Yasuhito Shirai

Three Distinct Mechanisms for Translocation and Activation of the δ Subspecies of Protein Kinase C

ABSTRACT We expressed δ subspecies of protein kinase C (δ-PKC) fused with green fluorescent protein (GFP) in CHO-K1 cells and observed the movement of this fusion protein in living cells after three different stimulations. The δ-PKC–GFP fusion protein had enzymological characteristics very similar to those of the native δ-PKC and was present throughout the cytoplasm in CHO-K1 cells. ATP at 1 mM caused a transient translocation of δ-PKC–GFP to the plasma membrane approximately 30 s after the stimulation and a sequent retranslocation to the cytoplasm within 3 min. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 μM), induced a slower translocation of δ-PKC–GFP, and the translocation was unidirectional. Concomitantly, the kinase activity of δ-PKC–GFP was increased by these two stimulations, when the kinase activity of the immunoprecipitated δ-PKC–GFP was measured in vitro in the absence of PKC activators such as phosphatidylserine and diacylglycerol. Hydrogen peroxide (H2O2; 5 mM) failed to translocate δ-PKC–GFP but increased its kinase activity more than threefold. δ-PKC–GFP was strongly tyrosine phosphorylated when treated with H2O2 but was tyrosine phosphorylated not at all by ATP stimulation and only slightly by TPA treatment. Both TPA and ATP induced the translocation of δ-PKC–GFP even after treatment with H2O2. Simultaneous treatment with TPA and H2O2 further activated δ-PKC–GFP up to more than fivefold. TPA treatment of cells overexpressing δ-PKC–GFP led to an increase in the number of cells in G2/M phase and of dikaryons, while stimulation with H2O2 increased the number of cells in S phase and induced no significant change in cell morphology. These results indicate that at least three different mechanisms are involved in the translocation and activation of δ-PKC.

Ceramide-induced Apoptosis by Translocation, Phosphorylation, and Activation of Protein Kinase Cδ in the Golgi Complex

Protein kinase C (PKC), a Ca2+/phospholipid-dependent protein kinase, is known as a key enzyme in various cellular responses, including apoptosis. However, the functional role of PKC in apoptosis has not been clarified. In this study, we focused on the involvement of PKCδ in ceramide-induced apoptosis in HeLa cells and examined the importance of spatiotemporal activation of the specific PKC subtype in apoptotic events. Ceramide-induced apoptosis was inhibited by the PKCδ-specific inhibitor rottlerin and also was blocked by knockdown of endogenous PKCδ expression using small interfering RNA. Ceramide induced the translocation of PKCδ to the Golgi complex and the concomitant activation of PKCδ via phosphorylation of Tyr311 and Tyr332 in the hinge region of the enzyme. Unphosphorylatable PKCδ (mutants Y311F and Y332F) could translocate to the Golgi complex in response to ceramide, suggesting that tyrosine phosphorylation is not necessary for translocation. However, ceramide failed to activate PKCδ lacking the C1B domain, which did not translocate to the Golgi complex, but could be activated by tyrosine phosphorylation. These findings suggest that ceramide translocates PKCδ to the Golgi complex and that PKCδ is activated by tyrosine phosphorylation in the compartment. Furthermore, we utilized species-specific knockdown of PKCδ by small interfering RNA to study the significance of phosphorylation of Tyr311 and Tyr332 in PKCδ for ceramide-induced apoptosis and found that phosphorylation of Tyr311 and Tyr332 is indispensable for ceramide-induced apoptosis. We demonstrate here that the targeting mechanism of PKCδ, dual regulation of both its activation and translocation to the Golgi complex, is critical for the ceramide-induced apoptotic event.

Reactive Oxygen Species Derived from NOX1/NADPH Oxidase Enhance Inflammatory Pain

The involvement of reactive oxygen species (ROS) in an augmented sensitivity to painful stimuli (hyperalgesia) during inflammation has been suggested, yet how and where ROS affect the pain signaling remain unknown. Here we report a novel role for the superoxide-generating NADPH oxidase in the development of hyperalgesia. In mice lacking Nox1 (Nox1−/Y), a catalytic subunit of NADPH oxidase, thermal and mechanical hyperalgesia was significantly attenuated, whereas no change in nociceptive responses to heat or mechanical stimuli was observed. In dorsal root ganglia (DRG) neurons of Nox1+/Y, pretreatment with chemical mediators bradykinin, serotonin, or phorbol 12-myristate 13-acetate (PMA) augmented the capsaicin-induced calcium increase, whereas this increase was significantly attenuated in DRG neurons of Nox1−/Y. Concomitantly, PMA-induced translocation of PKCε was markedly perturbed in Nox1−/Y or Nox1+/Y DRG neurons treated with ROS-scavenging agents. In cells transfected with tagged PKCε, hydrogen peroxide induced translocation and a reduction in free sulfhydryls of full-length PKCε but not of the deletion mutant lacking the C1A domain. These findings indicate that NOX1/NADPH oxidase accelerates the translocation of PKCε in DRG neurons, thereby enhancing the TRPV1 activity and the sensitivity to painful stimuli.

Subtype-Specific Translocation of the δ Subtype of Protein Kinase C and Its Activation by Tyrosine Phosphorylation Induced by Ceramide in HeLa Cells

ABSTRACT We investigated the functional roles of ceramide, an intracellular lipid mediator, in cell signaling pathways by monitoring the intracellular movement of protein kinase C (PKC) subtypes fused to green fluorescent protein (GFP) in HeLa living cells. C2-ceramide but not C2-dihydroceramide induced translocation of δPKC-GFP to the Golgi complex, while αPKC- and ζPKC-GFP did not respond to ceramide. The Golgi-associated δPKC-GFP induced by ceramide was further translocated to the plasma membrane by phorbol ester treatment. Ceramide itself accumulated to the Golgi complex where δPKC was translocated by ceramide. Gamma interferon also induced the δPKC-specific translocation from the cytoplasm to the Golgi complex via the activation of Janus kinase and Mg2+-dependent neutral sphingomyelinase. Photobleaching studies showed that ceramide does not evoke tight binding of δPKC-GFP to the Golgi complex but induces the continuous association and dissociation of δPKC with the Golgi complex. Ceramide inhibited the kinase activity of δPKC-GFP in the presence of phosphatidylserine and diolein in vitro, while the kinase activity of δPKC-GFP immunoprecipitated from ceramide-treated cells was increased. The immunoprecipitated δPKC-GFP was tyrosine phosphorylated after ceramide treatment. Tyrosine kinase inhibitor abolished the ceramide-induced activation and tyrosine phosphorylation of δPKC-GFP. These results suggested that gamma interferon stimulation followed by ceramide generation through Mg2+-dependent sphingomyelinase induced δPKC-specific translocation to the Golgi complex and that translocation results in δPKC activation through tyrosine phosphorylation of the enzyme.

An essential role of the aPKC-Aurora A-NDEL1 pathway in neurite elongation by modulation of microtubule dynamics.

Orchestrated remodelling of the cytoskeketon is prominent during neurite extension. In contrast with the extensive characterization of actin filament regulation, little is known about the dynamics of microtubules during neurite extension. Here we identify an atypical protein kinase C (aPKC)–Aurora A–NDEL1 pathway that is crucial for the regulation of microtubule organization during neurite extension. aPKC phosphorylates Aurora A at Thr 287 (T287), which augments interaction with TPX2 and facilitates activation of Aurora A at the neurite hillock, followed by phosphorylation of NDEL1 at S251 and recruitment. Suppression of aPKC, Aurora A or TPX2, or disruption of Ndel1, results in severe impairment of neurite extension. Analysis of microtubule dynamics with a microtubule plus-end marker revealed that suppression of the aPKC–Aurora A–NDEL1 pathway resulted in a significant decrease in the frequency of microtubule emanation from the microtubule organizing centre (MTOC), suggesting that Aurora A acts downstream of aPKC. These findings demonstrate a surprising role of aPKC–Aurora A–NDEL1 pathway in microtubule remodelling during neurite extension.

Subtype-specific Translocation of Diacylglycerol Kinase α and γ and Its Correlation with Protein Kinase C

We examined the translocation of diacylglycerol kinase (DGK) α and γ fused with green fluorescent protein in living Chinese hamster ovary K1 cells (CHO-K1) and investigated temporal and spatial correlations between DGK and protein kinase C (PKC) when both kinases are overexpressed. DGKα and γ were present throughout the cytoplasm of CHO-K1 cells. Tetradecanoylphorbol 13-acetate (TPA) induced irreversible translocation of DGKγ, but not DGKα, from the cytoplasm to the plasma membrane. The (TPA)-induced translocation of DGKγ was inhibited by the mutation of C1A but not C1B domain of DGKγ and was not inhibited by staurosporine. Arachidonic acid induced reversible translocation of DGKγ from the cytoplasm to the plasma membrane, whereas DGKα showed irreversible translocation to the plasma membrane and the Golgi network. Purinergic stimulation induced reversible translocation of both DGKγ and α to the plasma membrane. The timing of the ATP-induced translocation of DGKγ roughly coincided with that of PKCγ re-translocation from the membrane to the cytoplasm. Furthermore, re-translocation of PKCγ was obviously hastened by co-expression with DGKγ and was blocked by an inhibitor of DGK (R59022). These results indicate that DGK shows subtype-specific translocation depending on extracellular signals and suggest that PKC and DGK are orchestrated temporally and spatially in the signal transduction.

Enzymological Analysis of Mutant Protein Kinase Cγ Causing Spinocerebellar Ataxia Type 14 and Dysfunction in Ca2+ Homeostasis

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease caused by mutations in protein kinase Cγ (PKCγ). Interestingly, 18 of 22 mutations are concentrated in the C1 domain, which binds diacylglycerol and is necessary for translocation and regulation of PKCγ kinase activity. To determine the effect of these mutations on PKCγ function and the pathology of SCA14, we investigated the enzymological properties of the mutant PKCγs. We found that wild-type PKCγ, but not C1 domain mutants, inhibits Ca2+ influx in response to muscarinic receptor stimulation. The sustained Ca2+ influx induced by muscarinic receptor ligation caused prolonged membrane localization of mutant PKCγ. Pharmacological experiments showed that canonical transient receptor potential (TRPC) channels are responsible for the Ca2+ influx regulated by PKCγ. Although in vitro kinase assays revealed that most C1 domain mutants are constitutively active, they could not phosphorylate TRPC3 channels in vivo. Single molecule observation by the total internal reflection fluorescence microscopy revealed that the membrane residence time of mutant PKCγs was significantly shorter than that of the wild-type. This fact indicated that, although membrane association of the C1 domain mutants was apparently prolonged, these mutants have a reduced ability to bind diacylglycerol and be retained on the plasma membrane. As a result, they fail to phosphorylate TRPC channels, resulting in sustained Ca2+ entry. Such an alteration in Ca2+ homeostasis and Ca2+-mediated signaling in Purkinje cells may contribute to the neurodegeneration characteristic of SCA14.

A role for PKC-ε in FcγR-mediated phagocytosis by RAW 264.7 cells

Protein kinase C (PKC) plays a prominent role in immune signaling, and the paradigms for isoform selective signaling are beginning to be elucidated. Real-time microscopy was combined with molecular and biochemical approaches to demonstrate a role for PKC-ɛ in Fcγ receptor (FcγR)–dependent phagocytosis. RAW 264.7 macrophages were transfected with GFP-conjugated PKC isoforms, and GFP movement was followed during phagocytosis of fluorescent IgG–opsonized beads. PKC-ɛ, but not PKC-δ, concentrated around the beads. PKC-ɛ accumulation was transient; apparent as a “flash” on target ingestion. Similarly, endogenous PKC-ɛ was specifically recruited to the nascent phagosomes in a time-dependent manner. Overexpression of PKC-ɛ, but not PKC-α, PKC-δ, or PKC-γ enhanced bead uptake 1.8-fold. Additionally, the rate of phagocytosis in GFP PKC-ɛ expressors was twice that of cells expressing GFP PKC-δ. Expression of the regulatory domain (ɛRD) and the first variable region (ɛV1) of PKC-ɛ inhibited uptake, whereas the corresponding PKC-δ region had no effect. Actin polymerization was enhanced on expression of GFP PKC-ɛ and ɛRD, but decreased in cells expressing ɛV1, suggesting that the ɛRD and ɛV1 inhibition of phagocytosis is not due to effects on actin polymerization. These results demonstrate a role for PKC-ɛ in FcγR-mediated phagocytosis that is independent of its effects on actin assembly.

Localization of group V phospholipase A2 in caveolin-enriched granules in activated P388D1 macrophage-like cells.

In murine P388D1 macrophages, the generation of prostaglandin E2 in response to long term stimulation by lipopolysaccharide involves the action of Group V secreted phospholipase A2 (PLA2), Group IV cytosolic PLA2 (cPLA2), and cyclooxygenase-2 (COX-2). There is an initial activation of cPLA2 that induces expression of Group V PLA2, which in turn induces both the expression of COX-2 and most of the arachidonic acid substrate for COX-2-dependent prostaglandin E2 generation. Because Group V PLA2 is a secreted enzyme, it has been assumed that after cellular stimulation, it must be released to the extracellular medium and re-associates with the outer membrane to release arachidonic acid from phospholipids. In the present study, confocal laser scanning microscopy experiments utilizing both immunofluorescence and green fluorescent protein-labeled Group V PLA2 shows that chronic exposure of the macrophages to lipopolysaccharide results in Group V PLA2 being associated with caveolin-2-containing granules close to the perinuclear region. Heparin, a cell-impermeable complex carbohydrate with high affinity for Group V PLA2, blocks that association, suggesting that the granules are formed by internalization of the Group V sPLA2 previously associated with the outer cellular surface. Localization of Group V PLA2 in perinuclear granules is not observed if the cells are treated with the Group IV PLA2 inhibitor methyl arachidonyl fluorophosphonate, confirming the important role for Group IV PLA2 in the activation process. Cellular staining with antibodies against COX-2 reveals the presence of COX-2-rich granules in close proximity to those containing Group V PLA2. Collectively, these results suggest that encapsulation of Group V PLA2 into granules brings the enzyme to the perinuclear envelope during cell activation where it may be closer to Group IV PLA2 and COX-2 for efficient prostaglandin synthesis.

Superoxide production at phagosomal cup/phagosome through βI protein kinase C during FcγR-mediated phagocytosis in microglia

Protein kinase C (PKC) plays a prominent role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcγR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. βI PKC, εPKC, and diacylglycerol kinase (DGK) β dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47phox, an essential cytosolic component of NADPH oxidase and a substrate for βI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O2−) production was profoundly inhibited by Gö6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O2− production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of βI PKC is involved in O2− production, and that O2− production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKβ plays a prominent role in regulation of O2− production during FcγR-mediated phagocytosis.