Abstract
A multifunctional Ca2+/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca2+/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca2+/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca2+/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca2+/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca2+/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca2+/calmodulin-dependent protein kinase of Candida albicans.
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Manalan AS, Klee CB: Calmodulin. Adv Cyclic Nucleotide Prot Phosphoryl Res 18: 227-278, 1984
Goldenring JR, Goanzalez B, McGurie JS Jr, De Lorenzo RJ: Purification and characterization of calmodulin dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubulin associated protein. J Biol Chem 258: 12632-12640, 1983
Schulman H: The multifunctional Ca2+/calmodulin dependent protein kinase. Adv Second Messenger Phosphoprot Res 22: 39-112, 1988
Scherer S, Magee PT: Genetics of Candida albicans. Microbiol Rev 54: 226-241 1990
Wood, Nugent: Inhibitory effects of chlorpromazine on candida species. Antimicrob Agents Chemother 27: 692-694, 1985
Datta A, Ganesan K, Natarajan K: Current trends in Candida albicans research. Adv Microb Physiol 30: 53-88, 1989
Iida H, Sakaguchi S, Yagawa Y, Anraku Y: Cell cycle control by Ca2+ in Saccharomyces cerevisiae. J Biol Chem 265: 21216-21222, 1990
Lu KP, Means RA: Regulation of cell cycle by calcium and calmodulin. Endocrin Rev 14: 40-58, 1993
Sharma S, Kaur H, Khuller GK: Cell cycle effects of phenothiazines: Trifluoperazine and chlorpromazine in Candida albicans. FEMS Microbiol Lett 199: 185-190, 2001
Sharma S, Khuller GK: Changes in the cellular composition of C. albicans resistant to miconazole. Ind J Biochem Biophys 33: 420-424, 1996
Roskoski JR: Assays of protein kinases. Meth Enzymol 99: 3-6, 1983
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970
Lowry OH, Rosebrough NJ, Farr AL, Randal RJ: Protein measurement with folinphenol reagent. J Biol Chem 193: 265-275, 1951
Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72: 248-256, 1976
Miyakawa T, Oka Y, Tsuchiya E, Fukui S: Saccharomyces cerevisiae protein kinase dependent on Ca2+ and calmodulin. J Bacteriol 171: 1417-1422, 1989
Londesborough J, Nuutinen M: Ca2+/calmodulin dependent protein kinase in Saccharomyces cerevisiae. FEBS Lett 219: 249-253, 1987
Bartelt DC, Fidel S, Farber LH, Wolff DJ, Hammell RL: Calmodulin dependent multifunctional protein kinase in Aspergilllus nidulans. Proc Natl Acad Sci USA 85: 3279-3283, 1988
Ulloa RM, Torres HN, Ochatt CM, Tellez-Inon MT: Ca2+/calmodulin dependent protein kinase activity in the ascomycetes Neurospora crassa. Mol Cell Biochem 102: 155-163, 1991
Maeno H, Reyes PL, Veda T, Rudolph SA, Greengard P: Autophosphorylation of adenosine 3′,5′-monophosphate dependent protein kinase from bovine brain. Arch Biochem Biophys 164: 551-559, 1974
Rosen OM, Erlichman J: Reversible autophosphorylation of a cyclic 3′,5′-AMP dependent protein kinase from bovine cardiac muscle. J Biol Chem 250: 7786-7794, 1975
de Jonge HR, Rosen OM: Self phosphorylation of cyclic guanosine 3′,5′-monophosphate dependent protein kinase from bovine lung: Effect of cyclic adenosine 3′,5′-monophosphate, cyclic guanosine 3′,5′-monophosphate and kinase. J Biol Chem 252: 2780-2783, 1977
Kikkawa V, Takai V, Minakuchi R, Inohara S, Nishizuka Y: Calcium activated, phospholipid dependent protein kinase from rat brain. J Biol Chem 257: 13341-13348, 1982
Hathaway DR, Adelstien RS: Human platelet myosin light chain kinase requires the calcium binding protein calmodulin for activity. Proc Natl Acad Sci USA 76: 1653-1657, 1979
Kameshita I, Fujisawa H: Autophosphorylation of calmodulin dependent protein kinase from rat cerebral cortex. J Biochem 113: 583-590, 1993
Chatila T, Anderson KA, Ho N, Means AR: A unique phosphorylation dependent mechanism for the activation of calcium/calmodulin dependent protein kinase type IV/GR. J Biol Chem 27: 21542-21548, 1996
Hidaka H, Kobayashi R: Pharmacology of protein kinase inhibitors. Annu Rev Pharmacol Toxicol 32: 377-392, 1992
Yuasa T, Muto S: Ca2+ dependent protein kinase form the halotolerant green alga Dunaliella tertiolecta: Partial purification and Ca2+ dependent association of the enzyme to the microsomes. Arch Biochem Biophys 296: 175-182, 1992
DeRiemer SA, Kaczmavek LK, Lai Y, McGuinness TL, Greengard P: Calcium/calmodulin dependent protein phosphorylation in the nervous system of Aplysia. J Neurosci 4: 1618-1625, 1984
Miyano O, Kameshita I, Fujisawa H: Purification and characterization of a brain specific multifunctional calmodulin dependent protein kinase from rat cerebellum. J Biol Chem 267: 1198-1203, 1992
Ito T, Mochizuki H, Kato M, Nimura Y, Hanai T, Usuda N, Hidaka H: Ca2+/calmodulin dependent protein kinase V: Tissue distribution and immunohistochemical localization in rat brain. Arch Biochem Biophys 312: 278-284, 1994
Ishida A, Kitani T, Okuno S, Fujisawa H: Inactivation of Ca2+/calmodulin protein kinase II by Ca2+/calmodulin. J Biochem 115: 1075-1082, 1994
Gundersen RE, Nelson DL: A novel Ca2+/dependent protein kinase from Paramecium tetraurelia. J Biol Chem 262: 4602-4609, 1987
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Dhillon, N.K., Sharma, S. & Khuller, G. Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans . Mol Cell Biochem 252, 183–191 (2003). https://doi.org/10.1023/A:1025596008765
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DOI: https://doi.org/10.1023/A:1025596008765