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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.

Show MeSH
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.
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pone-0004766-g007: 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.

Mentions: Our data shows that the C-terminus of CKIε stabilizes some substrate interactions, specifically in the absence of motifs that are required for binding (Fig. 6A and B). However, autophosphorylation of the C-terminus has also been shown to inhibit CKIε's activity towards protein targets [6], [7], [53], [54]. We hypothesized that this effect might be partially mediated by a change in affinity for substrates. When CKIε is allowed to fully autophosphorylate, neither mPer1 nor xDsh bind to it (Fig. 7). In addition, hyperphosphorylated CKIε no longer binds to mAxin, a protein that is able to bind to both CKIα and CKIε. Hyperphosphorylation of CKIε's C-terminus thus inhibits the binding of substrate proteins, and may act as a regulatory mechanism to control phosphorylation of targets.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2651596&req=5

pone-0004766-g007: 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.
Mentions: Our data shows that the C-terminus of CKIε stabilizes some substrate interactions, specifically in the absence of motifs that are required for binding (Fig. 6A and B). However, autophosphorylation of the C-terminus has also been shown to inhibit CKIε's activity towards protein targets [6], [7], [53], [54]. We hypothesized that this effect might be partially mediated by a change in affinity for substrates. When CKIε is allowed to fully autophosphorylate, neither mPer1 nor xDsh bind to it (Fig. 7). In addition, hyperphosphorylated CKIε no longer binds to mAxin, a protein that is able to bind to both CKIα and CKIε. Hyperphosphorylation of CKIε's C-terminus thus inhibits the binding of substrate proteins, and may act as a regulatory mechanism to control phosphorylation of targets.

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