Abstract
Murine embryo fibroblasts (C3H 10T1/2) which were genetically engineered to overproduce the beta 1 isoform of protein kinase C (PKC-beta 1) were used to obtain homogeneous preparations of PKC-beta 1 for the purpose of characterizing the specific structural and functional properties of this isoform. Fractionation of PKC activity from these cells by hydroxyapatite chromatography produced one major peak, which represented 93% of the total cellular PKC activity and was not detected in control cells. This major peak of activity was shown by Western-blotting analysis with a beta 1-specific antiserum to be the overproduced beta 1-isoform, and exhibited a band at 77 kDa. The functional properties of the overproduced PKC-beta 1 were established with regard to phospholipid-dependence, Ca2(+)-dependence, responsiveness to a phorbol ester tumour promoter, activation by arachidonic acid (plus Ca2+), and inhibition by known PKC inhibitors. From these studies we conclude that PKC-beta 1 overproduced by C3H 10T1/2 cells exhibits the structural and functional properties previously ascribed to native PKC. Furthermore, these data provide the first definitive biochemical characteristics of this isoform of PKC.
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Selected References
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- Adriaenssens P. I., Bixler C. J., Anderson M. W. Isolation and quantitation of DNA-bound benzo(a)pyrene metabolites: comparison of hydroxyapatite and precipitation procedures. Anal Biochem. 1982 Jun;123(1):162–169. doi: 10.1016/0003-2697(82)90637-6. [DOI] [PubMed] [Google Scholar]
- Arcoleo J. P., Weinstein I. B. Activation of protein kinase C by tumor promoting phorbol esters, teleocidin and aplysiatoxin in the absence of added calcium. Carcinogenesis. 1985 Feb;6(2):213–217. doi: 10.1093/carcin/6.2.213. [DOI] [PubMed] [Google Scholar]
- Castagna M., Takai Y., Kaibuchi K., Sano K., Kikkawa U., Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem. 1982 Jul 10;257(13):7847–7851. [PubMed] [Google Scholar]
- Coussens L., Parker P. J., Rhee L., Yang-Feng T. L., Chen E., Waterfield M. D., Francke U., Ullrich A. Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. Science. 1986 Aug 22;233(4766):859–866. doi: 10.1126/science.3755548. [DOI] [PubMed] [Google Scholar]
- Hidaka H., Inagaki M., Kawamoto S., Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry. 1984 Oct 9;23(21):5036–5041. doi: 10.1021/bi00316a032. [DOI] [PubMed] [Google Scholar]
- House C., Kemp B. E. Protein kinase C contains a pseudosubstrate prototope in its regulatory domain. Science. 1987 Dec 18;238(4834):1726–1728. doi: 10.1126/science.3686012. [DOI] [PubMed] [Google Scholar]
- Housey G. M., Johnson M. D., Hsiao W. L., O'Brian C. A., Murphy J. P., Kirschmeier P., Weinstein I. B. Overproduction of protein kinase C causes disordered growth control in rat fibroblasts. Cell. 1988 Feb 12;52(3):343–354. doi: 10.1016/s0092-8674(88)80027-8. [DOI] [PubMed] [Google Scholar]
- Huang K. P., Huang F. L., Nakabayashi H., Yoshida Y. Biochemical characterization of rat brain protein kinase C isozymes. J Biol Chem. 1988 Oct 15;263(29):14839–14845. [PubMed] [Google Scholar]
- Huang K. P., Nakabayashi H., Huang F. L. Isozymic forms of rat brain Ca2+-activated and phospholipid-dependent protein kinase. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8535–8539. doi: 10.1073/pnas.83.22.8535. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kikkawa U., Ogita K., Ono Y., Asaoka Y., Shearman M. S., Fujii T., Ase K., Sekiguchi K., Igarashi K., Nishizuka Y. The common structure and activities of four subspecies of rat brain protein kinase C family. FEBS Lett. 1987 Nov 2;223(2):212–216. doi: 10.1016/0014-5793(87)80291-0. [DOI] [PubMed] [Google Scholar]
- Kikkawa U., Ono Y., Ogita K., Fujii T., Asaoka Y., Sekiguchi K., Kosaka Y., Igarashi K., Nishizuka Y. Identification of the structures of multiple subspecies of protein kinase C expressed in rat brain. FEBS Lett. 1987 Jun 15;217(2):227–231. doi: 10.1016/0014-5793(87)80668-3. [DOI] [PubMed] [Google Scholar]
- Kirschmeier P. T., Housey G. M., Johnson M. D., Perkins A. S., Weinstein I. B. Construction and characterization of a retroviral vector demonstrating efficient expression of cloned cDNA sequences. DNA. 1988 Apr;7(3):219–225. doi: 10.1089/dna.1988.7.219. [DOI] [PubMed] [Google Scholar]
- Kosaka Y., Ogita K., Ase K., Nomura H., Kikkawa U., Nishizuka Y. The heterogeneity of protein kinase C in various rat tissues. Biochem Biophys Res Commun. 1988 Mar 30;151(3):973–981. doi: 10.1016/s0006-291x(88)80461-3. [DOI] [PubMed] [Google Scholar]
- Krauss R. S., Housey G. M., Johnson M. D., Weinstein I. B. Disturbances in growth control and gene expression in a C3H/10T1/2 cell line that stably overproduces protein kinase C. Oncogene. 1989 Aug;4(8):991–998. [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
- McCaffrey P. G., Rosner M. R., Kikkawa U., Sekiguchi K., Ogita K., Ase K., Nishizuka Y. Characterization of protein kinase C from normal and transformed cultured murine fibroblasts. Biochem Biophys Res Commun. 1987 Jul 15;146(1):140–146. doi: 10.1016/0006-291x(87)90702-9. [DOI] [PubMed] [Google Scholar]
- Nakadate T., Jeng A. Y., Blumberg P. M. Comparison of protein kinase C functional assays to clarify mechanisms of inhibitor action. Biochem Pharmacol. 1988 Apr 15;37(8):1541–1545. doi: 10.1016/0006-2952(88)90016-0. [DOI] [PubMed] [Google Scholar]
- Nishizuka Y. Studies and perspectives of protein kinase C. Science. 1986 Jul 18;233(4761):305–312. doi: 10.1126/science.3014651. [DOI] [PubMed] [Google Scholar]
- Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature. 1988 Aug 25;334(6184):661–665. doi: 10.1038/334661a0. [DOI] [PubMed] [Google Scholar]
- Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
- O'Brian C. A., Liskamp R. M., Solomon D. H., Weinstein I. B. Inhibition of protein kinase C by tamoxifen. Cancer Res. 1985 Jun;45(6):2462–2465. [PubMed] [Google Scholar]
- O'Brian C. A., Ward N. E., Weinstein I. B., Bull A. W., Marnett L. J. Activation of rat brain protein kinase C by lipid oxidation products. Biochem Biophys Res Commun. 1988 Sep 30;155(3):1374–1380. doi: 10.1016/s0006-291x(88)81293-2. [DOI] [PubMed] [Google Scholar]
- Ono Y., Fujii T., Ogita K., Kikkawa U., Igarashi K., Nishizuka Y. Identification of three additional members of rat protein kinase C family: delta-, epsilon- and zeta-subspecies. FEBS Lett. 1987 Dec 21;226(1):125–128. doi: 10.1016/0014-5793(87)80564-1. [DOI] [PubMed] [Google Scholar]
- Parker P. J., Coussens L., Totty N., Rhee L., Young S., Chen E., Stabel S., Waterfield M. D., Ullrich A. The complete primary structure of protein kinase C--the major phorbol ester receptor. Science. 1986 Aug 22;233(4766):853–859. doi: 10.1126/science.3755547. [DOI] [PubMed] [Google Scholar]
- Pelosin J. M., Vilgrain I., Chambaz E. M. A single form of protein kinase C is expressed in bovine adrenocortical tissue, as compared to four chromatographically resolved isozymes in rat brain. Biochem Biophys Res Commun. 1987 Aug 31;147(1):382–391. doi: 10.1016/s0006-291x(87)80133-x. [DOI] [PubMed] [Google Scholar]
- Sekiguchi K., Tsukuda M., Ase K., Kikkawa U., Nishizuka Y. Mode of activation and kinetic properties of three distinct forms of protein kinase C from rat brain. J Biochem. 1988 May;103(5):759–765. doi: 10.1093/oxfordjournals.jbchem.a122343. [DOI] [PubMed] [Google Scholar]
- Sekiguchi K., Tsukuda M., Ogita K., Kikkawa U., Nishizuka Y. Three distinct forms of rat brain protein kinase C: differential response to unsaturated fatty acids. Biochem Biophys Res Commun. 1987 Jun 15;145(2):797–802. doi: 10.1016/0006-291x(87)91035-7. [DOI] [PubMed] [Google Scholar]
- Takai Y., Kishimoto A., Kikkawa U., Mori T., Nishizuka Y. Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated, phospholipid-dependent protein kinase system. Biochem Biophys Res Commun. 1979 Dec 28;91(4):1218–1224. doi: 10.1016/0006-291x(79)91197-5. [DOI] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weinstein I. B. Growth factors, oncogenes, and multistage carcinogenesis. J Cell Biochem. 1987 Mar;33(3):213–224. doi: 10.1002/jcb.240330308. [DOI] [PubMed] [Google Scholar]
- Weinstein I. B. The origins of human cancer: molecular mechanisms of carcinogenesis and their implications for cancer prevention and treatment--twenty-seventh G.H.A. Clowes memorial award lecture. Cancer Res. 1988 Aug 1;48(15):4135–4143. [PubMed] [Google Scholar]