Conventional Protein Kinase C Isoforms Research Articles

Platelet Activation Pathways Controlling Reversible Integrin αIIbβ3 Activation.

Background Agonist-induced platelet activation, with the integrin αIIbβ3 conformational change, is required for fibrinogen binding. This is considered reversible under specific conditions, allowing a second phase of platelet aggregation. The signaling pathways that differentiate between a permanent or transient activation state of platelets are poorly elucidated. Objective To explore platelet signaling mechanisms induced by the collagen receptor glycoprotein VI (GPVI) or by protease-activated receptors (PAR) for thrombin that regulate time-dependent αIIbβ3 activation. Methods Platelets were activated with collagen-related peptide (CRP, stimulating GPVI), thrombin receptor-activating peptides, or thrombin (stimulating PAR1 and/or 4). Integrin αIIbβ3 activation and P-selectin expression was assessed by two-color flow cytometry. Signaling pathway inhibitors were applied before or after agonist addition. Reversibility of platelet spreading was studied by microscopy. Results Platelet pretreatment with pharmacological inhibitors decreased GPVI- and PAR-induced integrin αIIbβ3 activation and P-selectin expression in the target order of protein kinase C (PKC) > glycogen synthase kinase 3 > β-arrestin > phosphatidylinositol-3-kinase. Posttreatment revealed secondary αIIbβ3 inactivation (not P-selectin expression), in the same order, but this reversibility was confined to CRP and PAR1 agonist. Combined inhibition of conventional and novel PKC isoforms was most effective for integrin closure. Pre- and posttreatment with ticagrelor, blocking the P2Y 12 adenosine diphosphate (ADP) receptor, enhanced αIIbβ3 inactivation. Spreading assays showed that PKC or P2Y 12 inhibition provoked a partial conversion from filopodia to a more discoid platelet shape. Conclusion PKC and autocrine ADP signaling contribute to persistent integrin αIIbβ3 activation in the order of PAR1/GPVI > PAR4 stimulation and hence to stabilized platelet aggregation. These findings are relevant for optimization of effective antiplatelet treatment.

Trace amine-associated receptor 1 regulation of Kv1.4 channels in trigeminal ganglion neurons contributes to nociceptive behaviors

BackgroundTrace amines, such as tyramine, are endogenous amino acid metabolites that have been hypothesized to promote headache. However, the underlying cellular and molecular mechanisms remain unknown.MethodsUsing patch-clamp recording, immunostaining, molecular biological approaches and behaviour tests, we elucidated a critically functional role of tyramine in regulating membrane excitability and pain sensitivity by manipulating Kv1.4 channels in trigeminal ganglion (TG) neurons.ResultsApplication of tyramine to TG neurons decreased the A-type K+ current (IA) in a manner dependent on trace amine-associated receptor 1 (TAAR1). Either siRNA knockdown of Gαo or chemical inhibition of βγ subunit (Gβγ) signaling abrogated the response to tyramine. Antagonism of protein kinase C (PKC) prevented the tyramine-induced IA response, while inhibition of conventional PKC isoforms or protein kinase A elicited no such effect. Tyramine increased the membrane abundance of PKCθ in TG neurons, and either pharmacological or genetic inhibition of PKCθ blocked the TAAR1-mediated IA decrease. Furthermore, PKCθ-dependent IA suppression was mediated by Kv1.4 channels. Knockdown of Kv1.4 abrogated the TAAR1-induced IA decrease, neuronal hyperexcitability, and pain hypersensitivity. In a mouse model of migraine induced by electrical stimulation of the dura mater surrounding the superior sagittal sinus, blockade of TAAR1 signaling attenuated mechanical allodynia; this effect was occluded by lentiviral overexpression of Kv1.4 in TG neurons.ConclusionThese results suggest that tyramine induces Kv1.4-mediated IA suppression through stimulation of TAAR1 coupled to the Gβγ-dependent PKCθ signaling cascade, thereby enhancing TG neuronal excitability and mechanical pain sensitivity. Insight into TAAR1 signaling in sensory neurons provides attractive targets for the treatment of headache disorders such as migraine.

Abstract P2-24-03: Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation

Abstract Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation Sayantani Sarkar Bhattacharya1, Taylor L. Witter1, Anh Q. T. Cong1, Elizabeth Bruinsma2, Swaathi Jayaraman2, Matthew P. Goetz2, John R. Hawse1, and Matthew J. Schellenberg1 1Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN USA 55905 2Department of Oncology, Mayo Clinic, Rochester, MN USA 55905 Background: Z-Endoxifen (ENDX), the active metabolite of tamoxifen (TAM) and a selective estrogen receptor modulator (SERM), exhibited high antitumor activity in endocrine-resistant hormone receptor-positive breast and other gynecologic cancer. ENDX has also been shown to be a protein kinase C (PKC) inhibitor. PKCs participate in diverse cellular functions and their activity is often elevated in breast tumors. Guided by mechanistic insights from our recently determined crystal structure of PKCβI, we sought to determine the effects of ENDX on PKCβI in a breast cancer cell line model. Methods: To determine how ENDX regulates PKC activity, we used a Z’LYTE kinase activity assay. This Fluorescence Resonance Energy Transfer (FRET) based biochemical method can detect differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. We probed changes in activity for conventional and novel PKCs, as well as the purified catalytic domain of conventional PKCs in vitro. Alongside, as kinase activity of PKC relies on its spatial assembly, therefore we studied its intracellular localization using live cell confocal imaging. MCF7 cells expressing YFP-tagged PKCβI were grown in a glass bottom chamber and treated with ENDX and other modulators for relevant time and doses for this study. Images were taken using Zeiss LSM 780 confocal laser scanning microscope and analyzed in Zeiss-ZEN microscope software and GraphPad Prism 9. Results: Our data from an in vitro kinase assay indicates that ENDX inhibits the kinase activity of conventional (PKCβI) and novel (PKC𝛿) PKC isoforms with a similar IC50, however PKCβI catalytic domain is less sensitive to ENDX. We also identified a multi-domain mechanism of PKC inhibition through an allosteric inhibitory mechanism. Our live cell imaging study demonstrated that ENDX promotes the recruitment of PKCβI to the cell membrane in both a dose and time-dependent manner. Moreover, this translocation can also be mitigated by co-treatment with inhibitors of the PKC-regulator PHLPP phosphatases. Conclusion: Taken together, these results suggest the allosteric effects of ENDX trigger PKCβI recruitment to the cell membrane, yet since ENDX also inhibits kinase activity it suggests that ENDX triggers a non-productive interaction with the enzyme and "breaks" the well-known mechanism of PKC activation upon binding the cell membrane. Furthermore, ENDX is likely to trigger dephosphorylation and ultimately degradation, suggesting that ENDX represents a new mechanistic basis for targeting and downregulating PKC in cancer cells. Hence, the current study provides an integrated pattern of highly specific treatments regimen that can exploit PKCβI as a repurposing clinical target in breast cancer. Citation Format: Sayantani Sarkar Bhattacharya, Taylor L. Witter, Anh T. Cong, Elizabeth S. Bruinsma, Swaathi Jayaraman, Matthew P. Goetz, John Hawse, Matthew Schellenberg. Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-03.

Protein kinase C isoforms mediate the formation of neutrophil extracellular traps.

Neutrophils release extracellular traps (NETs) in response to numerous pathogenic microbes as the last suicidal resource (NETosis) in the fight against infection. Apart from the host defense function, NETs play an essential role in the pathogenesis of various autoimmune, inflammatory and malignant diseases. Therefore, understanding the molecular mechanisms of NETosis is important for regulating the aberrant or excessive NET release. Protein kinase C (PKC) is a serine/threonine kinase which is involved in various neutrophil functions, however, little is known about its implication in NETosis activated by various physiological and pharmacological stimuli. Since there are conventional, novel and atypical PKC isoforms (α, βI, βII, δ, and ζ) found in human neutrophils, we investigated their impact in NETosis, oxidative burst and spreading applying pharmacological approach. Using specific inhibitors of PKC isoforms, we showed that PKCβ, PKCδ, and PKCζ are involved in the oxidative burst, spreading and NETosis activated by calcium ionophore A23187, while only PKCβ is implicated in these functions activated by phorbol 12-myristate 13-acetate (PMA). The data obtained in our study might help in the development of new drugs useful for the treatment of autoimmune and inflammatory diseases associated with NETs.

Protein Kinase‐C Inhibition Within the Periaqueductal Gray Counteracts the Development of Morphine Antinociceptive Tolerance

The ventrolateral periaqueductal gray (vlPAG) is an important midbrain region that regulates the descending pain modulatory system and is implicated in the development of antinociceptive tolerance to opioid analgesics. Protein Kinase-C (PKC) is a Ser/Thr kinase that phosphorylates the μ-opioid receptor (MOPr) following morphine activation. This phosphorylation is responsible for desensitization of the receptor, resulting in the termination of G-Protein signaling. It is speculated that PKC-mediated desensitization of the MOPr does not readily lead to the internalization of the receptor, thus contributing to tolerance. Previous studies have shown that intracerebroventricular administration of PKC inhibitors prevented the development of morphine tolerance in mice. Further, a non-specific PKC/GRK inhibitor was able to block the development of morphine tolerance in the vlPAG. However, the contribution of PKC to morphine tolerance in the vlPAG has not been directly explored. Using immunofluorescence staining, we observed increased phosphorylated PKC that is co-localized with the MOPr at the cell membrane of vlPAG neurons in morphine-tolerant animals. Further, we assessed the development of morphine antinociceptive tolerance following pre-treatment with the PKC inhibitor, Gö-7874, which targets conventional PKC isoforms. Repeated microinjections of the compound into the vlPAG of wild-type mice in combination with morphine led to a reduction in the development of morphine tolerance. Interestingly, repeated microinjections of Gö-7874 alone led to an enhancement of subsequent morphine antinociception. Lastly, morphine-induced changes in intracellular cAMP levels were assessed in-vitro, following pre-treatment with Gö-7874. This study contributes to the understanding of PKC-mediated MOPr desensitization processes leading to morphine tolerance.

Role of prelimbic cortex PKC and PKM\u03b6 in fear memory reconsolidation and persistence following reactivation

The persistence of newly acquired memories is supported by the activity of PKMζ, an atypical isoform of protein kinase C (PKC). Whether the activity of conventional and atypical PKC isoforms contributes to reactivated memories to persist is still unknown. Similarly, whether memory reactivation is a prerequisite for interventions to be able to change memory persistence is scarcely investigated. Based on the above, we examined the role of conventional and atypical PKC isoforms in the prelimbic cortex in reconsolidation and persistence of a reactivated contextual fear memory in male Wistar rats. It is shown that (i) inhibiting the PKC activity with chelerythrine or the PKMζ activity with ZIP impaired the persistence of a reactivated memory for at least 21 days; (ii) ZIP given immediately after memory reactivation affected neither the reconsolidation nor the persistence process. In contrast, when given 1 h later, it impaired the memory persistence; (iii) chelerythrine given immediately after memory reactivation impaired the reconsolidation; (iv) omitting memory reactivation prevented the chelerythrine- and ZIP-induced effects: (v) the ZIP action is independent of the time elapsed between its administration and the initial memory test. The results indicate that prelimbic cortex PKC and PKMζ are involved in memory reconsolidation and persistence.

Melatonin-mediated inhibition of Cav3.2 T-type Ca2+ channels induces sensory neuronal hypoexcitability through the novel protein kinase C-eta isoform.

Recent studies implicate melatonin in the antinociceptive activity of sensory neurons. However, the underlying mechanisms are still largely unknown. Here, we identify a critical role of melatonin in functionally regulating Cav3.2 T-type Ca2+ channels (T-type channel) in trigeminal ganglion (TG) neurons. Melatonin inhibited T-type channels in small TG neurons via the melatonin receptor 2 (MT2 receptor) and a pertussis toxin-sensitive G-protein pathway. Immunoprecipitation analyses revealed that the intracellular subunit of the MT2 receptor coprecipitated with Gαo . Both shRNA-mediated knockdown of Gαo and intracellular application of QEHA peptide abolished the inhibitory effects of melatonin. Protein kinase C (PKC) antagonists abolished the melatonin-induced T-type channel response, whereas inhibition of conventional PKC isoforms elicited no effect. Furthermore, application of melatonin increased membrane abundance of PKC-eta (PKCη ) while antagonism of PKCη or shRNA targeting PKCη prevented the melatonin-mediated effects. In a heterologous expression system, activation of MT2 receptor strongly inhibited Cav3.2 T-type channel currents but had no effect on Cav3.1 and Cav3.3 current amplitudes. The selective Cav3.2 response was PKCη dependent and was accompanied by a negative shift in the steady-state inactivation curve. Furthermore, melatonin decreased the action potential firing rate of small TG neurons and attenuated the mechanical hypersensitivity in a mouse model of complete Freund's adjuvant-induced inflammatory pain. These actions were inhibited by T-type channel blockade. Together, our results demonstrated that melatonin inhibits Cav3.2 T-type channel activity through the MT2 receptor coupled to novel Gβγ -mediated PKCη signaling, subsequently decreasing the membrane excitability of TG neurons and pain hypersensitivity in mice.

Protein kinase C downregulation induces senescence via FoxO3a inhibition in HCT116 and HEK293 cells

We investigated the impact of protein kinase C (PKC) on cellular senescence. The PKC activity and expression of conventional PKC (cPKC) and atypical PKC (aPKC) isoforms decreased during replicative senescence in IMR-90 cells. Forced inhibition of cPKC or aPKC induced the activation of senescence markers, including senescence-associated β-galactosidase activity and reactive oxygen species (ROS)-p53-p21Cip1/WAF1 axis in HCT116 and HEK293 cells. PKC inhibition triggered the nuclear exportation of FoxO3a via stimulation of AKT-mediated phosphorylation of FoxO3a, and thereby decreased the transcription of FoxO3a target genes. Conversely, ectopic expression of the PKC isoforms led to stimulation of the nuclear import of FoxO3a and expression of the FoxO3a target genes. Ectopic FoxO3a expression attenuated ROS accumulation and senescent phenotypes induced by PKC inhibition. Therefore, this study suggests for the first time that downregulation of PKC induces senescence through the AKT-FoxO3a-ROS-p53-p21Cip1/WAF1 pathway in HCT116 and HEK293 cells.

Aeromonas hydrophila-induced alterations in cytosolic calcium activate pro-apoptotic cPKC-MEK1/2-TNFα axis in infected headkidney macrophages of Clarias gariepinus

Alterations in intracellular-calcium (Ca2+)i homeostasis is critical to Aeromonas hydrophila-induced headkidney macrophages (HKM) apoptosis of Clarias gariepinus, though the implications are poorly understood. Here, we describe the role of intermediate molecules of Ca2+−signaling pathway that are involved in HKM apoptosis. We observed phosphoinositide-3-kinase/phospholipase C is critical for (Ca2+)i release in infected HKM. Heightened protein kinase-C (PKC) activity and phosphorylation of MEK1/2-ERK1/2 was noted which declined in presence of 2-APB, Go6976 and PD98059, inhibitors to IP3-receptor, conventional PKC isoforms (cPKC) and MEK1/2 respectively implicating Ca2+/cPKC/MEK-ERK1/2 axis imperative in A. hydrophila-induced HKM apoptosis. Significant tumor necrosis factor-α (TNFα) production and its subsequent reduction in presence of MEK-ERK1/2 inhibitor U0126 suggested TNFα production downstream to cPKC-mediated signaling via MEK1/2-ERK1/2 pathway. RNAi and inhibitor studies established the role of TNFα in inducing caspase-8-mediated apoptosis of infected HKM. We conclude, alterations in A. hydrophila-induced (Ca2+)i alterations activate cPKC-MEK1/2-ERK1/2-TNFα signaling cascade triggering HKM apoptosis.

Imaging Regulatory Lipids and Protein Kinase C at Sites of Exocytosis

The control of exocytosis is physiologically essential. In vitro SNARE proteins are sufficient to drive model membrane fusion, but in cells there are over twenty additional proteins and lipids that work together to drive efficient, fast, and timely release of vesicular cargo. Many of these important accessory proteins are controlled via phosphorylation, and protein kinase C (PKC) has previously been linked to the modulation of exocytosis. We use total internal reflection fluorescence microscopy to observe the spatiotemporal dynamics of dozens of proteins and lipids relative to single sites of exocytosis in living endocrine INS-1 cells. INS-1 cells are a model system for insulin secretion. We use a vesicle cargo marker NPY-GFP to identify where exocytosis occurs and tag proteins-of-interest with a red fluorescent protein. Lipid species are visualized using lipid sensors: fluorescent proteins fused to protein domains with known specificity for single lipids. After stimulating exocytosis using ionomycin, we observe a transient accumulation of PIP2- and DAG-sensors at exocytic sites centered on the time of membrane fusion and only lasting a few seconds. Intriguingly, we observe a concomitant recruitment of conventional PKC isoforms with kinetics broadly similar to PIP2 and DAG. Novel and atypical PKCs do not appear to be recruited. PIP2, DAG, and PKC recruitment only occur at sites of exocytosis and not at control docked-vesicles nearby to sites of exocytosis. PKC is known to target several critical exocytic proteins, including munc18 and dynamin-1, and we are currently investigating whether these targets are phosphorylated in response to the recruitment of PKC that we observe. These data suggest that a regulatory lipid cascade may recruit and activate PKC to sites of exocytosis to regulate membrane fusion or cargo release.

Suppression of PKC-α attenuates TNF-α-evoked cerebral barrier breakdown via regulations of MMP-2 and plasminogen–plasmin system

Ischaemic stroke, accompanied by neuroinflammation, impairs blood–brain barrier integrity through a complex mechanism involving both protein kinase C (PKC) and urokinase. Using an in vitro model of human blood–brain barrier (BBB) composed of brain microvascular endothelial cells (HBMEC) and astrocytes, this study assessed the putative roles of these elements in BBB damage evoked by enhanced availability of pro-inflammatory cytokine, TNF-α. Treatment of HBMEC with TNF-α significantly increased the mRNA and protein expressions of all plasminogen–plasmin system (PPS) components, namely tissue plasminogen activator, urokinase, urokinase plasminogen activator receptor and plasminogen activator inhibitor-1 and also the activities of urokinase, total PKC and extracellular MMP-2. Inhibition of urokinase by amiloride abated the effects of TNF-α on BBB integrity and MMP-2 activity without affecting that of total PKC. Conversely, pharmacological inhibition of conventional PKC isoforms dramatically suppressed TNF-α-induced overactivation of urokinase. Knockdown of PKC-α gene via specific siRNA in HBMEC suppressed the stimulatory effects of TNF-α on protein expression of all PPS components, MMP-2 activity, DNA fragmentation rates and pro-apoptotic caspase-3/7 activities. Establishment of co-cultures with BMEC transfected with PKC-α siRNA attenuated the disruptive effects of TNF-α on BBB integrity and function. This was partly due to elevations observed in expression of a tight junction protein, claudin-5 and partly to prevention of stress fibre formation. In conclusion, specific inhibition of PKC-α in cerebral conditions associated with exaggerated release of pro-inflammatory cytokines, notably TNF-α may be of considerable therapeutic value and help maintain endothelial cell viability, appropriate cytoskeletal structure and basement membrane.

Phorbol Myristate Acetate Suppresses Breast Cancer Cell Growth via Down‐regulation of P‐Rex1 Expression

Phosphatidylinositol‐3,4,5‐trisphosphate‐dependent Rac exchange factor 1 (P‐Rex1) is highly overexpressed in estrogen receptor‐positive and ErbB2‐overexpressing human luminal breast tumors. The present study was to investigate the role and mechanism underlying phorbol‐12‐myristate‐13‐acetate (PMA), a protein kinase C (PKC) activator, down‐regulation of P‐Rex1 in breast cancer cells. P‐Rex1 mRNA and protein expression were determined by real‐time PCR and western blot analysis, respectively. Cell growth was analyzed by counting cells number and 5‐bromo‐2′‐deoxyuridine (BrdU) cell proliferation assay. The translocation of PKC was examined by immunofluorescence staining. Our results showed that treatment with PMA caused a time‐ and concentration‐dependent decrease in P‐Rex1 protein levels in breast cancer MCF‐7 and BT‐474 cells with a maximum reduction of 87.2 ± 1.1 and 57.0 ± 8.6%, respectively, at a concentration of 10 ng/ml. Interestingly, either PMA treatment or silence of P‐Rex1 by siRNA inhibits MCF‐7 and BT‐474 cell growth by 60–80%, which can be attenuated by restoring P‐Rex1 expression. MCF‐7 and BT‐474 cells express PMA‐sensitive conventional (α and β) and novel (δ, ɛ and η) PKC isoforms. Immunofluorescence staining analysis of breast cancer cells showed translocation of PKCs from cytosol to the plasma membrane and/or nucleus, an indication of their activation upon PMA stimulation. The PKC general inhibitor Gö6983, but not the conventional PKC isoform inhibitor Gö6976, blocked PMA inhibition of P‐Rex1 expression and breast cancer cell growth. Expression of a constitutively active PKCɛ mutant suppressed P‐Rex1 expression and breast cancer cell growth, which was blocked by Gö6983 treatment. In contrast, expression of wild‐type PKCɛ, wild‐type or constitutively active PKCδ and PKCη had no effect. Together, our data suggest that PMA suppresses breast cancer cell growth via P‐Rex1 down‐regulation and PKCɛ activation plays a key role in this regulation. Our study may provide a novel strategy for development of chemotherapeutic agents for P‐Rex1‐overexpressing breast cancer patients that develop resistance to anti‐estrogen and/or anti‐ErbB2 therapies.Support or Funding InformationSupported by grants from NIH (5P20GM103489 and R01HL116849), Nebraska State LB595 research program and the National Natural Science Foundation of China (31100595 and 31300683).

Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-releasing Hormone

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1-7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of Gq/11 proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Gö 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer.

Opposing roles of conventional and novel PKC isoforms in Hippo-YAP pathway regulation

LETTER TO THE EDITOR Cell Research (2015) 25:985-988. © 2015 IBCB, SIBS, CAS All rights reserved 1001-0602/15 $ 32.00 www.nature.com/cr npg Opposing roles of conventional and novel PKC isoforms in Hippo-YAP pathway regulation Cell Research (2015) 25:985-988. doi:10.1038/cr.2015.88; published online 24 July 2015 Dear Editor, The Hippo-YAP pathway is an evolutionally conserved signaling module that controls tissue growth during de- velopment and its dysregulation causes cancer [1]. Core components of the Hippo pathway include a kinase cas- cade comprising MST1/2 and LATS1/2 kinases, in which MST1/2 phosphorylates and activates LATS1/2 [2]. The major downstream effectors of the Hippo pathway are the transcriptional co-activators YAP and TAZ, which are phosphorylated and inhibited by LATS1/2 [3, 4]. Un- phosphorylated YAP/TAZ localizes in the nucleus and promotes target gene expression through binding to the TEAD family transcription factors [5, 6]. Protein kinase C (PKC) controls a broad range of biological processes and can be classified into three sub-groups based on sequence homology and activation mechanisms: the Ca 2+ - and dia- cylglycerol (DAG)-dependent conventional PKC (cPKC α, βI, βII, and γ), the DAG-dependent novel PKC (nPKC δ, θ, e, and η), and the Ca 2+ - and DAG-independent atypical PKC (aPKC ι and ζ) [7]. Recent studies have established that extracellular diffusible signals act through G-protein coupled receptors (GPCRs) to regulate the Hippo-YAP pathway [8, 9]. PKC represents one of the major effectors downstream of GPCRs (especially Gq/11-coupled recep- tors). This led us to investigate whether PKC regulates the Hippo-YAP pathway. We used the DAG analog TPA to activate PKC in HE- K293A cells and observed that TPA induced a rapid and robust YAP dephosphorylation as determined by western blotting using the phospho-specific (serine 127) YAP an- tibody and by differential electrophoretic mobility shift on phos-tag-containing gels. Similarly, TPA also induced TAZ dephosphorylation as indicated by a faster electro- phoretic migration (Figure 1A). TPA-induced YAP de- phosphorylation was also observed in HeLa and U251MG cells (Supplementary information, Figure S1A and S1B). Phosphorylated YAP localizes in the cytoplasm, whereas dephosphorylated YAP translocates to the nucleus to pro- mote target gene expression. Consistent with this model, TPA induced YAP nuclear localization in HEK293A cells (Figure 1B). Moreover, GO6983, an inhibitor of both cPKC and nPKC, effectively blocked TPA-induced YAP/ TAZ dephosphorylation (Figure 1A). These observations suggest a role of PKC in YAP/TAZ activation. Next, we examined whether activation of PKC by physiological stimuli would activate YAP/TAZ. We found that addition of acetylcholine to U251MG cells, which express the Gq/11-coupled muscarinic acetylcholine re- ceptor M3, resulted in significant YAP dephosphorylation (Figure 1C). Inhibition of either M3 receptor by 4-DAMP or PKC by GO6983 suppressed acetylcholine-induced YAP dephosphorylation (Figure 1C), suggesting that YAP activation by acetylcholine is mediated by the M3 recep- tor and PKC. Moreover, Gq/11 knockdown in U251MG cells suppressed YAP dephosphorylation by acetylcho- line (Supplementary information, Figure S1C). Collec- tively, these data indicate that PKC acts downstream of Gq/11-coupled receptors to activate YAP. In the efforts to examine the effect of TPA on YAP/ TAZ phosphorylation in different cell types, we were surprised that TPA induced a dramatic YAP/TAZ phos- phorylation in Swiss3T3 cells (Figure 1D), a response completely opposite to what was observed in HEK293A and several other cell lines (Figure 1A and Supplementa- ry information, Figure S1A and S1B). Notably, TPA-in- duced YAP/TAZ phosphorylation could be blocked by GO6983 (Figure 1D), indicating that PKC activation was also responsible for the increase of YAP/TAZ phosphory- lation in Swiss3T3 cells. Similar effects were observed in MEF cells and the lung cancer A549 cells (Supplementary information, Figure S1D and S1E). Consistent with the increased YAP phosphorylation, TPA treatment promoted a YAP cytoplasmic localization in MEF cells (Figure 1E). Although seemingly paradoxical, the above results are very interesting and show that PKC can either positively or negatively regulate YAP activity in a cell-type-depen- dent manner. The opposing effects of TPA on YAP phosphorylation observed in different cell types prompted us to speculate that different PKC isoforms may exert opposite effects on YAP regulation. To test this, individual cPKC or nPKC isoform was co-transfected with YAP into HEK293A cells and TPA-induced YAP phosphorylation/dephosphoryla-

Role of protein kinase C in agonist-induced desensitization of 5-HT1A receptor coupling to calcium channels in F11 cells

The 5-Hydroxytriptamine 1A receptor (5-HT1A) is expressed both as a pre- and post-synaptic receptor in neurons. The presynaptic receptor preferentially desensitizes compared to post-synaptic receptors, suggesting different underlying mechanisms of agonist-induced desensitization. Using F11 cells as a model of post-synaptic neurons, the present study examined the role of protein kinase C (PKC) and protein kinase A (PKA) in desensitization of the 5-HT1A-receptor by agonist. Desensitization in whole cell experiments was dependent on internal [Ca2+] and was blocked by chelation of intracellular Ca2+. Using the perforated patch technique, desensitization was reduced when Ba2+ was used as the conducting cation. Selective inhibitors of conventional PKC isoforms prevented 5-HT-induced desensitization, whereas an inhibitor of PKA did not. In cells in which 3 PKC/PKA sites located in the third intracellular loop (i3) of the 5-HT1A receptor were mutated (i3, T229A-S253G-T343A), 5-HT-mediated desensitization was reduced (and abolished in the absence of intracellular Ca2+). In cells in which a fourth mutation was added (T149 in the second i2 loop), the cells responded similarly to the triple mutants suggesting that phosphorylation of T149 does not contribute greatly to the desensitization induced by 5-HT-mediated activation of PKC. Thus agonist-induced uncoupling of the 5-HT1A-receptor is PKC-dependent, but requires a different set of phosphorylation sites than phorbol ester-mediated PKC activation, suggesting differential recruitment of PKC. Furthermore, these studies reveal that 5-HT1A-receptor desensitization utilizes a different kinase in F11 cells and serotonergic neurons, which may in part account for their differential sensitivity in vivo.

Phorbol ester reduces ethanol excitation of dopaminergic neurons of the ventral tegmental area: involvement of protein kinase C theta

Neurons of the ventral tegmental area (VTA) play a key role in the rewarding and reinforcing effects of drugs of abuse, including alcohol. Ethanol directly increases the firing rate of dopaminergic (DAergic) VTA neurons, but modulation of the firing rate of DAergic VTA neurons can be controlled by a number of factors, including some that are under the control of protein kinase C (PKC). Application of phorbol esters activates PKC and the present study assessed the effect of a phorbol ester, phorbol 12-myristate 13-acetate (PMA), on ethanol-induced excitation of DA VTA neurons. Ethanol-induced excitation of DAergic VTA neurons was reduced significantly in the presence of PMA. This action of PMA was antagonized by chelerythrine chloride, a non-selective antagonist of PKC, but not by moderate concentrations of antagonists of conventional PKC isoforms (Gö6976 and Gö6983). A PKC δ/θ inhibitor antagonized PMA-induced reduction of ethanol excitation. Since PKCδ antagonist Gö6983 did not antagonize the effect of PMA on ethanol excitation, the PMA reduction of ethanol excitation is most likely to be mediated by PKCθ. Antagonists of intracellular calcium pathways were ineffective in antagonizing PMA action on ethanol excitation, consistent with the lack of calcium dependence of PKCθ. In summary, ethanol-induced excitation of VTA neurons is attenuated in the presence of PMA, and this attenuation appears to be mediated by PKCθ. This novel mechanism for interfering with ethanol activation of reward-related neurons could provide a new target for pharmacotherapy to ameliorate alcoholism.

The effects of a 1.8 GHz continuous electromagnetic fields on mucociliary transport of human nasal mucosa

The aim of this study was to investigate the effects of a 1.8 GHz continuous electromagnetic fields (EMF) on human nasal mucociliary transport, and to determine the pathophysiology of ciliary beat frequency (CBF) during an EMF-induced change. Human nasal mucosa cells were exposed to a 1.8 GHz EMF (SAR=1.0 W/kg), and CBF was analyzed using an optical flow technique with the peak detection method. The 1.8 GHz-exposed group showed a decreased CBF when compared to the control group. In the cytotoxicity assay, difference in survival rates was not found between the two groups. In the EMF-exposed group, protein kinase C (PKC) activity was increased during a PKC activity assay. The broad PKC inhibitor, Calphostin C abolished the EMF-induced decrease of CBF. The EMF-induced decrease of CBF was abolished by GF 109203X, a novel PKC (nPKC) isoform inhibitor, whereas the decrease was not attenuated by Gö-6976, a specific inhibitor of conventional PKC (cPKC) isoform. EMF may inhibit CBF via an nPKC-dependent mechanism. Therefore, we have confirmed that EMF could decrease CBF by increasing PKC activity.

Abstract 287: Identification of a Novel PKCα Isoform in Heart Transcriptome via RNA Deep Sequencing

The Protein kinase C (PKC) family of Ca2+ and /or lipid-activated AGC kinases are important regulators of multiple cellular responses including cell growth and programmed cell death. Among these, PKCα is the predominant conventional PKC isoform in heart and has been implicated to have a major impact on cardiac function, hypertrophic response and heart failure. Earlier studies have revealed that the conserved AGC-C-terminal tail containing active site cleft is responsible for regulating AGC kinase activity. The phosphorylation events in AGC-C-terminal tail mediate the activity of kinase by regulating kinase maturation, translocation and substrate interaction. In a genome-wide RNA-Seq analysis in mouse heart, we have identified a novel isoform of PKCα-- due to the insertion of a novel exon in its C-terminal tail. This novel exon has a highly conserved sequence from mouse to human, suggesting a conserved role in PKCα regulation. Tissue analysis showed that this PKCα isoform with Novel Exon (PKCαNE) has enriched expression in heart and skeletal muscle, indicating the involvement of a muscle specific alternative splicing event. The insertion of this novel exon lies between the kinase C-Lobe Tether and Active Site Tether. Biochemistry study in cells showed that both PKCα and PKCαNE were activated and translocated from cytosol to membrane and nucleus in response to PMA. However, upon IGF-1 treatment, PKCαNE showed no activation and could not translocate to either membrane or nucleus in contrast to PKCα. To further explore the mechanism for PKCαNE activation, we have determined the phosphorylation events of this novel isoform. Interestingly, the PKCαNE showed a decreased phosphorylation level at the Turn Motif , which could directly impact its activation and maturation. In summary, we have identified a novel isoform of PKCα that is enriched in heart and skeletal muscle. The PKCαNE isoform has different activation profile and its biological significance in cell signaling in physiology and diseases remains to be further explored.

Desmosomal adhesiveness is developmentally regulated in the mouse embryo and modulated during trophectoderm migration

During embryonic development tissues remain malleable to participate in morphogenetic movements but on completion of morphogenesis they must acquire the toughness essential for independent adult life. Desmosomes are cell–cell junctions that maintain tissue integrity especially where resistance to mechanical stress is required. Desmosomes in adult tissues are termed hyper-adhesive because they adhere strongly and are experimentally resistant to extracellular calcium chelation. Wounding results in weakening of desmosomal adhesion to a calcium-dependent state, presumably to facilitate cell migration and wound closure. Since desmosomes appear early in mouse tissue development we hypothesised that initial weak adhesion would be followed by acquisition of hyper-adhesion, the opposite of what happens on wounding. We show that epidermal desmosomes are calcium-dependent until embryonic day 12 (E12) and become hyper-adhesive by E14. Similarly, trophectodermal desmosomes change from calcium-dependence to hyper-adhesiveness as blastocyst development proceeds from E3 to E4.5. In both, development of hyper-adhesion is accompanied by the appearance of a midline between the plasma membranes supporting previous evidence that hyper-adhesiveness depends on the organised arrangement of desmosomal cadherins. By contrast, adherens junctions remain calcium-dependent throughout but tight junctions become calcium-independent as desmosomes mature. Using protein kinase C (PKC) activation and PKCα−/− mice, we provide evidence suggesting that conventional PKC isoforms are involved in developmental progression to hyper-adhesiveness. We demonstrate that modulation of desmosomal adhesion by PKC can regulate migration of trophectoderm. It appears that tissue stabilisation is one of several roles played by desmosomes in animal development.

Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na+channels

Na(+) current derived from expression of the cardiac isoform SCN5A is reduced by receptor-mediated or direct activation of protein kinase C (PKC). Previous work has suggested a possible role for loss of Na(+) channels at the plasma membrane in this effect, but the results are controversial. In this study, we tested the hypothesis that PKC activation acutely modulates the intracellular distribution of SCN5A channels and that this effect can be visualized in living cells. In human embryonic kidney cells that stably expressed SCN5A with green fluorescent protein (GFP) fused to the channel COOH-terminus (SCN5A-GFP), Na(+) currents were suppressed by an exposure to PKC activation. Using confocal microscopy, colocalization of SCN5A-GFP channels with the plasma membrane under control and stimulated conditions was quantified. A separate population of SCN5A channels containing an extracellular epitope was immunolabeled to permit temporally stable labeling of the plasma membrane. Our results demonstrated that Na(+) channels were preferentially trafficked away from the plasma membrane by PKC activation, with a major contribution by Ca(2+)-sensitive or conventional PKC isoforms, whereas stimulation of protein kinase A (PKA) had the opposite effect. Removal of the conserved PKC site Ser(1503) or exposure to the NADPH oxidase inhibitor apocynin eliminated the PKC-mediated effect to alter channel trafficking, indicating that both channel phosphorylation and ROS were required. Experiments using fluorescence recovery after photobleaching demonstrated that both PKC and PKA also modified channel mobility in a manner consistent with the dynamics of channel distribution. These results demonstrate that the activation of protein kinases can acutely regulate the intracellular distribution and molecular mobility of cardiac Na(+) channels in living cells.