Oxidative Activation of Protein Kinase Cγ through the C1 Domain
2005; Elsevier BV; Volume: 280; Issue: 14 Linguagem: Inglês
10.1074/jbc.m407762200
ISSN1083-351X
AutoresDingbo Lin, Dolores J. Takemoto,
Tópico(s)Redox biology and oxidative stress
ResumoThe accumulation of reactive oxygen species (ROS, for example H2O2) is linked to several chronic pathologies, including cancer and cardiovascular and neurodegenerative diseases (Gate, L., Paul, J., Ba, G. N., Tew, K. D., and Tapiero, H. (1999) Biomed. Pharmacother. 53, 169–180). Protein kinase C (PKC) γ is a unique isoform of PKC that is found in neuronal cells and eye tissues. This isoform is activated by ROS such as H2O2. Mutations (H101Y, G118D, S119P, and G128D) in the PKCγ Cys-rich C1B domain caused a form of dominant non-episodic cerebellar ataxia in humans (Chen, D.-H., Brkanac, Z., Verlinde, C. L. M. J., Tan, X.-J., Bylenok, L., Nochli, D., Matsushita, M., Lipe, H., Wolff, J., Fernandez, M., Cimino, P. J., Bird, T. D., and Raskind, W. H. (2003) Am. J. Hum. Genet. 72, 839–849; van de Warrenburg, B. P. C., Verbeek, D. S., Piersma, S. J., Hennekam, F. A. M., Pearson, P. L., Knoers, N. V. A. M., Kremer, H. P. H., and Sinke, R. J. (2003) Neurology 61, 1760–1765). This could be due to a failure of the mutant PKCγ proteins to be activated by ROS and to subsequently inhibit gap junctions. The purpose of this study was to demonstrate the cellular mechanism of activation of PKCγ by H2O2 and the resultant effects on gap junction activity. H2O2 stimulated PKCγ enzyme activity independently of elevations in cellular diacylglycerol, the natural PKC activator. Okadaic acid, a phosphatase inhibitor, did not affect H2O2-stimulated PKCγ activity, indicating that dephosphorylation was not involved. The reductant, dithiothreitol, abolished the effects of H2O2, suggesting a direct oxidation of PKCγ at the Cys-rich C1 domain. H2O2 induced the C1 domain of PKCγ to translocate to plasma membranes, whereas the C2 domain did not. Direct effects of H2O2 on PKCγ were demonstrated using two-dimensional SDS-PAGE. Results demonstrated that PKCγ formed disulfide bonds in response to H2O2. H2O2-activated PKCγ was targeted into caveolin-1- and connexin 43-containing lipid rafts, and the PKCγ phosphorylated the connexin 43 gap junction proteins on Ser-368. This resulted in disassembly of connexin 43 gap junction plaques and decreased gap junction activity. Results suggested that H2O2 caused oxidation of the C1 domain, activation of the PKCγ, and inhibition of gap junctions. This inhibition of gap junctions could provide a protection to cells against oxidative stress. The accumulation of reactive oxygen species (ROS, for example H2O2) is linked to several chronic pathologies, including cancer and cardiovascular and neurodegenerative diseases (Gate, L., Paul, J., Ba, G. N., Tew, K. D., and Tapiero, H. (1999) Biomed. Pharmacother. 53, 169–180). Protein kinase C (PKC) γ is a unique isoform of PKC that is found in neuronal cells and eye tissues. This isoform is activated by ROS such as H2O2. Mutations (H101Y, G118D, S119P, and G128D) in the PKCγ Cys-rich C1B domain caused a form of dominant non-episodic cerebellar ataxia in humans (Chen, D.-H., Brkanac, Z., Verlinde, C. L. M. J., Tan, X.-J., Bylenok, L., Nochli, D., Matsushita, M., Lipe, H., Wolff, J., Fernandez, M., Cimino, P. J., Bird, T. D., and Raskind, W. H. (2003) Am. J. Hum. Genet. 72, 839–849; van de Warrenburg, B. P. C., Verbeek, D. S., Piersma, S. J., Hennekam, F. A. M., Pearson, P. L., Knoers, N. V. A. M., Kremer, H. P. H., and Sinke, R. J. (2003) Neurology 61, 1760–1765). This could be due to a failure of the mutant PKCγ proteins to be activated by ROS and to subsequently inhibit gap junctions. The purpose of this study was to demonstrate the cellular mechanism of activation of PKCγ by H2O2 and the resultant effects on gap junction activity. H2O2 stimulated PKCγ enzyme activity independently of elevations in cellular diacylglycerol, the natural PKC activator. Okadaic acid, a phosphatase inhibitor, did not affect H2O2-stimulated PKCγ activity, indicating that dephosphorylation was not involved. The reductant, dithiothreitol, abolished the effects of H2O2, suggesting a direct oxidation of PKCγ at the Cys-rich C1 domain. H2O2 induced the C1 domain of PKCγ to translocate to plasma membranes, whereas the C2 domain did not. Direct effects of H2O2 on PKCγ were demonstrated using two-dimensional SDS-PAGE. Results demonstrated that PKCγ formed disulfide bonds in response to H2O2. H2O2-activated PKCγ was targeted into caveolin-1- and connexin 43-containing lipid rafts, and the PKCγ phosphorylated the connexin 43 gap junction proteins on Ser-368. This resulted in disassembly of connexin 43 gap junction plaques and decreased gap junction activity. Results suggested that H2O2 caused oxidation of the C1 domain, activation of the PKCγ, and inhibition of gap junctions. This inhibition of gap junctions could provide a protection to cells against oxidative stress. Oxidative stress is closely related to aging and a diverse range of diseases in humans (1.McCord J.M. Am. J. Med. 2000; 108: 652-659Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar, 2.Martindale J.L. Holbrook N.J. J. Cell. Physiol. 2002; 192: 1-15Crossref PubMed Scopus (1942) Google Scholar). Accumulation of reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; Cx43, connexin 43; Cav-1, caveolin-1; pY14-Cav-1, phosphorylated Cav-1 on tyrosine 14; PKC, protein kinase C; PLCγ, phospholipase C γ; ERK, extracellular signal-regulated kinase; pERK, phosphorylated ERK; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; siRNA, single interference RNA; PBS, phosphate-buffered saline; Me2SO, dimethyl sulfoxide; DTT, dithiothreitol; OA, okadaic acid; Cal C, calphostin C; IGF-1, insulin-like growth factor-1; DAG, diacylglycerol; EGFP, enhanced green fluorescent protein; TPA, 12-O-tetradecanoylphorbol-13-acetate; PMSF, phenylmethanesulfonyl fluoride; Mes, 2-(N-morpholino)ethanesulfonic acid; GFP, green fluorescent protein. may be responsible for some chronic pathologies, including cancer and cardiovascular and neurodegenerative diseases (3.Gate L. Paul J. Ba G.N. Tew K.D. Tapiero H. Biomed. Pharmacother. 1999; 53: 169-180Crossref PubMed Scopus (313) Google Scholar). For instance, Alzheimer disease is a neurodegenerative disorder associated with oxidative stress, and H2O2 is implicated in this disease (4.Milton N.G. Drugs Aging. 2004; 21: 81-100Crossref PubMed Scopus (114) Google Scholar). H2O2 is an uncharged and freely diffusible reactive oxygen species that is relatively high in brain (4.Milton N.G. Drugs Aging. 2004; 21: 81-100Crossref PubMed Scopus (114) Google Scholar). Recently, platelet-derived growth factor, a normal cell growth factor, has been reported to cause an elevation in cellular H2O2 (5.Chen K.C.-W. Zhou Y. Xing K. Krysan K. Lou M.F. Exp. Eye Res. 2004; 78: 1057-1067Crossref PubMed Scopus (69) Google Scholar). Thus, the identification of H2O2 sensors in cells should be very critical to understanding the pathobiology of human diseases caused by oxidative stress (6.Stone J.R. Arch. Biochem. Biophys. 2004; 422: 119-124Crossref PubMed Scopus (161) Google Scholar). One mechanism by which oxidative damage is signaled to adjacent cells is through open gap junctions that would pass apoptotic signals to adjacent cells. Gap junctions are clusters of channels that maintain homeostasis by intercellular exchange of ions, small metabolites, and cell signaling molecules (7.Wei C.-J. Xu X. Lo C.W. Annu. Rev. Cell Dev. Biol. 2004; 20: 811-838Crossref PubMed Scopus (335) Google Scholar). A gap junction channel is made of two hemi-channels called connexons, and each connexon consists of six membrane-spanning connexin protein molecules. Either closure or disassembly of gap junction channels causes inhibition of gap junction activity as determined through both gap junction activity assay (by scrape loading/dye transfer analysis) and cell surface gap junction plaques (by immuno-labeling assay) (8.Lin D. Boyle D. Takemoto D. Investig. Ophthalmol. Vis. Sci. 2003; 44: 1160-1168Crossref PubMed Scopus (53) Google Scholar). Previous publications (9.Huang R.P. Peng A. Hossain M.Z. Fan Y. Jagdale A. Boynton A.L. Carcinogenesis. 1999; 20: 485-492Crossref PubMed Scopus (57) Google Scholar, 10.Cho J.H. Cho S.D. Hu H. Kim S.H. Lee Y.S. Kang K.S. Carcinogenesis. 2002; 23: 1163-1169Crossref PubMed Scopus (110) Google Scholar) suggested that H2O2 induced hyperphosphorylation of connexins and inhibition of gap junctions. This inhibition may be due to closure of gap junction channels (11.Hossain M.Z. Jagdale A.B. Ao P. Boynton A.L. J. Cell. Physiol. 1999; 179: 87-96Crossref PubMed Scopus (49) Google Scholar, 12.Todt I. Ngezahayo A. Ernst A. Kolb H.A. J. Membr. Biol. 2001; 181: 107-114Crossref PubMed Scopus (20) Google Scholar). In contrast, H2O2 also is reported to increase gap junctional communication in astrocytes, and prolonged treatment with H2O2 consequently caused cell death (13.Rouach N. Calvo C.F. Duquennoy H. Glowinski J. Giaume C. Glia. 2004; 45: 28-38Crossref PubMed Scopus (32) Google Scholar). Connexin proteins have numerous kinase target sites, and Ser-368 is the site of phosphorylation of Cx43 by PKC (14.Lampe P.D. TenBroek E.M. Burt J.M. Kurata W.E. Jognson R.G. Lau A.F. J. Cell Biol. 2000; 149: 1503-1512Crossref PubMed Scopus (470) Google Scholar). Protein kinase C (PKC) comprises a family of serine/threonine kinases that contain at least 11 isoforms and can be found in most cell types (15.Newton A.C. Chem. Rev. 2001; 101: 2353-2364Crossref PubMed Scopus (837) Google Scholar). These isoforms are divided into three groups. Conventional PKCs are activated by both diacylglycerol (DAG) and calcium. Novel PKCs are calcium-independent but can still be stimulated by DAG. Atypical PKCs are independent of both calcium and DAG. PKCγ is a conventional isoform of PKC and is required for brain cells, peripheral nerves, retina, and lens (16.Oancea E. Meyer T. 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The presence of PKCγ in brain tissues appears to prevent brain ischemia and is a target for ischemic preconditioning (22.Aronowski J. Grotta J. Strong R. Waxman M. J. Cereb. Blood Flow Metab. 2000; 20: 343-349Crossref PubMed Scopus (46) Google Scholar, 23.Cardell M. Wieloch T. J. Neurochem. 1993; 61: 1308-1314Crossref PubMed Scopus (100) Google Scholar). Missense mutations (H101Y, G118D, S119P, G128D, or F643L) in PKCγ cause dominant non-episodic cerebellar ataxia in humans, suggesting that PKCγ-related and polyglutamine-related neurodegeneration may have a common pathway for neuronal cell damage and death in hereditary ataxia (24.Chen D.-H. Brkanac Z. Verlinde C.L.M.J. Tan X.-J. Bylenok L. Nochli D. Matsushita M. Lipe H. Wolff J. Fernandez M. Cimino P.J. Bird T.D. Raskind W.H. Am. J. Hum. Genet. 2003; 72: 839-849Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 25.van de Warrenburg B.P.C. Verbeek D.S. Piersma S.J. Hennekam F.A.M. Pearson P.L. Knoers N.V.A.M. Kremer H.P.H. Sinke R.J. Neurology. 2003; 61: 1760-1765Crossref PubMed Scopus (90) Google Scholar, 26.Stevanin G. Hahn V. Lohmann E. Bouslam N. Gouttard M. Soumphonphakdy C. Welter M.-L. Ollagnon-Roman E. Lemainque A. Ruberg M. Brice A. Durr A. Arch. Neurol. 2004; 61: 1242-1248Crossref PubMed Scopus (83) Google Scholar). Since oxidative damage is known to be involved in neurodegeneration, proteins that respond to oxidative stress would be critical for the health of neural tissues. We have observed that PKCγ is present in lens epithelial cells and is the primary sensor of changes in diacylglycerol (DAG) at low or physiological levels (8.Lin D. Boyle D. Takemoto D. Investig. Ophthalmol. Vis. Sci. 2003; 44: 1160-1168Crossref PubMed Scopus (53) Google Scholar). The PKCγ regulatory domain contains C1 and C2 motifs. PKCγ binds calcium at a C2 domain and DAG at a C1 domain (15.Newton A.C. Chem. Rev. 2001; 101: 2353-2364Crossref PubMed Scopus (837) Google Scholar, 27.Cho W. J. Biol. Chem. 2001; 276: 32407-32410Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). NMR structural analyses reveal that the C1 domain of PKCγ, like PKCα, has two zinc-finger motifs that are enriched with Cys residues and are called C1A and C1B (28.Xu R. Pawelczyk T. Xia T. Brown S. Biochemistry. 1997; 36: 10709-10717Crossref PubMed Scopus (122) Google Scholar). Both C1A and C1B domains of PKCγ have high affinity for DAG and are exposed, whereas only the C1A domain of PKCα has high affinity for DAG binding (29.Ananthanarayanan B. Stahelin R.V. Digman M.A. Cho W. J. Biol. Chem. 2003; 278: 46886-46894Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Both C1A and C1B domains are involved in DAG-stimulated PKCγ activation at basal intracellular calcium levels (8.Lin D. Boyle D. Takemoto D. Investig. Ophthalmol. Vis. Sci. 2003; 44: 1160-1168Crossref PubMed Scopus (53) Google Scholar, 29.Ananthanarayanan B. Stahelin R.V. Digman M.A. Cho W. J. Biol. Chem. 2003; 278: 46886-46894Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The C1 and C2 domains anchor PKC to plasma membrane, which in turn causes conformational changes and consequent protein activation (30.Voet D. Voet J.G. Biochemistry: Biomolecules, Mechanisms of Enzyme Action, and Metabolism,3rd Ed. 1. John Wiley & Sons, Inc, Hoboken, NJ2004Google Scholar). Sublethal exposure of cells to H2O2 causes numerous changes in the oxidation state of proteins (31.Cumming R.C. Andon N.L. Haynes P.A. Park M. Fischer W.H. Schubert D. J. Biol. Chem. 2004; 279: 21749-21758Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). PKCγ is a logical candidate for redox modification by H2O2 at sublethal doses through oxidation of the Cys residues within the C1 domain. This would result in PKCγ activation and could cause inhibition of gap junctions, a cell-protective mechanism. Here we show that 100 μm H2O2 activates PKCγ through the C1 domain. The C1 domain of PKCγ translocated to membranes upon H2O2 stimulation. Oxidation of PKCγ by H2O2 resulted in off-diagonal migration of the proteins, suggestive of disulfide bond formation. H2O2-activated PKCγ was targeted to caveolin-1 (Cav-1)- and connexin 43 (Cx43)-containing lipid rafts, and the PKCγ interacted with Cx43 gap junction proteins and Cav-1 and consequently phosphorylated Cx43 on Ser-368. Moreover, activation of PKCγ by H2O2 decreased Cx43 gap junction plaques and gap junction activity. Thus, oxidation of the PKCγ C1 domain may be responsible for the H2O2-induced activation of this enzyme and in subsequent inhibition of gap junctions. These results demonstrate that PKCγ is an oxidative stress-sensing protein that provides a protective effect for cells through inhibition of gap junctions. Materials—pEGFP-N3 vector and the monoclonal antibodies against flotillin, PKCα, PKCγ, Cx43, Cav-1, pY14-Cav-1, phosphotyrosine, and GFP were purchased from BD Biosciences. Rabbit polyclonal PKCγ phospho-Thr-514 antibody was purchased from Abcam (Cambridge, MA). Polyclonal rabbit anti-phosphothreonine, anti-phosphoserine, anti-Cx43, and anti-pS368-Cx43 were purchased from Chemicon (Temecula, CA). Rabbit anti-PLCγ1 and protein A/G PLUS-agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). pERK(Thr-202/Tyr-204) and ERK1/2 polyclonal antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase and the PepTag nonradioactive protein kinase C assay system were purchased from Promega (Madison, WI). Mouse anti-α-tubulin was purchased from Zymed Laboratories Inc. Dulbecco's modified Eagle's medium (low glucose), RPMI 1640 medium, trypsin-EDTA, gentamicin, penicillin/streptomycin, and Lipofectamine were purchased from Invitrogen. Dimethyl sulfoxide (Me2SO), dithiothreitol (DTT), H2O2, sodium fluoride (NaF), calcium chloride (CaCl2), and Takara Ex TaqDNA polymerase were purchased from Fisher. Fetal bovine serum was purchased from Atlanta Biologicals (Norcross, GA). Infinity™ cholesterol liquid stable reagent was purchased from Thermo Electron Corp. (Louisville, CO). Okadaic acid (OA), calphostin C (Cal C), phenylmethanesulfonyl fluoride (PMSF), insulin-like growth factor-I (IGF-I), Ponceau S solution, and protease inhibitor mixture were from Sigma. Protein molecular weight marker was purchased from Bio-Rad. Phorbol-12-myristate-13-acetate (TPA) and 4α-phorbol, 12,13-didecanoate, inactive phorbol ester, were purchased from Calbiochem. Diacylglycerol (DAG) assay kit was from Amersham Biosciences. QuikChange site-directed mutagenesis kit and StrataClean resin were purchased from Stratagene (La Jolla, CA). Alexa Fluor 466 and 568, lucifer yellow, rhodamine dextran, and SlowFade antifade were purchased from Molecular Probes (Eugene, OR). RNAiFect siRNA transfection reagent and PKCγ siRNA were purchased from Qiagen (Valencia, CA). Neuron 2A cells were purchased from American Type Culture Collection (Manassas, VA). Cell Culture—N/N1003A rabbit lens epithelial cells were cultured in Dulbecco's modified Eagle's medium (low glucose) supplemented with 10% fetal bovine serum and 50 μg/ml gentamicin, 0.05 unit/ml penicillin, 50 μg/ml streptomycin, pH 7.4, at 37 °C in an atmosphere of 90% air and 10% CO2. Neuron 2A (N2A) cells were cultured in RPMI1640 medium supplemented with 8% fetal bovine serum and 50 μg/ml gentamicin, 0.05 unit/ml penicillin, 50 μg/ml streptomycin, pH 7.4, at 37 °C in an atmosphere of 90% air and 10% CO2. When they reached 95% confluency, the cells were used for experiments after 2 h of serum starvation. PKCγ Activity Assay—Specific PKCγ activity was analyzed by use of the PepTag assay kit as described (32.Zhou J. Fariss R.N. Zelenka P.S. J. Biol. Chem. 2003; 278: 5388-5398Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 33.Lin D. Lobell S. Jewell A. Takemoto D.J. Mol. Vis. 2004; 10: 688-695PubMed Google Scholar). Equal protein amounts of whole cell extracts were immunoprecipitated with PKCγ antisera at 4 °C for 4 h as described previously (8.Lin D. Boyle D. Takemoto D. Investig. Ophthalmol. Vis. Sci. 2003; 44: 1160-1168Crossref PubMed Scopus (53) Google Scholar). Immunoprecipitated PKCγ-agarose bead complexes were recovered and incubated with PKC reaction mixture (25 μl) according to the manufacturer's instructions. The reactions were stopped by heating at 100 °C for 10 min, and fluorescent phospho-PepTag peptides (phosphorylated by PKCγ) were resolved by 0.8% agarose gel electrophoresis and visualized under UV light. The phosphorylated peptide bands were excised, and their fluorescence intensities were quantified by spectrophotometry at 570 nm. Endogenous Diacylglycerol Assay—Sample preparation and radioenzymatic assays were performed according to the manufacturer's instructions described previously (8.Lin D. Boyle D. Takemoto D. Investig. Ophthalmol. Vis. Sci. 2003; 44: 1160-1168Crossref PubMed Scopus (53) Google Scholar). Briefly, total lipids extracted with chloroform/methanol were used as the substrates for diacylglycerol (DAG) kinase. The reaction product, phosphatidic acid, traced by 32P, was separated by TLC gel, and consequently the TLC gels were exposed to x-ray film overnight. The spots corresponding to [32P]phosphatidic acid were isolated and quantitated by scintillation counting. Cellular DAG levels were calculated from DAG standard curves. Western Blot and Immunoprecipitation—Cells were collected and lysed on ice with cell lysis buffer followed by homogenization and sonication. The cell lysis buffer contains 20 mm Tris-HCl, pH 7.5, 0.5 mm EDTA, 0.5 mm EGTA, 0.5% Triton X-100, 0.1% protease inhibitor mixture, 5 mm NaF, and 2 mm PMSF. After centrifugation at 12,000 × g for 20 min, the supernatants were collected and used as whole cell extracts. Western blotting and immunoprecipitation were carried out as described previously (8.Lin D. Boyle D. Takemoto D. Investig. Ophthalmol. Vis. Sci. 2003; 44: 1160-1168Crossref PubMed Scopus (53) Google Scholar). Redox Two-dimensional SDS-PAGE—Formation of disulfide bonds was determined by “redox two-dimensional SDS-PAGE” as described (31.Cumming R.C. Andon N.L. Haynes P.A. Park M. Fischer W.H. Schubert D. J. Biol. Chem. 2004; 279: 21749-21758Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar) with modification. Whole cell extracts were treated with nonreducing SDS sample buffer (50 mm Tris-HCl, pH 6.8, 2% SDS, 0.05% bromphenol blue, and 10% glycerol) for 3 min at 85 °C before loading. Proteins (100 μg) were resolved by 8% SDS-PAGE in the first dimension. After electrophoresis, the gel lanes were cut and immersed in SDS sample buffer containing 100 mm DTT for 1 h at room temperature. Each gel strip was then horizontally applied to the top of another 8% gel and was bridged by 0.6% agarose gel in 120 mm Tris-HCl, pH 6.8. After electrophoresis, separated proteins in gels were transferred to nitrocellulose membranes that were subsequently stained in the Ponceau S solution to show prominent diagonal lines of proteins. After that, the membranes were immunoblotted with anti-PKCγ, PLCγ1, α-tubulin, or Cav-1 antisera, and immunoreacted proteins were visualized by ECL solution as described previously (8.Lin D. Boyle D. Takemoto D. Investig. Ophthalmol. Vis. Sci. 2003; 44: 1160-1168Crossref PubMed Scopus (53) Google Scholar, 34.Lin D. Zhou J. Zelenka P. Takemoto D. Investig. Ophthalmol. Vis. Sci. 2003; 44: 5259-5268Crossref PubMed Scopus (84) Google Scholar). Sucrose Gradient Centrifugation and Isolation of Lipid Rafts—Lipid rafts-enriched membrane fractions were prepared as described previously (34.Lin D. Zhou J. Zelenka P. Takemoto D. Investig. Ophthalmol. Vis. Sci. 2003; 44: 5259-5268Crossref PubMed Scopus (84) Google Scholar). Briefly, cells from three 75-cm2 flasks were collected and sonicated in cell lysis buffer containing 1% Triton X-100. Whole cell extracts were mixed with an equal volume of 80% sucrose in Mes-NaCl buffer containing 25 mm Mes, pH 6.5, 150 mm NaCl, 0.1% protease inhibitor mixture, 5 mm NaF, and 2 mm PMSF, loaded at the bottoms of 12-ml ultracentrifuge tubes, and then overlaid with 8 ml of a 5–35% continuous sucrose gradient in Mes-NaCl buffer containing 0.2% concentration of protease inhibitor mixture, 10 mm NaF, and 4 mm PMSF. The samples were ultracentrifuged at 245,000 × g for 22 h at 4 °C with a Beckman swinging bucket rotor SW41 Ti. Fractions (1 ml each) were collected from the top of each gradient (12 fractions total). Protein samples were precipitated with 10% trichloroacetic acid, separated by 10% SDS-PAGE, and immuno-visualized by Western blotting. In order to investigate protein interactions in lipid rafts, sucrose gradient fractions 3–6 were collected, combined, and further solubilized by sonication with addition of 0.1% SDS. The mixtures were used as Cav-1-containing lipid raft samples for coimmunoprecipitation assays (34.Lin D. Zhou J. Zelenka P. Takemoto D. Investig. Ophthalmol. Vis. Sci. 2003; 44: 5259-5268Crossref PubMed Scopus (84) Google Scholar). Measurement of Cholesterol Content in the Sucrose Gradient Fractions—Sucrose gradient fractions were isolated as described above. Cholesterol content was measured as described (35.Chen X. Resh M.D. J. Biol. Chem. 2002; 277: 49631-49637Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Total lipids in these fractions were extracted with 2:1 methanol/chloroform, followed by 1 ml of chloroform and 1 ml of water. Chloroform phase (containing lipids) was dried under nitrogen. Dry lipids were resuspended in isopropyl alcohol, and membrane cholesterol was assayed using Infinity cholesterol liquid stable reagent according to the manufacturer's instructions. Plasmid Construction, Transfection, and Fluorescent Microscopy— By using rat PKCγ:EGFP plasmid DNA as a template (34.Lin D. Zhou J. Zelenka P. Takemoto D. Investig. Ophthalmol. Vis. Sci. 2003; 44: 5259-5268Crossref PubMed Scopus (84) Google Scholar), PKCγ fragments of the C1 (amino acid sequence: 36–150) or C2 (amino acid sequence: 170–260) domains were amplified by PCR to introduce both BglII sites at the N termini and EcoRI sites at the C termini and subcloned into pEGFP-N3 vectors, respectively. The sequences were confirmed by sequencing. The oligonucleotide sequences were designed as follows: C1 domain, forward primer 5′-GA AGATCT ATG CAC AAG TTC ACC GCT CGT-3′ and reverse primer 5′-CG GAATTC GCA AAG GGA GGG CAC GCT-3′; C2 domain, forward primer 5′-GA AGATCT ATG GAT GAG ATC CAT ATT ACT GT-3′ and reverse primer 5′-CG GAATTC GGA CAT GGC ACC CAT GAA GTC A-3′. Ataxia mutation H101Y was made using PKCγ:EGFP plasmid DNA as a template completely following the provided instructions for the QuikChange site mutation kit. The primer sequence is as follows with the mutation nucleotides underlined: PKCγ H101Y, 5′-C GAC CCT CGC AAC AAG TAC AAG TTC CGT CTG CAC AGC-3′. Connexin 43 Ser-368 (Cx43S368A) point mutation was made using Cx43:EGFP plasmid DNA as a template. The primer sequence as follows with mutation nucleotides underlined: Cx43S368A, 5′-CCT TCC AGC AGA GCC GCC AGC GCC AGC AGC AG-3′. Plasmid DNA transient transfection into 80% confluent N/N1003A cells or neuro-2A (N2A) cells was performed by Lipofectamine transfection reagent according to the manufacturer's protocol. The GFP fluorescence and relocalization of GFP fusion proteins were checked using an ECLIPSE E600 Nikon fluorescent microscope (Tokyo, Japan). Protein Translocation Analysis—Confluent cells were homogenized with extraction buffer containing 50 mm Tris-HCl, pH 7.5, 20 mm MgCl2, 0.1% protease inhibitor mixture, 5 mm NaF, and 2 mm PMSF. Soluble and membrane fractions were separated by centrifugation at 100,000 × g for 1 h at 4 °C. The pellets were resuspended with extraction buffer. Proteins were resolved by SDS-PAGE and Western blot as described (8.Lin D. Boyle D. Takemoto D. Investig. Ophthalmol. Vis. Sci. 2003; 44: 1160-1168Crossref PubMed Scopus (53) Google Scholar). Western blot bands were quantified by UN-Scan-It software (Silk Scientific, Orem, UT) and expressed as pixel intensities. The average and/or total pixel intensity was analyzed and graphed from three separate experiments. The C1:EGFP and C2:EGFP fusion proteins from transient transfected cells were concentrated using Strata-Clean resin before loading. Endogenous PKCγ Knockdown—Endogenous PKCγ was knocked down by siRNA targeting to PKCγ DNA sequence 5′-AAC GGT GTA AAG CCA CGC TAA A-3′. The PKCγ siRNA was transfected into cells using RNAiFect siRNA transfection reagent according to the instructions provided. Knockdown of PKCγ was monitored by Western blot. Measurement of Cell Surface Gap Junctional Cx43 Plaques—Determination of endogenous Cx43 gap junction plaques was performed as described previously (8.Lin D. Boyle D. Takemoto D. Investig. Ophthalmol. Vis. Sci. 2003; 44: 1160-1168Crossref PubMed Scopus (53) Google Scholar, 34.Lin D. Zhou J. Zelenka P. Takemoto D. Investig. Ophthalmol. Vis. Sci. 2003; 44: 5259-5268Crossref PubMed Scopus (84) Google Scholar). Briefly, the cells were fixed with 2.5% paraformaldehyde for 5 min and labeled with anti-Cx43 for 2 h at room temperature. After washing, the fixed cells were incubated with the secondary antisera that were attached to a fluorochrome and had specific excitation and emission wavelength. Alexa Fluor 568 is goat anti-rabbit antibody and has an excitation/emission wavelength of 578:603. The cells were then mounted onto slides and examined using a Nikon scanning confocal microscope. We photographed 10 points per slide, three slides for each treatment, and the examples are shown. For quantitation, the cell surface Cx43 plaques (larger than 1 μm in length) from single cells in each image were counted. The number of Cx43 plaques was expressed as mean ± S.E. Values of p ≤ 0.05 were considered to be statistically significant (*). Gap junction plaque formation of Cx43:EGFP or Cx43S368A:EGFP in transfected N2A cells was confirmed by a Nikon scanning confocal microscope. Large gap junction plaques from single cells in each image were counted. The number of plaques was expressed as mean ± S.E. Values of p ≤ 0.05 were considered to be statistically significant (*). Gap Junction Activity Assay—N/N1003A cell gap junction activity was measured by the scrape loading/dye transfer assay as described previously (8.Lin D. Boyle D. Takemoto D. Investig. Ophthalmol. Vis. Sci. 2003; 44: 1160-1168Crossref PubMed Scopus (53) Google Scholar, 36.Nguyen A. Boyle D. Wagner L. Shinohara T. Takemoto D. Exp. Eye Res. 2003; 76: 565-572Crossref PubMed Scopus (25) Google Scholar) with some modifications. Briefly, after H2O2 treatment, cells were rinsed with phosphate-buffered saline (PBS) and then 2.5 μl of both 1% Lucifer Yellow and 0.75% rhodamine dextran in PBS were added at the center of the coverslip, and two cuts crossing each other were made passing through the dye. After incubation with the dye for 1 min, these cells were rinsed with PBS and incubated with normal growth medium for an additional 10 min to allow dye transfer. The cells were then fixed in 2.5% paraformaldehyde
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