Abstract
Role of protein kinase C (PKC) isotypes in the regulation of neutrophil function are not clearly known. In the present study we purified the β-PKC and ζ-PKC isotypes from human neutrophil. Both the isotypes are immunoreactive only to their respective antibodies. ζ-PKC was further confirmed by RT-PCR using specific primer. Co-factor requirements for both the kinases were found to be different when DG and ceramide were used as second messenger. Selective substrate specificities were determined for both β and ζ-PKC using isotype specific pseudosubstrates viz., [Ser25]PKC [19-31] and [Ser119]PKC[113-130] respectively. Endogenous protein phosphorylation by purified β-PKC and ζ-PKC showed their functional differences in neutrophil. β-PKC phosphorylated 13, 15, 19, 33, 36, 47, 80 and 92 kDa proteins and ζ-PKC phosphorylated 19, 22, 42, 47, 75 and 87 kDa proteins, only exception was the phosphorylation of 47 kDa protein which had been phosphorylated by both the kinases. Differences in phosphorylation between β-PKC and ζ-PKC clearly indicate the selective role for these PKC isotypes in the activation sequences of neutrophil.
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Nishizuka Y: The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334: 662-665, 1988
Kikkawa U, Kishimoto A, Nishizuta Y: The Protein Kinase C Family: Heterogeneity and Its Implications. Annu. Rev. Biochem. 58: 31-34, 1989
Coussens L, Parker PJ, Rhee L, Yang-Feng TL, Chen E, Waterfield MD, Francek U, Ullrich A: Substrate recognition by Ceramide–activated protein kinase–Evidence that kinase activity is proline directed. Science 233: 859-866, 1986
Marais RM, Parker PJ: Purification and characterisation of bovine brain protein kinase C isotypes α, β, and γ. Eur. J. Biochem. 182: 129-137, 1989
Borner C, Guadagno SN, Fabbro D, Weinstein IB: Expression of Four Protein Kinase C isoforms in Rat Fibroblasts. J. Biol. Chem. 267: 12892-12898, 1992
Akita Y, Ohno S, Konno Y, Yano A, Suzuki K: Expression and Properties of Two Distinct Classes of the Phorbol Ester Receptor Family, Four conventional Protein Kinase C Types and a Novel Protein Kinase C. J. Biol. Chem. 265: 354-362, 1990
Ono Y, Fuji T, Ogita K, Kikkawa U, Igarashi K, Nishizuka Y: The Structure Expression and Properties of Additional Members of the Protein Kinase C Family. J. Biol. Chem. 263: 6927-6932, (1988)
Ohno Y, Akita Y, Konno, Y, Imajoh S, Suzuki K: A Novel Phorbol Ester Receptor/ Protein Kinase, nPKC, Distinctly Related to the protein Kinase C Family. Cell, 53: 731-741, 1988
Schaap D, Parker PJ: Expression, Purification and Characterisation of Protein Kinase C-ε. J. Biol. Chem. 265: 7301-7307, 1990
Osada S, Mizuno K, Saido TC, Akita Y, Suzuki K, Kuroki T, Ohno S: A phorbol ester receptor/protein kinase, nPKCeta, a new member of the protein kinase C family predominantly expressed in lung and skin. J. Biol. Chem. 265: 22434-22440, 1990
Baier G, Telford D, Giampa L, Coggeshall KM, Bitterlich GB, Isakov N, Altman A: Molecular Cloning and Characterization of PKC theta, a Novel Member of the Protein Kinase C (PKC) Gene Family Exposed Predominantly in Hematopoitic Cells. J. Biol. Chem. 268: 4997-5004, 1993
Dekker LV, Parker PJ: Protein Kinase C–a question of specificity. Trends Biochem. Sci. 19: 73-77, 1994
Nishizuka Y: Protein Kinase C and lipid signaling for sustained cellular responses. FASEB J. 9: 484-496, 1995
McPhail LC, Harvath L: Signal Transduction in neutrophil oxidative metabolism and chemotaxis, Natural Immune System. In J.S. Abramson, J.G. Wheeler (eds). The Neutrophil. Oxford University Press, Oxford, 63-107, 1993
Lu DJ, Furuya W, Grinstein S: Involvement of multiple kinases in neutrophil activation. Cell 19: 43-349, 1993
DeChatelet LR, Shirley PS, Johnston RB Jr.: Effect of phorbol myristate acetate on the oxidative metabolism of human polymorpho nuclear leukocytes. Blood. 47 (4): 545-554, 1976
Fujita I, Irita K, Takeshieg K, Minakami S: Diacyl glycerol, 1-oleoyl-2-acetyl-glycerol, stimulates superoxide generation from human neutrophils. Biochem. Biophys. Res. Commun. 120: 318-324, 1984
Naccache PH, Molski MM, Sha'afi RI: Polymyxin B inhibits phorbol 12-myristate 13-acetate, but not chemotactic factor, induced effects in rabbit neutrophil. FEBS Lett. 193: 227-230, 1985
Cox CC, Dougherty RW, Ganong BR, Bell RM, Niedel J E and Snyderman R: Differential stimulation of the respiratory burst and lysosomal enzyme secretion in human polymorphonuclear leukocytes by synthetic diacylglycerols. J. Immunol. 136: 4611-4616, 1986
Wright TM, Hoffman RD, Nishijima J, Jakoi L, Snyderman R, Shin HS: Leukocyte chemoattraction by 1,2-diacylglycerol. Proc. Natl. Acad. Sci. 85:1869-1873, 1988
Clark RA, Volpp BD, Leidal KG, Neuseef WM: Two cytosolic components of the neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation. J. Clin. Invest. 85: 714-721, 1990
Volpp BD, Nauseef WM, Donelson JF, Moser DR, Clark RA: Cloning of the cDNA and functional expression of the 47-Kilodalton cytosolic component of human neutrophil respiratory burst oxidase. Proc. Natl. Acad. Sci. 86: 7195-7199, 1989
Lomax KJ, Leto TL, Nunoi H, Gallin JI, Malech HL: Recombinant 47-Kilodalton cytosolic factor restores NADPH oxidase in chronic granulomatous disease. Science 245: 409-412, 1989
Clark RA, Malech HL, Gallin JI, Nunoi H, Volpp BD, Pearson DW, Nauseef WM, Curnutte JT: Genetic varients of Chronic Granulomatous disease: Prevalences of two cytosolic components of NADPH oxidase system. (1989) N. Engl. J. Med. 331, 647-652
Segal AW, Heyworth PG, Cockroft S, Barrowman MM: Stimulated neutrophils from patients with autosomal recessive chronic granulomatous disease fail to phosphorylate a Mr-44,000 protein. Nature. 316: 547-549, 1985
Heyworth PG, Curnutte JT, Nauseef WM, Volpp BD, Pearson DW, Rosen H, Clark RA: Neutrophil nicotinamide adenine dinucletide phosphate oxidase assembly. Translocation of p47-phox requires interaction between p47-phox and cytochrome b558. J. Clin. Invest. 87: 352-356, 1991
Nauseef WM, Volpp BD, McCormick S, Leidal KG, Clark RA: Assembly of the neutropihl respiratory burst oxidase. J. Biol. Chem. 266: 5911-5917, 1991
Majumdar S, Rossi MW, Fujiki T, Phillips WA, Disa S, Queen CF, Johnston RB Jr, Rosen OM, Corkey BE, Korchak HM: Protein Kinase C isotypes and signaling in human neutrophils J. Biol. Chem. 266: 9285-9294, 1991
Majumdar S, Kane LH, Rossi MW, Volpp BD, Nauseef WM, Korchak HM: Protein Kinase C isotypes and signal-transduction in human neutrophils: Selective substrate specificity of Calciumdependent β-PKC and novel calcium-independent nPKC. Biochim. Biophys. Acta. 1176, 276-286, 1993
Pontremoli S, Meloni E, Sparatore B, Michetti M, Salamino F, Horecker BL: Isozymes of Protein Kinase C in Human Neutrophils and Their Modification by Two Endogenous Proteinases. J. Biol. Chem. 265: 706-712, 1990
Phillips WA, Fujiki T, Rossi MW, Korchak HM, Johnston RB Jr: Influence of Calcium on the subcellular Distribution of Protein Kinase C in Human neutrophils. J. Biol. Chem. 264: 8361-8365, 1989
Kent JD, Sergeant S, Burnes DJ and McPhail LC: Identification and regulation of protein kinase C δ in human neutrophil. J. Immunol. 147: 4641-4647, 1996
Boyum A: Isolation of mononuclear cells and granulocyte from human blood. Scand. J. Clin. Lab. Invest. 21, Suppl 97: 77-89, 1968
Korchak HM, Vienne K, Rutherford LE, Wilkenfeld C, Finkelstein MC, Weissmann G: Stimulus response coupling in human neutrophil. III. Differential requirements for receptor occupancy in neutrophil response to a chemoattractant. J. Biol. Chem. 259: 4076-4082, 1984
Benjamin LJ, Lindsay AM, Barnes PJ, Giembycz MA: Protein Kinase C isozymes in airway smooth muscle. Biochem. J 324: 167-175, 1997
Laemmli UK: Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature 227: 680-685, 1970
Ways D, Cook P, Webstar C, Parker P: Effect of Phorbol ester on PKC ζ. J. Biol. Chem. 267: 4799-4805, 1992
Nakanishi H, Brewer KA, Exton JH: Activation of ζ-isozymes of protein kinase C by phosphatidylinositol 3,4,5-triphosphate. J. Biol. Chem. 268: 13-16, 1993
Ono Y, Fujii T, Ogita K, Kikkawa U, Igarashi K, Nishizuka Y: Protein Kinase C ζ subspecies from rat brain: Its structure, expression and properties. (cDNA/gene expression). Proc. Natl. Acad. Sci. 86: 3099-3103, 1989
De BP, Gupta S, Gupta S, Banerjee AK: Cellular protein kinase C isoform ζ regulates human parainfluenza virus type 3 replication. Proc. Natl. Acad. Sci. 92(11): 5204-5208, 1995
Dang PM, Hakim J, Perianin A: Immunochemical identification and translocation of Protein Kinase C zeta in human neutrophil. FEBS Lett. 349: 338-342, 1994
Brindley DN, Abousalham A, Kikuchi Y, Wang C, Waggoner DW (1996) Biochem. Cell. Biol. 74, 469-476
Hannun YA: The sphingomyelin cycle and the second messenger function of ceramide. J. Biol. Chem. 269(5): 3125-3128, 1994
Kemp BE, Pearson RB, House C, Robinsosn PJ, Means AR (1989) Cell Signal. 1, 303-311
Lozano J, Berra E, Municio MM, Diaz-Meco MT, Dominguez I, Sanz L, Moscat J: Protein Kinase C ζ isoform is critical for κBdependent Promoter Activation by Sphingomyelinase. J. Biol. Chem. 269 (30): 19200-02, 1994
Hannun AY, Obeid ML: Ceramide: An intracellular signal for apoptosis. TIBS. 20: 73-77, 1995
Diaz-Meco MT, Dominguez I, Sanz L, Dent P, Lozano J, Municio MM, Berra E, Hay RT, Sturgill TW, Moscat J: ζPKC induced phosphorylation and inactivation of IκB-α in vitro. EMBO J. 13 (12): 2842-2848, 1994
House C, Wettenhall RE, Kemp BE: The influence of basic residues on the substrate specificity of protein kinase C. J. Biol. Chem. 262 (2): 772-777, 1987
Muller G, Ayoub M, Storz P, Rennecke J, Fabbro D, Pfizenmaier K: PKC μ is a molecular switch in signal transduction of TNF-α, bifunctionally regulated by ceramide and arachidonic acid. EMBO J. 14(9): 1961-1969, 1995
Heyworth PG, Karnovsky ML, Badway JA: Protein Phosphorylation Associated with Synergistic Stimulation of Neutrophils. J. Biol. Chem. 264: 14935-14939, 1989
Stokoe D, Campbell DG, Nakielny S, Hidaka H, Leevers SJ, Marshall C, Cohen P: MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase. EMBO J 11: 3985-3994, 1992
Ballester R, Furth ME, Rosen OM: Phorbol Ester–and Protein Kinase C-mediated phosphorylation of the Cellular Kirsten Ras Gene Product. J. Biol. Chem. 262: 2688-2695, 1987
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Das, S., Bhattacharyya, S., Ghosh, S. et al. Signal transduction mechanism in human neutrophil: comparative study between the ζ and β-protein kinase c isotypes. Mol Cell Biochem 203, 143–151 (2000). https://doi.org/10.1023/A:1007097220890
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DOI: https://doi.org/10.1023/A:1007097220890