title: Phosphoproteome Profiling Reveals Circadian Clock Regulation of Posttranslational Modifications in the Murine Hippocampus.
abstract: The circadian clock is an endogenous oscillator that drives daily rhythms in physiology, behavior, and gene expression. The underlying mechanisms of circadian timekeeping are cell-autonomous and involve oscillatory expression of core clock genes that is driven by interconnecting transcription-translation feedback loops (TTFLs). Circadian clock TTFLs are further regulated by posttranslational modifications, in particular, phosphorylation. The hippocampus plays an important role in spatial memory and the conversion of short- to long-term memory. Several studies have reported the presence of a peripheral oscillator in the hippocampus and have highlighted the importance of circadian regulation in memory formation. Given the general importance of phosphorylation in circadian clock regulation, we performed global quantitative proteome and phosphoproteome analyses of the murine hippocampus across the circadian cycle, applying spiked-in labeled reference and high accuracy mass spectrometry (MS). Of the 3,052 proteins and 2,868 phosphosites on 1,368 proteins that were accurately quantified, 1.7% of proteins and 5.2% of phosphorylation events exhibited time-of-day-dependent expression profiles. The majority of circadian phosphopeptides displayed abrupt fluctuations at mid-to-late day without underlying rhythms of protein abundance. Bioinformatic analysis of cyclic phosphorylation events revealed their diverse distribution in different biological pathways, most notably, cytoskeletal organization and neuronal morphogenesis. This study provides the first large-scale, quantitative MS analysis of the circadian phosphoproteome and proteome of the murine hippocampus and highlights the significance of rhythmic regulation at the posttranslational level in this peripheral oscillator. In addition to providing molecular insights into the hippocampal circadian clock, our results will assist in the understanding of genetic factors that underlie rhythms-associated pathological states of the hippocampus.
authors.affiliation: Faculty of Medicine, Department of Biochemistry, Microbiology and Immunology, Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, ON, Canada; Department of Chemistry, National Dong Hwa University, Shoufeng, Hualien, Taiwan.
authors.forename: Cheng-Kang
authors.initials: CK
authors.lastname: Chiang
authors.affiliation: Faculty of Medicine, Department of Biochemistry, Microbiology and Immunology, Ottawa Institute of Systems Biology, University of Ottawa , Ottawa, ON , Canada.
authors.forename: Bo
authors.initials: B
authors.lastname: Xu
authors.affiliation: Department of Biology, University of Toronto Mississauga , Mississauga, ON , Canada.
authors.forename: Neel
authors.initials: N
authors.lastname: Mehta
authors.affiliation: Faculty of Medicine, Department of Biochemistry, Microbiology and Immunology, Ottawa Institute of Systems Biology, University of Ottawa , Ottawa, ON , Canada.
authors.forename: Janice
authors.initials: J
authors.lastname: Mayne
authors.affiliation: Faculty of Medicine, Department of Biochemistry, Microbiology and Immunology, Ottawa Institute of Systems Biology, University of Ottawa , Ottawa, ON , Canada.
authors.forename: Warren Y L
authors.initials: WY
authors.lastname: Sun
authors.affiliation: Faculty of Medicine, Department of Biochemistry, Microbiology and Immunology, Ottawa Institute of Systems Biology, University of Ottawa , Ottawa, ON , Canada.
authors.forename: Kai
authors.initials: K
authors.lastname: Cheng
authors.affiliation: Faculty of Medicine, Department of Biochemistry, Microbiology and Immunology, Ottawa Institute of Systems Biology, University of Ottawa , Ottawa, ON , Canada.
authors.forename: Zhibin
authors.initials: Z
authors.lastname: Ning
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian , China.
authors.forename: Jing
authors.initials: J
authors.lastname: Dong
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian , China.
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
authors.affiliation: Department of Biology, University of Toronto Mississauga , Mississauga, ON , Canada.
authors.forename: Hai-Ying Mary
authors.initials: HM
authors.lastname: Cheng
authors.affiliation: Faculty of Medicine, Department of Biochemistry, Microbiology and Immunology, Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, ON, Canada; Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, ON, Canada; Canadian Institute for Advanced Research, Toronto, ON, Canada.
authors.forename: Daniel
authors.initials: D
authors.lastname: Figeys
keywords: circadian rhythm; hippocampus; kinase–substrate relations; phosphorylation; quantitative proteome and phosphoproteome analysis