This action of low LET protons on developing vessels was surprisingly effective (50% inhibition with 40 cGy exposure) and together with the effect of high LET ions may represent a significant hazard in the space environment.
The morphology of developing vessels 5 days after exposure showed significant differences that suggest distinct mechanisms of inhibition. Cells exposed to protons failed to make connections with other cells. Conversely, cells exposed to Fe ions extended cellular processes and made connections to other cells but did not develop a central lumen. The microtubule and actin cytoskeletons showed differences indicating that motility at the extending tips of endothelial cells is inhibited by protons but not Fe ions. Actin-rich protrusive structures that contain bundled and dynamic microtubules showed a 65% decrease when exposed to high-energy protons but not with the same dose of high-energy Fe ions. Since protein kinase C (PKC) has long been known to stimulate angiogenesis, we hypothesized that rescue of the capillary phenotype after proton exposure would be possible by stimulating PKC before irradiation. One-day-old vessel cultures were treated with 30 and 60 nM phorbol ester (PMA) 15 min before irradiation. Stimulation of PKC restored capillary formation in proton-treated cultures but not in Fe ion-treated cultures. More specifically, stimulation of PKC by PMA was able to restore the tip motility that was inhibited by low LET ions [ 1].
Further studies with various charged particles showed that low LET ion particles (Proton and He ions) with an LET lower or equal to 1 keV/μm inhibit vasculogenesis in the same way as protons. Higher LET charged particles (Silicon 1GeV, Oxygen 250 MeV and 1 GeV and Carbon 290 MeV and 1 GeV) with an LET ≥8 keV/μm inhibit vasculogenesis in the same way as Fe ions.
In conclusion, we have shown that low and high LET ions inhibit the formation of brain capillaries by different mechanisms. For low LET ions, inhibition involves regulation of PKC-dependent motile tips leading to a failure of cellular processes to migrate through the matrix and meet up with other processes. For high LET ions, the cells fail to complete angiogenesis by not migrating and forming tubular structures. This complexity of response opens up possibilities of greater control over angiogenesis and the resulting pathologies during coincident exposure or therapy. For exposure in space, knowledge of these mechanisms will enable more precise risk assessment and mitigation strategies. For radiotherapy, treatment could be manipulated to utilize the radiation effectively.
doi:10.1093/jrr/rrt172
PMCID: PMC3941500
angiogenesis; vasculogenesis; radiation; charged particles
18.  Activation of the Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Pathway by Conventional, Novel, and Atypical Protein Kinase C Isotypes 
Molecular and Cellular Biology  1998;18(2):790-798.
Phorbol ester treatment of quiescent Swiss 3T3 cells leads to cell proliferation, a response thought to be mediated by protein kinase C (PKC), the major cellular receptor for this class of agents. We demonstrate here that this proliferation is dependent on the activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade. It is shown that dominant-negative PKC-α inhibits stimulation of the ERK/MAPK pathway by phorbol esters in Cos-7 cells, demonstrating a role for PKC in this activation. To assess the potential specificity of PKC isotypes mediating this process, constitutively active mutants of six PKC isotypes (α, β1, δ, ɛ, η, and ζ) were employed. Transient transfection of these PKC mutants into Cos-7 cells showed that members of all three groups of PKC (conventional, novel, and atypical) are able to activate p42 MAPK as well as its immediate upstream activator, the MAPK/ERK kinase MEK-1. At the level of Raf, the kinase that phosphorylates MEK-1, the activation cascade diverges; while conventional and novel PKCs (isotypes α and η) are potent activators of c-Raf1, atypical PKC-ζ cannot increase c-Raf1 activity, stimulating MEK by an independent mechanism. Stimulation of c-Raf1 by PKC-α and PKC-η was abrogated for RafCAAX, which is a membrane-localized, partially active form of c-Raf1. We further established that activation of Raf is independent of phosphorylation at serine residues 259 and 499. In addition to activation, we describe a novel Raf desensitization induced by PKC-α, which acts to prevent further Raf stimulation by growth factors. The results thus demonstrate a necessary role for PKC and p42 MAPK activation in 12-O-tetradecanoylphorbol-13-acetate induced mitogenesis and provide evidence for multiple PKC controls acting on this MAPK cascade.
PMCID: PMC108790  PMID: 9447975
19.  Regulation of Transcriptional Networks by PKC Isozymes: Identification of c-Rel as a Key Transcription Factor for PKC-Regulated Genes 
PLoS ONE  2013;8(6):e67319.
Background
Activation of protein kinase C (PKC), a family of serine-threonine kinases widely implicated in cancer progression, has major impact on gene expression. In a recent genome-wide analysis of prostate cancer cells we identified distinctive gene expression profiles controlled by individual PKC isozymes and highlighted a prominent role for PKCδ in transcriptional activation.
Principal Findings
Here we carried out a thorough bioinformatics analysis to dissect transcriptional networks controlled by PKCα, PKCδ, and PKCε, the main diacylglycerol/phorbol ester PKCs expressed in prostate cancer cells. Despite the remarkable differences in the patterns of transcriptional responsive elements (REs) regulated by each PKC, we found that c-Rel represents the most frequent RE in promoters regulated by all three PKCs. In addition, promoters of PKCδ-regulated genes were particularly enriched with REs for CREB, NF-E2, RREB, SRF, Oct-1, Evi-1, and NF-κB. Most notably, by using transcription factor-specific RNAi we were able to identify subsets of PKCδ-regulated genes modulated by c-Rel and CREB. Furthermore, PKCδ-regulated genes condensed under the c-Rel transcriptional regulation display significant functional interconnections with biological processes such as angiogenesis, inflammatory response, and cell motility.
Conclusion/Significance
Our study identified candidate transcription factors in the promoters of PKC regulated genes, in particular c-Rel was found as a key transcription factor in the control of PKCδ-regulated genes. The deconvolution of PKC-regulated transcriptional networks and their nodes may greatly help in the identification of PKC effectors and have significant therapeutics implications.
doi:10.1371/journal.pone.0067319
PMCID: PMC3694964  PMID: 23826267
20.  Protein Kinase C and Extracellular Signal-Regulated Kinase Regulate Movement, Attachment, Pairing and Egg Release in Schistosoma mansoni 
Protein kinases C (PKCs) and extracellular signal-regulated kinases (ERKs) are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using ‘smart’ antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated) throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance.
Author Summary
Parasitic blood flukes, also called schistosomes, cause human schistosomiasis, a neglected tropical disease and major public health problem in developing countries, especially sub-Saharan Africa. Sustainable control of schistosomiasis is difficult, mainly because the complex life cycle of the parasite involves a freshwater snail host, and the ability of the parasite to evade the immune response of the human host and to survive for many years. Little is yet known about the cellular mechanisms in schistosomes and how they regulate parasite homeostasis, development and behaviour. In this paper, the nature of intracellular signalling by protein kinases C (PKCs) and extracellular signal-regulated kinases (ERKs) in schistosomes is studied and these proteins are found to be vital for the coordination of processes fundamental to parasite survival, such as muscular activity and reproductive function. Our results contribute to an understanding of molecular events regulating schistosome function and identify PKCs and ERKs as possible targets for the development of new chemotherapeutic treatments against schistosomiasis.
doi:10.1371/journal.pntd.0002924
PMCID: PMC4055629  PMID: 24921927
21.  Effects of the selective bisindolylmaleimide protein kinase C inhibitor GF 109203X on P-glycoprotein-mediated multidrug resistance. 
British Journal of Cancer  1996;74(6):897-905.
Inhibition of protein kinase C (PKC) is discussed as a new approach for overcoming multidrug resistance (MDR) in cancer chemotherapy. For evaluation of this concept we applied the bisindolylmaleimide GF 109203X, which shows a highly selective inhibition of PKC isozymes alpha, beta 1, beta 2, gamma, delta and epsilon in vitro. The efficacy of this compound in modulation of MDR was examined using several P-glycoprotein (P-gp)-overexpressing cell lines including a MDR1-transfected HeLa clone, and was compared with the activities of dexniguldipine-HCI (DNIG) and dexverapamil-HC1 (DVER), both of which essentially act via binding to P-gp. As PKC alpha has been suggested to play a major role in P-gp-mediated MDR, cell lines exhibiting different expression levels of this PKC isozyme were chosen. On crude PKC preparations or in a cellular assay using a cfos(-711)CAT-transfected NIH 3T3 clone, the inhibitory qualities of the bisindolylmaleimide at submicromolar concentrations were demonstrated. At up 1 microM final concentrations of the PKC inhibitor GF 109203X, a concentration at which many PKC isozymes should be blocked substantially, no cytotoxic or MDR-reversing effects whatsoever were seen, as monitored by 72 h tetrazolium-based colorimetric MTT assays or a 90 min rhodamine 123 accumulation assay. Moreover, depletion of PKC alpha by phorbol ester in HeLa-MDR1 transfectants had no influence on rhodamine 123 accumulation after 24 or 48 h. MDR reversal activity of GF 109203X was seen at higher final drug concentrations, however. Remarkably, [3H]vinblastine-sulphate binding competition experiments using P-gp-containing crude membrane preparations demonstrated similar dose dependencies as found for MDR reversion by the three modulators, i.e. decreasing efficacy in the series dexniguldipine-HCl > dexverapamil-HCl > GF 109203X. Similar interaction with the P-gp in the micromolar concentration range was revealed by competition of GF 109203X with photoincorporation of [3H]azidopine into P-gp-containing crude membrane preparations. No significant effect of the PKC inhibitor on MDR1 expression was seen, which was examined by cDNA-PCR. Thus, the bisindolylmaleimide GF 109203X probably influences MDR mostly via direct binding to P-gp. Our work identifies the bisindolylmaleimide GF 109203X as a new type of drug interacting with P-gp directly, but does not support the concept of a major contribution of PKC to a P-gp-associated MDR, at least using the particular cellular model systems and the selective, albeit general, PKC inhibitor GF 109203X.
Images
PMCID: PMC2074754  PMID: 8826855
22.  A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle. 
Molecular and Cellular Biology  1992;12(9):3930-3938.
A new protein kinase C (PKC)-related cDNA with unique tissue distribution has been isolated and characterized. This cDNA encodes a protein, nPKC theta, which consists of 707 amino acid residues and showed the highest sequence similarity to nPKC delta (67.0% in total). nPKC theta has a zinc-finger-like cysteine-rich sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are common in all PKC family members. However, nPKC theta lacks a putative Ca2+ binding region (C2 region) that is seen only in the conventional PKC subfamily (cPKC alpha, -beta I, -beta II, and -gamma) but not in the novel PKC subfamily (nPKC delta, -epsilon, -zeta, and -eta). Northern (RNA) blot analyses revealed that the mRNA for nPKC theta is expressed predominantly in skeletal muscle. Furthermore, nPKC theta mRNA is the most abundantly expressed PKC isoform in skeletal muscle among the nine PKC family members. nPKC theta expressed in COS1 cells serves as a phorbol ester receptor. By the use of an antipeptide antibody specific to the D2-D3 region of the nPKC theta sequence, nPKC theta was recognized as a 79-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in mouse skeletal muscle extract and also in an extract from COS1 cells transfected with an nPKC theta cDNA expression plasmid. Autophosphorylation of immunoprecipitated nPKC theta was observed; it was enhanced by phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate but attenuated by the addition of Ca2+. These results clearly demonstrate that nPKC theta should be considered a member of the PKC family of proteins that play crucial roles in the signal transduction pathway.
Images
PMCID: PMC360273  PMID: 1508194
23.  δPKC inhibition or εPKC activation repairs endothelial vascular dysfunction by regulating eNOS post-translational modification 
The balance between endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) and reactive oxygen species (ROS) production determines endothelial-mediated vascular homeostasis. Activation of protein kinase C (PKC) has been linked to imbalance of the eNOS/ROS system, which leads to endothelial dysfunction. We previously found that selective inhibition of delta PKC (δPKC) or selective activation of epsilon PKC (εPKC) reduces oxidative damage in the heart following myocardial infarction. In this study we determined the effect of these PKC isozymes in the survival of coronary endothelial cells (CVEC). We demonstrate here that serum deprivation of CVEC increased eNOS-mediated ROS levels, activated caspase-3, reduced Akt phosphorylation and cell number. Treatment with either the δPKC inhibitor, δV1-1, or the εPKC activator, ψεRACK, inhibited these effects, restoring cell survival through inhibition of eNOS activity. The decrease in eNOS activity coincided with specific de-phosphorylation of eNOS at Ser1179, and eNOS phosphorylation at Thr497 and Ser116. Furthermore, δV1-1 or ψεRACK induced physical association of eNOS with caveolin-1, an additional marker of eNOS inhibition, and restored Akt activation by inhibiting its nitration. Together our data demonstrate that 1) in endothelial dysfunction, ROS and reactive nitrogen species (RNS) formation result from uncontrolled eNOS activity mediated by activation of δPKC or inhibition of εPKC 2) inhibition of δPKC or activation of εePKC correct the perturbed phosphorylation state of eNOS, thus increasing cell survival. Since endothelial health ensures better tissue perfusion and oxygenation, treatment with a δPKC inhibitor and/or an εPKC activator in diseases of endothelial dysfunction should be considered.
doi:10.1016/j.yjmcc.2009.11.002
PMCID: PMC3760592  PMID: 19913548
24.  Mast cells and εPKC: A role in cardiac remodeling in hypertension-induced heart failure 
Heart failure (HF) is a chronic syndrome in which pathological cardiac remodeling is an integral part of the disease and mast cell (MC) degranulation-derived mediators have been suggested to play a role in its progression. Protein kinase C (PKC) signaling is a key event in the signal transduction pathway of MC degranulation. We recently found that inhibition of εPKC slows down the progression of hypertension-induced HF in salt-sensitive Dahl rats fed a high-salt diet. We therefore determined whether εPKC inhibition affects MC degranulation in this model. Six week-old male Dahl rats were fed with a high-salt diet to induce systemic hypertension, which resulted in concentric left ventricular hypertrophy at the age of 11 weeks, followed by myocardial dilatation and HF at the age of 17 weeks. We administered εV1-2 an εPKC-selective inhibitor peptide (3 mg/Kg/day), δV1-1, a δPKC-selective inhibitor peptide (3 mg/Kg/day), TAT (negative control; at equimolar concentration; 1.6 mg/Kg/day) or olmesartan (angiotensin receptor blocker [ARB] as a positive control; 3mg/Kg/day) between 11 weeks and 17 weeks. Treatment with εV1-2 attenuated cardiac MC degranulation without affecting MC density, myocardial fibrosis, microvessel patency, vascular thickening and cardiac inflammation in comparison to TAT- or δV1-1-treatment. Treatment with ARB also attenuated MC degranulation and cardiac remodeling, but to a lesser extent when compared to εV1-2. Finally, εV1-2 treatment inhibited MC degranulation in isolated peritoneal MCs. Together, our data suggest that εPKC inhibition attenuates pathological remodeling in hypertension-induced HF, at least in part, by preventing cardiac MC degranulation.
doi:10.1016/j.yjmcc.2008.08.009
PMCID: PMC2657602  PMID: 18804478
Mast cell degranulation; protein kinase C; PKC-selective inhibitor peptide; cardiac remodeling; heart failure
25.  DNA-dependent protein kinase catalytic subunit modulates the stability of c-Myc oncoprotein 
Molecular Cancer  2008;7:32.
Background
C-Myc is a short-lived oncoprotein that is destroyed by ubiquitin-mediated proteolysis. Dysregulated accumulation of c-Myc commonly occurs in human cancers. Some of those cases with the dysregulated c-Myc protein accumulation are attributed to gene amplification or increased mRNA expression. However, the abnormal accumulation of c-Myc protein is also a common finding in human cancers with normal copy number and transcription level of c-Myc gene. It seems that the mechanistic dysregulation in the control of c-Myc protein stabilization is another important hallmark associated with c-Myc accumulation in cancer cells. Here we report a novel mechanistic pathway through which DNA-dependent protein kinase catalytic subunit (DNA-PKcs) modulates the stability of c-Myc protein.
Results
Firstly, siRNA-mediated silencing of DNA-PKcs strikingly downregulated c-Myc protein levels in HeLa and HepG2 cells, and simultaneously decreased cell proliferation. The c-Myc protein level in DNA-PKcs deficient human glioma M059J cells was also found much lower than that in DNA-PKcs efficient M059K cells. ATM deficiency does not affect c-Myc expression level. Silencing of DNA-PKcs in HeLa cells resulted in a decreased stability of c-Myc protein, which was associated the increasing of c-Myc phosphorylation on Thr58/Ser62 and ubiquitination level. Phosphorylation of Akt on Ser473, a substrate of DNA-PKcs was found decreased in DNA-PKcs deficient cells. As the consequence, the phosphorylation of GSK3 β on Ser9, a negatively regulated target of Akt, was also decreased, and which led to activation of GSK 3β and in turn phosphorylation of c-Myc on Thr58. Moreover, inhibition of GSK3 activity by LiCl or specific siRNA molecules rescued the downregulation of c-Myc mediated by silencing DNA-PKcs. Consistent with this depressed DNA-PKcs cell model, overexpressing DNA-PKcs in normal human liver L02 cells, by sub-chronically exposing to very low dose of carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), increased c-Myc protein level, the phosphorylation of Akt and GSK3 β, as well as cell proliferation. siRNA-mediated silencing of DNA-PKcs in this cell model reversed above alterations to the original levels of L02 cells.
Conclusion
A suitable DNA-PKcs level in cells is necessary for maintaining genomic stability, while abnormal overexpression of DNA-PKcs may contribute to cell proliferation and even oncogenic transformation by stabilizing the c-Myc oncoprotein via at least the Akt/GSK3 pathway. Our results suggest DNA-PKcs a novel biological role beyond its DNA repair function.
doi:10.1186/1476-4598-7-32
PMCID: PMC2383926  PMID: 18426604

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