Recent studies have shown that casein kinase I (CKI) is an essential regulator of the mammalian circadian clock. However, the detailed mechanisms by which CKI regulates each component of the circadian negative-feedback loop have not been fully defined. We show here that mPer proteins, negative limbs of the autoregulatory loop, are specific substrates for CKI and CKI␦. The CKI phosphorylation of mPer1 and mPer3 proteins results in their rapid degradation, which is dependent on the ubiquitin-proteasome pathway. Moreover, CKI and CKI␦ are able to induce nuclear translocation of mPer3, which requires its nuclear localization signal. The mutation in potential phosphorylation sites on mPer3 decreased the extent of both nuclear translocation and degradation of mPer3 that are stimulated by CKI. CKI and CKI␦ affected the inhibitory effect of mPer proteins on the transcriptional activity of BMAL1-CLOCK, but the inhibitory effect of mCry proteins on the activity of BMAL1-CLOCK was unaffected. These results suggest that CKI and CKI␦ regulate the mammalian circadian autoregulatory loop by controlling both protein turnover and subcellular localization of mPer proteins.In most living organisms, behavioral and physiological processes display ϳ24-h rhythms that are controlled by circadian pacemakers (6,15,22,37). Although these rhythms persist in constant conditions, fluctuations of the natural environment entrain rhythms to precisely 24-h periods. The circadian clock is made up of three components: an input pathway adjusting the time, a central oscillator generating the circadian signal, and an output pathway manifesting itself in circadian physiology and behavior (10,12,23,25). In mammals, the master circadian pacemaker is located in the suprachiasmatic nucleus, which controls neural and humoral signals that either drive output rhythms or synchronize peripheral oscillators with the day-night cycle (3,38).Autoregulatory feedback loops of gene expression are believed to provide the rhythm-generating mechanisms. Since several clock genes are conserved in flies and mammals, the fundamental mechanism may be evolutionarily conserved (40). In mammals, the positive limb of the feedback loop is composed of CLOCK and BMAL1 (2,4,8,16), while the negative limb is composed of cryptochrome proteins (Cry1 and Cry2) and period proteins (Per1, Per2, and Per3) (1,5,27,29,31,32,33,35,41).In Drosophila melanogaster, doubletime (dbt) was identified as a kinase, which is thought to phosphorylate dPer (Drosophila Period) (17, 21). Missense mutations in dbt result in an altered circadian rhythm. Null alleles of dbt result in hypophosphorylation of dPer and arrhythmia. Dbt-mediated phosphorylation destabilizes dPer so that the level of dPer increases only when dTim (Drosophila Timeless) is increasing (17,21,24). In the Syrian hamster, the tau mutation is a spontaneous, semidominant mutation causing a short-period phenotype. Using a positional syntenic cloning strategy, the tau locus is revealed to encode casein kinase I ε (CKIε), a mammalian homolog o...

T he Ras/extracellular-signal-regulated kinase (ERK) mitogenactivated protein (MAP) kinase signaling pathway is among the key mechanisms that transmit signals from the cell surface to the nucleus.(1-7) A wide variety of extracellular stimuli induce sequential activation of three protein kinases, Raf, MEK and ERK, in the Ras/ERK signaling pathway (Fig. 1). ERK is a highly conserved serine/threonine kinase activated by MEK via phosphorylation on both threonine and tyrosine residues in the TEY sequence. Activated ERK phosphorylates both cytoplasmic and nuclear substrates, including many enzymes, cytoskeletal proteins and transcription factors. Recent studies have identified a number of Ras/ERK signaling-related proteins, such as scaffold proteins and inhibitor proteins of this pathway. These proteins provide variations in ERK signaling by modulating the duration, magnitude and subcellular compartmentalization of ERK activity. (3,8) Accumulating evidence suggests that such differences in ERK activity generate variations in signaling outputs that regulate cell fate decisions. Moreover, crosstalk with other pathways could also be crucial for determining signaling specificity. The Ras/ERK signaling pathway is known to regulate various cellular responses and, in particular, its role in cell cycle progression in G 1 phase and cell proliferation is well established. (9)(10)(11)(12) In addition, the pathway is activated constitutively in many types of cancer. Here we discuss recent findings, focusing on ERK signaling-mediated normal cell cycle progression and malignant transformation. ERK signaling regulatorsRecent studies have identified several negative regulators of the ERK signaling pathway and their action mechanisms have been analyzed. Among them, Sprouty, Spred and Sef were found to act as conserved inhibitors of the ERK signaling pathway. (13)(14)(15)(16)(17) More recent reports have demonstrated the detailed molecular mechanisms of action of these regulators (Fig. 1). Spred inhibits the ERK signaling pathway at the level of Raf by binding to Ras and Raf. (14) The inhibitory mechanisms of Sprouty and Sef have been controversial. Targets of Sprouty in the ERK signaling pathway are suggested to be Grb2, (18,19) Sos (19) and Raf1. (20) Hanafusa et al. showed that Sprouty1 and 2 become phosphorylated on a conserved tyrosine residue (Y53 in Sprouty1 and Y55 in Sprouty2) in their amino-terminal domain upon growth factor stimulation, and become bound to Grb2. (18) This binding prevents Grb2 from binding to either tyrosine-phosphorylated adaptor proteins or receptors, resulting in the inhibition of Ras/ERK signaling. This conserved tyrosine residue of Sprouty1 and 2 could be phosphorylated by Src family kinases and dephosphorylated by Shp2.(21-23) Other reports have shown that Sprouty2 becomes phosphorylated on the same conserved tyrosine residue upon epidermal growth factor (EGF) stimulation and binds to cCbl, the E3 ubiquitin ligase for the EGF receptor. (22,(24)(25)(26) This association leads to polyubiquitylation and...

A growing body of evidence suggests that Nrf1 is an inducible transcription factor that maintains cellular homeostasis. Under physiological conditions, Nrf1 is targeted to the endoplasmic reticulum (ER), implying that it translocates into the nucleus in response to an activating signal. However, the molecular mechanisms by which the function of Nrf1 is modulated remain poorly understood. Here, we report that two distinct degradation mechanisms regulate Nrf1 activity and the expression of its target genes. In the nucleus, ␤-TrCP, an adaptor for the SCF (Skp1-Cul1-F-box protein) ubiquitin ligase, promotes the degradation of Nrf1 by catalyzing its polyubiquitination. This activity requires a DSGLS motif on Nrf1, which is similar to the canonical ␤-TrCP recognition motif. The short interfering RNA (siRNA)-mediated silencing of ␤-TrCP markedly augments the expression of Nrf1 target genes, such as the proteasome subunit PSMC4, indicating that ␤-TrCP represses Nrf1 activation. Meanwhile, in the cytoplasm, Nrf1 is degraded and suppressed by the ER-associated degradation (ERAD) ubiquitin ligase Hrd1 and valosin-containing protein (VCP) under normal conditions. We identified a cytoplasmic degradation motif on Nrf1 between the NHB1 and NHB2 domains that exhibited species conservation. Thus, these results clearly suggest that both ␤-TrCP-and Hrd1-dependent degradation mechanisms regulate the transcriptional activity of Nrf1 to maintain cellular homeostasis.

SummaryMitogen-activated protein kinase (MAPK) pathways play central roles in controlling diverse cellular functions. They are finely regulated by several mechanisms, including scaffolding of their components, and phosphorylation/dephosphorylation and compartmentalization of MAPKs. A number of molecules have been identified as regulators involved in these mechanisms. They modulate the magnitude and the specificity of MAPK signaling, and thereby regulate the wide variety of signaling outputs. Recent studies have identified novel functions of the MAPK signaling pathways. It is becoming clear that strict regulation of the MAPK pathways underlies their manifold functions in numerous biological processes.

Posttranslational modifications of clock proteins are crucial to generating proper circadian rhythms of the correct length and amplitude. Here, we show that the protein kinase CK2 (casein kinase 2) plays a role in regulating the mammalian circadian clock. We found that inhibiting CK2 activity resulted in a decrease in the amplitude and an increase in the period of oscillations in circadian gene expression. CK2 specifically bound and phosphorylated PERIOD2 (PER2) and collaborated with the protein kinase CKIepsilon to promote PER2 degradation. We also identified a CK2 phosphorylation site (serine-53) in PER2, whose phosphorylation played a role in fine-tuning circadian rhythms and regulating PER2 stability but was dispensable for the cooperative effect of CK2 and CKIepsilon. Thus, our study identifies CK2 as a regulatory element of mammalian circadian rhythms and uncovers a role for CK2 in PER2 degradation.