Limits...
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
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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2651596&req=5

pone-0004766-g001: 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.

Mentions: Per1 and Dsh bind to CKIε in vivo, but they do not interact with the related isoform CKIα [23], [47]–[49]. In order to determine if these substrates bind to the two kinases with the corresponding affinities in vitro, we used a Glutathione S-Transferase (GST) pull-down system to follow protein-protein interactions. Due to protein instability and toxicity in E. coli, prior in vitro studies of CKIε have used a C-terminally truncated form of the protein called CKIεΔC [48], [50], [51], which eliminates the last 98 residues of the protein. Since we wanted to include all of the protein in our analysis, we developed an expression system for large quantities of pure full-length Xenopus CKIε fused to GST (CKIε-GST; see Methods). Using a similar strategy we also expressed full-length Xenopus CKIα fused to GST (CKIα-GST). Both kinases were catalytically active against β-catenin, indicating that they were both well folded (data not shown). Because CKIε autophosphorylation regulates substrate binding (see below), we used dephosphorylated CKIε for our binding studies except where otherwise noted. Using GST-pull-down assays we found that 35S-labelled Xenopus Dsh (xDsh) and mouse Per1 (mPer1) bound specifically to CKIε compared to CKIα (Fig. 1), recapitulating previously published in vivo results [19], [37], [46]–[48]. This provided us with a useful assay system to examine CKIε specificity for these substrates.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004766-g001: 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.
Mentions: Per1 and Dsh bind to CKIε in vivo, but they do not interact with the related isoform CKIα [23], [47]–[49]. In order to determine if these substrates bind to the two kinases with the corresponding affinities in vitro, we used a Glutathione S-Transferase (GST) pull-down system to follow protein-protein interactions. Due to protein instability and toxicity in E. coli, prior in vitro studies of CKIε have used a C-terminally truncated form of the protein called CKIεΔC [48], [50], [51], which eliminates the last 98 residues of the protein. Since we wanted to include all of the protein in our analysis, we developed an expression system for large quantities of pure full-length Xenopus CKIε fused to GST (CKIε-GST; see Methods). Using a similar strategy we also expressed full-length Xenopus CKIα fused to GST (CKIα-GST). Both kinases were catalytically active against β-catenin, indicating that they were both well folded (data not shown). Because CKIε autophosphorylation regulates substrate binding (see below), we used dephosphorylated CKIε for our binding studies except where otherwise noted. Using GST-pull-down assays we found that 35S-labelled Xenopus Dsh (xDsh) and mouse Per1 (mPer1) bound specifically to CKIε compared to CKIα (Fig. 1), recapitulating previously published in vivo results [19], [37], [46]–[48]. This provided us with a useful assay system to examine CKIε specificity for these substrates.

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