Casein kinase 1 isoform epsilon

Discovery

The human CK1ε was first isolated and cloned in 1995, officially identified as an isoform of the casein kinase 1 family. Three transcript variants encoding the same protein have been found for this gene in rat: CK1ε1, CK1ε2, and CK1ε3; and two have been found in humans. In humans, the CSNK1E gene localizes at 22q13.1 and consists of 12 exons.

Enzyme function

The protein encoded by the casein kinase 1 epsilon gene is a serine/threonine protein kinase and a member of the casein kinase I protein family, whose members have been implicated in the control of cytoplasmic and nuclear processes, including DNA replication and repair. Like other casein kinase 1 protein family members, casein kinase 1 epsilon recognizes the Ser(p)XXSer/Thr motif for phosphorylation. It is found in the cytoplasm as a monomer and can phosphorylate a variety of proteins, including itself. This autophosphorylation occurs in the protein's C-Terminal domain, a region believed to behave as a pseudosubstrate, and inhibits kinase activity.

The Circadian Clock

The Casein kinase 1 epsilon protein is part of the mammalian oscillator, a group of proteins that keep cells on a roughly 24-hour schedule. This oscillator, or "circadian clock," is made up of a transcription-translation negative feedback loop (TTNFL) in which several proteins work in tandem, each regulating the others' expression to generate a roughly 24-hour cycle of both mRNA and protein levels. The TTFNL also generates roughly 24-hour rhythms of outputs such as levels of cellular hormone release. Daily oscillations in protein and mRNA transcription have been observed in many cells, including the mammalian master clock known as the suprachiasmatic nucleus (SCN). However, unlike most circadian rhythm proteins, casein kinase 1 epsilon is constitutively active.

The most important proteins which make up the mammalian TTNFL are the Period (PER), and Cryptochrome (CRY) proteins, BMAL1, CLOCK, and casein kinase 1 epsilon. PER and CRY levels are regulated by negative feedback, meaning that they repress their own transcription. BMAL1 and CLOCK work to increase PER and CRY transcription by binding on the E-box domain upstream from the PER and CRY gene coding sequences. The level of PER and CRY proteins is regulated by casein kinase 1 through phosphorylation, which marks these proteins for degradation by the cell. Phosphorylation also hinders PER's ability to enter the nucleus by inducing a conformational change in its nuclear localization sequence. However, if CRY proteins bind to PER before this phosphorylation can occur, all three proteins stabilize into a complex that can enter the nucleus. Once inside the nucleus, PER and CRY can work to inhibit their own transcription, while casein kinase 1 epsilon works to modulate the activity of BMAL1 and CLOCK through phosphorylation.

As previously stated, the C-Terminal domain of casein kinase 1 epsilon behaves as a pseudosubstrate when phosphorylated, inhibiting kinase activity. The C-Terminal domain has also been shown to be dephosphorylated by phosphatases such as Protein phosphatase 1 (PP1) in vitro and cell culture, which regulates levels of active casein kinase in vivo. Current theory of circadian rhythms hypothesizes that this phosphorylation/dephosphorylation cycle of casein kinase 1 epsilon is important in modulation of the period of circadian rhythms in the cell, with increased phosphorylation decreasing casein kinase 1 epsilon activity (and subsequently increasing active CRY and PER) and dephosphorylation of casein kinase 1 epsilon resulting in a more active kinase (and lower levels of active CRY and PER).

In mice, casein kinase 1 epsilon has been shown to phosphorylate both PER1 and PER2, as well as CRY1 and CRY2. Casein kinase 1 results in a cyclic expression of mammalian oscillator proteins, resulting in a timekeeper (mammalian oscillator) for the cell:

Mutations to circadian function

In hamsters, the CK1ε-tau mutation was first discovered by Michael Menaker and colleagues while studying a laboratory shipment of Syrian hamsters. The prominent phenotype in the mutant hamsters was an unusually short free-running period — 22 hours in heterozygotes, and 20 hours in homozygotes for the mutation—making this allele semidominant. The gene was later mapped and identified by Joseph Takahashi and colleagues, which revealed a single base-pair C-to-T substitutional mutation in the hamster CK1ε gene. This single nucleotide polymorphism (SNP) results in an arginine-to-cysteine substitution in a phosphate recognition domain region of CK1ε, a highly conserved region of the gene across mammals. Presently, it is unclear how exactly the CK1ε-tau mutation results in a shorter free-running period. The CK1ε-tau mutation in hamsters was the first full description of a mammalian circadian mutant.

In humans, mutations affecting the PER2 phosphorylation site of the CK1ε gene results in Familial advanced sleep phase syndrome (FASPS). This mutation, which results in the loss of a single phosphate acceptor site on PER2, prevents CK1ε protein from binding to PER and leads to an unusually short circadian period. Therefore, while the mutation to the human Per2 gene results in a similarly shortened period as in the CK1ε-tau hamsters, the biological mechanisms behind each phenotype are a result of disruptions to different parts of the clock pathway.

Non-mammalian homologs

Two circadian rhythm functional homologs of this mammalian protein can be found in Drosophila melanogaster (fruit fly). Functional homologs refer to proteins sharing a similar function in another animal but that are not necessarily genetically similar.

One gene, coding for the protein Doubletime (abbreviated DBT), serves a similar purpose to casein kinase 1 epsilon in chronobiology, as it plays a role in the phosphorylation of PER. However its gene sequence shows no sequence homology. In addition, casein kinase 1 epsilon does not completely rescue circadian rhythms in fruit fly doubletime knockouts (dbt -/-), suggesting that these enzymes serve similar, but not identical, functions.

Another functional homolog, the Drosophila gene for glycogen synthase kinase 3 (GSK3), called shaggy and abbreviated sgg, codes for a protein which phosphorylates Timeless (TIM), the fruit fly CRY functional homolog. Like dbt, shaggy is not a sequence homolog to casein kinase 1 epsilon. Conversely, Gsk3 is also found in mammals, and mutants have been implicated in circadian rhythm abnormalities in patients suffering with bipolar disorder.

The Drosophila melanogaster genome contains other casein kinase 1 family enzymes, which are believed to serve no circadian function. However, a different casein kinase family enzyme, casein kinase 2 alpha, has been implicated in providing the initial phosphorylation of a serine residue that is recognized by both DBT and Shaggy for sequential PER and TIM phosphorylation.

Canonical Wnt pathway

The canonical Wnt Pathway involves the accumulation of β-catenin in the cytoplasm, which activates transcription factors. Casein kinase 1 epsilon, and related casein kinase 1 delta, is dephosphorylated in this pathway. Dephospho rylation of casein kinase 1 epsilon is likely achieved by Protein Phosphatase 2(PP2A), which increases both the enzymes' kinase activity in vivo. Casein kinase 1 epsilon and casein kinase 1 delta have been implicated in increasing β-catenin's stability in the cytoplasm, although studies of the mechanism for this stabilization are inconclusive. The current theory for how casein kinase 1 epsilon and/or casein kinase 1 delta function in this pathway is that both casein kinases either directly stabilize β-catenin though positive regulation, or that they indirectly stabilizes β-catenin through negative regulation of the β-catenin degradation complex (protease).

Cancer

Casein kinase 1 epsilon and delta are known to phosphorylate a tumor suppressor protein, p53 in vivo in both humans and murine, or old world rats. Casein kinase 1 epsilon is also implicated as indirectly causing cancer through its regulation of Yes-associated protein (YAP), an oncogene and regulator of organ size. After priming through phosphorylation by the serine/threonine kinase LATS, both casein kinase 1 epsilon and casein kinase 1 delta have been shown to phosphorylate YAP and marking it for ubiquitination and degradation.

Interactions

Casein kinase 1 epsilon has been shown to interact with PER1, PER2, CRY1, CRY2, BMAL1, CLOCK, NPAS2, and AXIN1.

Inhibitors

The use of these inhibitors has allowed for further study of the function of casein kinase 1 epsilon in a variety of processes including its regulation of circadian rhythms.