Circadian rhythms are endogenous self-sustained oscillations with approximately 24-hour rhythmicity that are manifested in various biochemical, physiological, and behavioral processes. The molecular mechanism of the circadian oscillator as a transcriptional/translational feedback loop is conserved among many species, including Neurospora, Arabidopsis, Drosophila, and mammals. Proteins composing the cellular clocks in all genetic model systems studied to date, including mammals, show striking and temporally distinct phosphorylation patterns. In the mammalian clock mechanism, two members of the casein kinase I (CKI) family play an important role in phosphorylating specific core clock proteins, a post-translational modification that ultimately leads to the turnover of the phosphorylated clock proteins by ubiquitin-mediated proteasomal degradation. Mutations in CKIe and CKI( have been shown to alter circadian rhythms at the cellular and behavioral levels in mammals. Indeed, mutations in CKI have been implicated as the cause of two human sleep/wake disorders. Thus, members of the mammalian CKI family represent important targets for further investigation to better understand the 24-hour dynamics of the circadian clock in mammals. The experiments proposed here seek to determine the structure/function relationships of the mammalian CKIe enzyme on the mammalian circadian clock through an in vitro random mutagenesis approach. By mutagenizing CKIe and then testing the effects of mutations on enzyme function, we seek to elucidate important structural features of CKIe necessary for its role in the cellular clock system.
Only recently has the importance of the mammalian circadian system to the regulation of such fundamental biological processes as the cell cycle and metabolism become apparent. A convergence of data from microarray studies, quantitative trait locus analysis, and mutagenesis screens demonstrates the pervasiveness of circadian regulation in biological systems. The importance of maintaining the internal temporal homeostasis conferred by the circadian system is revealed by animal models in which genes coding for core components of the clock result in disease, including cancer and disturbances to the sleep/wake cycle. The experiments proposed here focus on generating mutations in a kinase that plays an important role in the cellular circadian clock of mammals. By identifying mutations in this enzyme that affect clock function, we can better define the role of this protein in the mammalian clock system.
| Lowrey, Phillip L; Takahashi, Joseph S (2011) Genetics of circadian rhythms in Mammalian model organisms. Adv Genet 74:175-230 |
