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The Ca2+-independent, but diacylglycerol-regulated, novel protein kinase C (PKC) theta (θ) is highly expressed in hematopoietic cells where it participates in immune signaling and platelet function. Mounting evidence suggests that PKCθ may be involved in cancer, particularly blood cancers, breast cancer, and gastrointestinal stromal tumors, yet how to target this kinase (as an oncogene or as a tumor suppressor) has not been established. Here, we examine the effect of four cancer-associated mutations, R145H/C in the autoinhibitory pseudosubstrate, E161K in the regulatory C1A domain, and R635W in the regulatory C-terminal tail, on the cellular activity and stability of PKCθ. Live-cell imaging studies using the genetically-encoded fluorescence resonance energy transfer-based reporter for PKC activity, C kinase activity reporter 2 (CKAR2), revealed that the pseudosubstrate and C1A domain mutations impaired autoinhibition to increase basal signaling. This impaired autoinhibition resulted in decreased stability of the protein, consistent with the well-characterized behavior of Ca2+-regulated PKC isozymes wherein mutations that impair autoinhibition are paradoxically loss-of-function because the mutant protein is degraded. In marked contrast, the C-terminal tail mutation resulted in enhanced autoinhibition and enhanced stability. Thus, the examined mutations were loss-of-function by different mechanisms: mutations that impaired autoinhibition promoted the degradation of PKC, and those that enhanced autoinhibition stabilized an inactive PKC. Supporting a general loss-of-function of PKCθ in cancer, bioinformatics analysis revealed that protein levels of PKCθ are reduced in diverse cancers, including lung, renal, head and neck, and pancreatic. Our results reveal that PKCθ function is lost in cancer., (© 2024 The Author(s).)

Fluoride is unnecessary in the human body. Long-term fluoride exposure may lead to immune system abnormalities. However, the mechanism remains unclear. This study aim to explore the mechanism of fluoride interference in the immune system and also identify the key indicators of fluoride-induced immune damage. Questionnaires were used to collect basic information. Multiple linear analyses and other statistical methods were used in order to process the data. Flow cytometry was used to detect relevant immunomarkers and analyze immune damage. Simultaneously, Wistar rats and cell models exposed to fluoride were established to detect the effects of fluoride on immune homeostasis. The results showed that sex, residence time, smoking, and Corona Virus Disease 2019 (COVID-19) infection may indirectly influence fluoride-induced immune damage. In residents of fluoride-exposed areas, there was a significant decrease in CD3 + T lymphocytes and CD4 + and CD8 + cells and a downward trend in the CD4 + /CD8 + cell ratio. CD4 + CD8 + /CD4 + , regulatory T cells (Tregs), and Tregs/effector T cells (Teffs) ratios showed opposite changes. Fluoride inhibits T cell activation by inhibiting the expression and phosphorylation of Protein Kinase C-θ (PKC-θ), hinders the internalization of T cell receptors, and affects NF-kB and c-Jun protein expression, leading to homeostatic Treg/Teff imbalance in vivo and in vitro experiments. This study represents the first evidence suggesting that PKC-θ may be the key to immune imbalance in the body under fluoride exposure. It is possible that Tregs/Teffs cell ratio provide a reference point for the diagnosis and treatment of fluoride-induced immune damage., Competing Interests: Declaration of competing interest The author declares that he does not receive any payment from any source and that there is no conflict of interest with the content of this article. The author's unit is shown on the cover. The preparation of this review was carried out during the author's normal working hours. This manuscript was written entirely by the author, who is solely responsible for the preparation of this manuscript. The views and conclusions expressed therein are those of the author and are not necessarily those of any sponsoring entity. None of the authors have been previously involved in any litigation, advocacy, or regulatory activity related to the content of this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Abstract: The inhibitory surface receptor programmed cell death protein 1 (PD1) is a major target for antibody-based cancer immunotherapies. Nevertheless, a substantial number of patients fail to respond to the treatment or experience adverse effects. An improved understanding of intracellular pathways targeted by PD1 is thus needed to develop better predictive and prognostic biomarkers. Here, via unbiased phosphoproteome analysis of primary human T cells, we demonstrate that PD1 triggering inhibited the phosphorylation and physical association with protein kinase Cθ (PKCθ) of a variety of cytoskeleton-related proteins. PD1 blocked activation and recruitment of PKCθ to the forming immune synapse (IS) in a Src homology-2 domain-containing phosphatase-1/2 (SHP1/SHP2)-dependent manner. Consequently, PD1 engagement led to impaired synaptic phosphorylation of cytoskeleton-related proteins and formation of smaller IS. T-cell receptor induced phosphorylation of the PKCθ substrate and binding partner vimentin was long-lasting and it could be durably inhibited by PD1 triggering. Vimentin phosphorylation in intratumoral T cells also inversely correlated with the levels of the PD1 ligand, PDL1, in human lung carcinoma. Thus, PKCθ and its substrate vimentin represent important targets of PD1-mediated T-cell inhibition, and low levels of vimentin phosphorylation may serve as a biomarker for the activation of the PD1 pathway., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: The protein kinase C theta (PKCθ) has an important and non-redundant function downstream of the antigen receptor and co-receptor complex in T lymphocytes. PKCθ is not only essential for activation of NF-κB, AP-1 and NFAT and subsequent interleukin-2 expression, but also critical for positive selection and development of regulatory T lymphocytes in the thymus. Several domains regulate its activity, such as a pseudosubstrate sequence mediating an auto-inhibitory intramolecular interaction, the tandem C1 domains binding diacylglycerol, and phosphorylation at conserved tyrosine, threonine as well as serine residues throughout the whole length of the protein. To address the importance of the variable domain V1 at the very N-terminus, which is encoded by exon 2, a mutated version of PKCθ was analyzed for its ability to stimulate T lymphocyte activation., Methods: T cell responses were analyzed with promoter luciferase reporter assays in Jurkat T cells transfected with PKCθ expression constructs. A mouse line expressing mutated instead of wild type PKCθ was analyzed in comparison to PKCθ-deficient and wild type mice for thymic development and T cell subsets by flow cytometry and T cell activation by quantitative RT-PCR, luminex analysis and flow cytometry., Results: In cell lines, the exon 2-replacing mutation impaired the transactivation of interleukin-2 expression by constitutively active mutant form of PKCθ. Moreover, analysis of a newly generated exon 2-mutant mouse line (PKCθ-E2 mut ) revealed that the N-terminal replacement mutation results in an hypomorph mutant of PKCθ combined with reduced PKCθ protein levels in CD4 + T lymphocytes. Thus, PKCθ-dependent functions in T lymphocytes were affected resulting in impaired thymic development of single positive T lymphocytes in vivo. In particular, there was diminished generation of regulatory T lymphocytes. Furthermore, early activation responses such as interleukin-2 expression of CD4 + T lymphocytes were significantly reduced even though cell viability was not affected. Thus, PKCθ-E2 mut mice show a phenotype similar to conventional PKCθ-deficient mice., Conclusion: Taken together, PKCθ-E2 mut mice show a phenotype similar to conventional PKCθ-deficient mice. Both our in vitro T cell culture experiments and ex vivo analyses of a PKCθ-E2-mutant mouse line independently validate the importance of PKCθ downstream of the antigen-receptor complex for activation of CD4 + T lymphocytes.

Haemorrhagic disease caused by grass carp reovirus (GCRV) can result in large-scale death of young grass carp, leading to irreparable economic losses that seriously affect large-scale breeding. Protein kinase C (PKC, also known as PRKC) represents a family of serine/threonine protein kinases that includes multiple isozymes in many species. Among these, PKC-θ (PKC theta, also written as PRKCQ) is a novel isoform, mainly expressed in T cells, that is known to be involved in immune system function in mammals. To date, no research on immunological functions of fish Pkc-θ has been reported. To address this issue, we cloned the grass carp pkc-θ gene. Phylogenetic and syntenic analysis showed that this gene is the most evolutionarily conserved relative to zebrafish. Real-time quantitative PCR (RT-qPCR) indicated that pkc-θ was expressed at high levels in the gills and spleen of healthy grass carp. Infection with GCRV down regulated pkc-θ expression in the gills and spleen. Gene products that function upstream and downstream of pkc-θ were up regulated in the gill, but were down-regulated in the spleen. These results suggest that direct or indirect targeting of pkc-θ by GCRV may help the virus evade host immune defences in the spleen. Phorbol ester (PMA) treatment of Jurkat T cells induced translocation of grass carp Pkc-θ from the cytoplasm to the plasma membrane. This response to PMA suggests evolutionary conservation of an immune response function in fish Pkc-θ, as well as conservation of its sequence and structural domains. This study expanded our knowledge of the fish PKC gene family, and explored the role of pkc-θ in function of the grass carp immune system, providing new insights which may facilitate further studies of its biological functions., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Protein kinase C theta (PKCθ) plays a critical role in T cell signaling and is an attractive target for the treatment of T cell-mediated diseases such as transplant rejection and autoimmune disease. To identify PKCθ inhibitors, we focused on the 2,6-diamino-3-carbamoyl-5-cyanopyrazine derivative 2, which exhibited moderate PKCθ inhibitory activity. Optimization of 2 identified the 2,4-diamino-5-cyanopyrimidine derivative 16c, which exhibited potent PKCθ inhibitory activity and showed good selectivity against other PKC isozymes. Compound 16c prolonged graft survival in an in vivo rat heterotopic cardiac transplant model., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Abstract Background The protein kinase C theta (PKCθ) has an important and non-redundant function downstream of the antigen receptor and co-receptor complex in T lymphocytes. PKCθ is not only essential for activation of NF-κB, AP-1 and NFAT and subsequent interleukin-2 expression, but also critical for positive selection and development of regulatory T lymphocytes in the thymus. Several domains regulate its activity, such as a pseudosubstrate sequence mediating an auto-inhibitory intramolecular interaction, the tandem C1 domains binding diacylglycerol, and phosphorylation at conserved tyrosine, threonine as well as serine residues throughout the whole length of the protein. To address the importance of the variable domain V1 at the very N-terminus, which is encoded by exon 2, a mutated version of PKCθ was analyzed for its ability to stimulate T lymphocyte activation. Methods T cell responses were analyzed with promoter luciferase reporter assays in Jurkat T cells transfected with PKCθ expression constructs. A mouse line expressing mutated instead of wild type PKCθ was analyzed in comparison to PKCθ-deficient and wild type mice for thymic development and T cell subsets by flow cytometry and T cell activation by quantitative RT-PCR, luminex analysis and flow cytometry. Results In cell lines, the exon 2-replacing mutation impaired the transactivation of interleukin-2 expression by constitutively active mutant form of PKCθ. Moreover, analysis of a newly generated exon 2-mutant mouse line (PKCθ-E2mut) revealed that the N-terminal replacement mutation results in an hypomorph mutant of PKCθ combined with reduced PKCθ protein levels in CD4+ T lymphocytes. Thus, PKCθ-dependent functions in T lymphocytes were affected resulting in impaired thymic development of single positive T lymphocytes in vivo. In particular, there was diminished generation of regulatory T lymphocytes. Furthermore, early activation responses such as interleukin-2 expression of CD4+ T lymphocytes were significantly reduced even though cell viability was not affected. Thus, PKCθ-E2mut mice show a phenotype similar to conventional PKCθ-deficient mice. Conclusion Taken together, PKCθ-E2mut mice show a phenotype similar to conventional PKCθ-deficient mice. Both our in vitro T cell culture experiments and ex vivo analyses of a PKCθ-E2-mutant mouse line independently validate the importance of PKCθ downstream of the antigen-receptor complex for activation of CD4+ T lymphocytes.

Background: Previous finding using DNA microarray and bioinformatics analysis, we have reported some key factors which regulated gene expression and signaling pathways in injured sciatic nerve during Wallerian Degeneration (WD). This research is focused on protein kinase C theta (Prkcq) participates in the regulation of the WD process., Methods: In this study, we explored the molecular mechanism by which Prkcq in Schwann cells (SCs) affects nerve degeneration and regeneration in vivo and in vitro after rat sciatic nerve injury., Results: Study of the cross-sectional model showed that Prkcq expression decreased significantly during sciatic nerve repair. Functional analysis showed that upregulation and downregulation of Prkcq could affect the proliferation, migration and apoptosis of Schwann cells and lead to the expression of related factors through the activation of the β-catenin, c-fos, and p-c-jun/c-jun pathways., Conclusion: The study provides insights into the role of Prkcq in early WD during peripheral nerve degeneration and/or regeneration., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The PKC isozymes represent the most prominent family of signaling proteins mediating response to the ubiquitous second messenger diacylglycerol. Among them, PKCθ is critically involved in T-cell activation. Whereas all the other conventional and novel PKC isoforms have twin C1 domains with potent binding activity for phorbol esters, in PKCθ only the C1b domain possesses potent binding activity, with little or no activity reported for the C1a domain. In order to better understand the structural basis accounting for the very weak ligand binding of the PKCθ C1a domain, we assessed the effect on ligand binding of twelve amino acid residues which differed between the C1a and C1b domains of PKCθ. Mutation of Pro 9 of the C1a domain of PKCθ to the corresponding Lys 9 found in C1b restored in vitro binding activity for [ 3 H]phorbol 12,13-dibutyrate to 3.6 nM, whereas none of the other residues had substantial effect. Interestingly, the converse mutation in the C1b domain of Lys 9 to Pro 9 only diminished binding affinity to 11.7 nM, compared to 254 nM in the unmutated C1a. In confocal experiments, deletion of the C1b domain from full length PKCθ diminished, whereas deletion of the C1a domain enhanced 5-fold (at 100 nM PMA) the translocation to the plasma membrane. We conclude that the Pro 168 residue in the C1a domain of full length PKCθ plays a critical role in the ligand and membrane binding, while exchanging the residue (Lys 240 ) at the same position in C1b domain of full length PKCθ only modestly reduced the membrane interaction., (Copyright © 2018 Elsevier B.V. All rights reserved.)

T cell receptor signaling, together with cytokine-induced signals, can differentially regulate RNA processing to influence T helper versus regulatory T cell fate. Protein kinase C family members have been shown to function in alternative splicing and RNA processing in various cell types. T cell-specific protein kinase C theta, a molecular regulator of T cell receptor downstream signaling, has been shown to phosphorylate splicing factors and affect post-transcriptional control of T cell gene expression. In this study, we explored how using a synthetic cell-penetrating peptide mimic for intracellular anti-protein kinase C theta delivery fine-tunes differentiation of induced regulatory T cells through its differential effects on RNA processing. We identified protein kinase C theta signaling as a critical modulator of two key RNA regulatory factors, heterogeneous nuclear ribonucleoprotein L (hnRNPL) and protein-l-isoaspartate O-methyltransferase-1 (PCMT1), and loss of protein kinase C theta function initiated a "switch" in post-transcriptional organization in induced regulatory T cells. More interestingly, we discovered that protein-l-isoaspartate O- methyltransferase-1 acts as an instability factor in induced regulatory T cells, by methylating the forkhead box P3 (FOXP3) promoter. Targeting protein-l-isoaspartate O-methyltransferase-1 using a cell-penetrating antibody revealed an efficient means of modulating RNA processing to confer a stable regulatory T cell phenotype., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Regulatory T cells maintain immunological tolerance and dampen inflammatory responses. Administering regulatory T cells can prevent the immune-mediated tissue destruction of graft-versus-host disease, which frequently accompanies hematopoietic stem cell transfer. Neutralizing the T cell-specific kinase, protein kinase C theta, which promotes T cell effector functions and represses regulatory T cell differentiation, augments regulatory T cell immunosuppression and stability. We used a synthetic, cell-penetrating peptide mimic to deliver antibodies recognizing protein kinase C theta into primary human CD4 T cells. When differentiated ex vivo into induced regulatory T cells, treated cells expressed elevated levels of the regulatory T cell transcriptional regulator forkhead box P3, the surface-bound immune checkpoint receptor programmed death receptor-1, and pro-inflammatory interferon gamma, previously ascribed to a specific population of stable, highly suppressive human induced regulatory T cells. The in vitro suppressive capacity of these induced regulatory T cells was 10-fold greater than that of T cells differentiated without antibody delivery. When administered at the time of graft-versus-host disease induction, using a humanized mouse model, antibody-treated regulatory T cells were superior to non-treated T cells in attenuating lethal outcomes. This antibody delivery approach may overcome obstacles currently encountered using patient-derived regulatory T cells as a cell-based therapy for immune modulation., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Protein kinase C theta (PKCθ) plays a critical role in T cell signaling and has therapeutic potential for T cell-mediated diseases such as transplant rejection and rheumatoid arthritis. PKCθ inhibitors have emerged as effective immunomodulative agents for the prevention of transplant rejection. We previously reported that the 2,4-diamino-5-cyanopyrimidine derivative 2 was a potent PKCθ inhibitor; however, it exhibited CYP3A4 time-dependent inhibition (TDI). Here, we report the structural modification of compound 2 into 34 focusing on mitigating CYP3A4 TDI. Compound 34 exhibited potent in vitro activity with mitigated CYP3A4 TDI and efficacy in vivo transplant model., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Breast cancer (BC) is the most commonly diagnosed cancer confronting women worldwide. Crocin, a glycosylated carotenoid extracted from Crocus sativus L., possesses anti-cancer and anti-inflammatory activities. This study tried to explore the influences of crocin on proliferation and inflammation of BC cells, and to investigate the possible mechanism. The protein levels of protein kinase C theta (PRKCQ) and nuclear factor kappa B (NF-κB) p-p65 and p65 were examined using western blot analysis. The potential targets of crocin were predicted using the PharmMapper database. Cell viability and proliferation were determined utilizing CCK-8 and EdU incorporation assays, respectively. Inflammation was assessed by detecting the levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) using RT-qPCR and ELISA. Results showed that crocin inhibited NF-κB activation and suppressed cell viability and proliferation in BC cells. Crocin caused a significant reduction of levels of TNF-α and IL-1β, suggesting that crocin suppressed inflammation in BC cells. NF-κB inhibition decreased proliferation and inflammation in BC cells. Additionally, PRKCQ was identified as a potential target of crocin according to PharmMapper database. Crocin treatment inhibited the activation of NF-κB in BC cells by reducing PRKCQ expression. Mechanistically, PRKCQ-dependent activation of NF-κB pathway reversed the effects of crocin on the proliferation and inflammation in BC cells. In conclusion, crocin inhibited NF-κB-mediated inflammation and proliferation in BC cells through reducing PRKCQ expression., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

We previously reported a facile preparation method of 3-substituted-2,6-difluoropyridines, which were easily converted to 2,3,6-trisubstituted pyridines by nucleophilic aromatic substitution with good regioselectivity and yield. In this study, we demonstrate the synthetic utility of 3-substituted-2,6-difluoropyridines in drug discovery via their application in the synthesis of various 2,3,6-trisubstituted pyridines, including macrocyclic derivatives, as novel protein kinase C theta inhibitors in a moderate to good yield. This synthetic approach is useful for the preparation of 2,3,6-trisubstituted pyridines, which are a popular scaffold for drug candidates and biologically attractive compounds., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The activation of conventional T cells upon T cell receptor stimulation critically depends on protein kinase C theta (PKCθ). However, its role in regulatory T (Treg) cell function has yet to be fully elucidated. Using siRNA or the potent and PKC family-selective pharmacological inhibitor AEB071, we could show that murine Treg-mediated suppression in vitro is independent of PKCθ function. Likewise, Treg cells of PKCθ-deficient mice were fully functional, showing a similar suppressive activity as wild-type CD25+CD4+ T cells in an in vitro suppression assay. Furthermore, in vitro-differentiated wild-type and PKCθ-deficient iTreg cells showed comparable Foxp3 expression as well as suppressive activity. However, we observed a reduced percentage of Foxp3+CD25+ CD4+ T cells in the lymphatic organs of PKCθ-deficient mice. Taken together, our results suggest that while PKCθ is involved in Treg cell differentiation in vivo, it is dispensable for Treg-mediated suppression.

Secretion of interleukin-10 (IL-10) by CD4+ T cells is an essential immunoregulatory mechanism. The work presented here assesses the role of the signaling molecule protein kinase C theta (PKCθ) in the induction of IL-10 expression in CD4+ T cells. Using wildtype and PKCθ-deficient Tg4 T cell receptor transgenic mice, we implemented a well-described protocol of repeated doses of myelin basic protein (MBP)Ac1-9[4Y] antigen to induce Tr1-like IL-10+ T cells. We find that PKCθ is required for the efficient induction of IL-10 following antigen administration. Both serum concentrations of IL-10 and the proportion of IL-10+ T cells were reduced in PKCθ-deficient mice relative to wildtype mice following [4Y] treatment. We further characterized the T cells of [4Y] treated PKCθ-deficient Tg4 mice and found reduced expression of the transcription factors cMaf, Nfil3 and FoxP3 and the surface receptors PD-1 and Tim3, all of which have been associated with the differentiation or function of IL-10+ T cells. Finally, we demonstrated that, unlike [4Y] treated wildtype Tg4 T cells, cells from PKCθ-deficient mice were unable to suppress the priming of naïve T cells in vitro and in vivo. In summary, we present data demonstrating a role for PKCθ in the induction of suppressive, IL-10-secreting T cells induced in TCR-transgenic mice following chronic antigen administration. This should be considered when contemplating PKCθ as a suitable drug target for inducing immune suppression and graft tolerance., Competing Interests: The authors have declared that no competing interests exist.

Objective: Neutrophils have an important role in the rapid innate immune response, and the release or active secretion of elastase from neutrophils is linked to various inflammatory responses. Purpose of this study was to determine how the human neutrophil elastase affects the interleukin-10 (IL-10) response in peripheral blood mononuclear cells (PBMC). Materials and Methods: In this prospective study, changes in IL-10 messenger RNA (mRNA) and protein expression levels in monocytes derived from human PBMCs were investigated after stimulation with human neutrophil elastase (HNE). A set of inhibitors was used for examining the pathways for IL-10 production induced by HNE. Results: Reverse transcription polymerase chain reaction (RT-PCR) showed that stimulation with HNE upregulated IL-10 mRNA expression by monocytes, while the enzyme-linked immunosorbent assay (ELISA) revealed an increase of IL-10 protein level in the culture medium. A phospholipase C inhibitor (U73122) partially blunted the induction of IL-10 mRNA expression by HNE, while IL-10 mRNA expression was significantly reduced by a protein kinase C (PKC) inhibitor (Rottlerin). A calcium chelator (3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester: TMB-8) inhibited the response of IL-10 mRNA to stimulation by HNE. In addition, pretreatment with a broad-spectrum PKC inhibitor (Ro-318425) partly blocked the response to HNE. Finally, an inhibitor of PKC theta/delta abolished the increased level of IL-10 mRNA expression. Conclusion: These results indicate that HNE mainly upregulates IL-10 mRNA expression and protein production in moncytes via a novel PKC theta/delta, although partially via the conventional PKC pathway.

The PKC-θ isoform of protein kinase C is selectively expressed in T lymphocytes and plays an important role in the T cell antigen receptor (TCR)-triggered activation of mature T cells, T cell proliferation, and the subsequent release of cytokines such as interleukin-2 (IL-2). Herein, we report the synthesis and structure-activity relationship (SAR) of a novel series of PKC-θ inhibitors. Through a combination of structure-guided design and exploratory SAR, suitable replacements for the basic C4 amine of the original lead ( 3 ) were identified. Property-guided design enabled the identification of appropriately substituted C2 groups to afford potent analogs with metabolic stability and permeability to support in vivo testing. With exquisite general kinase selectivity, cellular inhibition of T cell activation as assessed by IL-2 expression, a favorable safety profile, and demonstrated in vivo efficacy in models of acute and chronic T cell activation with oral dosing, CC-90005 ( 57 ) was selected for clinical development.

Protein Kinase C theta (PKCθ) is a serine/threonine kinase that belongs to the novel PKC subfamily. In normal tissue, its expression is restricted to skeletal muscle cells, platelets and T lymphocytes in which PKCθ controls several essential cellular processes such as survival, proliferation and differentiation. Particularly, PKCθ has been extensively studied for its role in the immune system where its translocation to the immunological synapse plays a critical role in T cell activation. Beyond its physiological role in immune responses, increasing evidence implicates PKCθ in the pathology of various diseases, especially autoimmune disorders and cancers. In this review, we discuss the implication of PKCθ in various types of cancers and the PKCθ-mediated signaling events controlling cancer initiation and progression. In these types of cancers, the high PKCθ expression leads to aberrant cell proliferation, migration and invasion resulting in malignant phenotype. The recent development and application of PKCθ inhibitors in the context of autoimmune diseases could benefit the emergence of treatment for cancers in which PKCθ has been implicated.

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 (ICa,L) and to explore the regulatory mechanisms of this effect in adult ventricular myocytes. Results: NF-1 reversibly decreased ICa,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-Gβ antibody as well as pertussis toxin pretreatment abolished the inhibitory effects of NF-1 on ICa,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 (PKCθ), and PKCθ inhibition abolished the decrease in ICa,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 Gi/o-protein and the novel PKCθ isoform in adult ventricular myocytes.

The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) induces transition of the epithelial MCF-7 cell line to a mesenchymal phenotype. A subset of the resulting mesenchymal cells has surface markers characteristics of a cancer stem cell (CSC) population. We profiled the transcriptome changes associated with the epithelial to mesenchymal transition and those that occurred in the CSC subset. Using a siRNA knockdown strategy, we examined the extent to which these changes were dependent on the PKC family member, PKC-θ. The importance of the cytoplasmic signaling role of this kinase is well established and in this study, we have shown by PKC-θ ChIP-sequencing analysis that this kinase has a dual role with the ability to also associate with chromatin on a subset of PKC-θ dependent genes. In the associated manuscript (Zafar et al., 2014 [5]) we presented evidence for the first time showing that this nuclear role of PKC-θ is also important for gene induction and mesenchymal/CSC phenotype. Here we describe the analysis associated with the transcriptome and ChIP-seq data presented in Zafar et al. (2014) [5] and uploaded to NCBI Gene Expression Omnibus (GSE53335).

The activation of conventional T cells upon T cell receptor stimulation critically depends on protein kinase C theta (PKCθ). However, its role in regulatory T (Treg) cell function has yet to be fully elucidated. Using siRNA or the potent and PKC family-selective pharmacological inhibitor AEB071, we could show that murine Treg-mediated suppression in vitro is independent of PKCθ function. Likewise, Treg cells of PKCθ-deficient mice were fully functional, showing a similar suppressive activity as wild-type CD25+CD4+ T cells in an in vitro suppression assay. Furthermore, in vitro-differentiated wild-type and PKCθ-deficient iTreg cells showed comparable Foxp3 expression as well as suppressive activity. However, we observed a reduced percentage of Foxp3+CD25+ CD4+ T cells in the lymphatic organs of PKCθ-deficient mice. Taken together, our results suggest that while PKCθ is involved in Treg cell differentiation in vivo, it is dispensable for Treg-mediated suppression.

Secretion of interleukin-10 (IL-10) by CD4+ T cells is an essential immunoregulatory mechanism. The work presented here assesses the role of the signaling molecule protein kinase C theta (PKCθ) in the induction of IL-10 expression in CD4+ T cells. Using wildtype and PKCθ-deficient Tg4 T cell receptor transgenic mice, we implemented a well-described protocol of repeated doses of myelin basic protein (MBP)Ac1-9[4Y] antigen to induce Tr1-like IL-10+ T cells. We find that PKCθ is required for the efficient induction of IL-10 following antigen administration. Both serum concentrations of IL-10 and the proportion of IL-10+ T cells were reduced in PKCθ-deficient mice relative to wildtype mice following [4Y] treatment. We further characterized the T cells of [4Y] treated PKCθ-deficient Tg4 mice and found reduced expression of the transcription factors cMaf, Nfil3 and FoxP3 and the surface receptors PD-1 and Tim3, all of which have been associated with the differentiation or function of IL-10+ T cells. Finally, we demonstrated that, unlike [4Y] treated wildtype Tg4 T cells, cells from PKCθ-deficient mice were unable to suppress the priming of naïve T cells in vitro and in vivo. In summary, we present data demonstrating a role for PKCθ in the induction of suppressive, IL-10-secreting T cells induced in TCR-transgenic mice following chronic antigen administration. This should be considered when contemplating PKCθ as a suitable drug target for inducing immune suppression and graft tolerance.

HIV-1 infection cannot be cured due to reservoirs formed early after infection. Decreasing the massive CD4+ T cell activation that occurs at the beginning of the disease would delay reservoir seeding, providing a better prognosis for patients. CD4+ T cell activation is mediated by protein kinase C (PKC) theta (θ), which is involved in T-cell proliferation, as well as NF-κB, NF-AT, and AP-1 activation. We found that PKCθ activity increased viral replication, but also that HIV-1 induced higher activation of PKCθ in infected CD4+ T cells, creating a feedback loop. Therefore, specific inhibition of PKCθ activity could contribute to control HIV-1 replication. We tested the efficacy of seven PKCθ specific inhibitors to control HIV-1 replication in CD4+ T cells and selected two of the more potent and safer: CGX1079 and CGX0471. They reduced PKCθ phosphorylation at T538 and its translocation to the plasma membrane, which correlated with decreased HIV-1 retrotranscription through partial inhibition of SAMHD1 antiviral activity, rendering lower proviral integration. CGX1079 and CGX0471 also interfered with viral transcription, which would reduce the production of new virions, as well as the subsequent spread and infection of new targets that would increase the reservoir size. CGX1079 and CGX0471 did not completely abrogate T-cell functions such as proliferation and CD8-mediated release of IFN-γ in PBMCs from HIV-infected patients, thereby avoiding general immunosuppresion. Consequently, using PKCθ inhibitors as adjuvant of antiretroviral therapy in recently infected patients would decrease the pool of activated CD4+ T cells, thwarting proviral integration and reducing the reservoir size., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Protein kinase C theta, (PRKCQ/PKCθ) is a serine/threonine kinase that is highly expressed in a subset of triple-negative breast cancers (TNBC) and promotes their growth, anoikis resistance, epithelial-mesenchymal transition (EMT), and invasion. Here, we show that PRKCQ regulates the sensitivity of TNBC cells to apoptosis triggered by standard-of-care chemotherapy by regulating levels of pro-apoptotic Bim., Methods: To determine the effects of PRKCQ expression on chemotherapy-induced apoptosis, shRNA and cDNA vectors were used to modulate the PRKCQ expression in MCF-10A breast epithelial cells or triple-negative breast cancer cells (MDA-MB231Luc, HCC1806). A novel PRKCQ small-molecule inhibitor, 17k, was used to inhibit kinase activity. Viability and apoptosis of cells treated with PRKCQ cDNA/shRNA/inhibitor +/-chemotherapy were measured. Expression levels of Bcl2 family members were assessed., Results: Enhanced expression of PRKCQ is sufficient to suppress apoptosis triggered by paclitaxel or doxorubicin treatment. Downregulation of PRKCQ also enhanced the apoptosis of chemotherapy-treated TNBC cells. Regulation of chemotherapy sensitivity by PRKCQ mechanistically occurs via regulation of levels of Bim, a pro-apoptotic Bcl2 family member; suppression of Bim prevents the enhanced apoptosis observed with combined PRKCQ downregulation and chemotherapy treatment. Regulation of Bim and chemotherapy sensitivity is significantly dependent on PRKCQ kinase activity; overexpression of a catalytically inactive PRKCQ does not suppress Bim or chemotherapy-associated apoptosis. Furthermore, PRKCQ kinase inhibitor treatment suppressed growth, increased anoikis and Bim expression, and enhanced apoptosis of chemotherapy-treated TNBC cells, phenocopying the effects of PRKCQ downregulation., Conclusions: These studies support PRKCQ inhibition as an attractive therapeutic strategy and complement to chemotherapy to inhibit the growth and survival of TNBC cells.

Porcine epidemic diarrhea virus (PEDV), a member of the Alpha-coronavirus genus in the Coronaviridae family, induces acute diarrhea, vomiting, and dehydration in neonatal piglets. This study aimed to investigate the genetic dependencies of PEDV and identify potential therapeutic targets by using a single-guide RNA (sgRNA) lentiviral library to screen host factors required for PEDV infection. Protein kinase C θ (PKCθ), a calcium-independent member of the PKC family localized in the cell membrane, was found to be a crucial host factor in PEDV infection. The investigation of PEDV infection was limited in Vero and porcine epithelial cell-jejunum 2 (IPEC-J2) due to defective interferon production in Vero and the poor replication of PEDV in IPEC-J2. Therefore, identifying suitable cells for PEDV investigation is crucial. The findings of this study reveal that human embryonic kidney (HEK) 293T and L929 cells, but not Vero and IPEC-J2 cells, were suitable for investigating PEDV infection. PKCθ played a significant role in endocytosis and the replication of PEDV, and PEDV regulated the expression and phosphorylation of PKCθ. Apoptosis was found to be involved in PEDV replication, as the virus activated the PKCθ-B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) axis in HEK293T and L929 cells to increase viral endocytosis and replication via mitochondrial apoptosis. This study demonstrated the suitability of HEK293T and L929 cells for investigating PEDV infection and identified PKCθ as a host factor essential for PEDV infection. These findings provide valuable insights for the development of strategies and drug targets for PEDV infection.

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 (ICa,L) and to explore the regulatory mechanisms of this effect in adult ventricular myocytes., Results: NF-1 reversibly decreased ICa,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-Gβ antibody as well as pertussis toxin pretreatment abolished the inhibitory effects of NF-1 on ICa,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 (PKCθ), and PKCθ inhibition abolished the decrease in ICa,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 Gi/o-protein and the novel PKCθ isoform in adult ventricular myocytes., (© 2015 S. Karger AG, Basel.)

Protein kinases C (PKCs) are a family of serine/threonine kinases involved in various cellular processes, including proliferation, differentiation, cell survival, and apoptosis. Here, we report the identification, structure-activity relationship (SAR), and 3D-QSAR studies of 69 natural indolocarbazoles, including 15 new compounds, from marine streptomyces strains. Interestingly, we found that the chair conformational isomer of 7-oxo-staurosporine (compound 15 ) inhibited PKCθ more potently than the corresponding boat isomer. An evaluation of kinase selectivity and antitumor efficacy revealed that 15 was a potent dual PKCθ/δ inhibitor and that it could efficiently inhibit tumor growth in pancreatic cancer (PC) by inducing cellular apoptosis and suppressing the NF-κB/p-P65 pathway. In addition, we demonstrated that overexpression of p-PKCδ and p-P65 was associated with poor survival rates in patients with PC, and p-PKCθ expression also showed significant positive correlations with p-PKCδ and p-P65 levels. Finally, the PC patient-derived xenograft model further confirmed the potential anti-PC efficacy of 15 .

T-cell activation is a critical part of the adaptive immune system, enabling responses to foreign cells and external stimulus. In this process, T-cell antigen receptor (TCR) activation stimulates translocation of the downstream kinase PKCθ to the membrane, leading to NF-κB activation and thus transcription of relevant genes. However, the details of how PKCθ is recruited to the membrane remain enigmatic. It is known that annexin A5 (ANXA5), a calcium-dependent membrane-binding protein, has been reported to mediate PKCδ activation by interaction with PKCδ, a homologue of PKCθ, which implicates a potential role of ANXA5 involved in PKCθ signaling. Here we demonstrate that ANXA5 does play a critical role in the recruitment of PKCθ to the membrane during T-cell activation. ANXA5 knockout in Jurkat T cells substantially inhibited the membrane translocation of PKCθ upon TCR engagement and blocked the recruitment of CARMA1-BCL10-MALT1 signalosome, which provides a platform for the catalytic activation of IKKs and subsequent activation of canonical NF-κB signaling in activated T cells. As a result, NF-κB activation was impaired in ANXA5-KO T cells. T-cell activation was also suppressed by ANAX5 knockdown in primary T cells. These results demonstrated a novel role of ANXA5 in PKC translocation and PKC signaling during T-cell activation., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Hu et al.)

Activation of coagulation occurs in sepsis and contributes to the development of thrombosis. Platelet α-granule exocytosis plays an important role in septic coagulation abnormalities. The present study aimed to investigate the effects and the underlying mechanisms of exogenous carbon monoxide, carbon monoxide-releasing molecules II (CORM-2)-liberated CO, on suppressing platelet α-granule exocytosis in sepsis. It was shown that CORM-2 weakened α-granule membrane fusion with platelet plasma membrane and attenuated α-granule contents exocytosis in LPS-Induced platelet. Further studies revealed that CORM-2 suppressed the expression of integrin α IIb β 3 in platelets stimulated by LPS. This was accompanied by a decrease in production and phosphorylation of PKCθ and Munc18a, SNARE complex assembly and subsequently platelet α-granule exocytosis. Taken together, we suggested that the potential mechanism of suppressive effect of CORM-2 on LPS-induced platelet SNAREs complex assembly and α-Granule Exocytosis might involve integrin α IIb β 3 -mediated PKCθ/Munc18a pathway activation., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

In the present study, receptor induced 3D-QSAR model was developed for a set of 46 thieno[2,3-b]pyridine-5- carbonitrile PKC-θ inhibitors, to explore the structural requirements of the molecules necessary for PKC-θ inhibition. Since the chemical nature of the studied molecules was different from the crystal ligand of the selected protein, induced fit docking (IFD) protocol was employed to induce the conformational changes in the active site of the selected protein. Thereafter, all molecules were docked into the newly generated active site environment of the selected protein using glide docking program, and the 3D-QSAR analysis was performed in PHASE program utilizing the docking based alignment of the molecules. The best 3D-QSAR model was selected on the basis of the highest value of Q(2)test (0.600), and the selected model also showed high values of R(2)train , 0.915, Pearson-r, 0.801 and low value of SD, 0.241. The contour maps corresponding to the selected 3D-QSAR model, in combination with docking analysis, helped to explore the essential amino acid residues involved in binding, and structural requirements of the ligand molecules necessary for complementary fit with the active site of the protein. Therefore, the information revealed from the generated model can further be explored as a novel tool for the designing of new congener molecules that can serve as potential therapeutics for the treatment of various disease conditions associated with abnormal PKC-θ signalling.

We have previously shown that mice deficient in protein kinase C theta (PKCθ) have the ability to reject cardiac allografts, but are susceptible to tolerance induction. Here we tested role of B cells in assisting alloimmune responses in the absence of PKCθ. Mouse cardiac allograft transplantations were performed from Balb/c (H-2d) to PKCθ knockout (PKCθ(-/-)), PKCθ and B cell double-knockout (PBDK, H-2b) mice and wild-type (WT) C57BL/6 (H-2b) mice. PBDK mice spontaneously accepted the allografts with the inhibition of NF-κB activation in the donor cardiac allograft. Anti-B cell antibody (rituximab) significantly delayed allograft rejection in PKCθ(-/-), but not in WT mice. Co-transfer of PKCθ(-/-) T plus PKCθ(-/-) B cells or primed sera triggered allograft rejection in Rag1(-/-) mice, and only major histocompatibility complex class II-enriched B cells, but not class I-enriched B cells, were able to promote rejection. This, together with the inability of PKCθ(-/-) and CD28(-/-) double-deficient (PCDK) mice to acutely reject allografts, suggested that an effective cognate interaction between PKCθ(-/-) T and B cells for acute rejection is CD28 molecule dependent. We conclude that T-B cell interactions synergize with PKCθ(-/-) T cells to mediate acute allograft rejection., (© 2013 Steunstichting ESOT. Published by John Wiley & Sons Ltd.)