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1.  Protein kinase C mechanisms that contribute to cardiac remodelling 
Protein phosphorylation is a highly-regulated and reversible process that is precisely controlled by the actions of protein kinases and protein phosphatases. Factors that tip the balance of protein phosphorylation lead to changes in a wide range of cellular responses, including cell proliferation, differentiation and survival. The protein kinase C (PKC) family of serine/threonine kinases sits at nodal points in many signal transduction pathways; PKC enzymes have been the focus of considerable attention since they contribute to both normal physiological responses as well as maladaptive pathological responses that drive a wide range of clinical disorders. This review provides a background on the mechanisms that regulate individual PKC isoenzymes followed by a discussion of recent insights into their role in the pathogenesis of diseases such as cancer. We then provide an overview on the role of individual PKC isoenzymes in the regulation of cardiac contractility and pathophysiological growth responses, with a focus on the PKC-dependent mechanisms that regulate pump function and/or contribute to the pathogenesis of heart failure.
PMCID: PMC5024564  PMID: 27433023
myocardial remodelling; post translational modification; protein kinase C
2.  Mechanisms for redox-regulation of protein kinase C 
Protein kinase C (PKC) is comprised of a family of signal-regulated enzymes that play pleiotropic roles in the control of many physiological and pathological responses. PKC isoforms are traditionally viewed as allosterically activated enzymes that are recruited to membranes by growth factor receptor-generated lipid cofactors. An inherent assumption of this conventional model of PKC isoform activation is that PKCs act exclusively at membrane-delimited substrates and that PKC catalytic activity is an inherent property of each enzyme that is not altered by the activation process. This traditional model of PKC activation does not adequately explain the many well-documented actions of PKC enzymes in mitochondrial, nuclear, and cardiac sarcomeric (non-sarcolemmal) subcellular compartments. Recent studies address this dilemma by identifying stimulus-specific differences in the mechanisms for PKC isoform activation during growth factor activation versus oxidative stress. This review discusses a number of non-canonical redox-triggered mechanisms that can alter the catalytic properties and subcellular compartmentation patterns of PKC enzymes. While some redox-activated mechanisms act at structural determinants that are common to all PKCs, the redox-dependent mechanism for PKCδ activation requires Src-dependent tyrosine phosphorylation of a unique phosphorylation motif on this enzyme and is isoform specific. Since oxidative stress contributes to pathogenesis of a wide range of clinical disorders, these stimulus-specific differences in the controls and consequences of PKC activation have important implications for the design and evaluation of PKC-targeted therapeutics.
PMCID: PMC4477140  PMID: 26157389
protein kinase C; oxidative stress; tyrosine phosphorylation; Src; post-translational modifications
3.  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.
PMCID: PMC3547298  PMID: 23335926
protein kinase C; signal transduction; T cell activation; cell cycle; cyclin; cyclin-dependent kinase; cyclin-dependent kinase inhibitor
4.  Combined cardiomyocyte PKCδ and PKCε gene deletion uncovers their central role in restraining developmental and reactive heart growth 
Science signaling  2015;8(373):ra39.
Cell growth is orchestrated by changes in gene expression that respond to developmental and environmental cues. Among the signaling pathways that direct growth are enzymes of the protein kinase C (PKC) family, which are ubiquitous proteins belonging to three distinct subclasses: conventional PKCs, novel PKCs, and atypical PKCs. Functional overlap makes determining the physiological actions of different PKC isoforms difficult. We showed that two novel PKC isoforms, PKCδ and PKCε, redundantly govern stress-reactive and developmental heart growth by modulating the expression of cardiac genes central to stress-activated protein kinase and periostin signaling. Mice with combined postnatal cardiomyocyte-specific genetic ablation of PKCδ and germline deletion of PKCε (DCKO) had normally sized hearts, but their hearts had transcriptional changes typical of pathological hypertrophy. Cardiac hypertrophy and dysfunction induced by hemodynamic overloading were greater in DCKO mice than in mice with a single deletion of either PKCδ or PKCε. Furthermore, gene expression analysis of the hearts of DCKO mice revealed transcriptional derepression of the genes encoding the kinase ERK (extracellular signal–regulated kinase) and periostin. Mice with combined embryonic ablation of PKCδ and PKCε showed enhanced growth and cardiomyocyte hyperplasia that induced pathological ventricular stiffening and early lethality, phenotypes absent in mice with a single deletion of PKCδ or PKCε. Our results indicate that novel PKCs provide retrograde feedback inhibition of growth signaling pathways central to cardiac development and stress adaptation. These growth-suppressing effects of novel PKCs have implications for therapeutic inhibition of PKCs in cancer, heart, and other diseases.
PMCID: PMC4677479  PMID: 25900833
5.  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
6.  Rational design and validation of an anti-protein kinase C active-state specific antibody based on conformational changes 
Scientific Reports  2016;6:22114.
Protein kinase C (PKC) plays a regulatory role in key pathways in cancer. However, since phosphorylation is a step for classical PKC (cPKC) maturation and does not correlate with activation, there is a lack of tools to detect active PKC in tissue samples. Here, a structure-based rational approach was used to select a peptide to generate an antibody that distinguishes active from inactive cPKC. A peptide conserved in all cPKCs, C2Cat, was chosen since modeling studies based on a crystal structure of PKCβ showed that it is localized at the interface between the C2 and catalytic domains of cPKCs in an inactive kinase. Anti-C2Cat recognizes active cPKCs at least two-fold better than inactive kinase in ELISA and immunoprecipitation assays, and detects the temporal dynamics of cPKC activation upon receptor or phorbol stimulation. Furthermore, the antibody is able to detect active PKC in human tissue. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Thus, this antibody represents a reliable, hitherto unavailable and a valuable tool to study PKC activation in cells and tissues. Similar structure-based rational design strategies can be broadly applied to obtain active-state specific antibodies for other signal transduction molecules.
PMCID: PMC4766434  PMID: 26911897
7.  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
8.  Isoform-specific dynamic translocation of PKC by α1-adrenoceptor stimulation in live cells 
Protein kinase C (PKC) plays key roles in the regulation of signal transduction and cellular function in various cell types. At least ten PKC isoforms have been identified and intracellular localization and trafficking of these individual isoforms are important for regulation of enzyme activity and substrate specificity. PKC can be activated at downstream of Gq-protein coupled receptor (GqPCR) signaling and translocated to the various cellular compartments including plasma membrane (PM). Recent reports suggested that a different type of GqPCRs would activate different PKC isoforms (classic, novel and atypical PKCs) with different trafficking patterns. However, the knowledge of isoform-specific activation of PKC by each GqPCR is limited. α1-Adrenoceptor (α1-AR) is the one of the GqPCR highly expressed in the cardiovascular system. In this study, we examined the isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-AR stimulation (α1-ARS). Rat PKCα, βI, βII, δ, ε and ζ fused with GFP at C-term were co-transfected with human α1A-AR into HEK293T cells. The isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-ARS using phenylephrine was measured by confocal microscopy. Before stimulation, GFP-PKCs were localized at cytosolic region. α1-ARS strongly and rapidly translocated a classical PKC (cPKC), PKCα, (< 30s) to PM, with PKCα returning diffusively into the cytosol within 5 min. α1-ARS rapidly translocated other cPKCs, PKCβI and PKCβII, to the PM (<30s), with sustained membrane localization. One of novel PKCs (nPKCs), PKCε, but not another nPKC, PKCδ, was translocated by α1-AR stimulation to the PM (<30s) and its membrane localization was also sustained. Finally, α1-AR stimulation did not cause a diacylglycerol-insensitive atypical PKC, PKCζ translocation. Our data suggest that PKCα, β and ε activation may underlie physiological and pathophysiological responses of α1-AR signaling for the phosphorylation of membrane-associated substrates including ion-channel and transporter proteins in the cardiovascular system.
PMCID: PMC4564329  PMID: 26277396
9.  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.
PMCID: PMC2693016  PMID: 17548206
10.  Protein kinase C in the wood frog, Rana sylvatica: reassessing the tissue-specific regulation of PKC isozymes during freezing 
PeerJ  2014;2:e558.
The wood frog, Rana sylvatica, survives whole-body freezing and thawing each winter. The extensive adaptations required at the biochemical level are facilitated by alterations to signaling pathways, including the insulin/Akt and AMPK pathways. Past studies investigating changing tissue-specific patterns of the second messenger IP3 in adapted frogs have suggested important roles for protein kinase C (PKC) in response to stress. In addition to their dependence on second messengers, phosphorylation of three PKC sites by upstream kinases (most notably PDK1) is needed for full PKC activation, according to widely-accepted models. The present study uses phospho-specific immunoblotting to investigate phosphorylation states of PKC—as they relate to distinct tissues, PKC isozymes, and phosphorylation sites—in control and frozen frogs. In contrast to past studies where second messengers of PKC increased during the freezing process, phosphorylation of PKC tended to generally decline in most tissues of frozen frogs. All PKC isozymes and specific phosphorylation sites detected by immunoblotting decreased in phosphorylation levels in hind leg skeletal muscle and hearts of frozen frogs. Most PKC isozymes and specific phosphorylation sites detected in livers and kidneys also declined; the only exceptions were the levels of isozymes/phosphorylation sites detected by the phospho-PKCα/βII (Thr638/641) antibody, which remained unchanged from control to frozen frogs. Changes in brains of frozen frogs were unique; no decreases were observed in the phosphorylation levels of any of the PKC isozymes and/or specific phosphorylation sites detected by immunoblotting. Rather, increases were observed for the levels of isozymes/phosphorylation sites detected by the phospho-PKCα/βII (Thr638/641), phospho-PKCδ (Thr505), and phospho-PKCθ (Thr538) antibodies; all other isozymes/phosphorylation sites detected in brain remained unchanged from control to frozen frogs. The results of this study indicate a potential important role for PKC in cerebral protection during wood frog freezing. Our findings also call for a reassessment of the previously-inferred importance of PKC in other tissues, particularly in liver; a more thorough investigation is required to determine whether PKC activity in this physiological situation is indeed dependent on phosphorylation, or whether it deviates from the generally-accepted model and can be “overridden” by exceedingly high levels of second messengers, as has been demonstrated with certain PKC isozymes (e.g., PKCδ).
PMCID: PMC4157297  PMID: 25210662
Protein kinase C; Phosphorylation; Wood frog; Freeze-tolerance; Tissue-specific; Immunoblotting; Signal transduction; Adaptation; Catalytic competence; Second messenger
11.  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.
PMCID: PMC544167  PMID: 15643058
12.  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.
PMCID: PMC3204459  PMID: 20937286
13.  Common Mechanisms of Alzheimer’s Disease and Ischemic Stroke: The Role of Protein Kinase C in the Progression of Age-Related Neurodegeneration 
Ischemic stroke and Alzheimer’s disease (AD), despite being distinct disease entities, share numerous pathophysiological mechanisms such as those mediated by inflammation, immune exhaustion, and neurovascular unit compromise. An important shared mechanistic link is acute and chronic changes in protein kinase C (PKC) activity. PKC isoforms have widespread functions important for memory, blood-brain barrier maintenance, and injury repair that change as the body ages. Disease states accelerate PKC functional modifications. Mutated forms of PKC can contribute to neurodegeneration and cognitive decline. In some cases the PKC isoforms are still functional but are not successfully translocated to appropriate locations within the cell. The deficits in proper PKC translocation worsen stroke outcome and amyloid-β toxicity. Cross talk between the innate immune system and PKC pathways contribute to the vascular status within the aging brain. Unfortunately, comorbidities such as diabetes, obesity, and hypertension disrupt normal communication between the two systems. The focus of this review is to highlight what is known about PKC function, how isoforms of PKC change with age, and what additional alterations are consequences of stroke and AD. The goal is to highlight future therapeutic targets that can be applied to both the treatment and prevention of neurologic disease. Although the pathology of ischemic stroke and AD are different, the similarity in PKC responses warrants further investigation, especially as PKC-dependent events may serve as an important connection linking age-related brain injury.
PMCID: PMC4446718  PMID: 25114088
Alzheimer’s disease; blood-brain barrier; immune exhaustion; innate immunity; ischemic stroke; protein kinase C
14.  Nociceptive-induced Myocardial Remote Conditioning Is Mediated By Neuronal Gamma Protein Kinase C 
Basic research in cardiology  2013;108(5):10.1007/s00395-013-0381-x.
Deciphering the remote conditioning molecular mechanism may provide targets to develop therapeutics that can broaden the clinical application. To further investigate this, we tested whether two protein kinase C isozymes, the ubiquitously expressed epsilon PKC (εPKC) and the neuronal specific gamma PKC (γPKC), mediate nociceptive-induced remote myocardial conditioning.
Male Sprague-Dawley rats were used for both in vivo and ex vivo myocardial ischemia-reperfusion protocols. For the in vivo studies, using a surgical abdominal incision for comparison, applying only to the abdomen either bradykinin or the εPKC activator (ψεRACK) reduced myocardial infarct size (45±1%, 44±2%, respectively, versus incision: 43±2%, and control: 63±2%, P < 0.001). Western blot showed only εPKC, and not γPKC, is highly expressed in the myocardium. However, applying a selective γPKC inhibitor (γV5-3) to the abdominal skin blocked remote protection by any of these strategies.
Using an ex vivo isolated heart model without an intact nervous system, only selective εPKC activation, unlike a selective classical PKC isozyme activator (activating α, β, βII and γ), reduced myocardial injury. Importantly, the classical PKC isozyme activator given to the abdomen in vivo (with an intact nervous system including γPKC) during myocardial ischemia reduced infarct size as effectively as an abdominal incision or ψεRACK (45±1% versus 45±2% and 47±1%, respectively). The classical PKC activator-induced protection was also blocked by spinal cord surgical transection.
These findings identified potential remote conditioning mimetics, with these strategies effective even during myocardial ischemia. A novel mechanism of nociceptive-induced remote conditioning, involving γPKC, was also identified.
PMCID: PMC3856950  PMID: 23982492
infarct size; remote; incision; protein kinase C; gamma; epsilon
15.  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.
PMCID: PMC3568763  PMID: 22527640
PKC; PKA; Cardiac myocyte; Cardiac troponin I; Power output
16.  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.
•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
PMCID: PMC4052534  PMID: 24936444
Apoptosis; Endothelial cell; Hyperglycaemia; Protein kinase C; NADPH oxidase
17.  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.
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
18.  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.
PMCID: PMC2762617  PMID: 19885391
19.  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.
PMCID: PMC2814177  PMID: 19099253
melanoma; Wnt5A; Protein Kinase C (PKC)
20.  Differential expression of protein kinase C isoforms in coronary arteries of diabetic mice lacking the G-protein Gα11 
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.
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.
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.
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.
PMCID: PMC3024287  PMID: 21190563
21.  Protein kinase C, an elusive therapeutic target? 
Nature reviews. Drug discovery  2012;11(12):937-957.
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.
PMCID: PMC3760692  PMID: 23197040
22.  Protein kinase Cζ exhibits constitutive phosphorylation and phosphatidylinositol-3,4,5-triphosphate-independent regulation 
The Biochemical journal  2015;473(4):509-523.
Atypical protein kinase C (aPKC) isoenzymes are key modulators of insulin signalling, and their dysfunction correlates with insulin-resistant states in both mice and humans. Despite the engaged interest in the importance of aPKCs to type 2 diabetes, much less is known about the molecular mechanisms that govern their cellular functions than for the conventional and novel PKC isoenzymes and the functionally-related protein kinase B (Akt) family of kinases. Here we show that aPKC is constitutively phosphorylated and, using a genetically-encoded reporter for PKC activity, basally active in cells. Specifically, we show that phosphorylation at two key regulatory sites, the activation loop and turn motif, of the aPKC PKCζ in multiple cultured cell types is constitutive and independently regulated by separate kinases: ribosome-associated mammalian target of rapamycin complex 2 (mTORC2) mediates co-translational phosphorylation of the turn motif, followed by phosphorylation at the activation loop by phosphoinositide-dependent kinase-1 (PDK1). Live cell imaging reveals that global aPKC activity is constitutive and insulin unresponsive, in marked contrast to the insulin-dependent activation of Akt monitored by an Akt-specific reporter. Nor does forced recruitment to phosphoinositides by fusing the pleckstrin homology (PH) domain of Akt to the kinase domain of PKCζ alter either the phosphorylation or activity of PKCζ. Thus, insulin stimulation does not activate PKCζ through the canonical phosphatidylinositol-3,4,5-triphosphate-mediated pathway that activates Akt, contrasting with previous literature on PKCζ activation. These studies support a model wherein an alternative mechanism regulates PKCζ-mediated insulin signalling that does not utilize conventional activation via agonist-evoked phosphorylation at the activation loop. Rather, we propose that scaffolding near substrates drives the function of PKCζ.
PMCID: PMC4888060  PMID: 26635352
atypical protein kinase C; insulin; mTOR complex; phosphatidylinositol signalling; phosphatidylserine; phosphorylation
23.  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
24.  Anti-apoptotic effects of protein kinase C-δ and c-fos in cisplatin-treated thyroid cells 
British Journal of Pharmacology  2009;156(5):751-763.
Background and purpose:
We showed previously that cisplatin inititates a signalling pathway mediated by PKC-δ/extracellular signal-regulated kinase (ERK), important for maintaining viability in PC Cl3 thyroid cells. The studies described herein examined whether c-fos was associated with cisplatin resistance and the signalling link between c-fos and PKC-δ/ERK.
Experimental approach:
Cells were treated with various pharmacological inhibitors of PKCs and ERK, or were depleted of c-fos, PKC-δ, PKC-ε and caspase-3 by small interfering RNA (siRNA), then incubated with cisplatin and cytotoxicity assessed.
Key results:
Cisplatin provokes the induction of c-fos and the activation of conventional PKC-β, and novel PKC-δ and -ε. The cisplatin-provoked c-fos induction was decreased by Gö6976, a PKC-β inhibitor; by siRNA for PKC-δ- but not that for PKC-ε or by PD98059, a mitogen-activated protein kinase/ERK kinase inhibitor. Expression of c-fos was abolished by GF109203X, an inhibitor of all PKC isoforms, or by PD98059 plus Gö6976 or by PKC-δ-siRNA plus Gö6976. When c-fos expression was blocked by siRNA, cisplatin cytotoxicity was strongly enhanced with increased caspase-3 activation. In PKC-δ-depleted cells treated with cisplatin, caspase-3 activation was increased and cell viability decreased. In these PKC-δ-depleted cells, PD98059 did not affect caspase-3 activation.
Conclusions and implications:
In PC Cl3 cells, in the cell signalling pathways that lead to cisplatin resistance, PKC-δ controls ERK activity and, together with PKC-β, also the induction of c-fos. Hence, the protective role of c-fos in thyroid cells has the potential to provide new opportunities for therapeutic intervention.
PMCID: PMC2697754  PMID: 19254279
PKC-δ; c-fos; ERK; cisplatin; thyroid; PC Cl3
25.  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.
PMCID: PMC2925531  PMID: 20664637
bioinformatics; development; functional genomics; metabolic and regulatory networks; neuroscience

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