title: Cellular localization of CoPK12, a Ca(2+)/calmodulin-dependent protein kinase in mushroom Coprinopsis cinerea, is regulated by N-myristoylation.
abstract: Multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs) have been extensively studied in mammals, whereas fungus CaMKs still remain largely uncharacterized. We previously obtained CaMK homolog in Coprinopsis cinerea, designated CoPK12, and revealed its unique catalytic properties in comparison with the mammalian CaMKs. To further clarify the regulatory mechanisms of CoPK12, we investigated post-translational modification and subcellular localization of CoPK12 in this study. In C. cinerea, full-length CoPK12 (65 kDa) was fractionated in the membrane fraction, while the catalytically active fragment (46 kDa) of CoPK12 was solely detected in the soluble fraction by differential centrifugation. Expressed CoPK12-GFP was localized on the cytoplasmic and vacuolar membranes as visualized by green fluorescence in yeast cells. In vitro N-myristoylation assay revealed that CoPK12 is N-myristoylated at Gly-2 in the N-terminal position. Furthermore, calmodulin could bind not only to CaM-binding domain but also to the N-terminal myristoyl moiety of CoPK12. These results, taken together, suggest that the cellular localization and function of CoPK12 are regulated by protein N-myristoylation and limited proteolysis.
authors.affiliation: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-Cho, Kagawa 761-0795; Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521; and Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Kaneko
authors.affiliation: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-Cho, Kagawa 761-0795; Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521; and Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan.
authors.forename: Mitsuaki
authors.initials: M
authors.lastname: Tabuchi
authors.affiliation: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-Cho, Kagawa 761-0795; Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521; and Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Sueyoshi
authors.affiliation: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-Cho, Kagawa 761-0795; Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521; and Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan.
authors.forename: Atsuhiko
authors.initials: A
authors.lastname: Ishida
authors.affiliation: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-Cho, Kagawa 761-0795; Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521; and Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Utsumi
authors.affiliation: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-Cho, Kagawa 761-0795; Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521; and Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan kamesita@ag.kagawa-u.ac.jp.
authors.forename: Isamu
authors.initials: I
authors.lastname: Kameshita
mesh_terms: D000363:Agaricales / Q000166:cytology* / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021381:Protein Transport
chemical_list: D017871:Calcium-Calmodulin-Dependent Protein Kinases
keywords: Coprinopsis cinerea; N-myristoylation; calmodulin-dependent protein kinase; membrane association; mushroom