Abstract
Reversible protein phosphorylation has critical functions in the eukaryotic circadian negative feedback loops. In Neurospora, the FREQUENCY protein closes the circadian negative feedback loop by promoting the phosphorylation of its transcription activator, the WHITE COLLAR complex (WCC) and consequently inhibiting WCC activity. Here we show that protein phosphatase 4 is a novel component of the Neurospora clock by regulating both processes of the circadian negative feedback loop. The disruption of pp4 results in short period rhythms with low amplitude. In addition to its role in regulating FRQ phosphorylation and stability, PP4 also dephosphorylates and activates WCC. In contrast to PP2A, another phosphatase that activates WCC, PP4 has a major function in promoting nuclear entry of WCC. PKA, a WC kinase, inhibits WC nuclear localization. Furthermore, the FRQ-dependent WC phosphorylation promotes WCC cytosolic localization. Together, these results revealed WCC nucleocytoplasmic shuttling as an important step in the circadian negative feedback process and delineated the FRQ-dependent WCC inhibition as a two-step process: the inhibition of WCC DNA-binding activity followed by sequestration of WCC into the cytoplasm.
Highlights
Eukaryotic circadian oscillators consist of autoregulatory negative feedback loops in which there are positive and negative elements (Dunlap, 1999; Allada et al, 2001; Young and Kay, 2001; Sehgal, 2004)
We identified protein phosphatase 4 (PP4) as a critical component of the Neurospora circadian clock by & 2008 European Molecular Biology Organization regulating both arms of the circadian negative feedback loop
Disruption of pp4 results in a short period and low amplitude rhythm To further understand the role of protein phosphorylation in the Neurospora circadian clock, we carried out systematic deletion analyses of all potential serine/threonine protein phosphatases
Summary
Eukaryotic circadian oscillators consist of autoregulatory negative feedback loops in which there are positive and negative elements (Dunlap, 1999; Allada et al, 2001; Young and Kay, 2001; Sehgal, 2004). In these negative feedback loops, the positive elements activate the transcription of the negative elements, whereas the protein products of the negative elements inhibit the activity of the positive. FRQ and FRH (a FRQinteracting RNA helicase) form another complex (FFC) that represses frq transcription by inhibiting WCC activity through their physical interaction (Aronson et al, 1994a; Merrow et al, 1997; Denault et al, 2001; Cheng et al, 2001a, 2005; Froehlich et al, 2003; Schafmeier et al, 2005; He et al, 2006)
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