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PLoS One
2009 Jan 01;43:e4766. doi: 10.1371/journal.pone.0004766.
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Interactions between Casein kinase Iepsilon (CKIepsilon) and two substrates from disparate signaling pathways reveal mechanisms for substrate-kinase specificity.
Dahlberg CL
,
Nguyen EZ
,
Goodlett D
,
Kimelman D
.
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Members of the Casein Kinase I (CKI) family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIepsilon and two substrates from different signaling pathways. CKIepsilon, but not CKIalpha, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIalpha's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIepsilon does not determine Dishevelled's and Period's preference for CKIepsilon nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIepsilon with its substrates. We demonstrate that autophosphorylation of CKIepsilon's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding. The biochemical interactions between CKIepsilon and Disheveled, Period, and its own C-terminus lead to models that explain CKIepsilon's specificity and regulation.
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19274088
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Figure 1. Recombinant CKIε, but not CKIα, interacts with mPer1 and xDsh.(A) GST, GST-CKIε, or GST-CKIα was bound to glutathione sepharose, and then incubated with 35S-labeled mPer1 or xDsh. 10% of the mPer1 input and 25% of the xDsh input are shown. Coomassie stained gel shows the levels of GST fusion protein used for each pull-down. (B) Quantification of three independent experiments. Values are normalized against the amount of protein bound by GST-CKIε, and error bars represent standard deviation of the mean.
Figure 2. Protein constructs used in this work.CKIα and -ε are shown with their conserved kinase domains in gray and black, respectively (89% similarity, 75% identity). The arrow indicates the position of residue 295 in CKIε, and the non-conserved, charged region of the protein is colored red. The filled arrowhead indicates the position of residue 319, where CKIε is conventionally truncated. The C-terminus contains autophosphorylation sites and is colored yellow. The open arrowhead and white bars indicate the position of residues N275 and R279 (CKIε), and I283 and T287 (CKIα).
Figure 3. xDsh and mPer1 do not require CKIε's C-terminus for binding.(A, B) Purified GST, GST-CKIε, GST-CKIεΔC or GST-CKIεΔΔC were bound to glutathione sepharose and incubated with 35S-labeled xDsh or mPer1 as indicated.
Figure 4. Residues 275 and 279 regulate binding to xDsh and mPer1.(A) Space-filling representation of CKIδ (PDB ID 1CKJ, [50]). Residues shown in cyan and red are conserved between CKIε and CKIδ, but not CKIα. Red residues N275 and R279 are solvent accessible and are chemically distinct in CKIα. Orange shading shows the position of the ATP binding cleft. (B) Binding of 35S-labeled mPer1 and xDsh to GST-CKIεΔC, GST-CKIεΔC N275A/R279A, or GST-CKIεΔC N275I/R279T was performed. (C) Quantification of three independent experiments. Values are normalized against the amount of protein bound by GST-CKIεΔC.
Figure 5. The C-terminal tail enhances binding of xDsh and mPer1 to mutant CKIε.(A) GST, GST-CKIε, or GST-CKIε N275I/R279T was bound to glutathione sepharose and incubated with 35S-labeled xDsh or mPer1. (B) Quantification of three independent experiments. Values are normalized against the amount of protein bound by GST-CKIε.
Figure 6. A. Neither xDsh nor mPer1 bind strongly to CKIα→ε.GST, GST-CKIε (partially purified) or GST- CKIα→ε, was bound to glutathione sepharose and incubated with 35S-labeled xDsh or mPer1. (B) GST, GST-CKIε, or GST- CKIα→ε I283N/T287R, was bound to glutathione sepharose and incubated with 35S-labeled xDsh or mPer1. The bar graphs represent quantification of three independent experiments. Values are normalized against the amount of protein bound to GST-CKIε.
Figure 7. Autophosphorylation of CKIε inhibits binding by substrate and scaffolding proteins.Purified GST and GST-fusion proteins were bound to glutathione sepharose and bound GST-CKIε was incubated with SAP or ATP for 1 hour. Resin was incubated with 35S-Methionine-labeled xDsh, mAxin or mPer1. 10% of the mPer1and mAxin input and 25% of the xDsh input were run. Coomassie stained gel shows the levels of GST fusion protein used for each pull-down.
Figure 8. Analysis of the binding of the C-terminal tail.(A) Full-length CKIε was incubated with either SAP or ATP prior to reaction with the EDC crosslinker. Lane 2 shows that there is no change in the apparent molecular weight of dephosphorylated CKIε. In lane 4, there is marked change in the migration of autophosphorylated, crosslinked CKIε (bracketed). Asterisk shows a high molecular weight species that may correspond to SAP-CKIε oligomers (lane 2). (B) Space-filling models of CKIδ are shown. The APBS plugin for PyMol (DeLano Scientific LLC) was used to establish electrostatic potential of solvent exposed atoms. Positively charged areas are shaded blue and correspond to basic regions of the protein; negatively charged regions are red, and correspond to acidic areas. The highly basic groove that has been postulated to be a phosphate recognition region is conservered across the CKI family. The two identified cross-linked residues are indicated with an X. The cartoon line on the left side diagram shows the position of the first 20 amino acids of the tail based on the crosslinking data. The dotted line on the right side shows the proposed extension of the tail onto the backside of the kinase.
Figure 9. Model of inter- and intra-molecular interactions involving CKIε.Red blocked arrows represent inhibition of binding and green arrows represent positive binding interactions. (A–D) Back face of the kinase. (A) CKIε binds to Dsh and Per using different binding sites. (B) Mutation of CKIε N275 and R279 to the corresponding CKIα identity inhibits Dsh and Per binding; however, the C-terminal tail and at least one other residue in the kinase domain promote Per binding. (C) Changing two residues in CKIα to the CKIε identity along with adding CKIε's C-terminus enables Dsh to bind to CKIα. Per is unable to bind this chimeric kinase. (D) Binding of the autophosphorylated tail to the backside prevents the binding of substrates. Phosphorylated sites detected by mass spectrometry are shown on the tail. (E) View of the front side of the kinase. Left, CKIε's C-terminus is labile when it is not phosphorylated, and CKIε is able to bind to partners. Right, upon incubation with ATP, CKIε autophosphorylates and the C-terminus binds tightly to the back side of the kinase domain. This positions the peptide PEDLDRERREHDREER next to the active site and the phosphate recognition groove. The X's show identified crosslinks between the peptide and the kinase domain.
Albornoz,
The CK1 gene family: expression patterning in zebrafish development.
2007, Pubmed
Albornoz,
The CK1 gene family: expression patterning in zebrafish development.
2007,
Pubmed
Amit,
Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway.
2002,
Pubmed
Bae,
Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock.
2001,
Pubmed
Bilic,
Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation.
2007,
Pubmed
Bryja,
Wnt-3a utilizes a novel low dose and rapid pathway that does not require casein kinase 1-mediated phosphorylation of Dvl to activate beta-catenin.
2007,
Pubmed
Bryja,
Wnt-5a induces Dishevelled phosphorylation and dopaminergic differentiation via a CK1-dependent mechanism.
2007,
Pubmed
Burzio,
Biochemical and cellular characteristics of the four splice variants of protein kinase CK1alpha from zebrafish (Danio rerio).
2002,
Pubmed
Cegielska,
T-antigen kinase inhibits simian virus 40 DNA replication by phosphorylation of intact T antigen on serines 120 and 123.
1994,
Pubmed
Cegielska,
Autoinhibition of casein kinase I epsilon (CKI epsilon) is relieved by protein phosphatases and limited proteolysis.
1998,
Pubmed
Cong,
Casein kinase Iepsilon modulates the signaling specificities of dishevelled.
2004,
Pubmed
Cooper,
Casein kinase 1 regulates connexin-43 gap junction assembly.
2002,
Pubmed
Davidson,
Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction.
2005,
Pubmed
,
Xenbase
Fish,
Isolation and characterization of human casein kinase I epsilon (CKI), a novel member of the CKI gene family.
1995,
Pubmed
Flotow,
Phosphate groups as substrate determinants for casein kinase I action.
1990,
Pubmed
Flotow,
Role of acidic residues as substrate determinants for casein kinase I.
1991,
Pubmed
Gao,
Casein kinase I phosphorylates and destabilizes the beta-catenin degradation complex.
2002,
Pubmed
,
Xenbase
Gietzen,
Identification of inhibitory autophosphorylation sites in casein kinase I epsilon.
1999,
Pubmed
Graves,
Role of COOH-terminal phosphorylation in the regulation of casein kinase I delta.
1995,
Pubmed
Green,
Identification of four alternatively spliced isoforms of chicken casein kinase I alpha that are all expressed in diverse cell types.
1998,
Pubmed
,
Xenbase
Gross,
Casein kinase I: spatial organization and positioning of a multifunctional protein kinase family.
1998,
Pubmed
Ha,
Mechanism of phosphorylation-dependent binding of APC to beta-catenin and its role in beta-catenin degradation.
2004,
Pubmed
Hardin,
Essential and expendable features of the circadian timekeeping mechanism.
2006,
Pubmed
Hino,
Casein kinase I epsilon enhances the binding of Dvl-1 to Frat-1 and is essential for Wnt-3a-induced accumulation of beta-catenin.
2003,
Pubmed
,
Xenbase
Ishida,
Constitutive expression and delayed light response of casein kinase Iepsilon and Idelta mRNAs in the mouse suprachiasmatic nucleus.
2001,
Pubmed
Klein,
CKIepsilon/discs overgrown promotes both Wnt-Fz/beta-catenin and Fz/PCP signaling in Drosophila.
2006,
Pubmed
Klimowski,
Site-specific casein kinase 1epsilon-dependent phosphorylation of Dishevelled modulates beta-catenin signaling.
2006,
Pubmed
,
Xenbase
Knippschild,
The casein kinase 1 family: participation in multiple cellular processes in eukaryotes.
2005,
Pubmed
Lee,
Direct association between mouse PERIOD and CKIepsilon is critical for a functioning circadian clock.
2004,
Pubmed
Lein,
Genome-wide atlas of gene expression in the adult mouse brain.
2007,
Pubmed
Liu,
Mechanism of regulation of casein kinase I activity by group I metabotropic glutamate receptors.
2002,
Pubmed
Longenecker,
Three-dimensional structure of mammalian casein kinase I: molecular basis for phosphate recognition.
1996,
Pubmed
Longenecker,
Crystallographic studies of casein kinase I delta toward a structural understanding of auto-inhibition.
1998,
Pubmed
Loudon,
The biology of the circadian Ck1epsilon tau mutation in mice and Syrian hamsters: a tale of two species.
2007,
Pubmed
Lowrey,
Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau.
2000,
Pubmed
Mackey,
Biological Rhythms Workshop IA: molecular basis of rhythms generation.
2007,
Pubmed
Marin,
A noncanonical sequence phosphorylated by casein kinase 1 in beta-catenin may play a role in casein kinase 1 targeting of important signaling proteins.
2003,
Pubmed
Matsubayashi,
Biochemical characterization of the Drosophila wingless signaling pathway based on RNA interference.
2004,
Pubmed
McKay,
The casein kinase I family: roles in morphogenesis.
2001,
Pubmed
McKay,
The casein kinase I family in Wnt signaling.
2001,
Pubmed
Miyazaki,
Phosphorylation of clock protein PER1 regulates its circadian degradation in normal human fibroblasts.
2004,
Pubmed
Okamura,
A conserved docking motif for CK1 binding controls the nuclear localization of NFAT1.
2004,
Pubmed
Penton,
A mutational analysis of dishevelled in Drosophila defines novel domains in the dishevelled protein as well as novel suppressing alleles of axin.
2002,
Pubmed
Peters,
Casein kinase I transduces Wnt signals.
1999,
Pubmed
,
Xenbase
Price,
CKI, there's more than one: casein kinase I family members in Wnt and Hedgehog signaling.
2006,
Pubmed
Pulgar,
Optimal sequences for non-phosphate-directed phosphorylation by protein kinase CK1 (casein kinase-1)--a re-evaluation.
1999,
Pubmed
,
Xenbase
Rivers,
Regulation of casein kinase I epsilon and casein kinase I delta by an in vivo futile phosphorylation cycle.
1998,
Pubmed
Rothbächer,
Dishevelled phosphorylation, subcellular localization and multimerization regulate its role in early embryogenesis.
2000,
Pubmed
,
Xenbase
Sampietro,
Crystal structure of a beta-catenin/BCL9/Tcf4 complex.
2006,
Pubmed
,
Xenbase
Seifert,
Frizzled/PCP signalling: a conserved mechanism regulating cell polarity and directed motility.
2007,
Pubmed
Shirogane,
SCFbeta-TRCP controls clock-dependent transcription via casein kinase 1-dependent degradation of the mammalian period-1 (Per1) protein.
2005,
Pubmed
Singh,
Characterization of protein cross-links via mass spectrometry and an open-modification search strategy.
2008,
Pubmed
Strutt,
Planar polarity is positively regulated by casein kinase Iepsilon in Drosophila.
2006,
Pubmed
Swiatek,
Negative regulation of LRP6 function by casein kinase I epsilon phosphorylation.
2006,
Pubmed
Swiatek,
Regulation of casein kinase I epsilon activity by Wnt signaling.
2004,
Pubmed
,
Xenbase
Takano,
A missense variation in human casein kinase I epsilon gene that induces functional alteration and shows an inverse association with circadian rhythm sleep disorders.
2004,
Pubmed
Takano,
Cloning and characterization of rat casein kinase 1epsilon.
2000,
Pubmed
Trang,
Casein kinases I of the silkworm, Bombyx mori: their possible roles in circadian timing and developmental determination.
2006,
Pubmed
Vielhaber,
Casein kinase I: from obscurity to center stage.
2001,
Pubmed
Vielhaber,
Nuclear export of mammalian PERIOD proteins.
2001,
Pubmed
,
Xenbase
Virshup,
Reversible protein phosphorylation regulates circadian rhythms.
2007,
Pubmed
Xu,
Crystal structure of casein kinase-1, a phosphate-directed protein kinase.
1995,
Pubmed
Xu,
Functional consequences of a CKIdelta mutation causing familial advanced sleep phase syndrome.
2005,
Pubmed
Yin,
Dysbindin structural homologue CK1BP is an isoform-selective binding partner of human casein kinase-1.
2006,
Pubmed
Zeng,
The mouse Fused locus encodes Axin, an inhibitor of the Wnt signaling pathway that regulates embryonic axis formation.
1997,
Pubmed
,
Xenbase
Zeng,
A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation.
2005,
Pubmed
,
Xenbase
Zeng,
Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions.
2008,
Pubmed
,
Xenbase
Zhang,
Regulation of wingless signaling by the CKI family in Drosophila limb development.
2006,
Pubmed
,
Xenbase