CK1epsilon Research Articles

Casein Kinase 1δ/ε Inhibitor, PF670462 Attenuates the Fibrogenic Effects of Transforming Growth Factor-β in Pulmonary Fibrosis.

Transforming growth factor-beta (TGF-β) is a major mediator of fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). However, therapeutic global inhibition of TGF-β is limited by unwanted immunosuppression and mitral valve defects. We performed an extensive literature search to uncover a little-known connection between TGF-β signaling and casein kinase (CK) activity. We have examined the abundance of CK1 delta and epsilon (CK1δ/ε) in lung tissue from IPF patients and non-diseased controls, and investigated whether inhibition of CK1δ/ε with PF670462 inhibits pulmonary fibrosis. CK1δ/ε levels in lung tissue from IPF patients and non-diseased controls were assessed by immunohistochemistry. Anti-fibrotic effects of the CK1δ/ε inhibitor PF670462 were assessed in pre-clinical models, including acute and chronic bleomycin mouse models and in vitro experiments on spheroids made from primary human lung fibroblast cells from IPF and control donors, and human A549 alveolar-like adenocarcinoma-derived epithelial cells. Increased expression of CK1δ and ε in IPF lungs compared to non-diseased controls was accompanied by increased levels of the product, phospho-period 2. In vitro, PF670462 prevented TGF-β-induced epithelial-mesenchymal transition. The stiffness of IPF-derived spheroids was reduced by PF670462 and TGF-β-induced fibrogenic gene expression was inhibited. The CK1δ/ε inhibitor PF670462 administered systemically or locally by inhalation prevented both acute and chronic bleomycin-induced pulmonary fibrosis in mice. PF670462 administered in a ‘therapeutic’ regimen (day 7 onward) prevented bleomycin-induced lung collagen accumulation. Elevated expression and activity of CK1 δ and ε in IPF and anti-fibrogenic effects of the dual CK1δ/ε inhibitor, PF670462, support CK1δ/ε as novel therapeutic targets for IPF.

Abstract B264: IC261 induces cell cycle arrest and apoptosis of human cancer cells via a CK1δ/ε independent mechanism

Abstract Casein kinase 1 delta (CK1δ) and CK1 epsilon (CK1ε) regulate Wnt/β catenin signaling and their dysregulation has been implicated in a variety of human cancers. Kinome RNAi screens and as well as targeted studies have identified CK1δ and CK1ε as prosurvival factors in a variety of cancer cell lines. While RNAi knockdown of CK1δ or CK1ε displayed only modest growth inhibition of various cell lines, the CKIδ/ε selective inhibitor 3-[2,4,6-(trimethoxyphenyl) methylidenyl]-indolin-2-one (IC261)(with an IC50 in the 20–50 µM range) has proven even more effective at selective killing of cancer cells, even at submicromolar levels. This finding has been taken as supportive evidence for the role of CKIε and CKIδ in cancer. The recent development of a selective and nanomolar CKIδ/ε inhibitor, PF670462, affords an opportunity to further explore this hypothesis. We found that PF670462 effectively blocked CK1 kinase activity and suppressed Wnt/β-catenin signaling in both HEK293 cells and HT1080 fibrosarcoma cells at submicromolar concentrations. However, consistent with the results of siRNA knockdown of CK1ε, PF670462 failed to induce cancer cell death at these concentrations. In contrast, we found that while 100 nM IC261 did not inhibit of CK1ε activity nor block Wnt/ catenin signaling in vivo, it resulted in the rapid induction of G2/M arrest of cell cycle and apoptosis in human cancer, but not non-transformed, cells. Taken together, inactivation of CK1δ/ε and subsequent block of Wnt/β catenin signaling may not be sufficient to inhibit cancer cell survival. Furthermore, IC261 potently regulates additional cancer cell targets at concentrations much lower than that required for CK1 inhibition and these novel unknown targets appear to be important for the maintenance of human cancer cell survival. Identification of the IC261 targets may provide insights into the mechanism of selective cancer cell toxicity. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B264.

Upregulation of β‐Catenin Levels in Superior Frontal Cortex of Chronic Alcoholics

Chronic and excessive alcohol misuse results in neuroadaptive changes in the brain. The complex nature of behavioral, psychological, emotional, and neuropathological characteristics associated with alcoholism is likely a reflection of the network of proteins that are affected by alcohol-induced gene expression patterns in specific brain regions. At the molecular level, however, knowledge remains limited regarding alterations in protein expression levels affected by chronic alcohol abuse. Thus, novel techniques that allow a comprehensive assessment of this complexity will enable the simultaneous assessment of changes across a group of proteins in the relevant neural circuitry. A proteomics analysis was performed using antibody microarrays to determine differential protein levels in superior frontal cortices between chronic alcoholics and age- and gender-matched control subjects. Seventeen proteins related to the catenin signaling pathway were analyzed, including alpha-, beta-, and delta-catenins, their upstream activators cadherin-3 (type I cadherin) and cadherin-5 (type II cadherin), and 5 cytoplasmic regulators c-Src, CK1 epsilon, GSK-3beta, PP2A-C alpha, and APC, as well as the nuclear complex partner of beta-catenin CBP and 2 downstream genes Myc and cyclin D1. ILK, G(alpha1), G(beta1), and G(beta2), which are activity regulators of GSK-3beta, were also analyzed. Both alpha- and beta-catenin showed significantly increased levels, while delta-catenin did not change significantly, in chronic alcoholics. In addition, the level of the beta-catenin downstream gene product Myc was significantly increased. Average levels of the catenin regulators c-Src, CK1 epsilon, and APC were also increased in chronic alcoholics, but the changes were not statistically significant. Chronic and excessive alcohol consumption leads to an upregulation of alpha- and beta-catenin levels, which in turn increase downstream gene expressions such as Myc that is controlled by beta-catenin signaling. This study showed that the beta-catenin signal transduction pathway was upregulated by chronic alcohol abuse, and prompts further investigation of mechanisms underlying the upregulation of alpha- and beta-catenins in alcoholism, which may have considerable pathogenic and therapeutic relevance.

Setting Clock Speed in Mammals: The CK1ɛtau Mutation in Mice Accelerates Circadian Pacemakers by Selectively Destabilizing PERIOD Proteins

The intrinsic period of circadian clocks is their defining adaptive property. To identify the biochemical mechanisms whereby casein kinase1 (CK1) determines circadian period in mammals, we created mouse null and tau mutants of Ck1 epsilon. Circadian period lengthened in CK1epsilon-/-, whereas CK1epsilon(tau/tau) shortened circadian period of behavior in vivo and suprachiasmatic nucleus firing rates in vitro, by accelerating PERIOD-dependent molecular feedback loops. CK1epsilon(tau/tau) also accelerated molecular oscillations in peripheral tissues, revealing its global role in circadian pacemaking. CK1epsilon(tau) acted by promoting degradation of both nuclear and cytoplasmic PERIOD, but not CRYPTOCHROME, proteins. Together, these whole-animal and biochemical studies explain how tau, as a gain-of-function mutation, acts at a specific circadian phase to promote degradation of PERIOD proteins and thereby accelerate the mammalian clockwork in brain and periphery.

Anti-apoptotic and growth-stimulatory functions of CK1 delta and epsilon in ductal adenocarcinoma of the pancreas are inhibited by IC261 in vitro and in vivo

Background: Pancreatic ductal adenocarcinomas (PDACs) are highly resistant to treatment due to changes in various signalling pathways. CK1 isoforms play important regulatory roles in these pathways. Aims: We analysed the...

Identification of mPer1 Phosphorylation Sites Responsible for the Nuclear Entry

Casein kinase 1 epsilon (CK1 epsilon) is an essential component of the circadian clock in mammals and Drosophila. The phosphorylation of Period (Per) proteins by CK1 epsilon is believed to be implicated in their subcellular localization and degradation, but the precise mechanism by which CK1 epsilon affects Per proteins has not been determined. In this study, three putative CK1 epsilon phosphorylation motif clusters in mouse Per1 (mPer1) were identified, and the phosphorylation status of serine and threonine residues in these clusters was examined. Phosphorylation of residues within a region defined by amino acids 653-663 and in particular of Ser-661 and Ser-663, was identified as responsible for the nuclear translocation of mPer1. Furthermore, phosphorylation of these residues may influence the nuclear translocation of a clock protein complex containing mPer1. These findings indicate that mPer1 phosphorylation is a critical aspect of the circadian clock mechanism.

Temporal expression of seven clock genes in the suprachiasmatic nucleus and the pars tuberalis of the sheep: evidence for an internal coincidence timer.

The 24-h expression of seven clock genes (Bmal1, Clock, Per1, Per2, Cry1, Cry2, and CK1 epsilon ) was assayed by in situ hybridization in the suprachiasmatic nucleus (SCN) and the pars tuberalis (PT) of the pituitary gland, collected every 4 h throughout 24 h, from female Soay sheep kept under long (16-h light/8-h dark) or short (8-h light/16-h dark) photoperiods. Locomotor activity was diurnal, inversely related to melatonin secretion, and prolactin levels were increased under long days. All clock genes were expressed in the ovine SCN and PT. In the SCN, there was a 24-h rhythm in Clock expression, in parallel with Bmal1, in antiphase with cycles in Per1 and Per2; there was low-amplitude oscillation of Cry1 and Cry2. The waveform of only Per1 and Per2 expression was affected by photoperiod, with extended elevated expression under long days. In the PT, the high-amplitude 24-h cycles in the expression of Bmal1, Clock, Per1, Per2, Cry1, and Cry2, but not CK1 epsilon, were influenced by photoperiod. Per1 and Per2 peaked during the day, whereas Cry1 and Cry2 peaked early in the night. Hence, photoperiod via melatonin had a marked effect on the phase relationship between Per/Cry genes in the PT. This supports the conclusion that an "external coincidence model" best explains the way photoperiod affects the waveform of clock gene expression in the SCN, the central pacemaker, whereas an "internal coincidence model" best explains the way melatonin affects the phasing of clock gene expression in the PT to mediate the photoperiodic control of a summer or winter physiology.