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Inoue M.,Kurume University | Yasuda K.,Kurume University | Uemura H.,Nagasaki University | Yasaka N.,Kurume University | And 8 more authors.
Journal of Biochemistry | Year: 2013

Hetero- and homodimerization of 14-3-3 proteins demonstrate distinctive functions in mammals and plants. Trypanosoma brucei 14-3-3I and II (Tb14-3-3I and II) play pivotal roles in motility, cytokinesis and the cell cycle; however, the significance and the mechanism of Tb14-3-3 dimerization are remained to be elucidated. We found that ectopically expressed epitope-tagged Tb14-3-3I and II proteins formed hetero- and homodimers with endogenous Tb14-3-3I and II proteins. However, we also found the ability to form hetero- or homodimers between Tb14-3-3I and II proteins was clearly affected by the sequence and location of the epitope tag used. We found a blue native polyacrylamide gel electrophoresis system followed by western blotting may distinguish monomer from dimer structure, and stable from unstable conformation of Tb14-3-3. Combined with co-immunoprecipitation results, we revealed that Tb14-3-3 proteins mainly existed as heterodimeric form. Furthermore, co-overexpression of Tb14-3-3I and II proteins in T. brucei induced aberrant numbers of organelles in cells, but overexpression of either isoform alone rarely produced such morphology. These results suggest that heterodimers play more significant roles than homodimers not only in the maintenance of steady-state levels of the 14-3-3 proteins but also in the regulation of cytokinesis. © 2013 The Authors 2013. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.


Saito T.,Tokyo Metroplitan University | Yano M.,Tokyo Metroplitan University | Kawai Y.,Tokyo Metroplitan University | Asada A.,Tokyo Metroplitan University | And 4 more authors.
Journal of Biological Chemistry | Year: 2013

Cyclin-dependent kinase 5 (Cdk5) is a brain-specific membrane- bound protein kinase that is activated by binding to the p35 or p39 activator. Previous studies have focused on p35- Cdk5, and little is known regarding p39-Cdk5. The lack of functional understanding of p39-Cdk5 is due, in part, to the labile property of p39-Cdk5, which dissociates and loses kinase activity in nonionic detergent conditions. Here we investigated the structural basis for the instability of p39-Cdk5. p39 and p35 contain N-terminal p10 regions and C-terminal Cdk5 activation domains (AD). Although p35 and p39 show higher homology in the C-terminalADthan the N-terminal region, the difference in stability is derived from the C-terminal AD. Based on the crystal structures of the p25 (p35 C-terminal region including AD)- Cdk5 complex, we simulated the three-dimensional structure of the p39 AD-Cdk5 complex and found differences in the hydrogen bond network between Cdk5 and its activators. Three amino acids of p35, Asp-259, Asn-266, and Ser-270, which are involved in hydrogen bond formation with Cdk5, are changed to Gln, Gln, and Pro in p39. Because these three amino acids in p39 do not participate in hydrogen bond formation, we predicted that the number of hydrogen bonds between p39 and Cdk5 was reduced compared with p35 and Cdk5. Using substitution mutants, we experimentally validated that the difference in the hydrogen bond network contributes to the different properties between Cdk5 and its activators. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.


Yoshitane H.,University of Tokyo | Honma S.,Hokkaido University | Imamura K.,University of Tokyo | Nakajima H.,Daiichi Sankyo | And 10 more authors.
EMBO Reports | Year: 2012

The posttranslational regulation of mammalian clock proteins has been assigned a time-keeping function, but seems to have more essential roles. Here we show that c-Jun N-terminal kinase (JNK), identified by inhibitor screening of BMAL1 phosphorylation at Ser 520/Thr 527/Ser 592, confers dynamic regulation on the clock. Knockdown of JNK1 and JNK2 abrogates BMAL1 phosphorylation and lengthens circadian period in fibroblasts. Mice deficient for neuron-specific isoform JNK3 have altered behavioural rhythms, with longer free-running period and compromised phase shifts to light. The locomotor rhythms are insensitive to intensity variance of constant light, deviating from Aschoff's rule. Thus, JNK regulates a core characteristic of the circadian clock by controlling the oscillation speed and the phase in response to light. © 2012 European Molecular Biology Organization.


Isumi Y.,Daiichi Sankyo | Hirata T.,Daiichi Sankyo | Saitoh H.,Daiichi Sankyo | Miyakawa T.,Daiichi Sankyo | And 5 more authors.
Biochemical and Biophysical Research Communications | Year: 2011

We found a novel protein-protein interaction between ubiquitin-specific protease 15 (USP15) and skeletal muscle LIM protein 1 (SLIM1): USP15 and SLIM1 directly bound under cell-free conditions and co-immunoprecipitated from the lysates of the cells, where they were co-expressed; and USP15 deubiquitinated SLIM1, resulting in the increase of protein levels of SLIM1. Because SLIM1 is strongly implicated in the pathogenesis of myopathies and cardiomyopathies, we generated transgenic (TG) mice with cardiac-specific overexpression of human USP15. Heart weight to body weight ratios and mRNA levels of fetal gene markers in the heart were significantly higher in USP15-TG mice than in wild-type (WT) mice. Also, protein levels of endogenous murine SLIM1 in the heart were significantly higher in USP15-TG mice than in WT mice. Furthermore, the protein of alternatively spliced isoform of SLIM1 was only detected in the heart of USP15-TG mice, and mRNA levels of this isoform were higher as compared to WT mice. These results indicate that USP15 is involved in the regulation of hypertrophic responses in cardiac muscle through transcriptional and post-translational modulation of SLIM1. © 2011 Elsevier Inc.

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