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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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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).
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pone-0004766-g002: 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).

Mentions: CKIε and CKIα are 75% identical, and 89% similar in their kinase domains (Fig. 2). This high degree of conservation between the kinases suggested that full binding by mPer1 and xDsh to CKIε might depend on the long C-terminal tail, which is unique to CKIε. We first examined binding of mPer1 and xDsh to CKIεΔC, which truncates the kinase at residue 319 (Fig. 2). Figure 3A shows that xDsh and mPer bound to CKIε and CKIεΔC to the same extent, demonstrating that CKIε does not require the autophosphorylation region of the C-terminus to bind to substrate proteins.


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004766-g002: 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).
Mentions: CKIε and CKIα are 75% identical, and 89% similar in their kinase domains (Fig. 2). This high degree of conservation between the kinases suggested that full binding by mPer1 and xDsh to CKIε might depend on the long C-terminal tail, which is unique to CKIε. We first examined binding of mPer1 and xDsh to CKIεΔC, which truncates the kinase at residue 319 (Fig. 2). Figure 3A shows that xDsh and mPer bound to CKIε and CKIεΔC to the same extent, demonstrating that CKIε does not require the autophosphorylation region of the C-terminus to bind to substrate proteins.

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