Essential serine/threonine-protein kinase that regulates diverse cellular growth and survival processes including Wnt signaling, DNA repair and circadian rhythms. It can phosphorylate a large number of proteins. Casein kinases are operationally defined by their preferential utilization of acidic proteins such as caseins as substrates. Phosphorylates connexin-43/GJA1, MAP1A, SNAPIN, MAPT/TAU, TOP2A, DCK, HIF1A, EIF6, p53/TP53, DVL2, DVL3, ESR1, AIB1/NCOA3, DNMT1, PKD2, YAP1, PER1 and PER2. Central component of the circadian clock. May act as a negative regulator of circadian rhythmicity by phosphorylating PER1 and PER2, leading to retain PER1 in the cytoplasm. YAP1 phosphorylation promotes its SCF(beta-TRCP) E3 ubiquitin ligase-mediated ubiquitination and subsequent degradation. DNMT1 phosphorylation reduces its DNA-binding activity. Phosphorylation of ESR1 and AIB1/NCOA3 stimulates their activity and coactivation. Phosphorylation of DVL2 and DVL3 regulates WNT3A signaling pathway that controls neurite outgrowth. EIF6 phosphorylation promotes its nuclear export. Triggers down-regulation of dopamine receptors in the forebrain. Activates DCK in vitro by phosphorylation. TOP2A phosphorylation favors DNA cleavable complex formation. May regulate the formation of the mitotic spindle apparatus in extravillous trophoblast. Modulates connexin-43/GJA1 gap junction assembly by phosphorylation. Probably involved in lymphocyte physiology. Regulates fast synaptic transmission mediated by glutamate.
Casein kinase I epsilon/delta phosphorylates certain clock-related proteins as part of a complex arrangement of transcriptional/translational feedback loops that comprise the circadian oscillator in mammals. Pharmacologic inhibition leads to a delay of the oscillations with the magnitude of this effect dependent upon the timing of drug administration.
Phosphorylation of members of the connexin family of gap junction proteins has been correlated with gap junction assembly, but the mechanisms involved remain unclear. We have examined the role of casein kinase 1 (CK1) in connexin-43 (Cx43) gap junction assembly. Cellular co-immunoprecipitation experiments and in vitro CK1 phosphorylation reactions indicate that CK1 interacted with and phosphorylated Cx43, initially on serine(s) 325, 328, or 330. (32)P(i)-Metabolically labeled cells treated with CKI-7, a specific CK1 inhibitor, showed a reduction in Cx43 phosphorylation on site(s) that can be phosphorylated by CK1 in vitro. To examine CK1 function, normal rat kidney cells were treated with CKI-7, and Cx43 content was analyzed by Triton X-100 extraction, cell-surface biotinylation, and immunofluorescence. Western blot analysis indicated a slight increase in total Cx43, whereas gap junctional (Triton-insoluble) Cx43 decreased, and non-junctional plasma membrane Cx43 increased (as detected by cell surface biotinylation). Immunofluorescence experiments in the presence of CK1 inhibitor showed increases in Cx43 plasma membrane localization but not necessarily accumulation at cell-cell interfaces. Decreased gap junctional and phosphorylated Cx43 was also detected when cells were treated with IC261, a CK1 inhibitor specific for delta or epsilon isoforms. These data suggest CK1delta could regulate Cx43 gap junction assembly by directly phosphorylating Cx43.
Tau hyperphosphorylation precedes neuritic lesion formation in Alzheimer's disease, suggesting it participates in the tau fibrillization reaction pathway. Candidate tau protein kinases include members of the casein kinase 1 (CK1) family of phosphotransferases, which are highly overexpressed in Alzheimer's disease brain and colocalize with neuritic and granulovacuolar lesions. Here we characterized the contribution of one CK1 isoform, Ckidelta, to the phosphorylation of tau at residues Ser202/Thr205 and Ser396/Ser404 in human embryonic kidney 293 cells using immunodetection and fluorescence microscopy. Treatment of cells with membrane permeable CK1 inhibitor 3-[(2,3,6-trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261) lowered occupancy of Ser396/Ser404 phosphorylation sites by >70% at saturation, suggesting that endogenous CK1 was the major source of basal phosphorylation activity at these sites. Overexpression of Ckidelta increased CK1 enzyme activity and further raised tau phosphorylation at residues Ser202/Thr205 and Ser396/Ser404 in situ. Inhibitor IC261 reversed tau hyperphosphorylation induced by Ckidelta overexpression. Co-immunoprecipitation assays showed direct association of tau and Ckidelta in situ, consistent with tau being a Ckidelta substrate. Ckidelta overexpression also produced a decrease in the fraction of bulk tau bound to detergent-insoluble microtubules. These results suggest that Ckidelta phosphorylates tau at sites that modulate tau/microtubule binding, and that the expression pattern of Ckidelta in Alzheimer's disease is consistent with it playing an important role in tau aggregation.
p53 is a potent transcription factor which is regulated by sequential multisite phosphorylation and acetylation. In this paper, we identify threonine 18 of p53, a key site in regulating the interaction between p53 and its regulatory partner MDM2, as a novel site phosphorylated in vitro by purified recombinant casein kinase 1 (CK1) delta. Strikingly, phosphorylation of threonine 18 is dependent upon prior phosphorylation of serine 15. These data highlight an additional and physiologically important target residue for CK1 in p53 and suggest a potential mechanism by which sequential modification of a pivotal N-terminal residue in p53 may occur following stress-activated modification of serine 15.
Protein kinase D2 (PKD2), a member of the PKD family of serine/threonine kinases, is localized in various subcellular compartments including the nucleus where the kinase accumulates upon activation of G-protein-coupled receptors. We define three critical post-translational modifications required for nuclear accumulation of PKD2 in response to activation of the CCK2 receptor (CCK2R): phosphorylation at Ser706 and Ser710 within the activation loop by PKC eta leading to catalytic activity and phosphorylation at Ser244 within the zinc-finger domain, which is crucial for blocking nuclear export of active PKD2 by preventing its interaction with the Crm-1 export machinery. We identify CK1delta and epsilon as upstream activated kinases by CCK2R that phosphorylate PKD2 at Ser244. Moreover, nuclear accumulation of active PKD2 is a prerequisite for efficient phosphorylation of its nuclear substrate, HDAC7. Only nuclear, active PKD2 mediates CCK2R-induced HDAC7 phosphorylation and Nur77 expression. Thus, we define a novel, compartment-specific signal transduction pathway downstream of CCK2R that phosphorylates PKD2 at three specific sites, results in nuclear accumulation of the active kinase and culminates in efficient phosphorylation of nuclear PKD2 substrates in human gastric cancer cells.
We previously reported that phosphorylation of topoisomerase (topo) IIalpha at serine-1106 (Ser-1106) regulates enzyme activity and sensitivity to topo II-targeted drugs. In this study we demonstrate that phosphorylation of Ser-1106, which is flanked by acidic amino acids, is regulated in vivo by casein kinase (CK) Idelta and/or CKIepsilon, but not by CKII. The CKI inhibitors, CKI-7 and IC261, reduced Ser-1106 phosphorylation and decreased formation of etoposide-stabilized topo II-DNA cleavable complex. In contrast, the CKII inhibitor, 5,6-dichlorobenzimidazole riboside, did not affect etoposide-stabilized topo II-DNA cleavable complex formation. Since, IC261 specifically targets the Ca(2+)-regulated isozymes, CKIdelta and CKIepsilon, we examined the effect of down-regulating these enzymes on Ser-1106 phosphorylation. Down-regulation of these isozymes with targeted si-RNAs led to hypophosphorylation of the Ser-1106 containing peptide. However, si-RNA-mediated down-regulation of CKIIalpha and alpha' did not alter Ser-1106 phosphorylation. Furthermore, reduced phosphorylation of Ser-1106, observed in HRR25 (CKIdelta/epsilon homologous gene)-deleted Saccharomyces cerevisiae cells transformed with human topo IIalpha, was enhanced following expression of human CKIepsilon. Down-regulation of CKIdelta and CKIepsilon also led to reduced formation of etoposide stabilized topo II-DNA cleavable complex. These results provide strong support for an essential role of CKIdelta/epsilon in phosphorylating Ser-1106 in human topo IIalpha and in regulating enzyme function.
Tau in Alzheimer disease brain is highly phosphorylated and aggregated into paired helical filaments comprising characteristic neurofibrillary tangles. Here we have analyzed insoluble Tau (PHF-tau) extracted from Alzheimer brain by mass spectrometry and identified 11 novel phosphorylation sites, 10 of which were assigned unambiguously to specific amino acid residues. This brings the number of directly identified sites in PHF-tau to 39, with an additional six sites indicated by reactivity with phosphospecific antibodies to Tau. We also identified five new phosphorylation sites in soluble Tau from control adult human brain, bringing the total number of reported sites to nine. To assess which kinases might be responsible for Tau phosphorylation, we used mass spectrometry to determine which sites were phosphorylated in vitro by several kinases. Casein kinase 1delta and glycogen synthase kinase-3beta were each found to phosphorylate numerous sites, and each kinase phosphorylated at least 15 sites that are also phosphorylated in PHF-tau from Alzheimer brain. A combination of casein kinase 1delta and glycogen synthase kinase-3beta activities could account for over three-quarters of the serine/threonine phosphorylation sites identified in PHF-tau, indicating that casein kinase 1delta may have a role, together with glycogen synthase kinase-3beta, in the pathogenesis of Alzheimer disease.
Previously we determined that Dishevelled-2/3 (Dvl) mediate Wnt-3a-dependent neurite outgrowth in Ewing sarcoma family tumor cells. Here we report that neurite extension was associated with Dvl phosphorylation and that both were inhibited by the casein kinase 1 (CK1) δ/ε inhibitor IC261. Small interfering RNAs targeting either CK1δ or CK1ε decreased Dvl phosphorylation, but only knockdown of CK1δ blocked neurite outgrowth. CK1δ but not CK1ε was detected at the centrosome, an organelle associated with neurite formation. Deletion analysis mapped the centrosomal localization signal (CLS) of CK1δ to its C-terminal domain. A fusion protein containing the CLS and EGFP displaced full-length CK1δ from the centrosome and inhibited Wnt-3a-dependent neurite outgrowth. In contrast to wild-type CK1ε, a chimera comprised of the kinase domain of CK1ε and the CLS of CK1δ localized to the centrosome and rescued Wnt-3a-dependent neurite outgrowth suppressed by CK1δ knockdown. These results provide strong evidence that the centrosomal localization of CK1δ is required for Wnt-3a-dependent neuritogenesis.
Circadian pacemaking requires the orderly synthesis, posttranslational modification, and degradation of clock proteins. In mammals, mutations in casein kinase 1 (CK1) epsilon or delta can alter the circadian period, but the particular functions of the WT isoforms within the pacemaker remain unclear. We selectively targeted WT CK1epsilon and CK1delta using pharmacological inhibitors (PF-4800567 and PF-670462, respectively) alongside genetic knockout and knockdown to reveal that CK1 activity is essential to molecular pacemaking. Moreover, CK1delta is the principal regulator of the clock period: pharmacological inhibition of CK1delta, but not CK1epsilon, significantly lengthened circadian rhythms in locomotor activity in vivo and molecular oscillations in the suprachiasmatic nucleus (SCN) and peripheral tissue slices in vitro. Period lengthening mediated by CK1delta inhibition was accompanied by nuclear retention of PER2 protein both in vitro and in vivo. Furthermore, phase mapping of the molecular clockwork in vitro showed that PF-670462 treatment lengthened the period in a phase-specific manner, selectively extending the duration of PER2-mediated transcriptional feedback. These findings suggested that CK1delta inhibition might be effective in increasing the amplitude and synchronization of disrupted circadian oscillators. This was tested using arrhythmic SCN slices derived from Vipr2(-/-) mice, in which PF-670462 treatment transiently restored robust circadian rhythms of PER2::Luc bioluminescence. Moreover, in mice rendered behaviorally arrhythmic by the Vipr2(-/-) mutation or by constant light, daily treatment with PF-670462 elicited robust 24-h activity cycles that persisted throughout treatment. Accordingly, selective pharmacological targeting of the endogenous circadian regulator CK1delta offers an avenue for therapeutic modulation of perturbed circadian behavior.
The ability of three isoforms of protein kinase CK1 (alpha, gamma(1), and delta) to phosphorylate the N-terminal region of p53 has been assessed using either recombinant p53 or a synthetic peptide reproducing its 1-28 sequence. Both substrates are readily phosphoylated by CK1delta and CK1alpha, but not by the gamma isoform. Affinity of full size p53 for CK1 is 3 orders of magnitude higher than that of its N-terminal peptide (K (m) 0.82 muM vs 1.51 mM). The preferred target is S20, whose phosphorylation critically relies on E17, while S6 is unaffected despite displaying the same consensus (E-x-x-S). Our data support the concept that non-primed phosphorylation of p53 by CK1 is an isoform-specific reaction preferentially affecting S20 by a mechanism which is grounded both on a local consensus and on a remote docking site mapped to the K(221)RQK(224) loop according to modeling and mutational analysis.
The Yes-associated protein (YAP) transcription coactivator is a key regulator of organ size and a candidate human oncogene. YAP is inhibited by the Hippo pathway kinase cascade, at least in part via phosphorylation of Ser 127, which results in YAP 14-3-3 binding and cytoplasmic retention. Here we report that YAP is phosphorylated by Lats on all of the five consensus HXRXXS motifs. Phosphorylation of Ser 381 in one of them primes YAP for subsequent phosphorylation by CK1delta/epsilon in a phosphodegron. The phosphorylated phosphodegron then recruits the SCF(beta-TRCP) E3 ubiquitin ligase, which catalyzes YAP ubiquitination, ultimately leading to YAP degradation. The phosphodegron-mediated degradation and the Ser 127 phosphorylation-dependent translocation coordinately suppress YAP oncogenic activity. Our study identified CK1delta/epsilon as new regulators of YAP and uncovered an intricate mechanism of YAP regulation by the Hippo pathway via both S127 phosphorylation-mediated spatial regulation (nuclear-cytoplasmic shuttling) and the phosphodegron-mediated temporal regulation (degradation).
Eukaryotic initiation factor 6 (eIF6), a highly conserved protein from yeast to mammals, is essential for 60 S ribosome biogenesis and assembly. Both yeast and mammalian eIF6 are phosphorylated at Ser-174 and Ser-175 by the nuclear isoform of casein kinase 1 (CK1). The molecular basis of eIF6 phosphorylation, however, remains elusive. In the present work, we show that subcellular distribution of eIF6 in the nuclei and the cytoplasm of mammalian cells is mediated by dephosphorylation and phosphorylation, respectively. This nucleo-cytoplasmic shuttling is dependent on the phosphorylation status at Ser-174 and Ser-175 of eIF6. We demonstrate that Ca(2+)-activated calcineurin phosphatase binds to and promotes nuclear localization of eIF6. Increase in intracellular concentration of Ca(2+) leads to rapid translocation of eIF6 from the cytoplasm to the nucleus, an event that is blocked by specific calcineurin inhibitors cyclosporin A or FK520. Nuclear export of eIF6 is regulated by phosphorylation at Ser-174 and Ser-175 by the nuclear isoform of CK1. Mutation of eIF6 at the phosphorylatable Ser-174 and Ser-175 to alanine or treatment of cells with the CK1 inhibitor, D4476 inhibits nuclear export of eIF6 and results in nuclear accumulation of eIF6. Together, these results establish eIF6 as a substrate for calcineurin and suggest a novel paradigm for calcineurin function in 60 S ribosome biogenesis via regulating the nuclear accumulation of eIF6.
Hypoxia-inducible factor 1 (HIF-1), a transcriptional activator that mediates cellular response to hypoxia and a promising target of anticancer therapy, is essential for adaptation to low oxygen conditions, embryogenesis and tumor progression. HIF-1 is a heterodimer of HIF-1alpha, expression of which is controlled by oxygen levels as well as by various oxygen-independent mechanisms, and HIF-1beta (or ARNT), which is constitutively expressed. In this work, we investigate the phosphorylation of the N-terminal heterodimerization (PAS) domain of HIF-1alpha and identify Ser247 as a major site of in vitro modification by casein kinase 1delta (CK1delta). Mutation of this site to alanine, surprisingly, enhanced the transcriptional activity of HIF-1alpha, a result phenocopied by inhibition or small interfering RNA (siRNA)-mediated silencing of CK1delta under hypoxic conditions. Conversely, overexpression of CK1delta or phosphomimetic mutation of Ser247 to aspartate inhibited HIF-1alpha activity without affecting its stability or nuclear accumulation. Immunoprecipitation and in vitro binding experiments suggest that CK1-dependent phosphorylation of HIF-1alpha at Ser247 impairs its association with ARNT, a notion also supported by modeling the structure of the complex between HIF-1alpha and ARNT PAS-B domains. We suggest that modification of HIF-1alpha by CK1 represents a novel mechanism that controls the activity of HIF-1 during hypoxia by regulating the interaction between its two subunits.
Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxynucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We recently showed that dCK was activated in vivo by phosphorylation of Ser-74. However, the protein kinase responsible was not identified. Ser-74 is located downstream a Glu-rich region, presenting similarity with the consensus phosphorylation motif of casein kinase 1 (CKI), and particularly of CKI delta. We showed that recombinant CKI delta phosphorylated several residues of bacterially overexpressed dCK: Ser-74, but also Ser-11, Ser-15, and Thr-72. Phosphorylation of dCK by CKI delta correlated with increased activity reaching at least 4-fold. Site-directed mutagenesis demonstrated that only Ser-74 phosphorylation was involved in dCK activation by CKI delta, strengthening the key role of this residue in the control of dCK activity. However, neither CKI delta inhibitors nor CKI delta siRNA-mediated knock-down modified Ser-74 phosphorylation or dCK activity in cultured cells. Moreover, these approaches did not prevent dCK activation induced by treatments enhancing Ser-74 phosphorylation. Taken together, the data preclude a role of CKI delta in the regulation of dCK activity in vivo. Nevertheless, phosphorylation of dCK by CKI delta could be a useful tool for elucidating the influence of Ser-74 phosphorylation on the structure-activity relationships in the enzyme.
The serine/threonine-specific casein kinase I delta (CKIdelta) is ubiquitously expressed in all tissues, is p53 dependently induced in stress situations and plays an important role in various cellular processes. Our immunohistochemical analysis of the human placenta revealed strongest expression of CKIdelta in extravillous trophoblast cells and in choriocarcinomas. Investigation of the functional role of CKIdelta in an extravillous trophoblast hybrid cell line revealed that CKIdelta was constitutively localized at the centrosomes and the mitotic spindle. Inhibition of CKIdelta with the CKI-specific inhibitor IC261 led to structural alterations of the centrosomes, the formation of multipolar spindles, the inhibition of mitosis and, in contrast to other cell lines, the induction of apoptosis. Our findings indicate that CKIdelta plays an important role in the mitotic progression and in the survival of cells of trophoblast origin. Therefore, IC261 could provide a new tool in treating choriocarcinomas.
Oncogenesis in breast cancer often requires the overexpression of the nuclear receptor coactivator AIB1/SRC-3 acting in conjunction with estrogen receptor-alpha (ERalpha). Phosphorylation of both ERalpha and AIB1 has been shown to have profound effects on their functions. In addition, proteasome-mediated degradation plays a major role by regulating their stability and activity. CK1delta, a member of the ubiquitous casein kinase-1 family, is implicated in the progression of breast cancer. In this study, we show that both ERalpha and AIB1 are substrates for CK1delta in vitro, and identify a novel AIB1 phosphorylation site (S601) targeted by CK1delta, significant for the co-activator function of AIB1. CK1delta is able to interact with ERalpha and AIB1 in vivo, while overexpression of CK1delta in breast cancer cells results in an increased association of ERalpha with AIB1 as confirmed by co-immunoprecipitation assays from cell lysates. Using an siRNA-based approach, luciferase reporter assays and qRT-PCR, we observe that silencing of CK1delta leads to reduced ERalpha transcriptional activity, despite increased ERalpha levels, similarly to proteasome inhibition. We provide evidence that AIB1 protein levels are reduced by CK1delta silencing, in an estradiol-dependent manner; such destabilization can be inhibited by pre-treatment with the proteasome inhibitor MG132. We propose that differing activities adopted by ERalpha and AIB1 as a consequence of their interactions with and phosphorylation by CK1delta, particularly AIB1 stabilization, influence the transcriptional activity of ERalpha, and therefore have a role in breast cancer development.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionIntAct
Mdm2 is the major negative regulator of the p53 pathway. Here, we report that Mdm2 is rapidly degraded after DNA damage and that phosphorylation of Mdm2 by casein kinase I (CKI) at multiple sites triggers its interaction with, and subsequent ubiquitination and destruction, by SCF(beta-TRCP). Inactivation of either beta-TRCP or CKI results in accumulation of Mdm2 and decreased p53 activity, and resistance to apoptosis induced by DNA damaging agents. Moreover, SCF(beta-TRCP)-dependent Mdm2 turnover also contributes to the control of repeated p53 pulses in response to persistent DNA damage. Our results provide insight into the signaling pathways controlling Mdm2 destruction and further suggest that compromised regulation of Mdm2 results in attenuated p53 activity, thereby facilitating tumor progression.
Evidence
2:
Inferred from Physical InteractionUniProtKB
Casein kinase-1 is a family of ubiquitous eukaryotic protein kinases that frequently function in tandem with the ubiquitin modification system to modulate protein turnover and trafficking. In Alzheimer's disease, these enzymes colocalize with ubiquitinated lesions, including neurofibrillary tangles and granulovacuolar degeneration bodies, suggesting they also play a role in disease pathogenesis. To identify binding partners that potentially regulate or recruit these enzymes toward disease lesions, a Sos-recruitment yeast two-hybrid screen was performed with human Ckidelta (the casein kinase-1 isoform most closely linked to granulovacuolar degeneration bodies) and a human brain cDNA library. All interacting clones contained a single open reading frame termed casein kinase-1 binding protein (CK1BP). On the basis of sequence alignments, CK1BP was a structural homologue of the acidic domain of dysbindin, a component of the dystrophin-associated protein complex and the biogenesis of lysosome-related organelles complex-1. CK1BP interacted with full-length Ckidelta, the isolated Ckidelta catalytic domain, Ckigamma2, -gamma3, and -epsilon in the yeast two-hybrid system, and bound Ckidelta and -epsilon in pulldown assays but did not interact with Ckialpha. Interaction with the Ckidelta catalytic domain led to concentration-dependent inhibition of protein kinase activity in the presence of protein substrates tau and alpha-synuclein. Although intact dysbindin did not bind any CK1 isoform, deletion of its coiled-coil domain yielded a protein fragment that behaved much like CK1BP in two-hybrid screens. These data suggest that the acidic domain of dysbindin and its paralogs in humans may function to recruit casein kinase-1 isoforms to protein complexes involved in multiple biological functions.
Casein kinase-1 is a family of ubiquitous eukaryotic protein kinases that frequently function in tandem with the ubiquitin modification system to modulate protein turnover and trafficking. In Alzheimer's disease, these enzymes colocalize with ubiquitinated lesions, including neurofibrillary tangles and granulovacuolar degeneration bodies, suggesting they also play a role in disease pathogenesis. To identify binding partners that potentially regulate or recruit these enzymes toward disease lesions, a Sos-recruitment yeast two-hybrid screen was performed with human Ckidelta (the casein kinase-1 isoform most closely linked to granulovacuolar degeneration bodies) and a human brain cDNA library. All interacting clones contained a single open reading frame termed casein kinase-1 binding protein (CK1BP). On the basis of sequence alignments, CK1BP was a structural homologue of the acidic domain of dysbindin, a component of the dystrophin-associated protein complex and the biogenesis of lysosome-related organelles complex-1. CK1BP interacted with full-length Ckidelta, the isolated Ckidelta catalytic domain, Ckigamma2, -gamma3, and -epsilon in the yeast two-hybrid system, and bound Ckidelta and -epsilon in pulldown assays but did not interact with Ckialpha. Interaction with the Ckidelta catalytic domain led to concentration-dependent inhibition of protein kinase activity in the presence of protein substrates tau and alpha-synuclein. Although intact dysbindin did not bind any CK1 isoform, deletion of its coiled-coil domain yielded a protein fragment that behaved much like CK1BP in two-hybrid screens. These data suggest that the acidic domain of dysbindin and its paralogs in humans may function to recruit casein kinase-1 isoforms to protein complexes involved in multiple biological functions.
A cDNA clone coding for human casein kinase I (CK1) has been isolated and sequenced. The insert of 1911 bp contained an open reading frame of 415 amino acids. The entire amino acid sequence of human CK1 was 97% homologous to that of rat CK1 delta, and their sequences in the kinase domain (284 amino acid residues) were completely identical, predicting that the obtained cDNA is for a human homolog of the CK1 delta isoform (CSNKID). The considerable similarity in the amino acid sequence of the kinase domain of human CK1 delta to the Saccharomyces cerevisiae CK1, HRR25 (66%), and to the Saccharomyces pombe CK1, HHP1 (78%), which are involved in the repair of DNA strand break, supports the speculation that human CK1 delta might also act in DNA metabolism through excision and recombinational repair. The human CK1 delta gene was mapped to chromosome 17q25.2-q25.3 by fluorescence in situ hybridization and polymerase chain reaction analysis of the human/rodent hybrid cell panels.
Any process that modulates the frequency, rate or extent of gene expression such that an expression pattern recurs with a regularity of approximately 24 hours.
The process of restoring DNA after damage. Genomes are subject to damage by chemical and physical agents in the environment (e.g. UV and ionizing radiations, chemical mutagens, fungal and bacterial toxins, etc.) and by free radicals or alkylating agents endogenously generated in metabolism. DNA is also damaged because of errors during its replication. A variety of different DNA repair pathways have been reported that include direct reversal, base excision repair, nucleotide excision repair, photoreactivation, bypass, double-strand break repair pathway, and mismatch repair pathway.
A cDNA clone coding for human casein kinase I (CK1) has been isolated and sequenced. The insert of 1911 bp contained an open reading frame of 415 amino acids. The entire amino acid sequence of human CK1 was 97% homologous to that of rat CK1 delta, and their sequences in the kinase domain (284 amino acid residues) were completely identical, predicting that the obtained cDNA is for a human homolog of the CK1 delta isoform (CSNKID). The considerable similarity in the amino acid sequence of the kinase domain of human CK1 delta to the Saccharomyces cerevisiae CK1, HRR25 (66%), and to the Saccharomyces pombe CK1, HHP1 (78%), which are involved in the repair of DNA strand break, supports the speculation that human CK1 delta might also act in DNA metabolism through excision and recombinational repair. The human CK1 delta gene was mapped to chromosome 17q25.2-q25.3 by fluorescence in situ hybridization and polymerase chain reaction analysis of the human/rodent hybrid cell panels.
Any process that increases the rate, frequency, or extent of the Wnt receptor signaling pathway through beta-catenin, the series of molecular signals initiated by binding of a Wnt protein to a frizzled family receptor on the surface of the target cell, followed by propagation of the signal via beta-catenin, and ending with a change in transcription of target genes.
Several developmental pathways contribute to processes that regulate tissue growth and organ size. The Hippo pathway has emerged as one such critical regulator. However, how Hippo signaling is integrated with other pathways to coordinate these processes remains unclear. Here, we show that the Hippo pathway restricts Wnt/beta-Catenin signaling by promoting an interaction between TAZ and DVL in the cytoplasm. TAZ inhibits the CK1delta/epsilon-mediated phosphorylation of DVL, thereby inhibiting Wnt/beta-Catenin signaling. Abrogation of TAZ levels or Hippo signaling enhances Wnt3A-stimulated DVL phosphorylation, nuclear beta-Catenin, and Wnt target gene expression. Mice lacking Taz develop polycystic kidneys with enhanced cytoplasmic and nuclear beta-Catenin. Moreover, in Drosophila, Hippo signaling modulates Wg target gene expression. These results uncover a cytoplasmic function of TAZ in regulating Wnt signaling and highlight the role of the Hippo pathway in coordinating morphogenetic signaling with growth control.
Positive regulation of proteasomal ubiquitin-dependent protein catabolic processdefinition[GO:0032436]
Any process that activates or increases the frequency, rate or extent of the breakdown of a protein or peptide by hydrolysis of its peptide bonds, initiated by the covalent attachment of ubiquitin, and mediated by the proteasome.
Several developmental pathways contribute to processes that regulate tissue growth and organ size. The Hippo pathway has emerged as one such critical regulator. However, how Hippo signaling is integrated with other pathways to coordinate these processes remains unclear. Here, we show that the Hippo pathway restricts Wnt/beta-Catenin signaling by promoting an interaction between TAZ and DVL in the cytoplasm. TAZ inhibits the CK1delta/epsilon-mediated phosphorylation of DVL, thereby inhibiting Wnt/beta-Catenin signaling. Abrogation of TAZ levels or Hippo signaling enhances Wnt3A-stimulated DVL phosphorylation, nuclear beta-Catenin, and Wnt target gene expression. Mice lacking Taz develop polycystic kidneys with enhanced cytoplasmic and nuclear beta-Catenin. Moreover, in Drosophila, Hippo signaling modulates Wg target gene expression. These results uncover a cytoplasmic function of TAZ in regulating Wnt signaling and highlight the role of the Hippo pathway in coordinating morphogenetic signaling with growth control.
Casein kinase-1 is a family of ubiquitous eukaryotic protein kinases that frequently function in tandem with the ubiquitin modification system to modulate protein turnover and trafficking. In Alzheimer's disease, these enzymes colocalize with ubiquitinated lesions, including neurofibrillary tangles and granulovacuolar degeneration bodies, suggesting they also play a role in disease pathogenesis. To identify binding partners that potentially regulate or recruit these enzymes toward disease lesions, a Sos-recruitment yeast two-hybrid screen was performed with human Ckidelta (the casein kinase-1 isoform most closely linked to granulovacuolar degeneration bodies) and a human brain cDNA library. All interacting clones contained a single open reading frame termed casein kinase-1 binding protein (CK1BP). On the basis of sequence alignments, CK1BP was a structural homologue of the acidic domain of dysbindin, a component of the dystrophin-associated protein complex and the biogenesis of lysosome-related organelles complex-1. CK1BP interacted with full-length Ckidelta, the isolated Ckidelta catalytic domain, Ckigamma2, -gamma3, and -epsilon in the yeast two-hybrid system, and bound Ckidelta and -epsilon in pulldown assays but did not interact with Ckialpha. Interaction with the Ckidelta catalytic domain led to concentration-dependent inhibition of protein kinase activity in the presence of protein substrates tau and alpha-synuclein. Although intact dysbindin did not bind any CK1 isoform, deletion of its coiled-coil domain yielded a protein fragment that behaved much like CK1BP in two-hybrid screens. These data suggest that the acidic domain of dysbindin and its paralogs in humans may function to recruit casein kinase-1 isoforms to protein complexes involved in multiple biological functions.
Any process that modulates the frequency, rate or extent of a circadian rhythm. A circadian rhythm is a biological process in an organism that recurs with a regularity of approximately 24 hours.
The cellular process in which a signal is conveyed to trigger a change in the activity or state of a cell. Signal transduction begins with reception of a signal (e.g. a ligand binding to a receptor or receptor activation by a stimulus such as light), or for signal transduction in the absence of ligand, signal-withdrawal or the activity of a constitutively active receptor. Signal transduction ends with regulation of a downstream cellular process, e.g. regulation of transcription or regulation of a metabolic process. Signal transduction covers signaling from receptors located on the surface of the cell and signaling via molecules located within the cell. For signaling between cells, signal transduction is restricted to events at and within the receiving cell.
A cDNA clone coding for human casein kinase I (CK1) has been isolated and sequenced. The insert of 1911 bp contained an open reading frame of 415 amino acids. The entire amino acid sequence of human CK1 was 97% homologous to that of rat CK1 delta, and their sequences in the kinase domain (284 amino acid residues) were completely identical, predicting that the obtained cDNA is for a human homolog of the CK1 delta isoform (CSNKID). The considerable similarity in the amino acid sequence of the kinase domain of human CK1 delta to the Saccharomyces cerevisiae CK1, HRR25 (66%), and to the Saccharomyces pombe CK1, HHP1 (78%), which are involved in the repair of DNA strand break, supports the speculation that human CK1 delta might also act in DNA metabolism through excision and recombinational repair. The human CK1 delta gene was mapped to chromosome 17q25.2-q25.3 by fluorescence in situ hybridization and polymerase chain reaction analysis of the human/rodent hybrid cell panels.
The aggregation, arrangement and bonding together of a set of components to form the spindle, the array of microtubules and associated molecules that serves to move duplicated chromosomes apart.
Eur. J. Cell Biol. 79, 240-251 (2000)[PubMed:10826492]
Members of the casein kinase 1 family of serine/threonine kinases are highly conserved from yeast to mammals and seem to play an important role in vesicular trafficking, DNA repair, cell cycle progression and cytokinesis. We here report that in interphase cells of various mammalian species casein kinase 1 delta (CK1delta) specifically interacts with the trans Golgi network and cytoplasmic, granular particles that associate with microtubules. Furthermore, at mitosis CK1delta is recruited to the spindle apparatus and the centrosomes in cells, which have been exposed to DNA-damaging agents like etoposide or gammairradiation. In addition, determination of the affinity of CK1delta to different tubulin isoforms in immunoprecipitation-Western analysis revealed a dramatically enhanced complex formation between CK1delta and tubulins from mitotic extracts after introducing DNA damage. The high affinity of CK1delta to the spindle apparatus in DNA-damaged cells and its ability to phosphorylate several microtubule-associated proteins points to a regulatory role of CK1delta at mitosis.
The series of molecular signals initiated by binding of a Wnt protein to a frizzled family receptor on the surface of the target cell and ending with a change in cell state.
IEAUniProtKB KW
Enzymatic activity
This protein acts as an enzyme. It is known to catalyze the following reactions
EC 2.7.11.1: ATP + a protein ⇄ ADP + a phosphoprotein.
Casein kinase-1 is a family of ubiquitous eukaryotic protein kinases that frequently function in tandem with the ubiquitin modification system to modulate protein turnover and trafficking. In Alzheimer's disease, these enzymes colocalize with ubiquitinated lesions, including neurofibrillary tangles and granulovacuolar degeneration bodies, suggesting they also play a role in disease pathogenesis. To identify binding partners that potentially regulate or recruit these enzymes toward disease lesions, a Sos-recruitment yeast two-hybrid screen was performed with human Ckidelta (the casein kinase-1 isoform most closely linked to granulovacuolar degeneration bodies) and a human brain cDNA library. All interacting clones contained a single open reading frame termed casein kinase-1 binding protein (CK1BP). On the basis of sequence alignments, CK1BP was a structural homologue of the acidic domain of dysbindin, a component of the dystrophin-associated protein complex and the biogenesis of lysosome-related organelles complex-1. CK1BP interacted with full-length Ckidelta, the isolated Ckidelta catalytic domain, Ckigamma2, -gamma3, and -epsilon in the yeast two-hybrid system, and bound Ckidelta and -epsilon in pulldown assays but did not interact with Ckialpha. Interaction with the Ckidelta catalytic domain led to concentration-dependent inhibition of protein kinase activity in the presence of protein substrates tau and alpha-synuclein. Although intact dysbindin did not bind any CK1 isoform, deletion of its coiled-coil domain yielded a protein fragment that behaved much like CK1BP in two-hybrid screens. These data suggest that the acidic domain of dysbindin and its paralogs in humans may function to recruit casein kinase-1 isoforms to protein complexes involved in multiple biological functions.
Casein kinase-1 is a family of ubiquitous eukaryotic protein kinases that frequently function in tandem with the ubiquitin modification system to modulate protein turnover and trafficking. In Alzheimer's disease, these enzymes colocalize with ubiquitinated lesions, including neurofibrillary tangles and granulovacuolar degeneration bodies, suggesting they also play a role in disease pathogenesis. To identify binding partners that potentially regulate or recruit these enzymes toward disease lesions, a Sos-recruitment yeast two-hybrid screen was performed with human Ckidelta (the casein kinase-1 isoform most closely linked to granulovacuolar degeneration bodies) and a human brain cDNA library. All interacting clones contained a single open reading frame termed casein kinase-1 binding protein (CK1BP). On the basis of sequence alignments, CK1BP was a structural homologue of the acidic domain of dysbindin, a component of the dystrophin-associated protein complex and the biogenesis of lysosome-related organelles complex-1. CK1BP interacted with full-length Ckidelta, the isolated Ckidelta catalytic domain, Ckigamma2, -gamma3, and -epsilon in the yeast two-hybrid system, and bound Ckidelta and -epsilon in pulldown assays but did not interact with Ckialpha. Interaction with the Ckidelta catalytic domain led to concentration-dependent inhibition of protein kinase activity in the presence of protein substrates tau and alpha-synuclein. Although intact dysbindin did not bind any CK1 isoform, deletion of its coiled-coil domain yielded a protein fragment that behaved much like CK1BP in two-hybrid screens. These data suggest that the acidic domain of dysbindin and its paralogs in humans may function to recruit casein kinase-1 isoforms to protein complexes involved in multiple biological functions.
Exhibits substrate-dependent heparin activation. Drug-mediated inhibition leads to a delay of the oscillations with the magnitude of this effect dependent upon the timing of drug administration. Inhibited by phosphorylation. Repressed by 3-[(2,4,6-trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261), N-(2-Aminoethyl)-5-chloroisoquinoline-8-sulphonamide (CKI-7), 4-[4-(2,3-dihydro-benzo[1,4]dioxin-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl]benzamide (D4476), 3,4-Diaryl-isoxazoles and -imidazoles, and 4-(3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl) pyrimidin-2-ylamine (PF670462, PF670).
Casein kinase 1 delta and epsilon (CK1δ/ɛ) are key regulators of diverse cellular growth and survival processes including Wnt signaling, DNA repair and circadian rhythms. Recent studies suggest that they have an important role in oncogenesis. RNA interference screens identified CK1ɛ as a pro-survival factor in cancer cells in vitro and the CK1δ/ɛ-specific inhibitor IC261 is remarkably effective at selective, synthetic lethal killing of cancer cells. The recent development of the nanomolar CK1δ/ɛ-selective inhibitor, PF670462 (PF670) and the CK1ɛ-selective inhibitor PF4800567 (PF480) offers an opportunity to further test the role of CK1δ/ɛ in cancer. Unexpectedly, and unlike IC261, PF670 and PF480 were unable to induce cancer cell death. PF670 is a potent inhibitor of CK1δ/ɛ in cells; nanomolar concentrations are sufficient to inhibit CK1δ/ɛ activity as measured by repression of intramolecular autophosphorylation, phosphorylation of disheveled2 proteins and Wnt/β-catenin signaling. Likewise, small interfering RNA knockdown of CK1δ and CK1ɛ reduced Wnt/β-catenin signaling without affecting cell viability, further suggesting that CK1δ/ɛ inhibition may not be relevant to the IC261-induced cell death. Thus, while PF670 is a potent inhibitor of Wnt signaling, it only modestly inhibits cell proliferation. In contrast, while sub-micromolar concentrations of IC261 neither inhibited CK1δ/ɛ kinase activity nor blocked Wnt/β-catenin signaling in cancer cells, it caused a rapid induction of prometaphase arrest and subsequent apoptosis in multiple cancer cell lines. In a stepwise transformation model, IC261-induced killing required both overactive Ras and inactive p53. IC261 binds to tubulin with an affinity similar to colchicine and is a potent inhibitor of microtubule polymerization. This activity accounts for many of the diverse biological effects of IC261 and, most importantly, for its selective cancer cell killing.
Casein kinase I epsilon/delta phosphorylates certain clock-related proteins as part of a complex arrangement of transcriptional/translational feedback loops that comprise the circadian oscillator in mammals. Pharmacologic inhibition leads to a delay of the oscillations with the magnitude of this effect dependent upon the timing of drug administration.
The serine/threonine-specific casein kinase I delta (CKIdelta) is ubiquitously expressed in all tissues, is p53 dependently induced in stress situations and plays an important role in various cellular processes. Our immunohistochemical analysis of the human placenta revealed strongest expression of CKIdelta in extravillous trophoblast cells and in choriocarcinomas. Investigation of the functional role of CKIdelta in an extravillous trophoblast hybrid cell line revealed that CKIdelta was constitutively localized at the centrosomes and the mitotic spindle. Inhibition of CKIdelta with the CKI-specific inhibitor IC261 led to structural alterations of the centrosomes, the formation of multipolar spindles, the inhibition of mitosis and, in contrast to other cell lines, the induction of apoptosis. Our findings indicate that CKIdelta plays an important role in the mitotic progression and in the survival of cells of trophoblast origin. Therefore, IC261 could provide a new tool in treating choriocarcinomas.
Eukaryotic initiation factor 6 (eIF6), a highly conserved protein from yeast to mammals, is essential for 60 S ribosome biogenesis and assembly. Both yeast and mammalian eIF6 are phosphorylated at Ser-174 and Ser-175 by the nuclear isoform of casein kinase 1 (CK1). The molecular basis of eIF6 phosphorylation, however, remains elusive. In the present work, we show that subcellular distribution of eIF6 in the nuclei and the cytoplasm of mammalian cells is mediated by dephosphorylation and phosphorylation, respectively. This nucleo-cytoplasmic shuttling is dependent on the phosphorylation status at Ser-174 and Ser-175 of eIF6. We demonstrate that Ca(2+)-activated calcineurin phosphatase binds to and promotes nuclear localization of eIF6. Increase in intracellular concentration of Ca(2+) leads to rapid translocation of eIF6 from the cytoplasm to the nucleus, an event that is blocked by specific calcineurin inhibitors cyclosporin A or FK520. Nuclear export of eIF6 is regulated by phosphorylation at Ser-174 and Ser-175 by the nuclear isoform of CK1. Mutation of eIF6 at the phosphorylatable Ser-174 and Ser-175 to alanine or treatment of cells with the CK1 inhibitor, D4476 inhibits nuclear export of eIF6 and results in nuclear accumulation of eIF6. Together, these results establish eIF6 as a substrate for calcineurin and suggest a novel paradigm for calcineurin function in 60 S ribosome biogenesis via regulating the nuclear accumulation of eIF6.
In this study, we report on the discovery of isoxazole 1 as a potent dual inhibitor of p38alpha (IC(50) = 0.45 microM) and CK1delta (IC(50) = 0.23 microM). Because only a few effective small molecule inhibitors of CK1 have been described so far, we aimed to develop this structural class toward specific agents. Molecular modeling studies comparing p38alpha/CK1delta suggested an optimization strategy leading to design, synthesis, biological characterization, and SAR of highly potent compounds including 9 (IC(50) p38alpha = 0.006 microM; IC(50) CK1delta = 1.6 microM), 13 (IC(50) p38alpha = 2.52 microM; IC(50) CK1delta = 0.033 microM), 17 (IC(50) p38alpha = 0.019 microM; IC(50) CK1delta = 0.004 microM; IC(50) CK1epsilon = 0.073 microM), and 18 (CKP138) (IC(50) p38alpha = 0.041 microM; IC(50) CK1delta = 0.005 microM; IC(50) CK1epsilon = 0.447 microM) possessing differentiated specificity. Selected compounds were profiled over 76 kinases and evaluation of their cellular efficacy showed 18 (CKP138) to be a highly potent and dual-specific inhibitor of CK1delta and p38alpha.
Circadian pacemaking requires the orderly synthesis, posttranslational modification, and degradation of clock proteins. In mammals, mutations in casein kinase 1 (CK1) epsilon or delta can alter the circadian period, but the particular functions of the WT isoforms within the pacemaker remain unclear. We selectively targeted WT CK1epsilon and CK1delta using pharmacological inhibitors (PF-4800567 and PF-670462, respectively) alongside genetic knockout and knockdown to reveal that CK1 activity is essential to molecular pacemaking. Moreover, CK1delta is the principal regulator of the clock period: pharmacological inhibition of CK1delta, but not CK1epsilon, significantly lengthened circadian rhythms in locomotor activity in vivo and molecular oscillations in the suprachiasmatic nucleus (SCN) and peripheral tissue slices in vitro. Period lengthening mediated by CK1delta inhibition was accompanied by nuclear retention of PER2 protein both in vitro and in vivo. Furthermore, phase mapping of the molecular clockwork in vitro showed that PF-670462 treatment lengthened the period in a phase-specific manner, selectively extending the duration of PER2-mediated transcriptional feedback. These findings suggested that CK1delta inhibition might be effective in increasing the amplitude and synchronization of disrupted circadian oscillators. This was tested using arrhythmic SCN slices derived from Vipr2(-/-) mice, in which PF-670462 treatment transiently restored robust circadian rhythms of PER2::Luc bioluminescence. Moreover, in mice rendered behaviorally arrhythmic by the Vipr2(-/-) mutation or by constant light, daily treatment with PF-670462 elicited robust 24-h activity cycles that persisted throughout treatment. Accordingly, selective pharmacological targeting of the endogenous circadian regulator CK1delta offers an avenue for therapeutic modulation of perturbed circadian behavior.
J. Biol. Chem. 273, 15980-15984 (1998)[PubMed:9632646]
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 (Cegielska, A., Gietzen, K. F., Rivers, A., and Virshup, D. M. (1998) J. Biol. Chem. 273, 1357-1364). However, we now report that 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.
Tau hyperphosphorylation precedes neuritic lesion formation in Alzheimer's disease, suggesting it participates in the tau fibrillization reaction pathway. Candidate tau protein kinases include members of the casein kinase 1 (CK1) family of phosphotransferases, which are highly overexpressed in Alzheimer's disease brain and colocalize with neuritic and granulovacuolar lesions. Here we characterized the contribution of one CK1 isoform, Ckidelta, to the phosphorylation of tau at residues Ser202/Thr205 and Ser396/Ser404 in human embryonic kidney 293 cells using immunodetection and fluorescence microscopy. Treatment of cells with membrane permeable CK1 inhibitor 3-[(2,3,6-trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261) lowered occupancy of Ser396/Ser404 phosphorylation sites by >70% at saturation, suggesting that endogenous CK1 was the major source of basal phosphorylation activity at these sites. Overexpression of Ckidelta increased CK1 enzyme activity and further raised tau phosphorylation at residues Ser202/Thr205 and Ser396/Ser404 in situ. Inhibitor IC261 reversed tau hyperphosphorylation induced by Ckidelta overexpression. Co-immunoprecipitation assays showed direct association of tau and Ckidelta in situ, consistent with tau being a Ckidelta substrate. Ckidelta overexpression also produced a decrease in the fraction of bulk tau bound to detergent-insoluble microtubules. These results suggest that Ckidelta phosphorylates tau at sites that modulate tau/microtubule binding, and that the expression pattern of Ckidelta in Alzheimer's disease is consistent with it playing an important role in tau aggregation.
We previously reported that phosphorylation of topoisomerase (topo) IIalpha at serine-1106 (Ser-1106) regulates enzyme activity and sensitivity to topo II-targeted drugs. In this study we demonstrate that phosphorylation of Ser-1106, which is flanked by acidic amino acids, is regulated in vivo by casein kinase (CK) Idelta and/or CKIepsilon, but not by CKII. The CKI inhibitors, CKI-7 and IC261, reduced Ser-1106 phosphorylation and decreased formation of etoposide-stabilized topo II-DNA cleavable complex. In contrast, the CKII inhibitor, 5,6-dichlorobenzimidazole riboside, did not affect etoposide-stabilized topo II-DNA cleavable complex formation. Since, IC261 specifically targets the Ca(2+)-regulated isozymes, CKIdelta and CKIepsilon, we examined the effect of down-regulating these enzymes on Ser-1106 phosphorylation. Down-regulation of these isozymes with targeted si-RNAs led to hypophosphorylation of the Ser-1106 containing peptide. However, si-RNA-mediated down-regulation of CKIIalpha and alpha' did not alter Ser-1106 phosphorylation. Furthermore, reduced phosphorylation of Ser-1106, observed in HRR25 (CKIdelta/epsilon homologous gene)-deleted Saccharomyces cerevisiae cells transformed with human topo IIalpha, was enhanced following expression of human CKIepsilon. Down-regulation of CKIdelta and CKIepsilon also led to reduced formation of etoposide stabilized topo II-DNA cleavable complex. These results provide strong support for an essential role of CKIdelta/epsilon in phosphorylating Ser-1106 in human topo IIalpha and in regulating enzyme function.
Previously we determined that Dishevelled-2/3 (Dvl) mediate Wnt-3a-dependent neurite outgrowth in Ewing sarcoma family tumor cells. Here we report that neurite extension was associated with Dvl phosphorylation and that both were inhibited by the casein kinase 1 (CK1) δ/ε inhibitor IC261. Small interfering RNAs targeting either CK1δ or CK1ε decreased Dvl phosphorylation, but only knockdown of CK1δ blocked neurite outgrowth. CK1δ but not CK1ε was detected at the centrosome, an organelle associated with neurite formation. Deletion analysis mapped the centrosomal localization signal (CLS) of CK1δ to its C-terminal domain. A fusion protein containing the CLS and EGFP displaced full-length CK1δ from the centrosome and inhibited Wnt-3a-dependent neurite outgrowth. In contrast to wild-type CK1ε, a chimera comprised of the kinase domain of CK1ε and the CLS of CK1δ localized to the centrosome and rescued Wnt-3a-dependent neurite outgrowth suppressed by CK1δ knockdown. These results provide strong evidence that the centrosomal localization of CK1δ is required for Wnt-3a-dependent neuritogenesis.
Tau in Alzheimer disease brain is highly phosphorylated and aggregated into paired helical filaments comprising characteristic neurofibrillary tangles. Here we have analyzed insoluble Tau (PHF-tau) extracted from Alzheimer brain by mass spectrometry and identified 11 novel phosphorylation sites, 10 of which were assigned unambiguously to specific amino acid residues. This brings the number of directly identified sites in PHF-tau to 39, with an additional six sites indicated by reactivity with phosphospecific antibodies to Tau. We also identified five new phosphorylation sites in soluble Tau from control adult human brain, bringing the total number of reported sites to nine. To assess which kinases might be responsible for Tau phosphorylation, we used mass spectrometry to determine which sites were phosphorylated in vitro by several kinases. Casein kinase 1delta and glycogen synthase kinase-3beta were each found to phosphorylate numerous sites, and each kinase phosphorylated at least 15 sites that are also phosphorylated in PHF-tau from Alzheimer brain. A combination of casein kinase 1delta and glycogen synthase kinase-3beta activities could account for over three-quarters of the serine/threonine phosphorylation sites identified in PHF-tau, indicating that casein kinase 1delta may have a role, together with glycogen synthase kinase-3beta, in the pathogenesis of Alzheimer disease.
Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxynucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We recently showed that dCK was activated in vivo by phosphorylation of Ser-74. However, the protein kinase responsible was not identified. Ser-74 is located downstream a Glu-rich region, presenting similarity with the consensus phosphorylation motif of casein kinase 1 (CKI), and particularly of CKI delta. We showed that recombinant CKI delta phosphorylated several residues of bacterially overexpressed dCK: Ser-74, but also Ser-11, Ser-15, and Thr-72. Phosphorylation of dCK by CKI delta correlated with increased activity reaching at least 4-fold. Site-directed mutagenesis demonstrated that only Ser-74 phosphorylation was involved in dCK activation by CKI delta, strengthening the key role of this residue in the control of dCK activity. However, neither CKI delta inhibitors nor CKI delta siRNA-mediated knock-down modified Ser-74 phosphorylation or dCK activity in cultured cells. Moreover, these approaches did not prevent dCK activation induced by treatments enhancing Ser-74 phosphorylation. Taken together, the data preclude a role of CKI delta in the regulation of dCK activity in vivo. Nevertheless, phosphorylation of dCK by CKI delta could be a useful tool for elucidating the influence of Ser-74 phosphorylation on the structure-activity relationships in the enzyme.
Protein involved in the generation of rhythmic pattern of behaviors or activities, e.g. circadian rhythm which is a metabolic or behavioural rhythm within a cycle of 24 hours.
Protein involved in the Wnt signaling pathway. Wnts are a large family of cysteine-rich secreted glycoproteins that control development in organisms ranging from nematodes to mammals. Wnt genes are defined by sequence homology to the original members of the family, Wnt1 in the mouse and wingless (wg) in Drosophila. Wnt signaling is a very complex pathway which includes numerous ligands, receptors and transcriptional effectors. There is a well-characterized canonical pathway as well as diverse, less-characterized noncanonical pathways. Several components of Wnt signaling are implicated in the genesis of human cancer.
Protein which catalyzes the phosphorylation of serine or threonine residues on target proteins by using ATP as phosphate donor. Such phosphorylation may cause changes in the function of the target protein. Protein kinases share a conserved catalytic core common to both serine/ threonine and tyrosine protein kinases.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.