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. CKI (casein kinase I) has been postulated to prime PER2 for degradation. Supporting this idea, CKI inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKI inhibition also slows the degradation of PER2 in cells. CKI-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein ␤-TrCP to a specific site, and dominant negative ␤-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.Diverse organisms from prokaryotes to mammals coordinate behavioral and physiological rhythms with the daily dark-light cycle by means of a circadian clock. In mammals, the master circadian clock is located in the suprachiasmatic nucleus of the brain, and it entrains peripheral cell-autonomous clocks throughout the body. In mice, a positively acting heterodimeric transcription factor composed of the PAS-bHLH proteins CLOCK (CLK) and BMAL1 drives transcription of tissuespecific circadian output genes, as well as its own negative regulators, the Period (denoted mPer1, mPer2, and mPer3), and Cryptochrome (mCry1 and mCry2) genes. The mammalian PER and CRY proteins form multimeric complexes that enter the nucleus and repress the transcriptional activity of CLK/ BMAL1, modulating circadian output (reviewed in references 29 and 41). Additional stabilizing feedback loops, including inhibition of Bmal1 transcription by REV-ERB␣ (37), further contribute to the timing and robustness of the cycle. The daily rhythmic degradation of PERIOD proteins leading to derepression of CLK/BMAL1 is postulated to be critical to the proper functioning of the clock. Therefore, the mechanism and control of this process are of great interest.Genetic studies have identified CKIε (casein kinase Iε) as a key regulator of metazoan circadian rhythm and both genetic and biochemical studies suggest that the PER proteins are important substrates (reviewed in reference 10). CKIε was first implicated as a circadian regulator in Drosop...

Biological clocks with a period of Ϸ24 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 CKI 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 CKI tau increases kinase activity, and we verify this prediction experimentally. CKI 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.kinase ͉ systems biology ͉ phosphorylation ͉ PER ͉ degradation C ircadian rhythms govern key physiologic processes including sleep-wake cycles; glucose, lipid, and drug metabolism; heart rate; stress and growth hormones; and immunity, as well as basic cellular processes such as timing of the cell division cycle (1-6). The disruption of circadian rhythm causes significant physiologic stress, is frequently experienced in jet lag and night-shift work, and has been linked to bipolar disorder (7). Thus, circadian regulation of physiology has important consequences for health. A detailed quantitative model that makes clear, testable, and accurate predictions about the clock and how we may manipulate it can therefore have benefits for human health.Much of our understanding of clock components and their interactions began with the identification of mutations that affect circadian behavior (8, 9). In mammals, the original and most extensively studied circadian rhythm mutation is the semidominant tau, first described in 1988. Hamsters with this mutation show phase-advanced activity and have a circadian period of 20 h when homozygous mutant animals are isolated from time cues (9). This tau mutation has been identified as a missense mutation within the substrate recognition site of casein kinase I (denoted CKI tau ) (10). CKI and the closely related CKI␦ are widely expressed serine-threonine protein kinases implicated in development, circadian rhythms, and DNA metabolism (11). When tested in vitro on multiple substrates, CKI tau was shown to have a much reduced overall catalytic activity (10,12,13). This partial loss-of-function mutation and its phenotype have been difficult to reconcile with our current understanding of the molecular feedback loop that governs timing in mammalian cells (13) and recent empirical observations on clock function (14-16). For example, Dey et al. (1...

Autocrine EGF-receptor (EGFR) ligands are normally made as membrane-anchored precursors that are proteolytically processed to yield mature, soluble peptides. To explore the function of the membrane-anchoring domain of EGF, we expressed artificial EGF genes either with or without this structure in human mammary epithelial cells (HMEC). These cells require activation of the EGFR for cell proliferation. We found that HMEC expressing high levels of membrane- anchored EGF grew at a maximal rate that was not increased by exogenous EGF, but could be inhibited by anti–EGFR antibodies. In contrast, when cells expressed EGF lacking the membrane-anchoring domain (sEGF), their proliferation rate, growth at clonal densities, and receptor substrate phosphorylation were not affected by anti–EGFR antibodies. The sEGF was found to be colocalized with the EGFR within small cytoplasmic vesicles. It thus appears that removal of the membrane-anchoring domain converts autocrine to intracrine signaling. Significantly, sEGF inhibited the organization of HMEC on Matrigel, suggesting that spatial restriction of EGF access to its receptor is necessary for organization. Our results indicate that an important role of the membrane-anchoring domain of EGFR ligands is to restrict the cellular compartments in which the receptor is activated.

The Wnt signaling pathway is critical for embryonic development and is dysregulated in multiple cancers. Two closely related isoforms of casein kinase I (CKIdelta and epsilon) are positive regulators of this pathway. We speculated that mutations in the autoinhibitory domain of CKIdelta/epsilon might upregulate CKIdelta/epsilon activity and hence Wnt signaling and increase the risk of adenomatous polyps and colon cancer. Exons encoding the CKIepsilon and CKIdelta regulatory domains were sequenced from DNA obtained from individuals with adenomatous polyps and a family history of colon cancer unaffected by familial adenomatous polyposis or hereditary nonpolyposis colorectal cancer (HNPCC). A CKIdelta missense mutation, changing a highly conserved residue, Arg324, to His (R324H), was found in an individual with large and multiple polyps diagnosed at a relatively young age. Two findings indicate that this mutation is biologically active. First, ectopic ventral expression of CKIdelta(R324H) in Xenopus embryos results in secondary axis formation with an additional distinctive phenotype (altered morphological movements) similar to that seen with unregulated CKIepsilon. Second, CKIdelta(R324H) is more potent than wildtype CKIdelta in transformation of RKO colon cancer cells. Although the R324H mutation does not significantly change CKIdelta kinase activity in an in vitro kinase assay or Wnt/beta-catenin signal transduction as assessed by a beta-catenin reporter assay, it alters morphogenetic movements via a beta-catenin-independent mechanism in early Xenopus development. This novel human CKIdelta mutation may alter the physiological role and enhance the transforming ability of CKIdelta through a Wnt/beta-catenin independent mechanism and thereby influence colonic adenoma development.