FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1984867277> ?p ?o ?g. }

• W1984867277 endingPage "16664" @default.
• W1984867277 startingPage "16653" @default.
• W1984867277 abstract "A hallmark of the afflicted nervous tissue in amyotrophic lateral sclerosis is the presence of protein aggregates, which to a large extent contain the intermediate filament protein peripherin. Here we show that activation of protein kinase C (PKC) or overexpression of PKCϵ induces the aggregation of peripherin in cultured neuroblastoma cells with elevated amounts of peripherin. The formation of aggregates was coupled to an increased apoptosis, suggesting a functional link between these events. Both induction of aggregates and apoptosis were suppressed in cells that had been transfected with small interfering RNAs targeting PKCϵ. PKCϵ and peripherin associate as shown by co-immunoprecipitation, and the interaction is dependent on and mediated by the C1b domain of PKCϵ. The interaction was specific for PKCϵ since corresponding structures from other isoforms did not co-precipitate peripherin, with the exception for PKCη and -θ, which pulled down minute amounts. PKCϵ interacts with vimentin through the same structures but does not induce its aggregation. When the PKCϵ C1b domain is expressed in neuroblastoma cells together with peripherin, both phorbol ester-induced peripherin aggregation and apoptosis are abolished, supporting a model in which PKCϵ through its interaction with peripherin facilitates its aggregation and subsequent cell death. These events may be prevented by expressing molecules that bind peripherin at the same site as PKCϵ. A hallmark of the afflicted nervous tissue in amyotrophic lateral sclerosis is the presence of protein aggregates, which to a large extent contain the intermediate filament protein peripherin. Here we show that activation of protein kinase C (PKC) or overexpression of PKCϵ induces the aggregation of peripherin in cultured neuroblastoma cells with elevated amounts of peripherin. The formation of aggregates was coupled to an increased apoptosis, suggesting a functional link between these events. Both induction of aggregates and apoptosis were suppressed in cells that had been transfected with small interfering RNAs targeting PKCϵ. PKCϵ and peripherin associate as shown by co-immunoprecipitation, and the interaction is dependent on and mediated by the C1b domain of PKCϵ. The interaction was specific for PKCϵ since corresponding structures from other isoforms did not co-precipitate peripherin, with the exception for PKCη and -θ, which pulled down minute amounts. PKCϵ interacts with vimentin through the same structures but does not induce its aggregation. When the PKCϵ C1b domain is expressed in neuroblastoma cells together with peripherin, both phorbol ester-induced peripherin aggregation and apoptosis are abolished, supporting a model in which PKCϵ through its interaction with peripherin facilitates its aggregation and subsequent cell death. These events may be prevented by expressing molecules that bind peripherin at the same site as PKCϵ. Extensive neuronal cell death is an underlying problem of many neurodegenerative diseases. This is frequently accompanied with, and may in several instances be caused by, the formation of large aggregates of different proteins in the afflicted neuronal tissue. The perhaps most notable examples are the amyloid plaques and neurofibrillar tangles seen in Alzheimer disease (1Hardy J. Selkoe D.J. Science. 2002; 297: 353-356Crossref PubMed Scopus (10782) Google Scholar, 2Tanzi R.E. Bertram L. Cell. 2005; 120: 545-555Abstract Full Text Full Text PDF PubMed Scopus (1471) Google Scholar). These accumulations are supposedly generated as a consequence of misfolded proteins that are not properly degraded.Amyotrophic lateral sclerosis (ALS) 2The abbreviations used are: ALS, amyotrophic lateral sclerosis; ECFP, enhanced cyan fluorescent protein; GFP, green fluorescent protein; EGFP, enhanced GFP; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; z-VAD, benzylocarbonyl-Val-Ala-Asp-(O-methyl)fluoromethylketone; DMSO, dimethyl sulfoxide; siRNA, small interfering RNA; PBS, phosphate-buffered saline; ANOVA, analysis of variance. 2The abbreviations used are: ALS, amyotrophic lateral sclerosis; ECFP, enhanced cyan fluorescent protein; GFP, green fluorescent protein; EGFP, enhanced GFP; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; z-VAD, benzylocarbonyl-Val-Ala-Asp-(O-methyl)fluoromethylketone; DMSO, dimethyl sulfoxide; siRNA, small interfering RNA; PBS, phosphate-buffered saline; ANOVA, analysis of variance. is an adult-onset neurodegenerative disease characterized by a progressive death of upper and lower motor neurons present in the cerebral cortex, brainstem, and spinal cord. This leads to skeletal muscle atrophy, paralysis, and finally death (3Julien J.P. Beaulieu J.M. J. Neurol. Sci. 2000; 180: 7-14Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 4Guégan C. Przedborski S. The J. Clin. Investig. 2003; 111: 153-161Crossref PubMed Scopus (158) Google Scholar, 5Strong M.J. Kesavapany S. Pant H.C. J. Neuropathol. Exp. Neurol. 2005; 64: 649-664Crossref PubMed Scopus (173) Google Scholar, 6Xiao S. McLean J. Robertson J. Biochim. Biophys. Acta. 2006; 1762: 1001-1012Crossref PubMed Scopus (102) Google Scholar). As seen in many neurodegenerative diseases, different protein aggregates can be found in the afflicted neurons (5Strong M.J. Kesavapany S. Pant H.C. J. Neuropathol. Exp. Neurol. 2005; 64: 649-664Crossref PubMed Scopus (173) Google Scholar, 7He C.Z. Hays A.P. J. Neurol Sci. 2004; 217: 47-54Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). These frequently contain the intermediate filament proteins neurofilament and peripherin (8Migheli A. Pezzulo T. Attanasio A. Schiffer D. Lab. Investig. 1993; 68: 185-191PubMed Google Scholar, 9Corbo M. Hays A.P. J. Neuropathol. Exp. Neurol. 1992; 51: 531-537Crossref PubMed Scopus (132) Google Scholar). Peripherin is a class III intermediate filament that is specifically expressed in neuronal tissue and at particularly high levels in cells of the peripheral nervous system (10Portier M.M. de Néchaud B. Gros F. Dev. Neurosci. 1983; 6: 335-344Crossref PubMed Scopus (183) Google Scholar, 11Parysek L.M. Goldman R.D. J. Neurosci. 1988; 8: 555-563Crossref PubMed Google Scholar).Besides the findings in patients, there are a number of studies linking peripherin aggregates to ALS and/or motor neuron degeneration. Transgenic mice that overexpress peripherin exhibit motor neuron loss that is preceded by the appearance of peripherin inclusions in the cells (12Robertson J. Beaulieu J.M. Doroudchi M.M. Durham H.D. Julien J.P. Mushynski W.E. J. Cell Biol. 2001; 155: 217-226Crossref PubMed Scopus (103) Google Scholar, 13Beaulieu J.M. Nguyen M.D. Julien J.P. J. Cell Biol. 1999; 147: 531-544Crossref PubMed Scopus (195) Google Scholar) and by defective axonal transport (14Millecamps S. Robertson J. Larivière R. Mallet J. Julien J.P. J. Neurochem. 2006; 98: 926-938Crossref PubMed Scopus (43) Google Scholar). Furthermore, the aggregation of peripherin makes neurons more susceptible to cell death stimuli (12Robertson J. Beaulieu J.M. Doroudchi M.M. Durham H.D. Julien J.P. Mushynski W.E. J. Cell Biol. 2001; 155: 217-226Crossref PubMed Scopus (103) Google Scholar), suggesting a causal link between the formation of aggregates and cellular degeneration. The putative pathogenic role of the aggregates raises the possibility that their dissolution or a block of their further expansion may suppress the advancement of the disease. However, there is not much known about the molecular mechanisms underlying the formation of peripherin aggregates.In this study, we demonstrate that protein kinase C (PKC) promotes the aggregation of peripherin. PKC constitutes a serine/threonine kinase family that is subdivided in classical (PKCα, -βI, -βII, and -γ), novel (PKCδ, -ϵ, -η, and -θ), and atypical (PKCι and -ζ) PKC isoforms based on structural similarities (15Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1348) Google Scholar). The PKC isoforms are central regulators of a wide range of cellular processes. We have seen that PKCϵ induces outgrowth of neurites in many cell types of neural origin (16Zeidman R. Pettersson L. Sailaja P.R. Truedsson E. Fagerström S. Påhlman S. Larsson C. Int. J. Cancer. 1999; 81: 494-501Crossref PubMed Scopus (62) Google Scholar, 17Zeidman R. Löfgren B. Påhlman S. Larsson C. J. Cell Biol. 1999; 145: 713-726Crossref PubMed Scopus (110) Google Scholar, 18Ling M. Trollér U. Zeidman R. Lundberg C. Larsson C. Exp. Cell Res. 2004; 292: 135-150Crossref PubMed Scopus (32) Google Scholar). The effect is independent of the catalytic activity and mediated by the C1 domains in the regulatory domain of the molecule (17Zeidman R. Löfgren B. Påhlman S. Larsson C. J. Cell Biol. 1999; 145: 713-726Crossref PubMed Scopus (110) Google Scholar). In an approach to dissect the molecular mechanisms mediating this effect, we immunoprecipitated the neuritogenic PKCϵ structure and analyzed which proteins were co-precipitated. One of these was peripherin, and the interaction was confirmed for endogenous proteins. When analyzing a putative function of the interaction in the regulation of cellular morphology, we discovered that either activation of PKC or overexpression of PKCϵ markedly increase the number of cells with peripherin aggregates. Furthermore, the aggregate formation was accompanied by an increased apoptosis. Thus, the results in this study provide important clues to our understanding of the molecular basis for the formation of peripherin aggregates and its consequences for neuronal cell survival, a feature that is characteristic for ALS.EXPERIMENTAL PROCEDURESPlasmids—Expression vectors encoding full-length human PKC isoforms, isolated domains of PKC isoforms, or kinase-dead full-length PKCϵ (K438R mutation) fused to enhanced green fluorescent protein (EGFP) have been described previously (17Zeidman R. Löfgren B. Påhlman S. Larsson C. J. Cell Biol. 1999; 145: 713-726Crossref PubMed Scopus (110) Google Scholar, 18Ling M. Trollér U. Zeidman R. Lundberg C. Larsson C. Exp. Cell Res. 2004; 292: 135-150Crossref PubMed Scopus (32) Google Scholar, 19Schultz A. Jönsson J.I. Larsson C. Cell Death Differ. 2003; 10: 662-675Crossref PubMed Scopus (40) Google Scholar). To generate an expression vector encoding FLAG-tagged peripherin, cDNA was amplified with PCR using primers containing EcoRI and SalI sites and a peripherin full-length clone (RZPD Deutsches Ressourcenzentrum für Genomforschung GmbH, clone ID: IRAT p970D0254D6) as template. The cDNA was inserted into the p3XFLAG-CMV™-7.1 vector (Sigma) and sequenced to verify that no mutations had been introduced in the PCR.pCMV-script vectors (Stratagene) encoding either wild-type vimentin or mutants with N-terminal serine residues (Ser-4, -6, -7, -8, -9) mutated to alanine (vimentin Ala) or aspartate (vimentin Asp) were kindly provided by Dr. J. Ivaska (20Ivaska J. Vuoriluoto K. Huovinen T. Izawa I. Inagaki M. Parker P.J. EMBO J. 2005; 24: 3834-3845Crossref PubMed Scopus (209) Google Scholar). To produce ECFP-tagged peripherin or peripherin and EGFP as separate proteins, the FLAG-tagged peripherin cDNA was digested with EcoRI/SalI, and the fragment was inserted in either the pECFP-C1 expression vector (Clontech) or the pCMS-EGFP expression vector (Clontech).Cell Culture and Transfections—Human neuroblastoma cells SK-N-BE(2)C were grown in minimum essential medium (Sigma) supplemented with 10% fetal bovine serum (Euro-Clone), 100 IU/ml penicillin (Invitrogen), and 100 μg/μl streptomycin (Invitrogen). MDA-MB-231 breast carcinoma cells were grown in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum, 1% sodium pyruvate, 100 IU/ml penicillin, and 100 μg/μl streptomycin. Cells were kept at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air.For immunoprecipitation experiments, SK-N-BE(2)C or MDA-MB-231 cells were seeded at a density of 1.5–2 × 106 cells/100-mm cell culture dish. For immunofluorescence experiments, 200,000 SK-N-BE(2)C cells were seeded on glass coverslips in 35-mm cell culture dishes. For differentiation of SK-N-BE(2)C cells, 200,000 cells were seeded on glass coverslips in 35-mm cell culture dishes and incubated in 10 μm all-trans-retinoic acid (Sigma). Transfections were performed with 2 μg of DNA and 2 μl of Lipofectamine 2000 (Invitrogen) per ml Optimem I medium (Invitrogen) according to the supplier's protocol.For siRNA transfections 70,000 SK-N-BE(2)C cells were transfected on three consecutive days with 50 nm Stealth™ RNA interference oligonucleotides (Invitrogen) and 1.5 μl of Lipofectamine 2000 per ml Optimem I medium. Two off-target oligonucleotides with 44 and 48% GC-content, respectively, were used as controls. The PKCϵ oligonucleotides were 5′-CACAAGUUCGGUAUCCACAACUACA-3′ (siPKCϵ1), 5′-GCAAGGUCAUGUUGGCAGAACUCAA-3′ (siPKCϵ2), and 5′-CCACAAGUUCAUGGCCACCUAUCUU-3′ (siPKCϵ3). When indicated, cells were incubated with 16 nm 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma), 2 μm GF109203X (bisindolylmaleimide I; Calbiochem), or 20 μm benzylocarbonyl-Val-Ala-Asp-(O-methyl)fluoromethylketone (z-VAD, Sigma).Immunoprecipitation—Cells were treated as indicated in the protocol supplied with the μMACS epitope-tagged protein isolation kit (Militenyi Biotec). Cells were washed twice in ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer, supplied with the kit, and a complete protease inhibitor mixture (Roche Applied Science) for 30 min on ice. Lysates were cleared by centrifugation at 14,000 × g for 10 min at 4 °C and incubated either with anti-GFP-conjugated microbeads for 30 min or with 2 μg of anti-peripherin antibodies (Sigma) for 1 h prior to addition of protein G-coupled microbeads and an additional incubation for 30 min on rotation at 4 °C. Mouse IgG1 antibodies (ImmunoKontact) were used as controls. The immune complexes were recovered by applying the cell lysates on μ columns placed in the magnetic field of a μMACS separator and then washed and eluted with an elution buffer included in the kit.Identification of Co-precipitated Proteins—Immune complexes were obtained as described under the “Immunoprecipitation” section. Thereafter iodoacetamide was added to give a final concentration of 100 mm, and the precipitated proteins were incubated for 20 min in darkness. The samples were electrophoretically separated by SDS-PAGE using a 7.5% polyacrylamide gel. The gel was silver-stained as described previously (21Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7763) Google Scholar), first washed in 50% methanol with 5% acetic acid for 20 min and placed in 50% methanol for 10 min and then sensitized with 0.02% sodium thiosulfate before staining with 0.1% silver nitrate for 20 min at 4 °C and developed in a solution of 0.04% formalin and 2% sodium carbonate for less then 10 min followed by 5% acetic acid stopping the reaction. Bands in the lane with precipitate from SK-N-BE(2)C cells transfected with PKCϵ PSC1V3 and absent in the lane with precipitate from EGFP-transfected cells were excised and treated for subsequent in-gel digestion as described (22Hellman U. Silberring J. Ekman R Mass Spectrometry and Hyphenated Techniques in Neuropeptide Research. John Wiley & Sons, Inc, New York2002: 259-275Google Scholar). Briefly, after destaining and rehydration using neat acetonitrile, the samples were proteolyzed overnight using porcine modified trypsin (Promega). The generated peptides were analyzed by peptide mass fingerprinting using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (Ultraflex tandem time-of-flight, Bruker Daltonics). The instrument settings were optimized for analytes from 600 to 4500 Da and α-cyano-4-hydroxycinnamic acid was used as matrix. The peptide mass lists were used to search sequence databases for protein identification using Pro-Found at the PROWL web site as search engine.Immunoblotting—Proteins were electrophoretically separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). The membranes were incubated with primary monoclonal antibodies toward GFP (Zymed Laboratories Inc.), peripherin (Sigma), or vimentin (DakoCytomation) or with polyclonal antibodies toward PKCα, -βII, -δ, or -ϵ (Santa Cruz Biotechnology). The membranes were thereafter incubated with horseradish peroxidase-labeled secondary antibody (Amersham Biosciences), which was detected using the SuperSignal system (Biological Industries) as substrate. The chemoluminescence was captured with a charge-coupled device camera (Fujifilm).Immunofluorescence and Confocal Microscopy—16 h after transfection, cells were washed once in PBS, fixed with 4% paraformaldehyde in PBS for 4 min, washed twice in PBS, and thereafter permeabilized and blocked with 5% goat serum and 0.3% Triton X-100 in PBS for 30 min. Cells were incubated with primary antibodies toward peripherin (Sigma), vimentin (DAKO), or FLAG (Sigma) in PBS for 1 h. Following washes in PBS, cells were incubated with secondary Alexa Fluor 546-conjugated goat anti-mouse IgG antibody (Molecular Probes) diluted 1:800 in PBS for 1 h followed by extensive washes in PBS and mounted on object slides using 20 μl of PVA-DABCO (9.6% polyvinyl alcohol, 24% glycerol and 2.5% 1,4-diazabicyclo[2.2.2]octane in 67 mm Tris-HCl, pH 8.0). For phalloidin staining, cells were incubated with Alexa Fluor 546-conjugated Phalloidin (Molecular Probes) diluted 1:200 in PBS for 20 min after the secondary antibody incubation. The coverslips were studied by immunofluorescence microscopy or by confocal microscopy using a Bio-Rad Radiance 2000 confocal system fitted on a Nikon microscope with a ×60/NA 1.40 oil lens. Excitation wavelengths were 457 nm (ECFP), 488 nm (EGFP), and 543 nm (Alexa Fluor 546), and the emission filters used were HQ485/30 (ECFP), HQ515/30 (EGFP), and 600LP (Alexa Fluor 546). In aggregation experiments, 200 transfected cells, identified by the fluorescence of EGFP, were scored for aggregate contents.Analysis of Apoptosis—SK-N-BE(2)C cells were washed in PBS and fixed with 4% paraformaldehyde in PBS for 4 min and thereafter washed twice in PBS followed by incubation for 20 min in a DNA staining solution containing 3.5 μm Tris-HCl, pH 7.6, 10 mm NaCl, 5 μg/ml propidium iodide, 20 μg/ml RNase, and 0.1% v/v Nonidet P-40. The coverslips were examined by immunofluorescence microscopy where 200 transfected cells, identified by EGFP positivity, were counted and then scored for fragmented or condensed nuclei.RESULTSPeripherin Associates with PKCϵ in Neuroblastoma Cells—Our previous work has shown that PKCϵ, via its regulatory domain, induces neurites in a wide range of neural cell types. More specifically, the effect is mediated by a structure encompassing the two C1 domains and flanking residues in the pseudosubstrate and the V3 region (PKCϵ PSC1V3) (17Zeidman R. Löfgren B. Påhlman S. Larsson C. J. Cell Biol. 1999; 145: 713-726Crossref PubMed Scopus (110) Google Scholar, 23Ling M. Trollér U. Zeidman R. Stensman H. Schultz A. Larsson C. J. Biol. Chem. 2005; 280: 17910-17919Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Our hypothesis is therefore that PKCϵ exerts some of its biological effects via this structure, conceivably by interacting with other proteins. The identification of such interacting proteins could therefore provide a significant advance in our understanding of the mechanisms that mediate some PKCϵ effects.To identify proteins that interact with the PKCϵ PSC1V3 region, SK-N-BE(2)C neuroblastoma cells were transfected with a vector encoding this PKCϵ structure fused to EGFP (Fig. 1A). Cell lysates were thereafter immunoprecipitated with an anti-GFP antibody coupled to magnetic beads. The precipitate was separated with SDS-PAGE, and the gel was subjected to silver staining (Fig. 1B). As control, precipitates of lysates from cells transfected with empty EGFP vector were used to visualize proteins unspecifically precipitated. Silver staining revealed several bands that were only present in the lane with the PKCϵ PSC1V3 precipitate. One of these, representing a protein of ∼50 kDa was, following trypsin digestion and mass spectrometry analysis, identified as peripherin, a class III intermediate filament, with an estimated molecular mass of 54 kDa.To confirm the interaction, the association of endogenous PKCϵ with peripherin was investigated. Lysates from SK-N-BE(2)C neuroblastoma cells were immunoprecipitated using anti-peripherin antibodies, and PKCϵ was detected in the precipitate (Fig. 1C). Of the other classical and novel isoforms expressed in neuroblastoma cells, neither PKCβII nor PKCδ co-precipitated with peripherin. However, there was a faint band corresponding to PKCα. Isotype-matched control antibodies did not precipitate the PKC isoforms.The interaction of PKC with its binding partners has in several cases been shown to depend on the conformation of the PKC molecule. To investigate whether this is the case for the interaction of PKCϵ with peripherin, SK-N-BE(2)C cells were treated for 30 min with TPA prior to immunoprecipitation with antibodies against peripherin. When compared with untreated cells, no effect of TPA on the amount of co-precipitated PKCϵ could be discerned (Fig. 1C). Thus, the interaction of peripherin with PKCϵ is largely isoform-specific and not influenced by activation of PKC with phorbol esters.The Interaction Is Mediated via the C1b Domain in PKCϵ—Peripherin was found as a PKCϵ-binding protein by using a fragment of the regulatory domain, PSC1V3, suggesting that this is the site responsible for the association. However, it cannot be excluded that other parts of the PKCϵ molecule also participate in the association with peripherin. To compare the interaction capacity of different PKCϵ domains, SK-N-BE(2)C cells were transfected with vectors encoding either the regulatory or the catalytic domain together with a vector encoding full-length PKCϵ fused to EGFP (Fig. 2A). Immunoprecipitation of the GFP-tagged proteins revealed that peripherin binds to the regulatory domain and not to the catalytic domain of PKCϵ. The interaction with the isolated regulatory domain seemed to be stronger than the interaction with the holo-enzyme, perhaps implying that relevant structures are conformationally masked in the full-length protein.FIGURE 2The interaction with peripherin is mediated by the C1b domain of PKCϵ. SK-N-BE(2)C cells were transfected with vectors encoding EGFP fusions of either full-length PKCϵ (PKCϵFL), the PKCϵ regulatory domain (PKCϵRD), or the PKCϵ catalytic domain (PKCϵCD) (A); the PKCϵ C2 domain (PKCϵC2), the PKCϵ PSC1V3 domain (PKCϵPSC1V3), or a PKCϵ PSC1V3 variant where the PKCϵ C1b domain was exchanged for the PKCα C1b domain (PKCϵPSC1a(αC1b)V3)(B); or the PKCϵ regulatory domain (PKCϵRD), the PKCϵ C1a domain (PKCϵC1a), the PKCϵ C1b domain (PKCϵC1b), the tandem PKCϵ C1a and C1b domains together (PKCϵC1ab), or the PKCα C1b domain (PKCαC1b) (C). An empty EGFP vector was used as control. Lysates were immunoprecipitated using anti-GFP-conjugated magnetic beads and thereafter subjected to Western blotting using a peripherin antibody. The positions of molecular mass markers are shown to the left of the blots. Please note that the two rightmost constructs in the total cell lysate blot of B are weak and located immediately above an unspecific band and therefore hard to detect. The presence of the constructs are clearly seen in the blot with the precipitates.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further delineate the specific structures in the PKCϵ regulatory domain that mediate the interaction, the C2 domain and the PSC1V3 domain of PKCϵ fused to EGFP were expressed in SK-N-BE(2)C cells and immunoprecipitated (Fig. 2B). This demonstrated that the C2 domain does not contribute to the interaction with peripherin. In addition, a construct encoding PKCϵ PSC1V3 with the C1b domain exchanged for the PKCα C1b domain (PKCϵ PSC1a(αC1b)V3) (23Ling M. Trollér U. Zeidman R. Stensman H. Schultz A. Larsson C. J. Biol. Chem. 2005; 280: 17910-17919Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) was evaluated for peripherin interaction. The amount of peripherin that was co-precipitated with this chimera (Fig. 2B, lane 4) was substantially lower when compared with the amount that was precipitated with the pure PKCϵ construct. Furthermore, the isolated PKCϵ C1b domain co-precipitated with peripherin, whereas this was not observed for either the isolated PKCϵ C1a or the isolated PKCα C1b domain (Fig. 2C). Thus, the peripherin interaction seems to be mediated primarily by the PKCϵ C1b domain. However, the tandem PKCϵ C1aC1b domain displayed a stronger interaction than the isolated C1b domain (Fig. 2C), indicating that the C1a domain or structures flanking the C1b domain contribute to or promote the interaction.Peripherin Preferentially Binds to PKCϵ—To further investigate the isoform specificity of the interaction, SK-N-BE(2)C cells were transfected with vectors encoding full-length versions of PKCα, -βI, -βII, -δ, -ϵ, -η, and -θ fused to EGFP. The constructs were immunoprecipitated using the GFP tag, and the eluted immunoprecipitates were analyzed with Western blotting (Fig. 3A). The strongest association was seen with PKCϵ but PKCη also precipitated some peripherin.FIGURE 3PKCϵ is the isoform with the strongest interaction with peripherin. SK-N-BE(2)C cells were transfected with vectors encoding EGFP only or EGFP fused to the full-length versions (A); the regulatory domains (B); or the C1b domains (C) of the different PKC isoforms α, β, δ, ϵ, η, or θ. Cell lysates were immunoprecipitated (IP) using anti-GFP-conjugated magnetic beads and thereafter subjected to Western blotting using anti-peripherin antibody. The positions of the molecular mass markers for 66, 45, and 30 kDa are shown to the left of the blots. RD, regulatory domain.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We thereafter investigated whether the isolated regulatory domains from the previously mentioned isoforms can bind peripherin (Fig. 3B). Peripherin binds strongly to the regulatory domain of PKCϵ but not to the corresponding structure from PKCα, -β, or -δ. The low expression levels of the PKCη and -θ regulatory domains together with the faint peripherin bands in the precipitates indicate that these isoforms may also contain the structures necessary for association with peripherin.Next, the isolated C1b domains from PKCα and all novel PKC isoforms were compared in terms of association with peripherin (Fig. 3C). A substantial interaction with peripherin was observed for the PKCϵ C1b domain, although minor amounts of peripherin were detected in PKCη C1b and PKCθ C1b precipitates, reflecting the results with the corresponding regulatory domains (Fig. 3, A and B).Overexpressed Peripherin Aggregates in a PKC-dependent Manner—To analyze a putative functional importance of the interaction of PKCϵ with peripherin, we first overexpressed peripherin tagged to the FLAG epitope in SK-N-BE(2)C neuroblastoma cells and thereafter incubated the cells for 16 h with the PKC activator TPA and/or the PKC inhibitor GF109203X (Fig. 4, A and B). Transfected cells were then visualized by the green fluorescent protein, and exogenous peripherin was visualized with immunofluorescence toward the FLAG tag. Overexpression of peripherin resulted in the formation of large peripherin-positive aggregates in some cells. Exposure to TPA markedly increased the number of cells with aggregates (Fig. 4A, panel d) from 31 to 54% (Fig. 4B), and this effect was suppressed by GF109203X (Fig. 4A, panel f, and B). The peripherin aggregates are reminiscent of what is observed in nervous tissue of patients with ALS, and the PKC-mediated aggregation of peripherin may therefore shed mechanistic light on this disease (3Julien J.P. Beaulieu J.M. J. Neurol. Sci. 2000; 180: 7-14Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 6Xiao S. McLean J. Robertson J. Biochim. Biophys. Acta. 2006; 1762: 1001-1012Crossref PubMed Scopus (102) Google Scholar, 12Robertson J. Beaulieu J.M. Doroudchi M.M. Durham H.D. Julien J.P. Mushynski W.E. J. Cell Biol. 2001; 155: 217-226Crossref PubMed Scopus (103) Google Scholar).FIGURE 4Activation of PKC or overexpression of PKCϵ potentiates peripherin aggregation.A, SK-N-BE(2)C cells were co-transfected with an empty EGFP vector (panels a–f), full-length PKCϵ (PKCϵFL) fused to EGFP (panels g and h), or kinase-dead PKCϵ (PKCϵKD) fused to EGFP together with a vector encoding peripherin tagged to the FLAG epitope. The cells were thereafter treated with either 16 nm TPA alone (panels c and d) or TPA combined with 2 μm GF109203X (GFX, panels e and f) for 16 h. Exogenous peripherin was visualized by immunofluorescence with an anti-FLAG antibody. Cells were examined with a confocal microscope, and images show EGFP (panels a, c, and e,) PKCϵ-EGFP (panel g), and FLAG-tagged peripherin (panels b, d, f, and h). Cells treated with DMSO were used as controls (panels a and b and panels g and h). B, SK-N-BE(2)C cells transfected and treated as in A were examined with fluorescence microscopy, and 200 transfected cells, visualized by the EGFP fluorescence, were scored for the presence of peripherin aggregates. A Western blot demonstrating relative expression of PKCϵ-EGFP fusions is shown above the graph. Data (mean ± S.E., n = 3) shows the percentage of tr" @default.
• W1984867277 created "2016-06-24" @default.
• W1984867277 creator A5020503361 @default.
• W1984867277 creator A5032534504 @default.
• W1984867277 creator A5037114968 @default.
• W1984867277 date "2008-06-01" @default.
• W1984867277 modified "2023-10-16" @default.
• W1984867277 title "Protein Kinase Cϵ Binds Peripherin and Induces Its Aggregation, Which Is Accompanied by Apoptosis of Neuroblastoma Cells" @default.
• W1984867277 cites W1496021072 @default.
• W1984867277 cites W1592357554 @default.
• W1984867277 cites W1592463385 @default.
• W1984867277 cites W1963526979 @default.
• W1984867277 cites W1970857342 @default.
• W1984867277 cites W1976151026 @default.
• W1984867277 cites W1988103147 @default.
• W1984867277 cites W1989326240 @default.
• W1984867277 cites W1993859056 @default.
• W1984867277 cites W1994730834 @default.
• W1984867277 cites W2008452978 @default.
• W1984867277 cites W2008563656 @default.
• W1984867277 cites W2012016770 @default.
• W1984867277 cites W2013973620 @default.
• W1984867277 cites W2019868249 @default.
• W1984867277 cites W2021771327 @default.
• W1984867277 cites W2022367303 @default.
• W1984867277 cites W2030802378 @default.
• W1984867277 cites W2032786799 @default.
• W1984867277 cites W2033087568 @default.
• W1984867277 cites W2034334262 @default.
• W1984867277 cites W2047695690 @default.
• W1984867277 cites W2058546812 @default.
• W1984867277 cites W2061954667 @default.
• W1984867277 cites W2062383179 @default.
• W1984867277 cites W2065752107 @default.
• W1984867277 cites W2070243481 @default.
• W1984867277 cites W2071258325 @default.
• W1984867277 cites W2078594804 @default.
• W1984867277 cites W2081457844 @default.
• W1984867277 cites W2082055400 @default.
• W1984867277 cites W2091821388 @default.
• W1984867277 cites W2096386031 @default.
• W1984867277 cites W2096410114 @default.
• W1984867277 cites W2098662382 @default.
• W1984867277 cites W2104169720 @default.
• W1984867277 cites W2104969843 @default.
• W1984867277 cites W2107768418 @default.
• W1984867277 cites W2108296389 @default.
• W1984867277 cites W2110690118 @default.
• W1984867277 cites W2121217628 @default.
• W1984867277 cites W2131819352 @default.
• W1984867277 cites W2137511299 @default.
• W1984867277 cites W2151111659 @default.
• W1984867277 cites W2157978028 @default.
• W1984867277 cites W2158787641 @default.
• W1984867277 cites W4251091760 @default.
• W1984867277 doi "https://doi.org/10.1074/jbc.m710436200" @default.
• W1984867277 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18408015" @default.
• W1984867277 hasPublicationYear "2008" @default.
• W1984867277 type Work @default.
• W1984867277 sameAs 1984867277 @default.
• W1984867277 citedByCount "20" @default.
• W1984867277 countsByYear W19848672772012 @default.
• W1984867277 countsByYear W19848672772013 @default.
• W1984867277 countsByYear W19848672772014 @default.
• W1984867277 countsByYear W19848672772015 @default.
• W1984867277 countsByYear W19848672772021 @default.
• W1984867277 countsByYear W19848672772023 @default.
• W1984867277 crossrefType "journal-article" @default.
• W1984867277 hasAuthorship W1984867277A5020503361 @default.
• W1984867277 hasAuthorship W1984867277A5032534504 @default.
• W1984867277 hasAuthorship W1984867277A5037114968 @default.
• W1984867277 hasBestOaLocation W19848672771 @default.
• W1984867277 hasConcept C104317684 @default.
• W1984867277 hasConcept C153911025 @default.
• W1984867277 hasConcept C161238802 @default.
• W1984867277 hasConcept C184235292 @default.
• W1984867277 hasConcept C185592680 @default.
• W1984867277 hasConcept C190283241 @default.
• W1984867277 hasConcept C2776715637 @default.
• W1984867277 hasConcept C28521208 @default.
• W1984867277 hasConcept C54355233 @default.
• W1984867277 hasConcept C55493867 @default.
• W1984867277 hasConcept C81885089 @default.
• W1984867277 hasConcept C86803240 @default.
• W1984867277 hasConcept C90934575 @default.
• W1984867277 hasConcept C95444343 @default.
• W1984867277 hasConcept C97029542 @default.
• W1984867277 hasConceptScore W1984867277C104317684 @default.
• W1984867277 hasConceptScore W1984867277C153911025 @default.
• W1984867277 hasConceptScore W1984867277C161238802 @default.
• W1984867277 hasConceptScore W1984867277C184235292 @default.
• W1984867277 hasConceptScore W1984867277C185592680 @default.
• W1984867277 hasConceptScore W1984867277C190283241 @default.
• W1984867277 hasConceptScore W1984867277C2776715637 @default.
• W1984867277 hasConceptScore W1984867277C28521208 @default.
• W1984867277 hasConceptScore W1984867277C54355233 @default.
• W1984867277 hasConceptScore W1984867277C55493867 @default.
• W1984867277 hasConceptScore W1984867277C81885089 @default.

• next