We expressed the γ-subspecies of protein kinase C (γ-PKC) fused with green fluorescent protein (GFP) in various cell lines and observed the movement of this fusion protein in living cells under a confocal laser scanning fluorescent microscope. γ-PKC–GFP fusion protein had enzymological properties very similar to that of native γ-PKC. The fluorescence of γ-PKC– GFP was observed throughout the cytoplasm in transiently transfected COS-7 cells. Stimulation by an active phorbol ester (12-O-tetradecanoylphorbol 13-acetate [TPA]) but not by an inactive phorbol ester (4α-phorbol 12, 13-didecanoate) induced a significant translocation of γ-PKC–GFP from cytoplasm to the plasma membrane. A23187, a Ca2+ ionophore, induced a more rapid translocation of γ-PKC–GFP than TPA. The A23187-induced translocation was abolished by elimination of extracellular and intracellular Ca2+. TPA- induced translocation of γ-PKC–GFP was unidirected, while Ca2+ ionophore–induced translocation was reversible; that is, γ-PKC–GFP translocated to the membrane returned to the cytosol and finally accumulated as patchy dots on the plasma membrane. To investigate the significance of C1 and C2 domains of γ-PKC in translocation, we expressed mutant γ-PKC–GFP fusion protein in which the two cysteine rich regions in the C1 region were disrupted (designated as BS 238) or the C2 region was deleted (BS 239). BS 238 mutant was translocated by Ca2+ ionophore but not by TPA. In contrast, BS 239 mutant was translocated by TPA but not by Ca2+ ionophore. To examine the translocation of γ-PKC–GFP under physiological conditions, we expressed it in NG-108 cells, N-methyl-d-aspartate (NMDA) receptor–transfected COS-7 cells, or CHO cells expressing metabotropic glutamate receptor 1 (CHO/mGluR1 cells). In NG-108 cells , K+ depolarization induced rapid translocation of γ-PKC–GFP. In NMDA receptor–transfected COS-7 cells, application of NMDA plus glycine also translocated γ-PKC–GFP. Furthermore, rapid translocation and sequential retranslocation of γ-PKC–GFP were observed in CHO/ mGluR1 cells on stimulation with the receptor. Neither cytochalasin D nor colchicine affected the translocation of γ-PKC–GFP, indicating that translocation of γ-PKC was independent of actin and microtubule. γ-PKC–GFP fusion protein is a useful tool for investigating the molecular mechanism of γ-PKC translocation and the role of γ-PKC in the central nervous system.

The regulation of intracellular localization of AFX, a human Forkhead transcription factor, was studied. AFX was recovered as a phosphoprotein from transfected COS-7 cells growing in the presence of FBS, and the phosphorylation was eliminated by wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3-kinase. AFX was phosphorylated in vitro by protein kinase B (PKB), a downstream target of PI 3-kinase, but a mutant protein in which three putative phosphorylation sites of PKB had been replaced by Ala was not recognized by PKB. In Chinese hamster ovary cells (CHO-K1) cultured with serum, the AFX protein fused with green fluorescence protein (AFX-GFP) is localized mainly in the cytoplasm, and wortmannin induced transient nuclear translocation of the fusion protein. The AFX-GFP mutant in which all three phosphorylation sites had been replaced by Ala was detected exclusively in the cell nucleus. AFX-GFP was in the nucleus when the cells were infected with an adenovirus vector encoding a dominantnegative form of either PI 3-kinase or PKB, whereas the fusion protein stayed in the cytoplasm when the cells expressed constitutively active PKB. In CHO-K1 cells expressing AFX-GFP, DNA fragmentation was induced by the stable PI 3-kinase inhibitor LY294002, and the expression of the active form of PKB suppressed this DNA fragmentation. The phosphorylation site mutant of AFX-GFP enhanced DNA fragmentation irrespective of the presence and absence of PI 3-kinase inhibitor. These results indicate that the nuclear translocation of AFX is negatively regulated through its phosphorylation by PKB. P hosphatidylinositol (PI) 3-kinase mediates the signal from various growth factors to regulate cell proliferation and survival (1, 2). A Ser/Thr protein kinase, termed protein kinase B (PKB) or Akt, is identified as a downstream target of PI 3-kinase. This protein kinase is activated by interaction of its pleckstrin homology domain with PI 3-kinase products and/or by phosphorylation of its catalytic domain by some upstream protein kinases (3, 4). The potential role of PKB in insulin action has been explored extensively (2-4). In Caenorhabditis elegans, DAF-2, AGE-1, and Ce-Akt, which are homologues of the mammalian insulin receptor, p110 catalytic subunit of PI 3-kinase, and PKB, respectively, have been isolated (5-7). In this organism, DAF-16, a transcription factor containing the Forkhead motif, is a major downstream target of the AGE-1/ Ce-AKT signaling cascade (7-9). This protein is shown to mediate insulin-like metabolic and longevity signals, and genetic analysis reveals that the AGE-1/Ce-AKT pathway suppresses the activity of DAF-16 for gene transcription. DAF-16 contains three repeats of the consensus sequence for phosphorylation by PKB (10), ArgXaa-Arg-Xaa-Xaa-Ser/Thr, where Xaa is any amino acid, and thus this protein is thought to be a direct target of Ce-Akt (7).Some members of the Forkhead family of human transcription factors, FKHR (11), its related gene products (FKHRL1 and FKHR1) (12, 13), and AFX (14, 15), are structur...

The gamma isotype of protein kinase C (PKC gamma) is a member of the classical PKC (cPKC) subfamily which is activated by Ca(2+) and diacylglycerol in the presence of phosphatidylserine. Physiologically, PKC gamma is activated by a mechanism coupled with receptor-mediated breakdown of inositol phospholipid as other cPKC isotypes such as PKC alpha and PKC beta. PKC gamma is expressed solely in the brain and spinal cord and its localization is restricted to neurons, while PKC alpha and PKC beta are expressed in many tissues in addition to the brain. Within the brain, PKC gamma is the most abundant in the cerebellum, hippocampus and cerebral cortex, where the existence of neuronal plasticity has been demonstrated. Pharmacological and electrophysiological studies have shown that several neuronal functions, including long term potentiation (LTP) and long term depression (LTD), specifically require PKC gamma. Generation of mice deficient in PKC gamma provided more information regarding the physiological functions of this isotype. PKC gamma deficient mice (i) have modified long term potentiation (LTP) in hippocampus, (ii) exhibit mild deficits in spatial and contextual learning (iii) exhibit impaired motor coordination due to persistent multiple innervations of climbing fibers on Purkinje cells, (iv) show attenuation of opioid receptor activation, and (v) show decreased effects of ethanol on type A of gamma-aminobutyric acid (GABA) receptor. Furthermore, a point mutation in the PKC gamma gene may contribute to retinitis pigmentosa and Parkinsonian syndrome. This article reviews the specific functions of this neuron-specific isotype of PKC in neuronal signal transduction.

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease caused by mutations in protein kinase C␥ (PKC␥). Interestingly, 18 of 22 mutations are concentrated in the C1 domain, which binds diacylglycerol and is necessary for translocation and regulation of PKC␥ kinase activity. To determine the effect of these mutations on PKC␥ function and the pathology of SCA14, we investigated the enzymological properties of the mutant PKC␥s. We found that wild-type PKC␥, but not C1 domain mutants, inhibits Ca 2؉ influx in response to muscarinic receptor stimulation. The sustained Ca 2؉ influx induced by muscarinic receptor ligation caused prolonged membrane localization of mutant PKC␥. Pharmacological experiments showed that canonical transient receptor potential (TRPC) channels are responsible for the Ca 2؉ influx regulated by PKC␥. 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 PKC␥s 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 Ca 2؉ entry. Such an alteration in Ca 2؉ homeostasis and Ca 2؉ -mediated signaling in Purkinje cells may contribute to the neurodegeneration characteristic of SCA14.Autosomal dominant SCA14 is a genetically heterogenous group of neurodegenerative disorders characterized by progressive motor incoordination affecting the gait and limbs, cerebellar dysarthria, and nystagmus due to degeneration of cerebellar Purkinje cells. SCA14 is caused by missense or in-frame deletion mutations in the PRKCG gene encoding protein kinase C␥ (PKC␥) 2 (1). PKC␥ is a member of the PKC family that plays critical roles in many cellular functions, affecting diverse signal transduction pathways (2). PKC␥ is selectively expressed in neurons throughout the brain and is most abundant in cerebellar Purkinje cells (3), which specifically degenerate in SCA14 patients.One of the characteristic features of PKC␥ is its translocation from the cytoplasm to the plasma membrane (4). Translocation is a hallmark of enzyme activation and is triggered by the stimulation of G protein-coupled receptors. It is well known that activation of such receptors causes elevations of DAG and intracellular Ca 2ϩ (5). PKC␥ contains C1 and C2 domains in its regulatory domain (6). The C1 domain has two zinc-finger motifs, C1A and C1B, that contain highly conserved Cys residues that bind to diacylglycerol (DAG) and tumor promoting phorbol esters. The C2 domain is a Ca 2ϩ sensor that binds phosphatidylserine (PS) in the presence of elevated Ca 2ϩ . The C1 and C2 domains play crucial roles in PKC␥ transloca...

Protein kinase C (PKC) is known to be a key enzyme in signal transduction and is involved in the regulation of numerous cellular functions (30). PKC is activated by diacylglycerol (DG) produced by the receptor-coupled hydrolysis of membrane phosphoinositides (28, 30) and serves as the receptor for tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (TPA) (2, 28). The PKC family consists of at least 10 different subspecies that can be classified into three groups, classical, new, and atypical PKC (cPKC, nPKC, and aPKC, respectively), based on the structures of their regulatory domains) (29, 30). The differences in structure, enzymatic properties, and patterns of expression strongly suggest the specific functions of each subspecies of PKC, but the individual functions have not been fully clarified.The ␦ subspecies of PKC (␦-PKC) belongs to the nPKC group and is activated by DG in a calcium-independent manner (24,27,31,34). Phorbol ester treatment of NIH 3T3 cells overexpressing ␦-PKC produced significant changes in cell morphology and slowed cell growth (25), and TPA induced monocytic differentiation in 32D cells overexpressing ␦-PKC (26). Furthermore, treatment with phorbol ester of CHO cells overexpressing ␦-PKC induced cell division arrest (40), strongly suggesting that ␦-PKC is involved in the regulation of cell proliferation and differentiation. In addition to serine/threonine phosphorylation of ␦-PKC (1, 31-33), several extracellular signals induce the tyrosine phosphorylation of ␦-PKC (5,6,11,19,22,23,37). Denning et al. (5) observed the tyrosine phosphorylation of ␦-PKC among various PKC subspecies in cultured keratinocytes transformed with the Ha-v-ras gene. Stimulation of the platelet-derived growth factor receptor resulted in the tyrosine phosphorylation of ␦-PKC in myeloid progenitor cells (23). Treatment with phorbol ester also induced the tyrosine phosphorylation of ␦-PKC (22). ␦-PKC was tyrosine phosphorylated in vitro by c-Fyn (6, 22), c-Src (6, 11, 41) and growth factor receptors (6, 22); however, the effect of tyrosine phosphorylation on PKC activity has been controversial in these reports (5,6,11,22,23,38). Considering that ␦-PKC is tyrosine phosphorylated by TPA (22), which induced cell division arrest of CHO cells overexpressing ␦-PKC (40), the tyrosine phosphorylation of ␦-PKC seems to be related to the cell proliferation and differentiation. Recently, it was shown that H 2 O 2 treatment induces the tyrosine phosphorylation of ␦-PKC and that ␦-PKC is recovered as an activator-independent form from H 2 O 2 -treated cells (19). The physiological role of tyrosine phosphorylation of ␦-PKC by H 2 O 2 , however, has not been elucidated, and the functional differences between TPAand H 2 O 2 -induced activation of ␦-PKC have not been clarified.The PKC subspecies, especially of the cPKC and nPKC groups, are known to translocate from the cytosol to the membrane fraction upon activation (20). The translocation of another subspecies, ␥-PKC, was visualized in living cells by using ...

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 superoxidegenerating NADPH oxidase in the development of hyperalgesia. In mice lacking Nox1 (Nox1 Ϫ/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.

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.

Effects of fatty acids on translocation of the γ- and ε-subspecies of protein kinase C (PKC) in living cells were investigated using their proteins fused with green fluorescent protein (GFP). γ-PKC–GFP and ε-PKC–GFP predominated in the cytoplasm, but only a small amount of γ-PKC–GFP was found in the nucleus. Except at a high concentration of linoleic acid, all the fatty acids examined induced the translocation of γ-PKC–GFP from the cytoplasm to the plasma membrane within 30 s with a return to the cytoplasm in 3 min, but they had no effect on γ-PKC–GFP in the nucleus. Arachidonic and linoleic acids induced slow translocation of ε-PKC–GFP from the cytoplasm to the perinuclear region, whereas the other fatty acids (except for palmitic acid) induced rapid translocation to the plasma membrane. The target site of the slower translocation of ε-PKC–GFP by arachidonic acid was identified as the Golgi network. The critical concentration of fatty acid that induced translocation varied among the 11 fatty acids tested. In general, a higher concentration was required to induce the translocation of ε-PKC–GFP than that of γ-PKC–GFP, the exceptions being tridecanoic acid, linoleic acid, and arachidonic acid. Furthermore, arachidonic acid and the diacylglycerol analogue (DiC8) had synergistic effects on the translocation of γ-PKC–GFP. Simultaneous application of arachidonic acid (25 μM) and DiC8 (10 μM) elicited a slow, irreversible translocation of γ-PKC– GFP from the cytoplasm to the plasma membrane after rapid, reversible translocation, but a single application of arachidonic acid or DiC8 at the same concentration induced no translocation.These findings confirm the involvement of fatty acids in the translocation of γ- and ε-PKC, and they also indicate that each subspecies has a specific targeting mechanism that depends on the extracellular signals and that a combination of intracellular activators alters the target site of PKCs.