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Title: Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) interacts with neurofilament L and inhibits its filament association

Abstract

Ca{sup 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. - Highlights: • NFL was identified as a CaMKP-binding protein in an insoluble fraction of rat brain. • CaMKP bound to NFL in solution with a K{submore » d} value of 73 ± 17 nM. • A CaMKP-NFL complex was found in NGF-differentiated PC12 cells. • CaMKP-binding to NFL inhibited its filament association. • CaMKP may regulate network formation of neurofilaments in neurons.« less

Authors:
 [1];  [2]; ;  [1]; ;  [3];  [2]; ;  [1];  [1]
  1. Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 739-8521 (Japan)
  2. Department of Biochemistry, Asahikawa Medical University, Asahikawa, 078-8510 (Japan)
  3. Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa, 761-0795 (Japan)
Publication Date:
OSTI Identifier:
22606188
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 477; Journal Issue: 4; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ALBUMINS; BRAIN; CALCIUM IONS; CALMODULIN; CATTLE; ELECTRON MICROSCOPY; FILAMENTS; FLUORESCEIN; FLUORESCENCE; FLUORIDES; GROWTH FACTORS; MASS SPECTROSCOPY; NERVE CELLS; PEPTIDES; PHOSPHATASES; PHOSPHATES; RATS; TRITONS; TRITURUS

Citation Formats

Ozaki, Hana, Katoh, Tsuyoshi, Nakagawa, Ryoko, Ishihara, Yasuhiro, Sueyoshi, Noriyuki, Kameshita, Isamu, Taniguchi, Takanobu, Hirano, Tetsuo, Yamazaki, Takeshi, and Ishida, Atsuhiko, E-mail: aishida@hiroshima-u.ac.jp. Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) interacts with neurofilament L and inhibits its filament association. United States: N. p., 2016. Web. doi:10.1016/J.BBRC.2016.06.141.
Ozaki, Hana, Katoh, Tsuyoshi, Nakagawa, Ryoko, Ishihara, Yasuhiro, Sueyoshi, Noriyuki, Kameshita, Isamu, Taniguchi, Takanobu, Hirano, Tetsuo, Yamazaki, Takeshi, & Ishida, Atsuhiko, E-mail: aishida@hiroshima-u.ac.jp. Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) interacts with neurofilament L and inhibits its filament association. United States. doi:10.1016/J.BBRC.2016.06.141.
Ozaki, Hana, Katoh, Tsuyoshi, Nakagawa, Ryoko, Ishihara, Yasuhiro, Sueyoshi, Noriyuki, Kameshita, Isamu, Taniguchi, Takanobu, Hirano, Tetsuo, Yamazaki, Takeshi, and Ishida, Atsuhiko, E-mail: aishida@hiroshima-u.ac.jp. Fri . "Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) interacts with neurofilament L and inhibits its filament association". United States. doi:10.1016/J.BBRC.2016.06.141.
@article{osti_22606188,
title = {Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) interacts with neurofilament L and inhibits its filament association},
author = {Ozaki, Hana and Katoh, Tsuyoshi and Nakagawa, Ryoko and Ishihara, Yasuhiro and Sueyoshi, Noriyuki and Kameshita, Isamu and Taniguchi, Takanobu and Hirano, Tetsuo and Yamazaki, Takeshi and Ishida, Atsuhiko, E-mail: aishida@hiroshima-u.ac.jp},
abstractNote = {Ca{sup 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. - Highlights: • NFL was identified as a CaMKP-binding protein in an insoluble fraction of rat brain. • CaMKP bound to NFL in solution with a K{sub d} value of 73 ± 17 nM. • A CaMKP-NFL complex was found in NGF-differentiated PC12 cells. • CaMKP-binding to NFL inhibited its filament association. • CaMKP may regulate network formation of neurofilaments in neurons.},
doi = {10.1016/J.BBRC.2016.06.141},
journal = {Biochemical and Biophysical Research Communications},
number = 4,
volume = 477,
place = {United States},
year = {Fri Sep 02 00:00:00 EDT 2016},
month = {Fri Sep 02 00:00:00 EDT 2016}
}