Dataset Information

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

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.

SUBMITTER: Tang CT

PROVIDER: S-EPMC2705601 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Tang Chi-Tai CT Li Shaojie S Long Chengzu C Cha Joonseok J Huang Guocun G Li Lily L Chen She S Liu Yi Y

Proceedings of the National Academy of Sciences of the United States of America 20090608 26

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 addition ...[more]

PMID: 19506251

Similar Datasets

Project description:In the negative feedback loop driving the Neurospora circadian oscillator, the negative element, FREQUENCY (FRQ), inhibits its own expression by promoting phosphorylation of its heterodimeric transcriptional activators, White Collar-1 (WC-1) and WC-2. FRQ itself also undergoes extensive time-of-day-specific phosphorylation with over 100 phosphosites previously documented. Although disrupting individual or certain clusters of phosphorylation sites has been shown to alter circadian period lengths to some extent, it is still elusive how all the phosphorylations on FRQ control its activity. In this study, we systematically investigated the role in period determination of all 110 reported FRQ phosphorylation sites, using mutagenesis and luciferase reporter assays. Surprisingly, robust FRQ phosphorylation is still detected even when 84 phosphosites were eliminated altogether; further mutating another 26 phosphoresidues completely abolished FRQ phosphorylation. To identify phosphoresidue(s) on FRQ impacting circadian period length, a series of clustered frq phosphomutants covering all the 110 phosphosites were generated and examined for period changes. When phosphosites in the N-terminal and middle regions of FRQ were eliminated, longer periods were typically seen while removal of phosphorylation in the C-terminal tail resulted in extremely short periods, among the shortest reported. Interestingly, abolishing the 11 phosphosites in the C-terminal tail of FRQ not only results in an extremely short period, but also impacts temperature compensation (TC), yielding an overcompensated circadian oscillator. In addition, the few phosphosites in the middle of FRQ are also found to be crucial for TC. When different groups of FRQ phosphomutations were combined intramolecularly, expected additive effects were generally observed except for one novel case of intramolecular epistasis, where arrhythmicity resulting from one cluster of phosphorylation site mutants was restored by eliminating phosphorylation at another group of sites.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets