• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to project page

2017 Fiscal Year Research-status Report

新規睡眠制御分子SIK3シグナルカスケードの同定

Research Project

Project/Area Number 17K15592
Research InstitutionUniversity of Tsukuba

Principal Investigator

Ma Jing  筑波大学, 国際統合睡眠医科学研究機構, 研究員 (70793222)

Project Period (FY) 2017-04-01 – 2020-03-31
Keywordssleep/wakefulness / signal transduction / protein kinase / phosphoproteome
Outline of Annual Research Achievements

According to our research plan, we performed quantitative phosphoproteomic studies of whole mouse brains of sleep-deprived mice and Sleepy mutant mice. A combined proteome and phosphoproteome data for 9,410 mouse proteins and 62,384 phosphopeptides were examined. We identifies 80 mostly synaptic Sleep-Need-Index-PhosphoProteins (SNIPPs), whose phosphorylation states closely parallel changes of sleep need due to sleep-deprivation and Sleepy mutation. To examine whether SNIPPs are substrates of SLEEPY kinase, we compared the interactomes of SLEEPY and SIK3 by immunoprecipitation (IP) and mass spectrometric analysis using whole-brain lysates from the Flag-HA-Sik3+ and Flag-HA-Sik3Slp knock-in mice. Accordingly, SLEEPY preferentially associated with synaptic proteins, including 28 of 80 SNIPPs. The 28 SLEEPY-interacting SNIPPs contain 47 putative AMPK sites showing significant changes, of which 40 (85%) become hyper-phosphorylated, in Sleepy brains. Taken together, these observations suggest that SLEEPY may increase phosphorylation of SNIPPs by enhancing kinase-substrate association. Inhibition of SIK3 activity reduces phosphorylation state of SNIPPs and slow wave activity (SWA) during non-rapid-eye-movement sleep (NREMS), in both Sleepy and sleep-deprived wild-type mice. Our results suggest that SNIPPs accumulate/dissipate phosphorylation as the molecular substrate of sleep need.

Current Status of Research Progress
Current Status of Research Progress

1: Research has progressed more than it was originally planned.

Reason

We have finished (IP) and mass spectrometric analysis using whole-brain lysates from the Flag-HA-Sik3+ and Flag-HA-Sik3Slp knock-in mice. We also applied the “AMPK Motif Analyzer27” to predict 2,943 phosphopeptides as potential AMPK-like substrates in the Slp/WT phosphoproteome dataset. The 28 SLEEPY-interacting SNIPPs contain 47 putative AMPK sites showing significant changes, of which 40 (85%) become hyper-phosphorylated, in Sleepy brains. Taken together, these observations suggest that SLEEPY may increase phosphorylation of SNIPPs by enhancing kinase-substrate association.
We also attempted to rescue the phenotypes of Sleepy mice by intracerebroventricular (ICV) injection of HG-9-91-01 (HG) to inhibit SLEEPY/SIK3 kinase activity. Administration of HG significantly reduced phosphorylation of AMPK substrates, particularly those from 28 SLEEPY-interacting SNIPPs. Accordingly, inhibition of SLEEPY activity reduced phospho-state of SNIPPs and SWA. Similarly, inhibition of SIK3 activity reduced phosphorylation of AMPK substrates, phospho-state of SNIPPs and SWA of NREMS in sleep-deprived wild-type mice, suggesting a critical role of SIK3-SNIPPs in normal homeostatic sleep regulation.

Strategy for Future Research Activity

In new year, we will cross different contexts to identify key substrates of SLEEPY kinase. We will investigate the function of those Sleepy substrates through pharmacological and genetic approaches.

Causes of Carryover

New antibodies and genetic model are needed to identify key substrates of SLEEPY kinase, and investigate the functions.

  • Research Products

    (1 results)

All 2018

All Journal Article (1 results) (of which Int'l Joint Research: 1 results)

  • [Journal Article] Quantitative phosphoproteomic analysis of the molecular substrates of sleep need2018

    • Author(s)
      Zhiqiang Wang, Jing Ma, Chika Miyoshi, etc.
    • Journal Title

      Nature

      Volume: 印刷中 Pages: -

    • Int'l Joint Research

URL: 

Published: 2018-12-17  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi