Phosphorylation of the Neurospora clock protein FREQUENCY determines its degradation rate and strongly influences the period length of the circadian clock

Abstract

Under free running conditions, FREQUENCY (FRQ) protein, a central component of the Neurospora circadian clock, is progressively phosphorylated, becoming highly phosphorylated before its degradation late in the circadian day. To understand the biological function of FRQ phosphorylation, kinase inhibitors were used to block FRQ phosphorylation in vivo and the effects on FRQ and the clock observed. 6-dimethylaminopurine (a general kinase inhibitor) is able to block FRQ phosphorylation in vivo, reducing the rate of phosphorylation and the degradation of FRQ and lengthening the period of the clock in a dose-dependent manner. To confirm the role of FRQ phosphorylation in this clock effect, phosphorylation sites in FRQ were identified by systematic mutagenesis of the FRQ ORF. The mutation of one phosphorylation site at Ser-513 leads to a dramatic reduction of the rate of FRQ degradation and a very long period (>30 hr) of the clock. Taken together, these data strongly suggest that FRQ phosphorylation triggers its degradation, and the degradation rate of FRQ is a major determining factor for the period length of the Neurospora circadian clock. C ircadian clocks are found in almost all groups of organisms, and they control a wide variety of daily (Ϸ24-hr) endogenous (circadian) rhythms of molecular, physiological, and behavioral activities in these organisms (1). The identification of clock genes in several model systems and the understanding of how these genes are regulated have greatly enhanced our knowledge of how circadian clocks work at the molecular level (1-8). Common themes of clock mechanisms have begun to emerge: clock components comprising a core of the oscillator are part of a network of positive and negative interactions that establish a negative feedback loop that is essential for circadian oscillation (1, 9-16).The Neurospora circadian oscillator comprises an autoregulatory negative feedback cycle in which frq mRNA and FRQ protein are the central components (1,17,18). Mutations at the frq locus cause a variety of clock phenotypes: long and short period length (16-29 hr), arrhythmia, and loss of temperature and nutritional compensation of the clock; the deletion of the frq locus results in loss of normal circadian rhythmicity (18). Both frq mRNA and FRQ protein are rhythmically expressed in a daily fashion, and FRQ protein acts to repress the abundance of its own transcript (17,19,20). Importantly, the rhythmic expression of frq is essential for the negative feedback loop as shown by the facts that constitutive expression of frq results in the loss of the overt rhythm and step changes in frq expression reset the phase of the clock (17). Light and temperature, two of the most important environmental signals, reset the Neurospora clock by changing the levels of frq mRNA and FRQ protein (21,22).In addition to transcriptional control, frq is subject to several aspects of posttranscriptional regulation (23). First, two forms of FRQ protein are expressed because of initiation at alternative ATGs, a large form of 9...

Dependence of the period on the rate of protein degradation in minimal models for circadian oscillations

“…Among the multiple molecular mechanisms that influence the period of circadian rhythms, a major role is played by those that control clock protein degradation (Liu et al 2000;Eide et al 2005;Gallego et al 2006;Busino et al 2007;Reischl et al 2007). Here we compared three minimal models proposed for circadian clocks, showed that they predict opposite profiles of period as a function of the clock protein degradation rate, and indicated how to reconcile these contradictory predictions.…”

Molecular mechanism of light responses in Neurospora: from light-induced transcription to photoadaptation

“…We estimate that ∼40%-50% of WC-1 is degraded through this process. Phosphorylation-dependent protein degradation has been demonstrated for FRQ in Neurospora, a critical process regulating the circadian clock (Liu et al 2000;Gorl et al 2001;Yang et al 2002Yang et al , 2004He et al 2003).…”

Picking out parallels: plant circadian clocks in context

“…Intriguingly, phosphorylation of clock components is also a crucial part of both mammalian and Drosophila circadian feedback loops (Kloss et al 1998;Lowrey et al 2000). Furthermore, phosphorylation of FRQ is important in determining period length in Neurospora (Liu et al 2000).…”

“…Changes in protein phosphorylation levels can regulate cellular localization, protein stability and protein^protein interactions, and phosphorylation can be a prerequisite for DNA binding (Whitmarsh & Davis 2000). Intriguingly, phosphorylation of clock components is also a crucial part of both mammalian and Drosophila circadian feedback loops (Kloss et al 1998;Lowrey et al 2000). Furthermore, phosphorylation of FRQ is important in determining period length in Neurospora (Liu et al 2000).…”

“…In addition to transcriptional regulation of clock genes, posttranslational modi¢cations, such as protein phosphorylation, nuclear entry and targeted proteolysis, are involved in regulating the circadian period of activity of clock proteins and the clock (Edery et al 1994;Kloss et al 1998;Dunlap 1999;Liu et al 2000;Nishiwaki et al 2000). As discussed earlier (½ 2), CCA1 and LHY can regulate their own (and each other's) production through negative feedback loops.…”