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The casein kinase I (CKI) gene family is a rapidly enlarging group whose members have been implicated in the control of cytoplasmic and nuclear processes, including DNA replication and repair. A novel isoform of CKI from a human placental cDNA library has been cloned and characterized. The cDNA for this isoform, hCKI epsilon, predicts a basic polypeptide of 416 amino acids and a molecular mass of 47.3 kDa. It encodes a core kinase domain of 285 amino acids and a carboxyl-terminal tail of 123 amino acids. The kinase domain is 53%-98% identical to the kinase domains of other CKI family members and is most closely related to the delta isoform. Localization of the hCKI epsilon gene to chromosome 22q12-13 and the hCKI delta gene to chromosome 17q25 confirms that these are distinct genes in the CKI family. Northern blot analysis shows that hCKI epsilon is expressed in multiple human cell lines. Recombinant hCKI epsilon is an active enzyme that phosphorylates known CKI substrates, including a CKI-specific peptide substrate, and is inhibited by CKI-7, a CKI-specific inhibitor. A budding yeast isoform of CKI, HRR25, has been implicated in DNA repair responses. Expression of hCKI epsilon but not hCKI alpha rescues the slow-growth phenotype of a Saccharomyces cerevisiae strain with a deletion of HRR25. Human CKI epsilon is a novel CKI isoform with properties that overlap those of previously described CKI isoforms (Fish, 1995).

Casein kinase I epsilon (CKI epsilon) is a member of the CKI gene family, members of which are involved in the control of SV40 DNA replication, DNA repair, and cell metabolism. The mechanisms that regulate CKI epsilon activity and substrate specificity are not well understood. CKI epsilon, which contains a highly phosphorylated 123-amino acid carboxyl-terminal extension not present in CKI alpha, is substantially less active than CKI alpha in phosphorylating a number of substrates including SV40 large T antigen and is unable to inhibit the initiation of SV40 DNA replication. Two mechanisms for the activation of CKI epsilon have been identified: (1) a limited tryptic digestion of CKI epsilon produces a protease-resistant amino-terminal 39-kDa core kinase with several-fold enhanced activity; (2) phosphatase treatment of CKI epsilon activates CKI epsilon 5-20-fold toward T antigen. Similar treatment of a truncated form of CKI epsilon produces only a 2-fold activation. Notably, this activation is transient; reautophosphorylation leads to a rapid down-regulation of the kinase within 5 min. Phosphatase treatment also activates CKI epsilon toward the novel substrates I kappa B alpha and Ets-1. These mechanisms may serve to regulate CKI epsilon and related forms of CKI in the cell, perhaps in response to DNA damage (Cegielska, 1998).

Casein kinase I delta (CKIdelta) and casein kinase I epsilon (CKIepsilon) have been implicated in the response to DNA damage, but the understanding of how these kinases are regulated remains incomplete. In vitro, these kinases rapidly autophosphorylate, predominantly on their carboxyl-terminal extensions, and this autophosphorylation markedly inhibits kinase activity. However, while these kinases are able to autophosphorylate in vivo, they are actively maintained in the dephosphorylated, active state by cellular protein phosphatases. Treatment of cells with the cell-permeable serine/threonine phosphatase inhibitors okadaic acid or calyculin A leads to rapid increases in kinase intramolecular autophosphorylation. Since CKI autophosphorylation decreases kinase activity, this dynamic autophosphorylation/dephosphorylation cycle provides a mechanism for kinase regulation in vivo (Rivers, 1998).

Frequency (FRQ) is a crucial element of the circadian clock in Neurospora crassa. In the course of a circadian day FRQ is successively phosphorylated and degraded. Two PEST-like elements in FRQ, PEST-1 and PEST-2, are phosphorylated in vitro by recombinant CK-1a and CK-1b, two newly identified Neurospora homologs of casein kinase 1epsilon (Drosophila homolog: Doubletime). CK-1a is localized in the cytosol and the nuclei of Neurospora and it is in a complex with FRQ in vivo. Deletion of PEST-1 results in hypophosphorylation of FRQ and causes significantly increased protein stability. A strain harboring the mutant frqDeltaPEST-1 gene shows no rhythmic conidiation. Despite the lack of overt rhythmicity, frqDeltaPEST-1 RNA and FRQPEST-1 protein are rhythmically expressed and oscillate in constant darkness with a circadian period of 28 h. Thus, by deletion of PEST-1 the circadian period is lengthened and overt rhythmicity is dissociated from molecular oscillations of clock components (Gör, 2002).

The molecular oscillator that keeps circadian time is generated by a negative feedback loop. Nuclear entry of circadian regulatory proteins that inhibit transcription from E-box-containing promoters appears to be a critical component of this loop in both Drosophila and mammals. The Drosophila double-time gene product, a casein kinase Iepsilon (CKIepsilon) homolog, interacts with Drosophila Per and regulates circadian cycle length. Mammalian CKIepsilon binds to and phosphorylates the murine circadian regulator mPER1. Unlike both Drosophila Per and mPER2, mPER1 expressed alone in HEK 293 cells is predominantly a nuclear protein. Two distinct mechanisms appear to retard mPER1 nuclear entry: (1) coexpression of mPER2 leads to mPER1-mPER2 heterodimer formation and cytoplasmic colocalization; (2) coexpression of CKIepsilon leads to masking of the mPER1 nuclear localization signal and phosphorylation-dependent cytoplasmic retention of both proteins. CKIepsilon may regulate mammalian circadian rhythm by controlling the rate at which mPER1 enters the nucleus (Vielhaber, 2000).

An unexpected finding was that mPER1 expressed in HEK 293 cells is predominantly nuclear, while mPER2 is cytoplasmic. Coexpression of mPER1 with mPER2 or with active (but not inactive) CKIepsilon leads to accumulation of mPER1 in the cytoplasm rather than the nucleus. The CKI-dependent cytoplasmic localization requires a domain adjacent to the NLS in mPER1, implying that phosphorylation leads to a conformational change that masks the mPER1 NLS. These results suggest that both mPER2 and CKI can regulate mPER1 nuclear entry. The mechanism by which mPER2 keeps mPER1 in the cytoplasm appears to be distinct, and a study of the mPER1-mPER2 interaction is ongoing. Both mechanisms may allow for a delay in the negative regulation of circadian transcriptional activators such as CLOCK and BMAL1 (Vielhaber, 2000).

What mechanism finally allows nuclear entry of mPER protein complexes, leading to inhibition of CLOCK/BMAL1 activity? In Drosophila, heterodimerization of Per with tim allows nuclear import and subsequent inhibition of CLOCK/CYCLE transcription. However, there is no effect of mammalian TIM on mPER1 and mPER2 localization. In mammals, mCRY1 and mCRY2 have recently been shown to relocalize mPER1 and mPER2 proteins to the nucleus and efficiently repress transcription from E-box-containing promoters, although the mechanism by which mCRY proteins mediate this relocalization is not yet known. mCRY proteins may supply an NLS, although the data presented here raise the possibility that mCRY proteins could also allow unmasking of the mPER1 NLS by inhibition of CKI or recruitment of a specific phosphatase such as PP5 (Vielhaber, 2000).

Posttranslational regulation of clock proteins in mouse liver has been examined in vivo. The mouse PERIOD proteins (mPER1 and mPER2), CLOCK, and BMAL1 undergo robust circadian changes in phosphorylation. These proteins, the cryptochromes (mCRY1 and mCRY2), and casein kinase I epsilon (CKIepsilon) form multimeric complexes that are bound to DNA during negative transcriptional feedback. CLOCK:BMAL1 heterodimers remain bound to DNA over the circadian cycle. The temporal increase in mPER abundance controls the negative feedback interactions. Analysis of clock proteins in mCRY-deficient mice shows that the mCRYs are necessary for stabilizing phosphorylated mPER2 and for the nuclear accumulation of mPER1, mPER2, and CKIepsilon. in vivo evidence is provided that casein kinase I delta is a second clock relevant kinase (Lee, 2001).

These findings provide a novel mechanism by which the mPER proteins control the molecular clockwork; that is, the robust, high-amplitude oscillations in mPER protein abundance are necessary for perpetuating the circadian clock mechanism, since mPER proteins bring clock protein complexes into the nucleus at the proper time for negative transcriptional feedback. With rhythmic accumulation of either mPER1 or mPER2, the clock mechanism persists and drives circadian behavior for a period of time in constant conditions, as occurs when either mPer gene is targeted. Once in the nucleus, mPER2 appears to have the additional function of regulating Bmal1 transcription, leading to a more severe circadian phenotype with its disruption, compared with mPer1 disruption. When mPer1 and mPer2 are targeted together, the clock immediately ceases to function on placement in constant conditions, because the mPER rhythms are immediately disrupted (Lee, 2001).

Light-dependent transcriptional regulation of clock genes is a crucial step in the entrainment of the circadian clock. E4bp4, a vertebrate ortholog of Drosophila Vrille, is a light-inducible gene in the chick pineal gland, and it encodes a bZIP protein that represses transcription of cPer2, a chick pineal clock gene. Prolonged light period-dependent accumulation of E4BP4 protein is temporally coordinated with a delay of the rising phase of cPer2 in the morning. E4BP4 is phosphorylated progressively and then disappears in parallel with induced cPer2 expression. Characterization of E4BP4 revealed Ser182, a phosphoacceptor site located at the amino-terminal border of the Ser/Thr cluster, which forms the phosphorylation motifs for casein kinase 1 (CK1). This serine/threonine cluster is evolutionarily conserved from vertebrate E4BP4 to Drosophila Vrille. CK1 physically associates with E4BP4 and phosphorylates it. CK1-catalyzed phosphorylation of E4BP4 results in proteasomal proteolysis-dependent decrease of E4BP4 levels, while E4BP4 nuclear accumulation is attenuated by CK1 in a kinase activity-independent manner. CK1-mediated posttranslational regulation is accompanied by reduction of the transcriptional repression executed by E4BP4. These results not only demonstrate a phosphorylation-dependent regulatory mechanism for E4BP4 function but also highlight the role of CK1 as a negative regulator for E4BP4-mediated repression of cPer2 (Doi, 2004).

A novel mechanism controlling resetting speed of the circadian clock to environmental stimuli

Many aspects of mammalian physiology are driven through the coordinated action of internal circadian clocks. Clock speed (period) and phase (temporal alignment) are fundamental to an organism's ability to synchronize with its environment. In humans, lifestyles that disturb these clocks, such as shift work, increase the incidence of diseases such as cancer and diabetes. Casein kinases 1δ and ε are closely related clock components implicated in period determination. However, CK1δ is so dominant in this regard that it remains unclear what function CK1epsilon; normally serves. This study has revealed that CK1ε dictates how rapidly the clock is reset by environmental stimuli. Genetic disruption of CK1ε in mice enhances phase resetting of behavioral rhythms to acute light pulses and shifts in light cycle. This impact of CK1ε targeting is recapitulated in isolated brain suprachiasmatic nucleus and peripheral (lung) clocks during NMDA- or temperature-induced phase shift in association with altered PERIOD (PER) protein dynamics. Importantly, accelerated re-entrainment of the circadian system in vivo and in vitro can be achieved in wild-type animals through pharmacological inhibition of CK1ε. These studies therefore reveal a role for CK1ε in stabilizing the circadian clock against phase shift and highlight it as a novel target for minimizing physiological disturbance in shift workers (Pilorz, 2014).

The Doubletime homolog KIN-20 mainly regulates let-7 independently of its effects on the Period homolog LIN-42 in Caenorhabditis elegans

The Caenorhabditis elegans (C. elegans) heterochronic pathway, which regulates developmental timing, is thought to be an ancestral form of the circadian clock in other organisms. An essential member of this clock is the Period protein whose homolog, lin-42, in C. elegans is an important heterochronic gene. LIN-42 functions as a transcriptional repressor of multiple genes including the conserved lin-4 and let-7 microRNAs. Like other Period proteins, levels of LIN-42 oscillate throughout development. In other organisms this cycling is controlled in part by phosphorylation. KIN-20 is the C. elegans homolog of the Drosophila Period protein kinase Doubletime. Worms containing a large deletion in kin-20 have a significantly smaller brood size and develop slower than wild type C. elegans. This study analyzed the effect of kin-20 on lin-42 phenotypes and microRNA expression. kin-20 RNAi enhances loss-of-function lin-42 mutant phenotypes, and kin-20 mutant worms express lower levels of LIN-42. It was also shown that kin-20 is important for post-transcriptional regulation of mature let-7 and lin-4 microRNA expression. In addition, the increased level of let-7 found in lin-42n1089 mutant worms is not maintained after kin-20 RNAi treatment. Instead, let-7 is further repressed when levels of kin-20 and lin-42 are both decreased. Altogether these results suggest that though kin-20 regulates lin-42 and let-7 microRNA, it mainly affects let-7 microRNA expression independently of lin-42. These findings further understanding of the mechanisms by which these conserved circadian rhythmic genes interact to ultimately regulate rhythmic processes, developmental timing and microRNA biogenesis in C. elegans (Rhodehouse, 2018).

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