Setting the pace of the Neurospora circadian clock by multiple independent FRQ phosphorylation events

Chi Tai Tang, Shaojie Li, Chengzu Long, Joonseok Cha, Guocun Huang, Lily Li, She Chen, Yi Liu

Research output: Contribution to journalArticlepeer-review

62 Scopus citations

Abstract

Protein phosphorylation plays essential roles in eukaryotic circadian clocks. Like PERIOD in animals, the Neurospora core circadian protein FRQ is progressively phosphorylated and becomes extensively phosphorylated before its degradation. In this study, by using purified FRQ protein from Neurospora, we identified 43 in vivo FRQ phosphorylation sites by mass spectrometry analysis. In addition, we show that CK-1a and CKII are responsible for most FRQ phosphorylation events and identify an additional 33 phosphorylation sites by in vitro kinase assays. Whole-cell metabolic isotope labeling and quantitative MS analyses suggest that circadian oscillation of the FRQ phosphorylation profile is primarily due to progressive phosphorylation at the majority of these newly discovered phosphorylation sites. Furthermore, systematic mutations of the identified FRQ phosphorylation sites led to either long or short period phenotypes. These changes in circadian period are attributed to increases or decreases in FRQ stability, respectively. Together, this comprehensive study of FRQ phosphorylation reveals that regulation of FRQ stability by multiple independent phosphorylation events is a major factor that determines the period length of the clock. A model is proposed to explain how FRQ stability is regulated by multiple phosphorylation events.

Original languageEnglish (US)
Pages (from-to)10722-10727
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume106
Issue number26
DOIs
StatePublished - Jun 30 2009

Keywords

  • Casein kinase
  • Frequency
  • Mass spectrometry

ASJC Scopus subject areas

  • General

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