Role for Protein Kinase A in the Neurospora Circadian Clock by Regulating White Collar-Independent frequency Transcription through Phosphorylation of RCM-1

Abstract

Rhythmic activation and repression of clock gene expression is essential for the eukaryotic circadian clock functions. In the Neurospora circadian oscillator, the transcription of the frequency (frq) gene is periodically activated by the White Collar (WC) complex and suppressed by the FRQ-FRH complex. We previously showed that there is WC-independent frq transcription and its repression is required for circadian gene expression. How WC-independent frq transcription is regulated is not known. We show here that elevated protein kinase A (PKA) activity results in WC-independent frq transcription and the loss of clock function. We identified RCM-1 as the protein partner of RCO-1 and an essential component of the clock through its role in suppressing WC-independent frq transcription. RCM-1 is a phosphoprotein and is a substrate of PKA in vivo and in vitro. Mutation of the PKA-dependent phosphorylation sites on RCM-1 results in WC-independent transcription of frq and impaired clock function. Furthermore, we showed that RCM-1 is associated with the chromatin at the frq locus, a process that is inhibited by PKA. Together, our results demonstrate that PKA regulates frq transcription by inhibiting RCM-1 activity through RCM-1 phosphorylation.

ACKNOWLEDGMENTS

We thank Zhijun Wang, Zhipeng Zhou and Huijie Chen for critical reading of the manuscript, and we thank Huiqiang Lou and members of the He lab for helpful discussions and proofreading the manuscript. We also thank Xuemei Cao and Yan Gao for technical assistance.

This project is supported by grants from a project supported by the State Key Program of National Natural Science of China (31330004) and National Basic Research Program of China (973 Program) grant (2012CB947600) to Qun He and by grants from the National Institutes of Health (GM084283 and GM062591) and the Welch Foundation (I-1560) to Yi Liu.