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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 - PLoS ONE (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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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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.
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pone-0004766-g004: 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.

Mentions: To identify residues within the CKIε kinase domain that could confer specificity, we examined the structure of CKIδ. CKIδ is considered a very close relative of CKIε since they share 82% identity over the entire protein and 96% identity in their kinase domains, and since their cellular activities are indistinguishable [11], [13], [24], [48], [52]. Residues shown in cyan in Figure 4A are conserved between CKIε and CKIδ but are not conserved in CKIα. Two residues that are conserved in CKIε and CKIδ but not CKIα, and have solvent exposed side-chains, Asparagine 275 (N275) and Arginine 279 (R279), are shown in red (Fig. 4A). In CKIα, the corresponding residues have chemical properties that differ substantially from the CKIε residues (Asp→Ile and Arg→Thr), and therefore these residues could contribute specificity to CKIε-substrate interactions.


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 - PLoS ONE (2009)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651596&req=5

pone-0004766-g004: 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.
Mentions: To identify residues within the CKIε kinase domain that could confer specificity, we examined the structure of CKIδ. CKIδ is considered a very close relative of CKIε since they share 82% identity over the entire protein and 96% identity in their kinase domains, and since their cellular activities are indistinguishable [11], [13], [24], [48], [52]. Residues shown in cyan in Figure 4A are conserved between CKIε and CKIδ but are not conserved in CKIα. Two residues that are conserved in CKIε and CKIδ but not CKIα, and have solvent exposed side-chains, Asparagine 275 (N275) and Arginine 279 (R279), are shown in red (Fig. 4A). In CKIα, the corresponding residues have chemical properties that differ substantially from the CKIε residues (Asp→Ile and Arg→Thr), and therefore these residues could contribute specificity to CKIε-substrate interactions.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: The biochemical interactions between CKIepsilon and Disheveled, Period, and its own C-terminus lead to models that explain CKIepsilon's specificity and regulation.

Show MeSH