Protein Kinase C Signaling Research Articles

Protein Kinase C Activity is Increased in Human Coronary Smooth Muscle Cells following Exposure to 5‐FU Chemotherapy

Background5‐Fluorouracil (5‐FU) is the third most commonly used chemotherapeutic agent used to treat cancer but is strongly associated with a dose‐limiting cardiotoxicity. Cancer patients treated with 5‐FU chemotherapy are at risk of cardiotoxicity that manifests as alterations in the myocardial circulation such as resting or exertional angina, arrhythmias, myocardial infarction, and coronary vasospasm. However, little is known about the primary signaling mechanisms that contribute to cardiotoxicity with 5‐FU. Recent work has suggested that the Protein Kinase C (PKC) family of kinases plays a role in altered vasoconstrictive responses in vascular smooth muscle following exposure to 5‐FU. Further, PKC signaling in other pathologies is known to be influenced by reactive oxygen species (ROS) and 5‐FU chemotherapy is known to promote the production of ROS. Therefore, we hypothesized that 5‐FU administration would increase PKC activity in human coronary smooth muscle cells (HCSMCs).MethodsIn vitro, HCSMCs were incubated with 5‐FU (7×10−3M) for 3h, 1h, and 10 minutes. Following the respective incubation periods, media was aspirated in control and 5‐FU groups and the HCSMCs were scraped, homogenized in lysis buffer, and analyzed for protein content. The activity of PKC was assessed by a PKC activity assay kit.ResultsAnalysis revealed relative PKC activity was significantly increased in HCSMCs following 10 minutes of exposure to 5‐FU chemotherapy (6.52±0.36 versus 4.84±0.36; p=0.02) and was not different at 1 hour versus control and 3 hours versus control (1h 5.28±0.356 versus 4.84±0.36; p=0.82, 3h 5.67±0.36 versus 4.84±0.36; p=0.37).ConclusionsOur findings suggest 5‐FU exposure acutely upregulates PKC activity in coronary smooth muscle cells immediately following drug administration and the PKC pathway may have a mechanistic role in the altered vasoconstrictive response to 5‐FU as previously shown. These findings are imperative given the limited knowledge of mechanistic pathways that lead to vascular damage following 5‐FU exposure. Future studies will focus on reactive oxygen species as a regulator of PKC activity and specific isoforms involved in this pathway.

Neuroprotective Effects of Lithium in Chemotherapy‐induced Cognitive Impairments

Chemotherapy, a life‐saving treatment for cancer patients, is often associated with debilitating, sometimes irreversible, side effects. Approximately 40% of cancer survivors suffer from cognitive impairments, with symptoms including memory lapses, learning difficulties, and troubles with focusing, planning, multi‐tasking, and verbal communication. However, essential questions concerning the specific cognitive functions affected, the trajectories of cognitive changes, and the underlying mechanisms remain unclear. We aimed to investigate these questions by developing a mouse model of paclitaxel‐induced cognitive impairments. We also examined the potential neuroprotective effects of lithium, a compound previously shown to be beneficial in multiple neuropsychiatric and neurodegenerative disorders. Mice receiving four intraperitoneal injections of paclitaxel showed impaired short‐term spatial memory acquisition within a week of the last injection, which persisted for at least three weeks. Using Golgi‐Cox staining, we observed reduced dendritic arborization and spine density in the hippocampus and the cortex following paclitaxel injection, suggesting that these regions were particularly vulnerable. Importantly, lithium pretreatment completely rescued the behavioral and cellular deficits observed. Subsequently, we showed that paclitaxel treatment resulted in the upregulation of protein kinase C (PKC) both acutely and chronically, suggesting that dysregulated PKC signaling contributed to the neurological dysfunctions observed. In conclusion, we establish a molecular and cellular basis for paclitaxel‐induced cognitive impairments. Our findings also suggest a role for lithium as a protective agent to prevent cognitive impairments induced by paclitaxel and other chemotherapeutics.

Structural Basis of Protein Kinase C Tumor‐Promoting Response And Membrane Recruitment

Protein Kinase C (PKC) is a family of Ser/Thr kinases that regulate a multitude of cellular processes through participation in the phosphoinositide signaling pathway. The hallmark of their activation is translocation to lipid membranes. Dysregulation of PKC signaling has been implicated in cancer progression, cardiac disease, and Alzheimer’s disease. One of the major challenges in the field is to understand the structural basis of PKC activation and regulation ‐‐ at the level of atomic resolution.Here, we applied NMR and FRET spectroscopy to gain insight into the structure and protein‐membrane interactions of the regulatory domain of PKC that consist of three peripheral membrane modules: the tandem conserved homology‐1 (C1) and conserved homology‐2 (C2) domains. The C1 domains undergo membrane insertion upon binding their natural agonist, diacylglycerol, as well as tumor‐promoting phorbol esters and other small‐molecule agonists. C2 associates with anionic membranes upon binding Ca2+. Simultaneous association of all domains with membranes ensures the coincidence of four signals: Ca2+ ions, diacylglycerol, phosphatidylserine, and phosphatidylinositol‐4,5‐bisphosphate.Here, we report the first structure of the C1 domain complexed to a potent tumor‐promoting agent, phorbol 12,13‐dibutyrate (PDBu), in the membrane‐mimicking environment. The structure of the complex shows that C1 has to undergo a conformational change in order to capture the membrane‐embedded ligand. The structure also revealed the role of individual amino acids in the interactions with PDBu and membrane‐mimicking environment. To probe how different agonists modulate the interaction of C1 with membranes, we conducted NMR paramagnetic relaxation enhancement (PRE) experiments with bicelles doped with paramagnetic lipid. The PRE patterns revealed that the C1 domains adopt tilted rather than vertical orientation in the membrane. Our data on four different agonists indicate that C1 domains dictate the general orientation of the agonist complexes in the membrane, but the agonists determine the depth of insertion. This provides a plausible explanation for the differences in the potency of these ligands to achieve the acute and chronic activation response of PKC.To gain insight into the mechanism of coincidence detection, we developed protein‐to‐membrane FRET assay that we used to characterize the interplay of the C1 and C2 domains in the membrane association step. The FRET experiments revealed a clear picture of how tumor‐promoting agents can elicit constitutive activation of PKC through simultaneous recruitment of C1 domains to membranes and sensitization of the C2 domain to Ca2+.Support or Funding InformationNIH R01 GM108998; NSF CHE‐1905116; Welch Foundation A‐1784Agonist‐guided membrane interaction of C1B domain of PKCδ studied by paramagnetic NMR. (A) Schematic representation of the approach. (B) Chemical representations of the different agonists used in the study.Figure 1

The Implication of Androgens in the Presence of Protein Kinase C to Repair Alzheimer’s Disease-Induced Cognitive Dysfunction

Aging, as a major risk factor of memory deficiency, affects neural signaling pathways in hippocampus. In particular, age-dependent androgens deficiency causes cognitive impairments. Several enzymes like PKC are involved in memory deficiency. Indeed, PKC regulatory process mediates α-secretase activation to cleave APP in β-amyloid cascade and tau proteins phosphorylation mechanism. Androgens and cortisol regulate PKC signaling pathways, affecting the modulation of RACK1. Mitogen-activated protein kinase/ERK signaling pathway depends on CREB activity in hippocampal neurons and is involved in regulatory processes via PKC and androgens. Therefore, testosterone and PKC contribute in the neuronal apoptosis. The present review summarizes the current status of androgens, PKC, and their influence on cognitive learning. Inconsistencies in experimental investigations related to this fundamental correlation are also discussed, with emphasis on the mentioned contributors as the probable potent candidates for learning and memory improvement.

Protein Kinase C Isozymes and Autophagy during Neurodegenerative Disease Progression.

Protein kinase C (PKC) isozymes are members of the Serine/Threonine kinase family regulating cellular events following activation of membrane bound phospholipids. The breakdown of the downstream signaling pathways of PKC relates to several disease pathogeneses particularly neurodegeneration. PKC isozymes play a critical role in cell death and survival mechanisms, as well as autophagy. Numerous studies have reported that neurodegenerative disease formation is caused by failure of the autophagy mechanism. This review outlines PKC signaling in autophagy and neurodegenerative disease development and introduces some polyphenols as effectors of PKC isozymes for disease therapy.

The molecular mechanism of AhR-ARNT-XREs signaling pathway in the detoxification response induced by polycyclic aromatic hydrocarbons (PAHs) in clam Ruditapes philippinarum

The aryl hydrocarbon receptor (AhR) has been known primarily for its role in the regulation of several drug and xenobiotic metabolizing enzymes to mitigate environmental stresses. In this study, we interfere the expression of AhR gene to investigate the mechanism of AhR signaling pathway in the detoxification and antioxidation defense system that induced by Polycyclic Aromatic Hydrocarbons (PAHs) exposure by RNA interference (RNAi). The gene expressions of aryl hydrocarbon receptor nuclear translocator (ARNT), heat shock protein 90 (Hsp90) were evaluated after being exposed to benzo(a)pyrene (BaP) (4 μg/L) for 5 days and the positive correlations between AhR, ARNT, HSP90 indirectly indicating that AhR may have the ability to bind to ligands such as PAHs in Ruditapes philippinarum (R. philippinarum). Besides, the activities of detoxification enzymes were determined to investigate the role of AhR signaling pathway played in the metabolic detoxification. What's more, the gene expressions of protein kinase C (PKC) signaling pathway, mitogen-activated protein kinase (MAPKs) signaling pathway, NF-E2-related factor 2 (Nrf2) signaling pathway and antioxidant defense system indicated that AhR may regulate the Nrf2-Keap1 signaling pathway through Kelch-like ECH-associated protein-1 (Keap1) and MAPKs, PKC signaling pathways. In conclusion, adoption of RNA interference technology to explore the role of RpAhR gene played in the detoxification and antioxidation defense system under the PAHs stress at different time points can informe molecular endpoints for application towards ecotoxicology monitoring of bivalves.

Expression of protein kinase C in relation to the embryonic diapause process in the silkworm, Bombyx mori

In the present study, we investigated the expression of protein kinase C (PKC) signaling during the embryonic diapause process of Bombyx mori. PKC activity, determined using an antibody to phosphorylated substrates of PKC, was found to be significantly higher in developing eggs as compared to that of diapause eggs. In eggs whose diapause initiation was prevented by HCl, non-diapause eggs, and eggs in which diapause had been terminated by chilling of diapausing eggs at 5 °C for 70 days and then were transferred to 25 °C, PKC-dependent phosphorylation levels of multiple proteins showed dramatic stage-dependent increases compared to those of diapause eggs. Higher protein levels of PKC were also detected in developing eggs as compared to those of diapause eggs. Determination of PKC enzymatic activity during the middle embryonic stage showed higher PKC activity in developing eggs compared to diapause eggs, thus further confirming differential regulation of PKC signaling during the embryonic diapause process. Examination of temporal changes in mRNA expression levels of classical PKC (cPKC) and atypical PKC (aPKC) showed no difference between diapause and HCl-treated eggs. These results demonstrated that differential expressions of PKC signaling between diapause and developing eggs are related to the embryonic diapause process of B. mori.

Protein kinaseC promotes choline transporter‑like protein1 function via improved cell surface expression in immortalized human hepatic cells.

Choline is used to synthesize phospholipids and a lack of choline induces a number of liver-related diseases, including non-alcoholic steatohepatitis. The current study characterized the choline uptake system, at molecular and functional levels, in the immortalized human hepatic cell line, Fa2N-4, to identify the specific choline transporter involved in choline uptake. The present study also assesed whether choline deficiency or the inhibited choline uptake affected cell viability and apoptosis. Reverse transcription-quantitative polymerase chain reaction (PCR) revealed choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein expression in Fa2N-4 cells. [Methyl-3H]choline studies revealed choline uptake was saturable and mediated by a single transport system that functioned in a Na+-independent but pH-dependent manner, which was similar to CTL1. Hemicholinium-3 (HC-3), which is a choline uptake inhibitor, and choline deficiency inhibited cell viability, increased caspase-3 and −7 activities, and increased fluorescein isothiocyanate-Annexin V immunofluorescent staining indicated apoptosis. Immunofluorescent staining also revealed CTL1 and CTL2 localized in plasma and mitochondrial membranes, respectively. [Methyl-3H]choline uptake was enhanced by a protein kinase C (PKC) activator, phorbol-12-myristate 13-acetate (PMA). Immunofluorescence staining and western blot analysis demonstrated increased CTL1 expression on the cell membrane following PMA treatment. The results of current study indicated that extracellular choline is primarily transported via CTL1, relying on a direct H+ gradient that functions as a driving force in Fa2N-4 cells. Furthermore, it was hypothesized that CTL1 and the choline uptake system are strongly associated with cell survival, and that the choline uptake system is modulated by PKC signaling via increased CTL1 expression on the cell surface. These findings provide further insights into the pathogenesis of liver disease involving choline metabolism.

Ether lipid metabolism by AADACL1 regulates platelet function and thrombosis

AbstractWe previously reported the discovery of a novel lipid deacetylase in platelets, arylacetamide deacetylase-like 1 (AADACL1/NCEH1), and that its inhibition impairs agonist-induced platelet aggregation, Rap1 GTP loading, protein kinase C (PKC) activation, and ex vivo thrombus growth. However, precise mechanisms by which AADACL1 impacts platelet signaling and function in vivo are currently unknown. Here, we demonstrate that AADACL1 regulates the accumulation of ether lipids that impact PKC signaling networks crucial for platelet activation in vitro and in vivo. Human platelets treated with the AADACL1 inhibitor JW480 or the AADACL1 substrate 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG) exhibited decreased platelet aggregation, granule secretion, Ca2+ flux, and PKC phosphorylation. Decreased aggregation and secretion were rescued by exogenous adenosine 5′-diphosphate, indicating that AADACL1 likely functions to induce dense granule secretion. Experiments with P2Y12−/− and CalDAG GEFI−/− mice revealed that the P2Y12 pathway is the predominate target of HAG-mediated inhibition of platelet aggregation. HAG itself displayed weak agonist properties and likely mediates its inhibitory effects via conversion to a phosphorylated metabolite, HAGP, which directly interacted with the C1a domains of 2 distinct PKC isoforms and blocked PKC kinase activity in vitro. Finally, AADACL1 inhibition in rats reduced platelet aggregation, protected against FeCl3-induced arterial thrombosis, and delayed tail bleeding time. In summary, our data support a model whereby AADACL1 inhibition shifts the platelet ether lipidome to an inhibitory axis of HAGP accumulation that impairs PKC activation, granule secretion, and recruitment of platelets to sites of vascular damage.

Chronic activation of Mas-related gene receptors (Mrg) reduces the potency of morphine-induced analgesia via PKC pathway in naive rats

Mas oncogene-related gene receptors (Mrg) are uniquely distributed in small and medium cells of trigeminal and dorsal root ganglia (DRG). The physiological and pharmacological properties of Mrg are unknown. We have shown that intermittent activation of MrgC prevents and reverses morphine tolerance. Now we observed that intrathecal (i.t.) administration of the MrgC agonist bovine adrenal medulla 8–22 (BAM8-22, 3 nmol) for 3 and 6 days reduced the potency of morphine analgesia by 1.5 and 3.5 folds, respectively. Daily administration of BAM8-22 for 6 days also significantly decreased the tail flick latency. The administration of another MrgC agonist (Tyr6)-γ2-MSH-6–12 (MSH, 3 nmol) reduced morphine potency and the reduction was abolished following the co-administration of the protein kinase C (PKC) inhibitor chelerythrine chloride (CLT, 3 nmol). The chronic treatment with BAM8-22 or MSH increased the expression of PKC-gamma (PKCγ) in the cell membrane of spinal dorsal horn neurons and PKC-epsilon (PKCε) in the cell membrane and cytosol of DRG neurons. Moreover, the BAM8-22 treatment induced an increase in the expression of calcitonin gene-related peptide (CGRP) and neuronal nitric oxide synthase (nNOS) in small and medium cells in DRG. All of these responses were not seen when BAM8-22 or MSH was co-administered with the PKC inhibitor CLT (3 nmol) or GF-109203X (10 nmol). The present study suggested that the chronic activation of MrgC upregulated expressions of pronociceptive mediators via PKC signaling pathway leading to the suppression of antinociceptive property of morphine. These effects are opposite to those occurred when MrgC is activated acutely or moderately.

Protein kinase C is involved in the neuroprotective effect of berberine against intrastriatal injection of quinolinic acid-induced biochemical alteration in mice.

Protein kinase C (PKC) shows a neuronal protection effect in neurodegenerative diseases. In this study, we test whether berberine has a positive effect on the activity of PKC in quinolinic acid (QA)‐induced neuronal cell death. We used intrastriatal injections of QA mice model to test the effect of berberine on motor and cognitive deficits, and the PKC signalling pathway. Treatment with 50 mg/kg b.w of berberine for 2 weeks significantly prevented QA‐induced motor and cognitive impairment and related pathologic changes in the brain. QA inhibited the phosphorylation of PKC and its downstream molecules, GSK‐3β, ERK and CREB, enhanced the glutamate level and release of neuroinflammatory cytokines; these effects were attenuated by berberine. We used in vivo infusion of Go6983, a PKC inhibitor to disturb PKC activity in mice brain, and found that the effect of berberine to reverse motor and cognitive deficits was significantly reduced. Moreover, inhibition of PKC also blocked the anti‐excitotoxicity effect of berberine, which is induced by glutamate in PC12 cells and BV2 cells, as well as anti‐neuroinflammatory effect in LPS‐stimulated BV2 cells. Above all, berberine showed neuroprotective effect against QA‐induced acute neurotoxicity by activating PKC and its downstream molecules.

Specific miRNA-G Protein-Coupled Receptor Networks Regulate Sox9a/Sox9b Activities to Promote Gonadal Rejuvenation in Zebrafish.

Fertility and endocrine function rely on a tightly regulated synchronicity within the hypothalamic-pituitary-gonadal axis, for which the sex gonad serves as the primary source of sex steroid hormones and germ cells. To maintain hormonal stasis and fertility throughout the lifespan, inducing gonadal stem cell renewal is an attractive strategy. The follicle-stimulating hormone/cAMP/MAPK/Sox9 signaling axis and its regulated specific miRNAs are thought to regulate vertebrate gonadal development and sex differentiation, yet the regulatory networks are largely unknown. By genome-wide transcriptome mining and gonadal microinjections, we identify two G protein-coupled receptor (GPCR)-regulatory circuits: miR430a-Sox9a in the testis and miR218a-Sox9b in the ovary. Coinjection of a Sox9a-miR430a mixture promotes spermatogenesis, whereas Sox9b-miR218a mixture increases primordial ovarian follicles. Coimmunoprecipitation and mass spectrometry indicate that the two mixtures differentially modulate Sox9a/Sox9b multiple covalent modifications. We further reveal that miR430a and Sox9a synergistically activate testicular protein kinase C (PKC)/Akt signaling, whereas the miR218a and Sox9b mixture constrains ovary PKC/Akt signaling. pMIR-GFP reporter assay demonstrate that miR430a and miR218a target the 3' untranslated region (UTR) of four GPCR targets (lgr4, grk5l, grk4, and grp157). Knockdown of these GPCR genes or two Sox9 genes alters miR430a and miR218a regulation in the above gonad-specific PKC and Akt signaling pathways. These results establish two specific miRNA-GPCR-Sox9 networks and provide mechanistic insight into gonadal differentiation and rejuvenation. Stem Cells 2019;37:1189-1199.

Actin clearance promotes polarized dynein accumulation at the immunological synapse.

Immunological synapse (IS) formation between a T cell and an antigen-presenting cell is accompanied by the reorientation of the T cell centrosome toward the interface. This polarization response is thought to enhance the specificity of T cell effector function by enabling the directional secretion of cytokines and cytotoxic factors toward the antigen-presenting cell. Centrosome reorientation is controlled by polarized signaling through diacylglycerol (DAG) and protein kinase C (PKC). This drives the recruitment of the motor protein dynein to the IS, where it pulls on microtubules to reorient the centrosome. Here, we used T cell receptor photoactivation and imaging methodology to investigate the mechanisms controlling dynein accumulation at the synapse. Our results revealed a remarkable spatiotemporal correlation between dynein recruitment to the synaptic membrane and the depletion of cortical filamentous actin (F-actin) from the same region, suggesting that the two events were causally related. Consistent with this hypothesis, we found that pharmacological disruption of F-actin dynamics in T cells impaired both dynein accumulation and centrosome reorientation. DAG and PKC signaling were necessary for synaptic F-actin clearance and dynein accumulation, while calcium signaling and microtubules were dispensable for both responses. Taken together, these data provide mechanistic insight into the polarization of cytoskeletal regulators and highlight the close coordination between microtubule and F-actin architecture at the IS.

Abstract 2100: Resistance of gastric carcinoma cells towards c-met inhibition is mediated by compensatory HER3 upregulation involving SATB1 and PKC

Abstract Introduction: In patients with gastric cancer expressing high levels of c-met, inhibitors of the receptor tyrosine kinase (RTK) c-met are explored as targeted therapeutics. Unfortunately, clinical trials have failed to improve survival so far. This might be due to compensatory stimulation of other oncogenic RTKs. Here, we highlight the role of HER3 upregulation and its specific stimulation as resistance mechanism. On the mechanistic side, we demonstrate the genome regulator special AT-rich sequence-binding protein-1 (SATB1) as a critical mediator of this HER3 upregulation, with further manipulation being executed by contrary roles of Protein kinase C (PKC) and Phosphatidylinositol 3-kinase (PI3K). Methods: We used gastric cancer cell lines with a c-met amplification and evaluated the impact of pharmacological or siRNA-mediated c-met inhibition on cellular proliferation and survival. Moreover, we analyzed the expression of HER family receptors after inhibitor treatment. Furthermore, we used transient knockdown of SATB1 as well as inhibitors of PKC and PI3K to gain insight into signaling mechanisms. Results: The use of c-met kinase inhibitors or RNAi-induced downregulation of c-met led to a complete disruption of cellular proliferation. Without addition of c-met inhibitors, the investigated gastric cancer cells expressed HER1, HER2, and HER3 receptors, but no HER3 ligands. After pretreatment with c-met inhibitors, HER3 expression was markedly upregulated. Treatment of gastric cancer cells with the HER3 ligand beta-heregulin, usually secreted by cancer associated fibroblasts, partially reversed the anti-proliferative effects of c-met inhibitors. The induction of HER3 was reduced after pretreating cells with SATB1 siRNA. SATB1 is a known regulator of HER receptor expression in other tumor entities. The regulatory functions of SATB1 can be further modulated by phosphorylation via PKC or PI3K. Therefore, we investigated the effect of PKC and PI3K inhibition on HER3 induction. Of note, PKC inhibition markedly reduced HER3 upregulation, whereas PI3K inhibitors even increased HER3 levels. Conclusions: PKC and SATB1 are involved in the compensatory upregulation of HER3 upon c-met inhibition. Since this represents a critical resistance factor, interference with PKC and SATB1 signaling is a potential avenue to prevent resistance towards c-met inhibitors in gastric cancer. Citation Format: Robert Jenke, Thomas Büch, Miriam Rein, Stefanie Müller, Florian Lordick, Achim M. Aigner. Resistance of gastric carcinoma cells towards c-met inhibition is mediated by compensatory HER3 upregulation involving SATB1 and PKC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2100.

Protein kinase C activates multiple pathways to induce dendritic cell differentiation

Abstract Dendritic cell (DC) differentiation is a complex process that requires multiple signaling pathways to induce this response. Tumor derived factors can block this process, and one way in which this occurs is through repression of the protein kinase C (PKC) isoform PKCβ at the gene level. However, the role PKCβ plays in DC differentiation has yet to be fully characterized. To address this question, we used the K562 cell line, which our lab and others has shown to be capable of differentiating into DCs with PMA. Inhibition of either the ERK or NFκB pathways in the presence of PMA led to DCs that were unable to activate T-cells. Upon further examination of either pathway, we uncovered novel interactions for PKC signaling and ERK or NFκB during the early stages of DC differentiation. Multiple PKCs induced C-Raf activation, in part through RKIP repression. PKC signaling also induced changes in multiple ERK targets, including degradation of Foxo3a, an interaction not shown previously in DCs. PKCβ was the sole PKC isoform required for canonical NFκB signaling through a mechanism involving the CARD11 complex and PKCβ was required for CARD11 complex formation. However, the IKK kinase TAK1 was not required for downstream NFκB signaling, as opposed to its established role in B-cell and T-cell receptor induced NFκB signaling, indicating an alternative mechanism by which PKCβ induces NFκB signaling. PKCβ was also required for non-canonical NFκB signaling outside of the established TRAF and cIAP cascade. We also found crosstalk between the ERK and NFκB pathways at both protein-protein interaction and protein expression levels. Taken together, these results indicate PKC as an important signaling node for DC differentiation.

A novel conformation‐sensitive antibody‐based strategy to explore PKC signaling induced by opiate activation of μ‐opioid receptors

Opiates such as morphine and fentanyl are the most widely used analgesics for the treatment of pain; however, prolonged treatment with opiates decreases the responsiveness of opioid receptors, leading to the development of tolerance and opiate addiction. Recent alarming reports of the widespread misuse of fentanyl leading to the ‘Opioid Epidemic” have begun to refocus scientific investigations into the mechanisms of action of opioid receptors. Despite intense research efforts, the molecular mechanisms underlying opioid addiction remain unknown contributing to a public health challenge. Activation of opioid receptors leads to the initiation of a number of signaling cascades, resulting in enhanced protein phosphorylation. Among the various kinases, protein kinase C (PKC) has been reported to play a crucial role in the desensitization of opioid receptors. To date, the majority of studies with PKC have used genetic and/or pharmacological manipulation of PKC to explore the dynamics of PKC activation. In order to investigate the activation of endogenous PKC in native tissue, we developed conformational‐sensitive antibodies that are able to detect time‐ and ligand‐mediated activation of native PKC. We also generated antibodies to several substrates of PKC including pPP2A (regulatory subunit phosphoS90 PPP2R5D), phosphoSer143 MARCKS, and phosphoS375/T376 MOR. We used a high‐throughput microscopy approach to explore the temporal dynamics of opiate‐mediated PKC signaling and track the temporal dynamics of PKC activation in cells treated with PMA, morphine or fentanyl. Interestingly, in contrast to the PKC activation induced by morphine that is rapid but transient the activation by fentanyl is slow and sustained for a longer period of time. We are able to detect rapid activation of PKC in the striatum of animals acutely treated with morphine suggesting that the conformation‐sensitive activation, state sensitive anti‐PKC antibodies serve as hitherto unavailable, critical tools to investigate the role of PKC in opioid receptor signaling in particular and PKC‐mediated signaling in general.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Orexin facilitates GABAergic IPSCs via postsynaptic OX1 receptors coupling to the intracellular PKC signalling cascade in the rat cerebral cortex

Orexin has multiple physiological functions including wakefulness, appetite, nicotine intake, and nociception. The cerebral cortex receives abundant orexinergic projections and expresses both orexinergic receptor 1 (OX1R) and 2 (OX2R). However, little is known about orexinergic regulation of GABA-mediated inhibitory synaptic transmission. In the cerebral cortex, there are multiple GABAergic neural subtypes, each of which has its own morphological and physiological characteristics. Therefore, identification of presynaptic GABAergic neural subtypes is critical to understand orexinergic effects on GABAergic connections. We focused on inhibitory synapses at pyramidal neurons (PNs) from fast-spiking GABAergic neurons (FSNs) in the insular cortex by a paired whole-cell patch-clamp technique, and elucidated the mechanisms of orexin-induced IPSC regulation. We found that both orexin A and orexin B enhanced unitary IPSC (uIPSC) amplitude in FSN→PN connections without changing the paired-pulse ratio or failure rate. These effects were blocked by SB-334867, an OX1 receptor (OX1R) antagonist, but not by TCS-OX2-29, an OX2R antagonist. [Ala11, D-Leu15]-orexin B, a selective OX2R agonist, had little effect on uIPSCs. Variance-mean analysis demonstrated an increase in quantal content without a change in release probability or the number of readily releasable pools. Laser photolysis of caged GABA revealed that orexin A enhanced GABA-mediated currents in PNs. Downstream blockade of Gq/11 protein-coupled OX1Rs by IP3 receptor or protein kinase C (PKC) blockers and BAPTA injection into postsynaptic PNs diminished the orexin A-induced uIPSC enhancement. These results suggest that the orexinergic uIPSC enhancement is mediated via postsynaptic OX1Rs, which potentiate GABAA receptors through PKC activation.

Phospho-substrate profiling of Epac-dependent protein kinase C activity.

Exchange protein directly activated by cAMP (Epac) and protein kinase A are effectors for cAMP with distinct actions and regulatory mechanisms. Epac is a Rap guanine nucleotide exchange factor that activates Rap1; protein kinase C (PKC) is a major downstream target of Epac-Rap1 signaling that has been implicated in a variety of pathophysiological processes, including cardiac hypertrophy, cancer, and nociceptor sensitization leading to chronic pain. Despite the implication of both Epac and PKC in these processes, few downstream targets of Epac-PKC signaling have been identified. This study characterized the regulation of PKC activity downstream of Epac activation. Using an antibody that recognizes phospho-serine residues within the consensus sequence phosphorylated by PKC, we analyzed the 1-dimensional banding profile of PKC substrate protein phosphorylation from the Neuro2A mouse neuroblastoma cell line. Activation of Epac either indirectly by prostaglandin PGE2, or directly by 8-pCPT-2-O-Me-cAMP-AM (8pCpt), produced distinct PKC phospho-substrate protein bands that were suppressed by co-administration of the Epac inhibitor ESI09. Different PKC isoforms contributed to the induction of individual phospho-substrate bands, as determined using isoform-selective PKC inhibitors. Moreover, the banding profile after Epac activation was altered by disruption of the cytoskeleton, suggesting that the orchestration of Epac-dependent PKC signaling is regulated in part by interactions with the cytoskeleton. The approach described here provides an effective means to characterize Epac-dependent PKC activity.

Smoothelin-like 1 deletion enhances myogenic reactivity of mesenteric arteries with alterations in PKC and myosin phosphatase signaling

The role of the smoothelin-like 1 (SMTNL1) protein in mediating vascular smooth muscle contractile responses to intraluminal pressure was examined in resistance vessels. Mesenteric arterioles from wild type (WT) and SMTNL1 global knock-out (KO) mice were examined with pressure myography. SMTNL1 deletion was associated with enhanced myogenic tone in vessels isolated from male, but not female, mice. Intraluminal pressures greater than 40 mmHg generated statistically significant differences in myogenic reactivity between WT and KO vessels. No overt morphological differences were recorded for vessels dissected from KO animals, but SMTNL1 deletion was associated with loss of myosin phosphatase-targeting protein MYPT1 and increase in the myosin phosphatase inhibitor protein CPI-17. Additionally, we observed altered contractile responses of isolated arteries from SMTNL1 KO mice to phenylephrine, KCl-dependent membrane depolarization and phorbol 12,13-dibutyrate (PDBu). Using pharmacological approaches, myogenic responses of both WT and KO vessels were equally affected by Rho-associated kinase (ROCK) inhibition; however, augmented protein kinase C (PKC) signaling was found to contribute to the increased myogenic reactivity of SMTNL1 KO vessels across the 60–120 mmHg pressure range. Based on these findings, we conclude that deletion of SMTNL1 contributes to enhancement of pressure-induced contractility of mesenteric resistance vessels by influencing the activity of myosin phosphatase.

Rice black-streaked dwarf virus P10 suppresses protein kinase C in insect vector through changing the subcellular localization of LsRACK1.

Rice black-streaked dwarf virus (RBSDV) was known to be transmitted by the small brown planthopper (SBPH) in a persistent, circulative and propagative manner in nature. Here, we show that RBSDV major outer capsid protein (also known as P10) suppresses the protein kinase C (PKC) activity of SBPH through interacting with the receptor for activated protein kinase C 1 (LsRACK1). The N terminal of P10 (amino acids (aa) 1-270) and C terminal of LsRACK1 (aa 268-315) were mapped as crucial for the interaction. Confocal microscopy and subcellular fractionation showed that RBSDV P10 fused to enhanced green fluorescent protein formed vesicular structures associated with endoplasmic reticulum (ER) membranes in Spodoptera frugiperda nine cells. Our results also indicated that RBSDV P10 retargeted the initial subcellular localization of LsRACK1 from cytoplasm and cell membrane to ER and affected the function of LsRACKs to activate PKC. Inhibition of RACK1 by double stranded RNA-induced gene silencing significantly promoted the replication of RBSDV in SBPH. In addition, the PKC pathway participates in the antivirus innate immune response of SBPH. This study highlights that RACK1 negatively regulates the accumulation of RBSDV in SBPH through activating the PKC signalling pathway, and RBSDV P10 changes the subcellular localization of LsRACK1 and affects its function to activate PKC. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.