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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
Analysis of the binding of the C-terminal tail.(A) Full-length CKIε was incubated with either SAP or ATP prior to reaction with the EDC crosslinker. Lane 2 shows that there is no change in the apparent molecular weight of dephosphorylated CKIε. In lane 4, there is marked change in the migration of autophosphorylated, crosslinked CKIε (bracketed). Asterisk shows a high molecular weight species that may correspond to SAP-CKIε oligomers (lane 2). (B) Space-filling models of CKIδ are shown. The APBS plugin for PyMol (DeLano Scientific LLC) was used to establish electrostatic potential of solvent exposed atoms. Positively charged areas are shaded blue and correspond to basic regions of the protein; negatively charged regions are red, and correspond to acidic areas. The highly basic groove that has been postulated to be a phosphate recognition region is conservered across the CKI family. The two identified cross-linked residues are indicated with an X. The cartoon line on the left side diagram shows the position of the first 20 amino acids of the tail based on the crosslinking data. The dotted line on the right side shows the proposed extension of the tail onto the backside of the kinase.
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pone-0004766-g008: Analysis of the binding of the C-terminal tail.(A) Full-length CKIε was incubated with either SAP or ATP prior to reaction with the EDC crosslinker. Lane 2 shows that there is no change in the apparent molecular weight of dephosphorylated CKIε. In lane 4, there is marked change in the migration of autophosphorylated, crosslinked CKIε (bracketed). Asterisk shows a high molecular weight species that may correspond to SAP-CKIε oligomers (lane 2). (B) Space-filling models of CKIδ are shown. The APBS plugin for PyMol (DeLano Scientific LLC) was used to establish electrostatic potential of solvent exposed atoms. Positively charged areas are shaded blue and correspond to basic regions of the protein; negatively charged regions are red, and correspond to acidic areas. The highly basic groove that has been postulated to be a phosphate recognition region is conservered across the CKI family. The two identified cross-linked residues are indicated with an X. The cartoon line on the left side diagram shows the position of the first 20 amino acids of the tail based on the crosslinking data. The dotted line on the right side shows the proposed extension of the tail onto the backside of the kinase.

Mentions: We first looked for evidence of a conformational change by following differences in electrophoretic mobility of purified, hyperphosphorylated CKIε upon the addition of 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride (EDC), a zero-length crosslinker. When two or more proteins are crosslinked by EDC, the expected product is a band of approximately their additive molecular weights. In the case of CKIε, no high molecular weight bands appear, demonstrating that CKIε does not form dimers or higher-order oligomers in solution. Instead, the single band corresponding to CKIε broadened considerably, and showed faster mobility after EDC treatment as seen by PAGE analysis (Fig. 8A, lane 4). Interestingly, unphosphorylated CKIε did not show a change in mobility after crosslinking and it continued to migrate as a single band (Fig. 8A, lane 2). The broad band that appears after crosslinking suggests that the autophosphorylated CKIε occupies multiple conformations.


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)

Analysis of the binding of the C-terminal tail.(A) Full-length CKIε was incubated with either SAP or ATP prior to reaction with the EDC crosslinker. Lane 2 shows that there is no change in the apparent molecular weight of dephosphorylated CKIε. In lane 4, there is marked change in the migration of autophosphorylated, crosslinked CKIε (bracketed). Asterisk shows a high molecular weight species that may correspond to SAP-CKIε oligomers (lane 2). (B) Space-filling models of CKIδ are shown. The APBS plugin for PyMol (DeLano Scientific LLC) was used to establish electrostatic potential of solvent exposed atoms. Positively charged areas are shaded blue and correspond to basic regions of the protein; negatively charged regions are red, and correspond to acidic areas. The highly basic groove that has been postulated to be a phosphate recognition region is conservered across the CKI family. The two identified cross-linked residues are indicated with an X. The cartoon line on the left side diagram shows the position of the first 20 amino acids of the tail based on the crosslinking data. The dotted line on the right side shows the proposed extension of the tail onto the backside of the kinase.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004766-g008: Analysis of the binding of the C-terminal tail.(A) Full-length CKIε was incubated with either SAP or ATP prior to reaction with the EDC crosslinker. Lane 2 shows that there is no change in the apparent molecular weight of dephosphorylated CKIε. In lane 4, there is marked change in the migration of autophosphorylated, crosslinked CKIε (bracketed). Asterisk shows a high molecular weight species that may correspond to SAP-CKIε oligomers (lane 2). (B) Space-filling models of CKIδ are shown. The APBS plugin for PyMol (DeLano Scientific LLC) was used to establish electrostatic potential of solvent exposed atoms. Positively charged areas are shaded blue and correspond to basic regions of the protein; negatively charged regions are red, and correspond to acidic areas. The highly basic groove that has been postulated to be a phosphate recognition region is conservered across the CKI family. The two identified cross-linked residues are indicated with an X. The cartoon line on the left side diagram shows the position of the first 20 amino acids of the tail based on the crosslinking data. The dotted line on the right side shows the proposed extension of the tail onto the backside of the kinase.
Mentions: We first looked for evidence of a conformational change by following differences in electrophoretic mobility of purified, hyperphosphorylated CKIε upon the addition of 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride (EDC), a zero-length crosslinker. When two or more proteins are crosslinked by EDC, the expected product is a band of approximately their additive molecular weights. In the case of CKIε, no high molecular weight bands appear, demonstrating that CKIε does not form dimers or higher-order oligomers in solution. Instead, the single band corresponding to CKIε broadened considerably, and showed faster mobility after EDC treatment as seen by PAGE analysis (Fig. 8A, lane 4). Interestingly, unphosphorylated CKIε did not show a change in mobility after crosslinking and it continued to migrate as a single band (Fig. 8A, lane 2). The broad band that appears after crosslinking suggests that the autophosphorylated CKIε occupies multiple conformations.

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