CIPC-dependent phosphorylation of CLOCK and NPAS2 in circadian clockwork

摘要

Circadian rhythms are generated by the cell-autonomous circadian clock, in which the clock genes form a transcription/translation-based negative feedback loop. Basic helix-loop-helix (bHLH)-PAS (Period-Arylhydrocarbon receptor nuclear translocator-Single minded) transcription factors, CLOCK and BMAL1, form a complex and bind to E-box elements for activation of the transcription, which is suppressed by circadian binding with a variety of negative regulators. We found that CLOCK protein is mostly kept in the phosphorylated form over the day and is hyperphosphorylated in the suppression phase of E-box-dependent transcription. Ser38, Ser42, and Ser427 were identified as in vivo phosphorylation sites of CLOCK isolated from the mouse liver nuclei, and mutation analysis of the sites revealed that the phosphorylation at Ser38 and Ser42 reduces the DNA-binding ability of CLOCK-BMAL1 complex, thereby contributing to the suppression of E-box-dependent transactiva-tion. In NIH 3T3 cells, BMAL1 expression enhanced phosphorylation of coexpressed CLOCK protein, and the BMAL1-dependent phosphorylation of CLOCK was inhibited by coexpressed CRY2 or further stimulated by coexpression of CIPC (CLOCK-interacting protein, circadian). Functionally, phosphorylation of CLOCK facilitated its proteasomal degradation, highlighting the importance of CIPC in the negative regulation of E-box-dependent transcription. A paralog of CLOCK, neuronal PAS domain protein 2 (NPAS2), was also phosphorylated in BMAL1 -dependent manner, and CIPC expression further stimulated the NPAS2 phosphorylation. It is strongly suggested that the BMAL1-dependent and CIPC-stimulated phosphorylation of CLOCK and possibly NPAS2 represents an important regulatory mechanism underlying the circadian gene expression mediated by these transcription factor members.