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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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Related in: MedlinePlus

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.
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pone-0004766-g009: 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.

Mentions: The binding of xDsh to CKIε and not CKIα primarily requires the residues N275 and R279 (Fig. 9A). Mutation of these residues to either alanines or the CKIα identities, isoleucine and threonine respectively (Fig. 9B), prevented binding. Thus, the identity of these residues is essential, suggesting that Dsh directly contacts these residues. We also noted that the extent to which Dsh was phosphorylated (visualized by a change in electrophoretic mobility, Fig. 4) was different depending on the mutations that were made. The identities of the residues at positions 275 and 279 in CKIε may therefore also be important for correctly positioning Dsh for full phosphorylation. The C-terminal tail of CKIε provided some enhancement of xDsh binding and increased the level of phosphorylation by the mutant protein (Fig. 5), but it was not sufficient for binding. Strikingly, we saw full binding of xDsh to CKIα simply by changing the I283 and T287 to the CKIε identity and adding on the C-terminal tail (Fig. 9C). In the absence of the C-terminal tail, Dsh did not bind to the mutated CKIα (data not shown), which strengthens our hypothesis that the C-terminus of CKIε does contact Dsh, but is not required in for interactions with wild-type CKIε. These results allow us to fully explain the selectivity of CKIε for Dishevelled.


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651596&req=5

pone-0004766-g009: 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.
Mentions: The binding of xDsh to CKIε and not CKIα primarily requires the residues N275 and R279 (Fig. 9A). Mutation of these residues to either alanines or the CKIα identities, isoleucine and threonine respectively (Fig. 9B), prevented binding. Thus, the identity of these residues is essential, suggesting that Dsh directly contacts these residues. We also noted that the extent to which Dsh was phosphorylated (visualized by a change in electrophoretic mobility, Fig. 4) was different depending on the mutations that were made. The identities of the residues at positions 275 and 279 in CKIε may therefore also be important for correctly positioning Dsh for full phosphorylation. The C-terminal tail of CKIε provided some enhancement of xDsh binding and increased the level of phosphorylation by the mutant protein (Fig. 5), but it was not sufficient for binding. Strikingly, we saw full binding of xDsh to CKIα simply by changing the I283 and T287 to the CKIε identity and adding on the C-terminal tail (Fig. 9C). In the absence of the C-terminal tail, Dsh did not bind to the mutated CKIα (data not shown), which strengthens our hypothesis that the C-terminus of CKIε does contact Dsh, but is not required in for interactions with wild-type CKIε. These results allow us to fully explain the selectivity of CKIε for Dishevelled.

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
Related in: MedlinePlus