Isoforms Theta Research Articles

Selective small-molecule PKC-theta inhibitors effectively block TCR-mediated activation of naïve peripheral blood and tissue resident memory T cells

Abstract Immune mediated inflammatory diseases constitute a diverse set of illnesses in which an aberrant immune response damages self-tissue. While progress has been made in treating these conditions, there continues to be room for improvement. Patients diagnosed with T cell mediated disorders (TMDs) such as atopic dermatitis, rheumatoid arthritis and inflammatory bowel disease often respond well to broadly-acting immunosuppressive drugs, most of which come with significant side-effects, further highlighting the need for more targeted approaches. Compared to other family members, the protein kinase c - theta isoform (PKCt) expression is highly restricted to T cells, is critical to downstream signaling of the TCR complex, and represents an attractive therapeutic target for TMDs. Here, we report results from novel small molecule inhibitors (SMIs) which are highly specific for PKCt. In an in-vitro model of TCR-mediated activation of human PBMCs, our SMIs potently prevented activation of both CD4 and CD8 T cells without affecting cellular viability. Similarly, in in-vitro models of whole primary human skin biopsies under Th1, Th2, and Th17 inducing conditions, tissue resident memory T cells failed to activate and release pro-inflammatory cytokines after PKCt inhibition. Taken together, we have identified multiple novel SMIs of PKCt which are highly potent and highly selective, representing a potential new therapeutic strategy for patients with T-cell mediated inflammatory disease.

Abstract 140: Exploring Protein Kinase C Theta (PKCθ) Signaling In The Generation Of Procoagulant Platelet: Insights From Phosphoproteomics And Systems Analysis

Background: Platelet activation plays a pivotal role in physiological hemostasis and thrombosis. Protein Kinase C theta (PKCθ) isoforms exert significant regulatory influence on platelet activation. While specific PKC family members have well-defined roles in platelet function, the roles of PKCθ remain less explored. Objectives: To determine mechanisms of PKCθ signaling that orchestrate procoagulant platelet generation. Methods: Platelets were prepared from healthy human donors for ex vivo experiments. Platelet signaling and function were studied following stimulation with GPVI agonist crosslinked collagen-related peptide (CRP-XL). To investigate PKCθ inhibition mechanisms in platelets, small molecule inhibitors targeting the PKCθ (CC-90005 and C20) were employed. Results: From phosphoproteomics, we noted prominent phosphorylation on the C-terminus PKCθ at Ser685. Pretreatment of platelets with inhibitors specific PKC isoforms revealed that PKCα/β, PKCθ, and PKCΔ activities determine PKCθ Ser685 phosphorylation. From the PKC Inhibitors Activity Curves, we noted a robust and selective inhibitory impact of CC-90005 on PKCθ, with specificity surpassing that of C20. Parallel platelet function analyses revealed that inhibiting PKCθ had minimal impact on platelet spreading and aggregation. However, PKCθ inhibition in the presence of Ca 2+ upregulated platelet phosphatidylserine (PS) exposure, as measured by flow cytometry. This was accompanied by an increase in platelet procoagulant activity, where PKCθ inhibition reduced the lag time for fibrin formation. Furthermore, fluorescence microscopy also found that PKCθ inhibition significantly augmented the proportion of procoagulant platelets and microparticles. Additionally, compared to the control group, the use of cyclosporin A (CsA) reduced the production of procoagulant platelets, but the addition of CC-90005 or C20 rescued this phenomenon, with signal intensity surpassing that of the control group. Conclusions: Our study proposes a model wherein PKCΔ and PKCα/β activities induce the phosphorylation of PKCθ at S685, influencing mitochondrial calcium signaling and supporting procoagulant platelet generation.

Efficacy of a novel oral, potent, and highly selective PKCt inhibitor in the SCID T-cell transfer colitis model

Abstract Protein kinase C family theta isoform (PKCt) is a key kinase downstream of T-cell receptor (TCR) and CD28 co-signaling in T-cells, leading to T-cell activation and production of effector cytokines. As T-cells are key pathogenic cells in a multitude of chronic inflammatory diseases, inhibition of PKCt with an oral small molecule may provide a novel mechanism to treat chronic inflammatory diseases, such as colitis. In this study, we evaluated the efficacy of two orally bioavailable, potent, and highly selective PKCt inhibitors in the severe-combined-immunodeficient (SCID) T-cell transfer colitis model; this model is considered the most relevant to human inflammatory bowel disease (IBD), and displays characteristics of both Crohn’s disease (CD) and ulcerative colitis (UC). PKCt inhibition resulted in a dose-dependent prevention of colitis symptoms, including maintenance of body weight and stool score, with efficacy similar to an anti-IL-12 monoclonal antibody. Histological assessment and tissue cytokine and chemokine protein quantification of the colon were also performed. These data suggest that an oral PKCt inhibitor may have utility in the treatment of IBD, and other chronic inflammatory diseases driven by pathogenic T-cell activation.

MiR-128-1-5p inhibits cell proliferation and induces cell apoptosis via targeting PRKCQ in colorectal cancer

ABSTRACT Previous studies have indicated that miR-128 was downregulated in a variety of cancers including colorectal cancer (CRC). However, the role and the underlying molecular mechanisms of miR-128 in CRC still remain largely unknown. The aim of this study was to investigate the level of miR-128-1-5p in CRC patients and to explore both the effects and regulatory mechanisms of miR-128-1-5p in the malignancy of CRC. Real-time PCR and western blot were used to analyze the expression levels of miR-128-1-5p and the direct downstream target protein tyrosine kinase C theta isoform (PRKCQ). Cell Counting Kit-8, clone formation, TUNEL apoptosis assays, and subcutaneous tumor model were performed to investigate the malignant ability of colon cancer cells. A luciferase assay was performed to explore whether miR-128-1-5p could directly bind to 3’-UTR region of PRKCQ. In the present study, we detected the decreased expression and clinical significances of miR-128-1-5p in colorectal cancer tissues and cell lines. Functional experiments revealed that miR-128-1-5p inhibited cell proliferation and induced cell apoptosis and that PRKCQ was identified as a target of miR-128-1-5p and involved in miR-128-1-5p-mediated proliferation and apoptosis. In conclusion, our results showed that miR-128-1-5p reduced CRC growth by modulating PRKCQ expression and is a possible new therapeutic target for patients with CRC.

PRKCQ promotes oncogenic growth and anoikis resistance of a subset of triple-negative breast cancer cells.

BackgroundThe protein kinase C (PKC) family comprises distinct classes of proteins, many of which are implicated in diverse cellular functions. Protein tyrosine kinase C theta isoform (PRKCQ)/PKCθ, a member of the novel PKC family, may have a distinct isoform-specific role in breast cancer. PKCθ is preferentially expressed in triple-negative breast cancer (TNBC) compared to other breast tumor subtypes. We hypothesized that PRKCQ/PKCθ critically regulates growth and survival of a subset of TNBC cells.MethodsTo elucidate the role of PRKCQ/PKCθ in regulating growth and anoikis resistance, we used both gain and loss of function to modulate expression of PRKCQ. We enhanced expression of PKCθ (kinase-active or inactive) in non-transformed breast epithelial cells (MCF-10A) and assessed effects on epidermal growth factor (EGF)-independent growth, anoikis, and migration. We downregulated expression of PKCθ in TNBC cells, and determined effects on in vitro and in vivo growth and survival. TNBC cells were also treated with a small molecule inhibitor to assess requirement for PKCθ kinase activity in the growth of TNBC cells.ResultsPRKCQ/PKCθ can promote oncogenic phenotypes when expressed in non-transformed MCF-10A mammary epithelial cells; PRKCQ/PKCθ enhances anchorage-independent survival, growth-factor-independent proliferation, and migration. PKCθ expression promotes retinoblastoma (Rb) phosphorylation and cell-cycle progression under growth factor-deprived conditions that typically induce cell-cycle arrest of MCF-10A breast epithelial cells. Proliferation and Rb phosphorylation are dependent on PKCθ-stimulated extracellular signal-related kinase (Erk)/mitogen-activated protein kinase (MAPK) activity. Enhanced Erk/MAPK activity is dependent on the kinase activity of PKCθ, as overexpression of kinase-inactive PKCθ does not stimulate Erk/MAPK or Rb phosphorylation or promote growth-factor-independent proliferation. Downregulation of PRKCQ/PKCθ in TNBC cells enhances anoikis, inhibits growth in 3-D MatrigelTM cultures, and impairs triple-negative tumor xenograft growth. AEB071, an inhibitor of PKCθ kinase activity, also inhibits growth and invasive branching of TNBC cells in 3-D cultures, further supporting a role for PKCθ kinase activity in triple-negative cancer cell growth.ConclusionsEnhanced PRKCQ/PKCθ expression can promote growth-factor-independent growth, anoikis resistance, and migration. PRKCQ critically regulates growth and survival of a subset of TNBC. Inhibition of PKCθ kinase activity may be an attractive therapeutic approach for TNBC, a subtype in need of improved targeted therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0749-6) contains supplementary material, which is available to authorized users.

Nesfatin-1 Suppresses Cardiac L-type Ca2+ Channels Through Melanocortin Type 4 Receptor and the Novel Protein Kinase C Theta Isoform Pathway

Background/Aims: Nesfatin-1 (NF-1), an anorexic nucleobindin-2 (NUCB2)-derived hypothalamic peptide, acts as a peripheral cardiac modulator and it can induce negative inotropic effects. However, the mechanisms underlying these effects in cardiomyocytes remain unclear. Methods: Using patch clamp, protein kinase assays, and western blot analysis, we studied the effect of NF-1 on L-type Ca²⁺ currents (I_Ca,L) and to explore the regulatory mechanisms of this effect in adult ventricular myocytes. Results: NF-1 reversibly decreased I_Ca,L in a dose-dependent manner. This effect was mediated by melanocortin 4 receptor (MC4-R) and was associated with a hyperpolarizing shift in the voltage-dependence of inactivation. Dialysis of cells with GDP-β-S or anti-G_β antibody as well as pertussis toxin pretreatment abolished the inhibitory effects of NF-1 on I_Ca,L. Protein kinase C (PKC) antagonists abolished NF-1-induced responses, whereas inhibition of PKA activity or intracellular application of the fast Ca²⁺-chelator BAPTA elicited no such effects. Application of NF-1 increased membrane abundance of PKC theta isoform (PKC_θ), and PKC_θ inhibition abolished the decrease in I_Ca,L induced by NF-1. Conclusion: These data suggest that NF-1 suppresses L-type Ca²⁺ channels via the MC4-R that couples sequentially to the βγ subunits of G_i/o-protein and the novel PKC_θ isoform in adult ventricular myocytes.

Pro-oxidant and antioxidant responses in the liver and kidney of wild Goodea gracilis and their relation with halomethanes bioactivation

In mammals, it has been shown that halomethanes (HM) are bioactivated by enzymes such as CYP 2E1 and the theta isoform of GST to produce reactive metabolites. However, in fish, little information is available, although HM can form autochthonously in aquatic environments. This study assessed the effect of HM in dusky splitfin (Goodea gracilis) from three lakes of the Valley of Mexico by analysing specific HM biomarkers as well as a broad range of biomarkers. The concentration of HM was a function of its half-life (higher in deep waters), while its precursors and solar radiation are secondary factors that determine its concentration. The kidney showed higher basal metabolism than the liver, probably because of its function as a haematopoietic and filtration organ. Using integrated biological response version 2 (IBRv2), it was found that the hepatic and renal O₂· content is a pro-oxidant force capable of inducing oxidative stress (ROOH, TBARS and RC=O). Early damage was found to be dependent on low concentrations of HM in Major Lake, whereas late damage was observed in fish exposed to higher concentrations of HM in Zumpango Lake and Ancient Lake. The activities of enzymes involved in antioxidant defence seemed to be inefficient. The quantitative assessment of biomarkers (ANOVA) and the estimate of parameter A obtained from IBRv2 provided different information. However, the data support the greater predictive power of IBRv2, but it requires a series of interrelated biomarkers to infer these possibilities. G. gracilis presents marked patterns of adaptation, which are dependant on the HM concentrations in environmental mixtures, although the response is complex and many toxicants could induce similar responses.

PKCδ Negatively Regulates Primary Megakaryocyte Proliferation and Differentiation

AbstractAbstract 853Megakaryocytes are large, polyploid cells that give rise to platelets in the bone marrow and spleen. Megakaryocytes achieve their size and DNA content through a process known as endomitosis via signaling from the cytokine thrombopoietin (Tpo). We have previously determined that the novel Protein Kinase C isoforms theta (PKCθ) and delta (PKCδ) regulate a number of platelet functions including aggregation and secretion. However, the function of these two PKC isoforms in primary megakaryopoiesis has not yet been elucidated. Therefore we chose to utilize primary mouse megakaryocytes from WT, PKCδ−/− and PKCθ−/− mice to characterize the roles of PKCδ and PKCq in megakaryopoiesis. We were first able to determine via western blotting that megakaryocytes express more PKCd than either mononuclear bone marrow cells (p < 0.05) or progenitor cells isolated from bone marrow (p < 0.05). Deletion of PKCδ in mice caused an increase in white blood cell and platelet counts compared to WT mice (p < 005). However, deletion of PKCθ had no effect on murine blood cell counts. Observed increases in platelet counts in PKCδ−/− mice are due to increased platelet production as PKCδ−/− mice contain more thiazole orange positive platelets than WT mice (p < 0.05). Furthermore, we determined via flow cytometry that PKCδ−/− mice had more bone marrow megakaryocytes than WT mice (p < 0.05), although megakaryocyte DNA content was unaltered. Conversely, there was no alteration in megakaryocyte number or DNA content with PKCθ deletion. Interestingly, the increase in bone marrow-derived megakaryocyte count observed in PKCδ−/− mice was heightened following culture of bone marrow cells in 50ng/mL exogenous Tpo (Figure 1). Furthermore, in similar experiments, megakaryocyte DNA content was also enhanced in PKCδ−/− mice compared to WT mice (p < 0.05). PKCδ is an important pro-apoptotic protein, and heightened megakaryocyte number following culture could be due to reduced apoptosis in PKCδ−/− megakaryocytes. However, neither apoptosis nor necrosis was altered with PKCδ deletion as the number of AnnexinV+/7AAD- cells, and the number of AnnexinV+/7AAD+ cells, were not different from WT. Therefore, the observed increases in megakaryocyte number and DNA content could be due to elevated Tpo-induced signaling as ERK1/2 phosphorylation was heightened in PKCδ−/− megakaryocytes compared to WT, in response to exogenous Tpo (Figure 2). These data suggest that PKCδ is an important megakaryopoietic protein, which negatively regulates signaling induced by Tpo in megakaryocytes, while PKCθ is dispensable for primary mouse megakaryopoiesis. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Transmitter release in the neuromuscular synapse of the protein kinase C theta‐deficient adult mouse

We studied structural and functional features of the neuromuscular junction in adult mice (P30) genetically deficient in the protein kinase C (PKC) theta isoform. Confocal and electron microscopy shows that there are no differences in the general morphology of the endplates between PKC theta-deficient and wild-type (WT) mice. Specifically, there is no difference in the density of the synaptic vesicles. However, the myelin sheath is not as thick in the intramuscular nerve fibers of the PKC theta-deficient mice. We found a significant reduction in the size of evoked endplate potentials and in the frequency of spontaneous, asynchronous, miniature endplate potentials in the PKC theta-deficient neuromuscular preparations in comparison with the WT, but the mean amplitude of the spontaneous potentials is not different. These changes indicate that PKC theta has a presynaptic role in the function of adult neuromuscular synapses.

Decreased phosphorylation of delta and epsilon subunits of the acetylcholine receptor coincides with delayed postsynaptic maturation in PKC theta deficient mouse

Protein kinase C (PKC) activity is involved in the nicotinic acetylcholine receptor (nAChR) redistribution at the neuromuscular junction in vivo during postnatal maturation. Here we studied, in PKC theta (PKCθ) deficient mice (KO), how the theta isoform of PKC is involved in the nAChR cluster maturation that is accompanied by the developmental activity-dependent neuromuscular synapse elimination process. We found that axonal elimination and dispersion of nAChR from the postsynaptic plaques and its redistribution to form the mature postsynaptic apparatus were delayed but not totally suppressed in PKCθ deficient mice. Moreover, the delay in the maturation of the morphology of the nAChR clusters during the early postnatal synapse elimination period in the PKCθ deficient mice coincides with a reduction in the PKCθ-mediated phosphorylation on the delta subunit of the nAChR. In addition, we show evidence for PKCθ regulation of PKA in normally phosphorylating the epsilon subunit of nAChR. We have also found that the θ isoform of PKC is located on the postsynaptic component of the neuromuscular junction but is also expressed by motoneurons in the spinal cord and in the motor nerve terminals. The results allow us to hypothesize that a spatially specific and opposing action of PKCθ and PKA may result in activity-dependent alterations to synaptic connectivity at both the nerve inputs and the postsynaptic nAChR clusters.

PKC-delta induces cardiomyogenic gene expression in human adipose-derived stem cells

Stromal cells from fat tissues exhibit properties of mesenchymal stem cells from other sources with the ability to differentiate towards multiple cell types. However, effective differentiation of these mesenchymal cells, called adipose-derived stem cells (ADSCs), towards cardiomyogenic lineage has been limited to a small number of isolated clones in an extended culture. Previously, we reported that treatment with phorbol ester induces the expression of several cardiomyogenic genes in the absence of serum. This study was performed to identify the roles of PKC isoforms in cardiomyogenic gene expression of ADSCs. Treatment with 10 nM phorbol myristate acetate (PMA) for 24 h caused sustained increases in mRNA levels for various cardiomyogenic genes, such as Mef2C, cardiac actin and troponin, for at least 6 days following the drug removal. The use of various inhibitors specific for PKC isoforms demonstrated that the novel PKC-theta/delta isoforms mediate the PMA effects. RT-PCR revealed that ADSCs express significant mRNA for PKC-delta, but not theta isoform. Overexpression of cDNA for PKC-delta resulted in marked increases in cardiac mRNA expression. These results indicate that activation of PKC-delta induces the expression of multiple cardiomyogenic genes in ADSCs.

Targeting PKCtheta Inhibits Alloreactivity and Graft-Versus-Host Disease While Preserving Graft-Versus-Leukemia and Anti-Infection Activities.

Abstract 2458Poster Board II-435 Introduction:When used as therapy for hematopoietic malignances, allogeneic hematopoietic stem cell transplantation (HCT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells through immunologic mechanisms. However, graft-versus-host-disease (GVHD) is a potentially lethal complication of allogeneic BMT. Thus, inhibition of GVHD, while preserving GVL and protective responses against infectious agents, can enhance the therapeutic potential of BMT. GVHD is initiated by alloreactive donor T cells that recognize mismatched major and/or minor histocompatibility antigens and cause severe damage to hematopoietic and epithelial tissues. Protein kinase C theta isoform (PKCθ) is crucial in TCR signaling and regulates the nature of lymphocyte-specific effector responses. The aim of this study was to investigate the significance of PKCθ in donor T-cell-mediated GVHD, anti-leukemia, and antiviral infection. Methods:Our results were validated using clinically relevant murine bone marrow transplantation (BMT) models. GVHD was measured by recipient survival and clinical signs such as body weight loss. Leukemia/lymphoma was induced by intravenous injection of a luciferase expressing A20 clone (a B lymphoma cell line derived from BALB/c mice). Tumor invasion was quantitated using the Xenogen IVIS-200 bioluminescence imaging system. Murine cytomegalovirus (MCMV) infection was induced by intraperitoneally injecting MCMV in the recipients after GVHD was established. Results:Using 3 distinct mouse models of allogeneic BMT, we found that T cells lacking PKCθ are unable to undergo robust expansion and cause damage to recipient hematopoietic or epithelial tissues, demonstrating that an essential requirement for PKCθ in alloreactivity and GVHD development. In contrast, PKCθ-/- T cells retain the ability to respond to virus infection post-BMT. Mechanistically, absence of PKCθ raises the threshold for T cell activation that selectively impacts alloresponses, but T cell responses triggered by infection or following administration of antigen plus microbial adjuvant are relatively well preserved in the absence of PKCθ Furthermore, we evaluated the role of PKCθ in GVHD and GVL responses, and found that PKCθ-/- T cells have at least a 10-fold reduction in the ability to induce GVHD but only a ∼2.5-fold reduction in GVL compared to WT T cells. Thus, absence of PKCθ impacts GVHD responses more significantly than GVL responses. Conclusion:These findings validate PKCθ as a potentially unique therapeutic target that is required for GVHD induction but not for GVL or protective responses to infectious agents in allogeneic BMT. Disclosures:No relevant conflicts of interest to declare.

Involvement of Thyroid Hormones in the Alterations of T-Cell Immunity and Tumor Progression Induced by Chronic Stress

Stress alters the neuroendocrine system, immunity, and cancer. Although the classic stress hormones are glucocorticoids and catecholamines, thyroid hormones have also been related to stress. We recently reported that chronic restraint stress impairs T-cell mediated immunity and enhances tumor growth in mice. To study the participation of these hormones on the stress-induced alterations of the immune function and lymphoma growth, mice were subjected to acute or chronic stress, with or without thyroxin supplementation. Hormone levels, immune status, and cancer progression were evaluated. Differential endocrine alterations were observed in response to acute and chronic stress. Although corticosterone and noradrenaline levels were increased by acute stress, they were restored after prolonged exposure to the stressor. Instead, thyroid hormone levels were only reduced in chronically stressed animals in comparison with control subjects. Correlating, chronic but not acute stress impaired T-cell reactivity. Thyroxin replacement treatment of chronic restraint stress-exposed mice, which restored the euthyroid status, reversed the observed reduction of T-cell lymphoproliferative responses. Moreover, therapeutic thyroid replacement also reversed the alterations of lymphoma growth induced by chronic stress in syngeneic mice bearing tumors as well as Interleukin-2 production and specific cytotoxic response against tumor cells. Finally, we found that the isoforms theta and alpha of the protein kinase C are involved in these events. These results show for the first time that thyroid hormones are important neuroendocrine regulators of tumor evolution, most probably acting through the modulation of T-cell mediated immunity affected by chronic stress.

Identification, Characterization and Initial Hit-to-Lead Optimization of a Series of 4-Arylamino-3-Pyridinecarbonitrile as Protein Kinase C theta (PKCθ) Inhibitors

The protein kinase C (PKC) family of serine/threonine kinases is implicated in a wide variety of cellular processes. The PKC theta (PKCtheta) isoform is involved in TCR signal transduction and T cell activation and regulates T cell mediated diseases, including lung inflammation and airway hyperresponsiveness. Thus inhibition of PKCtheta enzyme activity by a small molecule represents an attractive strategy for the treatment of asthma. A PKCtheta high-throughput screening (HTS) campaign led to the identification of 4-(3-bromophenylamino)-5-(3,4-dimethoxyphenyl)-3-pyridinecarbonitrile 4a, a low microM ATP competitive PKCtheta inhibitor. Structure based hit-to-lead optimization led to the identification of 5-(3,4-dimethoxyphenyl)-4-(1H-indol-5-ylamino)-3-pyridinecarbonitrile 4p, a 70 nM PKCtheta inhibitor. Compound 4p was selective for inhibition of novel PKC isoforms over a panel of 21 serine/threonine, tyrosine, and phosphoinositol kinases, in addition to the conventional and atypical PKCs, PKCbeta, and PKCzeta, respectively. Compound 4p also inhibited IL-2 production in antiCD3/anti-CD28 activated T cells enriched from splenocytes.

Identification of 14-3-3 protein isoforms in human astrocytoma by immunohistochemistry

The 14-3-3 proteins are highly conserved, ubiquitous molecules involved in a variety of biologic events, such as cell cycle control, and apoptosis. In our previous study, it has been proved that they are expressed in primary human nervous system tumors. However, the isoform-specific expression of 14-3-3 protein is still need to be identified. This study is the first detection of 14-3-3 isoforms' specific expression in human astrocytoma. In the normal brain tissues, all the seven 14-3-3 isoforms' immunoreactivity was localized mainly in the neurons, while only weak expression of epsilon, zeta and theta was found in some glial cells. However, beta, epsilon, zeta, eta and theta isoforms' immunoreactivity was seen in the majority of astrocytoma samples and its immunoreactivity score was increased markedly with an increase in the pathologic grade of human astrocytomas. These results indicate that the five isoforms may play an important role in tumorigenesis of human astrocytoma.

Translocation of PKC theta by fatty acids causes hypothalamic insulin resistance.

Diets high in saturated fats (HFS) promote obesity and attenuate insulin-induced glucose uptake in peripheral tissues. Several potential mechanisms for this disruption have been proposed including altered intracellular signaling, gene expression and protein trafficking. Numerous recent studies have demonstrated that protein-kinase-C isoforms theta, delta and epsilon attenuate insulin and metabolic signaling in peripheral tissues. Because the product of fatty acid metabolism, diacylglyceride (DAG) can directly induce translocation of PKCs and thereby activate them, DAG and activated PKCs may represent a mechanism by which dietary fatty acids induce insulin resistance. Importantly, here we report that obesity and high-fat diets attenuate insulin signaling in the brain. Here we assessed the hypothesis that CNS PKC isoforms mediated the deleterious effects of HFS on CNS function. We found that consumption of HFS increased membrane localization of PKC theta and delta in rat hypothalamus. This was associated with impaired hypothalamic insulin signaling, including a reduction in activation of intracellular insulin signaling cascades, increased food intake, and the development of obesity. This effect was recapitulated both in vivo and in vitro by the addition of the phorbol-ester PMA, which induces PKC membrane-translocation. Collectively, these results suggest that saturated-fats can attenuate CNS insulin signaling, similar to peripheral insulin resistance, by directly influencing the trafficking of hypothalamic PKC isoforms. Therefore, PKC may represent a novel unifying mechanism by which diets high in saturated fats promote both peripheral and central metabolic disease.

The Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein Is Phosphorylated and Localizes in the Cytoplasm by 14-3-3-Mediated Translocation

The severe acute respiratory syndrome coronavirus(SARS-CoV) nucleocapsid (N) protein is one of the four structural proteins of the virus and is predicted to be a 46-kDa phosphoprotein. Our in silico analysis predicted N to be heavily phosphorylated at multiple residues. Experimentally, we have shown in this report that the N protein of the SARS-CoV gets serine-phosphorylated by multiple kinases, in both the cytoplasm and the nucleus. The phosphoprotein is stable and localizes in the cytoplasm and coprecipitates with the membrane fraction. Also, using specific inhibitors of phosphorylation and an in vitro phosphorylation assay, we show that the nucleocapsid protein is a substrate of cyclin-dependent kinase (CDK), glycogen synthase kinase, mitogen-activated protein kinase, and casein kinase II. Further, we show that the phosphorylated protein is translocated to the cytoplasm by binding to 14-3-3 (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein). 14-3-3 proteins are a family of highly conserved, ubiquitously expressed eukaryotic proteins that function primarily as adapters that modulate interactions between components of various cellular signaling and cell cycle regulatory pathways through phosphorylation-dependent protein-protein interactions. Coincidentally, the N protein was also found to downregulate the expression of the theta isoform of 14-3-3 (14-3-3theta), leading to the accumulation of phosphorylated N protein in the nucleus, in the absence of growth factors. Using short interfering RNA specific to 14-3-3theta we have inhibited its expression to show accumulation of phosphorylated N protein in the nucleus. Thus, the data presented here provide a possible mechanism for phosphorylation-dependent nucleocytoplasmic shuttling of the N protein. This 14-3-3-mediated transport of the phosphorylated N protein and its possible implications in interfering with the cellular machinery are discussed.

The role of the theta isoform of protein kinase C (PKC) in activity-dependent synapse elimination: evidence from the PKC theta knock-out mouse in vivo and in vitro.

PKC plays a critical role in competitive activity-dependent synapse modification at the neuromuscular synapse in vitro and in vivo. This action involves a reduction of the strength of inactive inputs to muscle cells that are activated by other inputs. A decrease of postsynaptic responsiveness and a loss of postsynaptic acetyl choline receptors account for the heterosynaptic loss in vitro. The loss is not seen in preparations in which PKC has been blocked pharmacologically. Here, we show that the loss does not occur in in vitro preparations made from animals genetically modified to lack the theta isoform of PKC. Synapse elimination in the newborn period in vivo is delayed but is eventually expressed in knock-out animals. PKC-dependent synapse reduction is suppressed in heterologous cultures combining normal nerve and PKC theta-deficient muscle, as might be expected from the postsynaptic locus of the changes that underlie the activity-dependent plasticity. Preparations in which PKC theta-deficient neurons innervated normal muscle also exhibited a marked deficit in PKC-deficient synapse reduction. The presynaptic action of PKC theta implied by this observation is blocked by TTX, and we propose that activity-related synapse strengthening is decreased by presynaptic PKC theta. Thus, PKC theta in both presynaptic and postsynaptic elements plays a critical role in activity-dependent synapse modulation and loss. We provide a model for activity-dependent synapse loss incorporating these findings.

A p53-independent G1 Cell Cycle Checkpoint Induced by the Suppression of Protein Kinase C α and θ Isoforms

The protein kinase C (PKC) family consists of multiple isoforms that are involved in the regulation of diverse cellular responses. Suppression of PKC induces growth arrest in various types of cells. However, the underlying molecular mechanisms have not been thoroughly investigated. In this report, we demonstrated that the concurrent inhibition, rather than separate inhibition, of phorbol ester-dependent PKC alpha and theta isoforms is crucial for the induction of G1 cell cycle arrest and that this negative cell cycle regulation is via p53-independent mechanisms. PKC suppression-mediated growth arrest is associated with the induction of cell cycle inhibitor p21WAF1/CIP1 and the occurrence of hypophosphorylated Rb. The G1 checkpoint induced by the suppression of PKC occurs not only in murine Swiss3T3 but also in p53-deficient cells and human lung cancer cells containing mutated p53. Luciferase and nuclear run-off assays demonstrated that p21WAF1/CIP1 is, in part, transcriptionally regulated in response to the suppression of PKC alpha and theta. However, the stability of p21 mRNA is also augmented after the addition of PKC alpha and theta antisense oligonucleotides, indicating the involvement of post-transcriptional mechanisms in p21WAF1/CIP1 expression. These data suggest the existence of a cell cycle checkpoint pathway regulated by PKC alpha and theta isoforms. Furthermore, our findings support the notion that G1 checkpoint control can be restored in tumor cells containing abnormal p53, by targeting the PKC-regulated p21WAF1/CIP1 induction.

The isoform-specific regulation of apoptosis by protein kinase C.

The process of apoptosis is regulated at several levels through phosphorylation by many different protein kinases. The protein kinase C (PKC) family, which comprises at least 10 isoforms with distinct means of regulation and tissue distribution patterns, have been shown to exert both inhibitory and stimulatory influences on apoptosis. This review details recent progress made in determining the roles played by individual PKC isoforms in the control of apoptosis, with reference to their target substrates and actions in different cell types. Although notable exceptions exist, the weight of evidence indicates that the alpha, beta, epsilon and atypical isoforms are anti-apoptotic in their action, whereas the delta and theta isoforms are usually involved in the promotion of apoptosis.