An opposite role for tau in circadian rhythms revealed by mathematical modeling.

Biological clocks with a period of approximately 24 h (circadian) exist in most organisms and time a variety of functions, including sleep-wake cycles, hormone release, bioluminescence, and core body temperature fluctuations. Much of our understanding of the clock mechanism comes from the identification of specific mutations that affect circadian behavior. A widely studied mutation in casein kinase I (CKI), the CKIepsilon(tau) mutant, has been shown to cause a loss of kinase function in vitro, but it has been difficult to reconcile this loss of function with the current model of circadian clock function. Here we show that mathematical modeling predicts the opposite, that the kinase mutant CKIepsilon(tau) increases kinase activity, and we verify this prediction experimentally. CKIepsilon(tau) is a highly specific gain-of-function mutation that increases the in vivo phosphorylation and degradation of the circadian regulators PER1 and PER2. These findings experimentally validate a mathematical modeling approach to a complex biological function, clarify the role of CKI in the clock, and demonstrate that a specific mutation can be both a gain and a loss of function depending on the substrate.

Casein kinase I in the mammalian circadian clock.

The circadian clock is characterized by daily fluctuations in gene expression, protein abundance, and posttranslational modification of regulatory proteins. The Drosophila PERIOD (dPER) protein is phosphorylated by the serine?threonine protein kinase, DOUBLETIME (DBT). Similarly, the murine PERIOD proteins, mPER1 and mPER2, are phosphorylated by casein kinase I epsilon (CKI), the mammalian homolog of DBT. CKIepsilon also phosphorylates and partially activates the transcription factor BMAL1. Given the variety of potential targets for CKIepsilon and other cellular kinases, the precise role of phosphorylation is likely to be a complex one. Biochemical analysis of these and other circadian regulatory proteins has proven to be a fruitful approach in determining how they function within the context of the molecular clockworks.

Control of mammalian circadian rhythm by CKIepsilon-regulated proteasome-mediated PER2 degradation.

The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKIepsilon (casein kinase Iepsilon) has been postulated to prime PER2 for degradation. Supporting this idea, CKIepsilon inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKIepsilon inhibition also slows the degradation of PER2 in cells. CKIepsilon-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein beta-TrCP to a specific site, and dominant negative beta-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.

The circadian regulatory proteins BMAL1 and cryptochromes are substrates of casein kinase Iepsilon.

The serine/threonine protein kinase casein kinase I epsilon (CKIepsilon) is a key regulator of metazoan circadian rhythm. Genetic and biochemical data suggest that CKIepsilon binds to and phosphorylates the PERIOD proteins. However, the PERIOD proteins interact with a variety of circadian regulators, suggesting the possibility that CKIepsilon may interact with and phosphorylate additional clock components as well. We find that CRY1 and BMAL1 are phosphoproteins in cultured cells. Mammalian PERIOD proteins act as a scaffold with distinct domains that simultaneously bind CKIepsilon and mCRY1 and mCRY2 (mCRY). mCRY is phosphorylated by CKIepsilon only when both proteins are bound to mammalian PERIOD proteins. BMAL1 is also a substrate for CKIepsilon in vitro, and CKIepsilon kinase activity positively regulates BMAL1-dependent transcription from circadian promoters in reporter assays. We conclude that CKIepsilon phosphorylates multiple circadian substrates and may exert its effects on circadian rhythm in part by a direct effect on BMAL1-dependent transcription.