Posttranslational mechanisms regulate the mammalian circadian clock.

Area of Science:

• Chronobiology
• Molecular Biology
• Genetics

Background:

• Circadian rhythms are endogenous biological processes that oscillate over approximately 24 hours.
• The core molecular clock machinery involves transcriptional-translational feedback loops.
• Posttranslational modifications, such as phosphorylation, play critical roles in regulating clock protein stability and function.

Purpose of the Study:

• To investigate the posttranslational regulation of core clock proteins in mouse liver.
• To elucidate the role of cryptochromes (mCRY1, mCRY2) in the stability and localization of clock proteins.
• To identify kinases involved in the circadian regulation of clock proteins.

Main Methods:

• In vivo analysis of clock protein phosphorylation in mouse liver.
• Co-immunoprecipitation to study protein complex formation.
• Analysis of clock protein behavior in cryptochrome-deficient mice.

Main Results:

• Mouse PERIOD proteins (mPER1, mPER2), CLOCK, and BMAL1 exhibit circadian-dependent phosphorylation.
• CLOCK:BMAL1 heterodimers bind DNA throughout the circadian cycle.
• Cryptochromes (mCRY1, mCRY2) are crucial for stabilizing phosphorylated mPER2 and facilitating the nuclear entry of mPER1, mPER2, and CKIepsilon.
• Casein kinase I delta is identified as a novel clock-relevant kinase.

Conclusions:

• Posttranslational regulation, particularly phosphorylation, is a key mechanism controlling mouse liver circadian clock function.
• Cryptochromes are essential for the stability and nuclear localization of PERIOD proteins and CKIepsilon.
• Casein kinase I delta represents a significant addition to the known kinases regulating the mammalian circadian clock.