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Structural and functional characterization of the human protein kinase ASK1.

Bunkoczi G, Salah E, Filippakopoulos P, Fedorov O, Müller S, Sobott F, Parker SA, Zhang H, Min W, Turk BE, Knapp S - Structure (2007)

Bottom Line: Here, we present the structure of the human ASK1 catalytic domain in complex with staurosporine.Reporter gene assays showed that all three identified in vitro autophosphorylation sites (Thr813, Thr838, Thr842) regulate ASK1 signaling, but site-directed mutants showed catalytic activities similar to wild-type ASK1, suggesting a regulatory mechanism independent of ASK1 kinase activity.The determined high-resolution structure of ASK1 and identified ATP mimetic inhibitors will provide a first starting point for the further development of selective inhibitors.

View Article: PubMed Central - PubMed

Affiliation: University of Oxford, Structural Genomics Consortium, Botnar Research Centre, Oxford OX3 7LD, United Kingdom.

ABSTRACT
Apoptosis signal-regulating kinase 1 (ASK1) plays an essential role in stress and immune response and has been linked to the development of several diseases. Here, we present the structure of the human ASK1 catalytic domain in complex with staurosporine. Analytical ultracentrifugation (AUC) and crystallographic analysis showed that ASK1 forms a tight dimer (K(d) approximately 0.2 microM) interacting in a head-to-tail fashion. We found that the ASK1 phosphorylation motifs differ from known ASK1 phosphorylation sites but correspond well to autophosphorylation sites identified by mass spectrometry. Reporter gene assays showed that all three identified in vitro autophosphorylation sites (Thr813, Thr838, Thr842) regulate ASK1 signaling, but site-directed mutants showed catalytic activities similar to wild-type ASK1, suggesting a regulatory mechanism independent of ASK1 kinase activity. The determined high-resolution structure of ASK1 and identified ATP mimetic inhibitors will provide a first starting point for the further development of selective inhibitors.

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Substrate Specificity of ASK1 and Identification of Autophosphorylation Sites(A) Phosphorylation motifs for ASK1. Biotinylated peptides bearing the indicated residue at the indicated position relative to a central Ser/Thr phosphoacceptor site were subjected to phosphorylation by ASK1 with radiolabeled ATP. Aliquots of each reaction were subsequently spotted onto a streptavidin membrane, which was washed, dried, and exposed to a phosphor screen. Shown is a representative array from three separate experiments. Quantified spot intensities representing the average of the three runs are provided in Table S2.(B) Consensus sequence determined from the peptide array data. The consensus sequence is shown in bold and alternative residues are indicated at each position by smaller italic letters. The site of phosphorylation is indicated in red and by a star (∗). The three autophosphorylation sites are also shown, and residues matching the consensus sequence are highlighted with yellow boxes.(C) Activation segments of the known ASK1 substrates of the MKK family of kinases. Residues phosphorylated by activation are highlighted in red.(D) Location of the three autophosphorylation sites in ASK1. Phosphorylated residues are labeled in red and are shown as spheres, whereas neighboring residues are labeled in blue and are indicated in ball-and-stick representation. The interacting ASK1 dimer is shown in green.
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fig5: Substrate Specificity of ASK1 and Identification of Autophosphorylation Sites(A) Phosphorylation motifs for ASK1. Biotinylated peptides bearing the indicated residue at the indicated position relative to a central Ser/Thr phosphoacceptor site were subjected to phosphorylation by ASK1 with radiolabeled ATP. Aliquots of each reaction were subsequently spotted onto a streptavidin membrane, which was washed, dried, and exposed to a phosphor screen. Shown is a representative array from three separate experiments. Quantified spot intensities representing the average of the three runs are provided in Table S2.(B) Consensus sequence determined from the peptide array data. The consensus sequence is shown in bold and alternative residues are indicated at each position by smaller italic letters. The site of phosphorylation is indicated in red and by a star (∗). The three autophosphorylation sites are also shown, and residues matching the consensus sequence are highlighted with yellow boxes.(C) Activation segments of the known ASK1 substrates of the MKK family of kinases. Residues phosphorylated by activation are highlighted in red.(D) Location of the three autophosphorylation sites in ASK1. Phosphorylated residues are labeled in red and are shown as spheres, whereas neighboring residues are labeled in blue and are indicated in ball-and-stick representation. The interacting ASK1 dimer is shown in green.

Mentions: In order to determine the sequence specificity of active site-mediated phosphorylation, we screened a peptide library with recombinant ASK1 (Figure 5A). The library was constructed to evaluate the contribution of all amino acid residues at each of nine positions (−5 to +4) surrounding a fixed central phosphoacceptor site (Hutti et al., 2004). The phosphorylation data revealed that ASK1 has a substantial preference for threonine over serine as a phospho-acceptor. ASK1 appears to be most selective at position +1 relative to the phosphorylation site, where it strongly prefers both aromatic and aliphatic hydrophobic residues in peptide substrates. We also observed strong phosphorylation of peptides bearing threonine residues in either the −2 or the +2 position. Because peptides in the library with fixed threonine residues can theoretically be phosphorylated at more than one site, these signals could reflect authentic selection for threonine at only one of the two positions. ASK1 also has secondary preferences for glutamine at the −2 position and for serine, arginine, and tyrosine at the +2 position. The kinase is not strongly selective at any of the other positions represented in the peptide library. The determined consensus motif is shown in Figure 5B.


Structural and functional characterization of the human protein kinase ASK1.

Bunkoczi G, Salah E, Filippakopoulos P, Fedorov O, Müller S, Sobott F, Parker SA, Zhang H, Min W, Turk BE, Knapp S - Structure (2007)

Substrate Specificity of ASK1 and Identification of Autophosphorylation Sites(A) Phosphorylation motifs for ASK1. Biotinylated peptides bearing the indicated residue at the indicated position relative to a central Ser/Thr phosphoacceptor site were subjected to phosphorylation by ASK1 with radiolabeled ATP. Aliquots of each reaction were subsequently spotted onto a streptavidin membrane, which was washed, dried, and exposed to a phosphor screen. Shown is a representative array from three separate experiments. Quantified spot intensities representing the average of the three runs are provided in Table S2.(B) Consensus sequence determined from the peptide array data. The consensus sequence is shown in bold and alternative residues are indicated at each position by smaller italic letters. The site of phosphorylation is indicated in red and by a star (∗). The three autophosphorylation sites are also shown, and residues matching the consensus sequence are highlighted with yellow boxes.(C) Activation segments of the known ASK1 substrates of the MKK family of kinases. Residues phosphorylated by activation are highlighted in red.(D) Location of the three autophosphorylation sites in ASK1. Phosphorylated residues are labeled in red and are shown as spheres, whereas neighboring residues are labeled in blue and are indicated in ball-and-stick representation. The interacting ASK1 dimer is shown in green.
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fig5: Substrate Specificity of ASK1 and Identification of Autophosphorylation Sites(A) Phosphorylation motifs for ASK1. Biotinylated peptides bearing the indicated residue at the indicated position relative to a central Ser/Thr phosphoacceptor site were subjected to phosphorylation by ASK1 with radiolabeled ATP. Aliquots of each reaction were subsequently spotted onto a streptavidin membrane, which was washed, dried, and exposed to a phosphor screen. Shown is a representative array from three separate experiments. Quantified spot intensities representing the average of the three runs are provided in Table S2.(B) Consensus sequence determined from the peptide array data. The consensus sequence is shown in bold and alternative residues are indicated at each position by smaller italic letters. The site of phosphorylation is indicated in red and by a star (∗). The three autophosphorylation sites are also shown, and residues matching the consensus sequence are highlighted with yellow boxes.(C) Activation segments of the known ASK1 substrates of the MKK family of kinases. Residues phosphorylated by activation are highlighted in red.(D) Location of the three autophosphorylation sites in ASK1. Phosphorylated residues are labeled in red and are shown as spheres, whereas neighboring residues are labeled in blue and are indicated in ball-and-stick representation. The interacting ASK1 dimer is shown in green.
Mentions: In order to determine the sequence specificity of active site-mediated phosphorylation, we screened a peptide library with recombinant ASK1 (Figure 5A). The library was constructed to evaluate the contribution of all amino acid residues at each of nine positions (−5 to +4) surrounding a fixed central phosphoacceptor site (Hutti et al., 2004). The phosphorylation data revealed that ASK1 has a substantial preference for threonine over serine as a phospho-acceptor. ASK1 appears to be most selective at position +1 relative to the phosphorylation site, where it strongly prefers both aromatic and aliphatic hydrophobic residues in peptide substrates. We also observed strong phosphorylation of peptides bearing threonine residues in either the −2 or the +2 position. Because peptides in the library with fixed threonine residues can theoretically be phosphorylated at more than one site, these signals could reflect authentic selection for threonine at only one of the two positions. ASK1 also has secondary preferences for glutamine at the −2 position and for serine, arginine, and tyrosine at the +2 position. The kinase is not strongly selective at any of the other positions represented in the peptide library. The determined consensus motif is shown in Figure 5B.

Bottom Line: Here, we present the structure of the human ASK1 catalytic domain in complex with staurosporine.Reporter gene assays showed that all three identified in vitro autophosphorylation sites (Thr813, Thr838, Thr842) regulate ASK1 signaling, but site-directed mutants showed catalytic activities similar to wild-type ASK1, suggesting a regulatory mechanism independent of ASK1 kinase activity.The determined high-resolution structure of ASK1 and identified ATP mimetic inhibitors will provide a first starting point for the further development of selective inhibitors.

View Article: PubMed Central - PubMed

Affiliation: University of Oxford, Structural Genomics Consortium, Botnar Research Centre, Oxford OX3 7LD, United Kingdom.

ABSTRACT
Apoptosis signal-regulating kinase 1 (ASK1) plays an essential role in stress and immune response and has been linked to the development of several diseases. Here, we present the structure of the human ASK1 catalytic domain in complex with staurosporine. Analytical ultracentrifugation (AUC) and crystallographic analysis showed that ASK1 forms a tight dimer (K(d) approximately 0.2 microM) interacting in a head-to-tail fashion. We found that the ASK1 phosphorylation motifs differ from known ASK1 phosphorylation sites but correspond well to autophosphorylation sites identified by mass spectrometry. Reporter gene assays showed that all three identified in vitro autophosphorylation sites (Thr813, Thr838, Thr842) regulate ASK1 signaling, but site-directed mutants showed catalytic activities similar to wild-type ASK1, suggesting a regulatory mechanism independent of ASK1 kinase activity. The determined high-resolution structure of ASK1 and identified ATP mimetic inhibitors will provide a first starting point for the further development of selective inhibitors.

Show MeSH
Related in: MedlinePlus