The Circadian tau Mutation in Casein Kinase 1 Is Part of a Larger Domain That Can Be Mutated to Shorten Circadian Period.

Drosophila Double-time (DBT) phosphorylates the circadian protein Period (PER). The period-altering mutation tau, identified in hamster casein kinase I (CKIε) and created in Drosophila DBT, has been shown to shorten the circadian period in flies, as it does in hamsters. Since CKI often phosphorylates downstream of previously phosphorylated residues and the tau amino acid binds a negatively charged ion in X-ray crystal structures, this amino acid has been suggested to contribute to a phosphate recognition site for the substrate. Alternatively, the tau amino acid may affect a nuclear localization signal (NLS) with which it interacts. We mutated the residues that were close to or part of the phosphate recognition site or NLS. Flies expressing DBT with mutations of amino acids close to or part of either of these motifs produced a shortening of period, suggesting that a domain, including the phosphate recognition site or the NLS, can be mutated to produce the short period phenotype. Mutation of residues affecting internally placed residues produced a longer period, suggesting that a specific domain on the surface of the kinase might generate an interaction with a substrate or regulator, with short periods produced when the interaction is disrupted.

Discovery of Inhibitor of Wnt Production 2 (IWP-2) and Related Compounds As Selective ATP-Competitive Inhibitors of Casein Kinase 1 (CK1) δ/ε.

Inhibitors of Wnt production (IWPs) are known antagonists of the Wnt pathway, targeting the membrane-bound O-acyltransferase porcupine (Porcn) and thus preventing a crucial Wnt ligand palmitoylation. Since IWPs show structural similarities to benzimidazole-based CK1 inhibitors, we hypothesized that IWPs could also inhibit CK1 isoforms. Molecular modeling revealed a plausible binding mode of IWP-2 in the ATP binding pocket of CK1δ which was confirmed by X-ray analysis. In vitro kinase assays demonstrated IWPs to be ATP-competitive inhibitors of wtCK1δ. IWPs also strongly inhibited the gatekeeper mutant M82FCK1δ. When profiled in a panel of 320 kinases, IWP-2 specifically inhibited CK1δ. IWP-2 and IWP-4 also inhibited the viability of various cancer cell lines. By a medicinal chemistry approach, we developed improved IWP-derived CK1 inhibitors. Our results suggest that the effects of IWPs are not limited to Porcn, but also might influence CK1δ/ε-related pathways.

Molecular basis for the regulation of the circadian clock kinases CK1δ and CK1ε.

CK1δ and CK1ε are unique in the casein kinase 1 family and play critical roles in a number of physiological intracellular pathways. In particular, these kinases are involved in composing the mammalian circadian clock by phosphorylating core clock proteins. Considering that CK1δ/ε phosphorylate other key biological molecules, such as ß-catenin and p53, understanding how the kinase activity is regulated would be greatly significant, since they are potential targets to develop pharmacological agents against cancer, pain, and circadian disorders. In this review, we summarize current knowledge attributed to kinase regulation including expression regulation, post-translational regulation, and kinase activity modulation by small molecules. Finally, we discuss how the kinase activity is regulated from a structural point of view.

A PER2-derived mechanism-based bisubstrate analog for casein kinase 1ε.

Casein kinase 1ε (CK1ε) plays an important regulatory role in various cellular processes including circadian rhythms. Mutations in CK1ε or the recognition site on its substrate PER2 result in modulation of the circadian period length. In particular, the tau mutation (R178C) in the catalytic domain of CK1ε was identified as the molecular basis for a dose-dependent heritable shortened circadian period in hamsters. However, the biochemical basis for the physiological effects of the tau mutant remains unclear. It has been reported that the tau mutation has reduced in vitro activity against some substrates but increased in vitro activity against other substrates. To better understand the effects of the CK1ε tau mutation, an ATP-phosphopeptide conjugate was synthesized to yield a transition-state bisubstrate analog. Kinase activity assays determined that the tau mutant has 80% reduced activity and a fourfold decrease in sensitivity to the bisubstrate analog compared to wild type. This confirms that Arg178 is important in the recognition of the preferred phosphosubstrates of CK1ε.

TDP-43 Phosphorylation by casein kinase Iε promotes oligomerization and enhances toxicity in vivo.

Dominant mutations in transactive response DNA-binding protein-43 (TDP-43) cause amyotrophic lateral sclerosis. TDP-43 inclusions occur in neurons, glia and muscle in this disease and in sporadic and inherited forms of frontotemporal lobar degeneration. Cytoplasmic localization, cleavage, aggregation and phosphorylation of TDP-43 at the Ser409/410 epitope have been associated with disease pathogenesis. TDP-43 aggregation is not a common feature of mouse models of TDP-43 proteinopathy, and TDP-43 is generally not thought to acquire an amyloid conformation or form fibrils. A number of putative TDP-43 kinases have been identified, but whether any of these functions to regulate TDP-43 phosphorylation or toxicity in vivo is not known. Here, we demonstrate that human TDP-43(Q331K) undergoes cytoplasmic localization and aggregates when misexpressed in Drosophila when compared with wild-type and M337V forms. Coexpression of Q331K with doubletime (DBT), the fly homolog of casein kinase Iε (CKIε), enhances toxicity. There is at best modest basal phosphorylation of misexpressed human TDP-43 in Drosophila, but coexpression with DBT increases Ser409/410 phosphorylation of all TDP-43 isoforms tested. Phosphorylation of TDP-43 in the fly is specific for DBT, as it is not observed using the validated tau kinases GSK-3ß, PAR-1/MARK2 or CDK5. Coexpression of DBT with TDP-43(Q331K) enhances the formation of high-molecular weight oligomeric species coincident with enhanced toxicity, and treatment of recombinant oligomeric TDP-43 with rat CKI strongly enhances its toxicity in mammalian cell culture. These data identify CKIε as a potent TDP-43 kinase in vivo and implicate oligomeric species as the toxic entities in TDP-43 proteinopathies.

RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-ß-catenin signaling.

Casein kinase 1 (CK1) members play key roles in numerous biological processes. They are considered "rogue" kinases, because their enzymatic activity appears unregulated. Contrary to this notion, we have identified the DEAD-box RNA helicase DDX3 as a regulator of the Wnt-ß-catenin network, where it acts as a regulatory subunit of CK1ε: In a Wnt-dependent manner, DDX3 binds CK1ε and directly stimulates its kinase activity, and promotes phosphorylation of the scaffold protein dishevelled. DDX3 is required for Wnt-ß-catenin signaling in mammalian cells and during Xenopus and Caenorhabditis elegans development. The results also suggest that the kinase-stimulatory function extends to other DDX and CK1 members, opening fresh perspectives for one of the longest-studied protein kinase families.

Structural basis for the potent and selective inhibition of casein kinase 1 epsilon.

Casein kinase 1 epsilon (CK1ε) and its closest homologue CK1δ are key regulators of diverse cellular processes. We report two crystal structures of PF4800567, a potent and selective inhibitor of CK1ε, bound to the kinase domains of human CK1ε and CK1δ as well as one apo CK1ε crystal structure. These structures provide a molecular basis for the strong and specific inhibitor interactions with CK1ε and suggest clues for further development of CK1δ inhibitors.

2-Benzamido-N-(1H-benzo[d]imidazol-2-yl)thiazole-4-carboxamide derivatives as potent inhibitors of CK1δ/ε.

In this study we identified two heterocyclic compounds (5 and 6) as potent and specific inhibitors of CK1δ (IC(50) = 0.040 and 0.042 µM, respectively). Whereas compound 5 exhibited fivefold higher affinity towards CK1δ than to CK1ε (IC(50) CK1ε = 0.199 µM), compound 6 also inhibited CK1ε (IC(50) = 0.0326 µM) in the same range as CK1δ. Selected compound 5 was screened over 442 kinases identifying 5 as a highly potent and selective inhibitor of CK1δ. X-ray analysis of 5 bound to CK1δ demonstrated its binding mode. In addition, characterization of 5 and 6 in a cell biological approach revealed the ability of both compounds to inhibit proliferation of tumor cell lines in a dose and cell line specific manner. In summary, our optimizations lead to the development of new highly selective CK1δ and ε specific inhibitors with biological activity.

Structural basis for the interaction between casein kinase 1 delta and a potent and selective inhibitor.

Casein kinase 1 delta (CK1δ) and its closest homologue CK1ε are key regulators of diverse cellular growth and survival processes such as Wnt signaling, DNA repair, and circadian rhythms. We report three crystal structures of the kinase domain of human CK1δ, one apo and two complexed with a potent and selective CK1δ/ε inhibitor PF670462 in two different crystal forms. These structures provide a molecular basis for the strong and specific inhibitor interactions and suggest clues for further development of CK1δ/ε inhibitors.

Casein kinase I epsilon somatic mutations found in breast cancer cause overgrowth in Drosophila.

We are using a candidate gene approach to identify genes contributing to cancer through somatic mutation. Somatic mutations were found in breast cancer samples in the human casein kinase I epsilon (CKIepsilon) gene, a homolog of the Drosophila gene dco in which certain point mutations lead to imaginal disc overgrowth. We therefore created fly genotypes in which the dco gene carried point mutations homologous to those discovered in CKIepsilon, and tested them in vivo. The results show that the most frequent mutation discovered in breast cancer, L39Q, causes a striking overgrowth phenotype in flies. Further experiments show that this mutation affects the newly recognized Fat/Warts signaling pathway, which controls organ size and shape in both flies and mammals. Another mutation, S101R, modifies the mutant phenotype so that the affected tissue disintegrates, mimicking more aggressive forms of breast cancer. Our results thus strongly support the conclusion that CKIepsilon mutations play important roles in breast carcinogenesis.

Breast cancer-specific mutations in CK1epsilon inhibit Wnt/beta-catenin and activate the Wnt/Rac1/JNK and NFAT pathways to decrease cell adhesion and promote cell migration.

INTRODUCTION: Breast cancer is one of the most common types of cancer in women. One of the genes that were found mutated in breast cancer is casein kinase 1 epsilon (CK1epsilon). Because CK1epsilon is a crucial regulator of the Wnt signaling cascades, we determined how these CK1epsilon mutations interfere with the Wnt pathway and affect the behavior of epithelial breast cancer cell lines. METHODS: We performed in silico modeling of various mutations and analyzed the kinase activity of the CK1epsilon mutants both in vitro and in vivo. Furthermore, we used reporter and small GTPase assays to identify how mutation of CK1epsilon affects different branches of the Wnt signaling pathway. Based on these results, we employed cell adhesion and cell migration assays in MCF7 cells to demonstrate a crucial role for CK1epsilon in these processes. RESULTS: In silico modeling and in vivo data showed that autophosphorylation at Thr 44, a site adjacent to the breast cancer point mutations in the N-terminal lobe of human CK1epsilon, is involved in positive regulation of the CK1epsilon activity. Our data further demonstrate that, in mammalian cells, mutated forms of CK1epsilon failed to affect the intracellular localization and phosphorylation of Dvl2; we were able to demonstrate that CK1epsilon mutants were unable to enhance Dvl-induced TCF/LEF-mediated transcription, that CK1epsilon mutants acted as loss-of-function in the Wnt/beta-catenin pathway, and that CK1epsilon mutants activated the noncanonical Wnt/Rac-1 and NFAT pathways, similar to pharmacological inhibitors of CK1. In line with these findings, inhibition of CK1 promoted cell migration as well as decreased cell adhesion and E-cadherin expression in the breast cancer-derived cell line MCF7. CONCLUSIONS: In summary, these data suggest that the mutations of CK1epsilon found in breast cancer can suppress Wnt/beta-catenin as well as promote the Wnt/Rac-1/JNK and Wnt/NFAT pathways, thus contributing to breast cancer development via effects on cell adhesion and migration. In terms of molecular mechanism, our data indicate that the breast cancer point mutations in the N-terminal lobe of CK1epsilon, which are correlated with decreased phosphorylation activities of mutated forms of CK1epsilon both in vitro and in vivo, interfere with positive autophosphorylation at Thr 44.

Drosophila and vertebrate casein kinase Idelta exhibits evolutionary conservation of circadian function.

Mutations lowering the kinase activity of Drosophila Doubletime (DBT) and vertebrate casein kinase Iepsilon/delta (CKIepsilon/delta) produce long-period, short-period, and arrhythmic circadian rhythms. Since most ckI short-period mutants have been isolated in mammals, while the long-period mutants have been found mostly in Drosophila, lowered kinase activity may have opposite consequences in flies and vertebrates, because of differences between the kinases or their circadian mechanisms. However, the results of this article establish that the Drosophila dbt mutations have similar effects on period (PER) protein phosphorylation by the fly and vertebrate enzymes in vitro and that Drosophila DBT has an inhibitory C-terminal domain and exhibits autophosphorylation, as does vertebrate CKIepsilon/delta. Moreover, expression of either Drosophila DBT or the vertebrate CKIdelta kinase carrying the Drosophila dbt(S) or vertebrate tau mutations in all circadian cells leads to short-period circadian rhythms. By contrast, vertebrate CKIdelta carrying the dbt(L) mutation does not lengthen circadian rhythms, while Drosophila DBT(L) does. Different effects of the dbt(S) and tau mutations on the oscillations of PER phosphorylation suggest that the mutations shorten the circadian period differently. The results demonstrate a high degree of evolutionary conservation of fly and vertebrate CKIdelta and of the functions affected by their period-shortening mutations.

Drosophila DBT lacking protein kinase activity produces long-period and arrhythmic circadian behavioral and molecular rhythms.

A mutation (K38R) which specifically eliminates kinase activity was created in the Drosophila melanogaster ckI gene (doubletime [dbt]). In vitro, DBT protein carrying the K38R mutation (DBT(K/R)) interacted with Period protein (PER) but lacked kinase activity. In cell culture and in flies, DBT(K/R) antagonized the phosphorylation and degradation of PER, and it damped the oscillation of PER in vivo. Overexpression of short-period, long-period, or wild-type DBT in flies produced the same circadian periods produced by the corresponding alleles of the endogenous gene. These mutations therefore dictate an altered "set point" for period length that is not altered by overexpression. Overexpression of the DBT(K/R) produced effects proportional to the titration of endogenous DBT, with long circadian periods at lower expression levels and arrhythmicity at higher levels. This first analysis of adult flies with a virtual lack of DBT activity demonstrates that DBT's kinase activity is necessary for normal circadian rhythms and that a general reduction of DBT kinase activity does not produce short periods.

Activation of 5'-AMP-activated kinase with diabetes drug metformin induces casein kinase Iepsilon (CKIepsilon)-dependent degradation of clock protein mPer2.

Metformin is one of the most commonly used first line drugs for type II diabetes. Metformin lowers serum glucose levels by activating 5'-AMP-activated kinase (AMPK), which maintains energy homeostasis by directly sensing the AMP/ATP ratio. AMPK plays a central role in food intake and energy metabolism through its activities in central nervous system and peripheral tissues. Since food intake and energy metabolism is synchronized to the light-dark (LD) cycle of the environment, we investigated the possibility that AMPK may affect circadian rhythm. We discovered that the circadian period of Rat-1 fibroblasts treated with metformin was shortened by 1 h. One of the regulators of the period length is casein kinase Iepsilon (CKIepsilon), which by phosphorylating and inducing the degradation of the circadian clock component, mPer2, shortens the period length. AMPK phosphorylates Ser-389 of CKIepsilon, resulting in increased CKIepsilon activity and degradation of mPer2. In peripheral tissues, injection of metformin leads to mPer2 degradation and a phase advance in the circadian expression pattern of clock genes in wild-type mice but not in AMPK alpha2 knock-out mice. We conclude that metformin and AMPK have a previously unrecognized role in regulating the circadian rhythm.

Regulation of wingless signaling by the CKI family in Drosophila limb development.

The Wingless (Wg)/Wnt signaling pathway regulates a myriad of developmental processes and its malfunction leads to human disorders including cancer. Recent studies suggest that casein kinase I (CKI) family members play pivotal roles in the Wg/Wnt pathway. However, genetic evidence for the involvement of CKI family members in physiological Wg/Wnt signaling events is lacking. In addition, there are conflicting reports regarding whether a given CKI family member functions as a positive or negative regulator of the pathway. Here we examine the roles of seven CKI family members in Wg signaling during Drosophila limb development. We find that increased CKIepsilon stimulates whereas dominant-negative or a null CKIepsilon mutation inhibits Wg signaling. In contrast, inactivation of CKIalpha by RNA interference (RNAi) leads to ectopic Wg signaling. Interestingly, hypomorphic CKIepsilon mutations synergize with CKIalpha RNAi to induce ectopic Wg signaling, revealing a negative role for CKIepsilon. Conversely, CKIalpha RNAi enhances the loss-of-Wg phenotypes caused by CKIepsilon null mutation, suggesting a positive role for CKIalpha. While none of the other five CKI isoforms can substitute for CKIalpha in its inhibitory role in the Wg pathway, several CKI isoforms including CG12147 exhibit a positive role based on overexpression. Moreover, loss of Gilgamesh (Gish)/CKIgamma attenuates Wg signaling activity. Finally, we provide evidence that several CKI isoforms including CKIalpha and Gish/CKIgamma can phosphorylate the Wg coreceptor Arrow (Arr), which may account, at least in part, for their positive roles in the Wg pathway.

Immunoreactivities to three circadian clock proteins in two ground crickets suggest interspecific diversity of the circadian clock structure.

The closely related crickets Dianemobius nigrofasciatus and Allonemobius allardi exhibit similar circadian rhythms and photoperiodic responses, suggesting that they possess similar circadian and seasonal clocks. To verify this assumption, antisera to Period (PER), Doubletime (DBT), and Cryptochrome (CRY) were used to visualize circadian clock neurons in the cephalic ganglia. Immunoreactivities referred to as PER-ir, DBT-ir, and CRY-ir were distributed mainly in the optic lobes (OL), pars intercerebralis (PI), dorsolateral protocerebrum, and the subesophageal ganglion (SOG). A system of immunoreactive cells in the OL dominates in D. nigrofasciatus, while immunoreactivities in the PI and SOG prevail in A. allardi. Each OL of D. nigrofasciatus contains 3 groups of cells that coexpress PER-ir and DBT-ir and send processes over the frontal medulla face to the inner lamina surface, suggesting functional linkage to the compound eye. Only 2 pairs of PER-ir cells (no DBT-ir) were found in the OL of A. allardi. Several groups of PER-ir cells occur in the brain of both species. The PI also contains DBT-ir and CRY-ir cells, but in A. allardi, most of the DBT-ir is confined to the SOG. Most immunoreactive cells in the PI and in the dorsolateral brain send their fibers to the contralateral corpora cardiaca and corpora allata. The proximity and, in some cases, proven identity of the PER-ir, DBT-ir, and CRY-ir perikarya are consistent with presumed interactions between the examined clock components. The antigens were always found in the cytoplasm, and no diurnal oscillations in their amounts were detected. The photoperiod, which controls embryonic diapause, the rate of larval development, and the wing length of crickets, had no discernible effect on either distribution or the intensity of the immunostaining.