title: Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) interacts with neurofilament L and inhibits its filament association.
abstract: Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) is a Ser/Thr phosphatase that belongs to the PPM family. Growing evidence suggests that PPM phosphatases including CaMKP act as a complex with other proteins to regulate cellular functions. In this study, using the two-dimensional far-western blotting technique with digoxigenin-labeled CaMKP as a probe, in conjunction with peptide mass fingerprinting analysis, we identified neurofilament L (NFL) as a CaMKP-binding protein in a Triton-insoluble fraction of rat brain. We confirmed binding of fluorescein-labeled CaMKP (F-CaMKP) to NFL in solution by fluorescence polarization. The analysis showed that the dissociation constant of F-CaMKP for NFL is 73 ± 17 nM (n = 3). Co-immunoprecipitation assay using a cytosolic fraction of NGF-differentiated PC12 cells showed that endogenous CaMKP and NFL form a complex in cells. Furthermore, the effect of CaMKP on self-assembly of NFL was examined. Electron microscopy revealed that CaMKP markedly prevented NFL from forming large filamentous aggregates, suggesting that CaMKP-binding to NFL inhibits its filament association. These findings may provide new insights into a novel mechanism for regulating network formation of neurofilaments during neuronal differentiation.
authors.affiliation: Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 739-8521, Japan.
authors.forename: Hana
authors.initials: H
authors.lastname: Ozaki
authors.affiliation: Department of Biochemistry, Asahikawa Medical University, Asahikawa, 078-8510, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Katoh
authors.affiliation: Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 739-8521, Japan.
authors.forename: Ryoko
authors.initials: R
authors.lastname: Nakagawa
authors.affiliation: Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 739-8521, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Ishihara
authors.affiliation: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa, 761-0795, Japan.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Sueyoshi
authors.affiliation: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa, 761-0795, Japan.
authors.forename: Isamu
authors.initials: I
authors.lastname: Kameshita
authors.affiliation: Department of Biochemistry, Asahikawa Medical University, Asahikawa, 078-8510, Japan.
authors.forename: Takanobu
authors.initials: T
authors.lastname: Taniguchi
authors.affiliation: Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 739-8521, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Hirano
authors.affiliation: Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 739-8521, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yamazaki
authors.affiliation: Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 739-8521, Japan. Electronic address: aishida@hiroshima-u.ac.jp.
authors.forename: Atsuhiko
authors.initials: A
authors.lastname: Ishida
mesh_terms: D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000737:chemistry* / Q000378:metabolism*; D016900:Neurofilament Proteins / Q000737:chemistry* / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D016716:PC12 Cells; D011485:Protein Binding; D051381:Rats; D014018:Tissue Distribution
chemical_list: D016900:Neurofilament Proteins; C044882:neurofilament protein L; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase
keywords: Interaction analysis; Intermediate filament; K(d); Mass spectrometry; Modulator; Neuron