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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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Effects of Mutation at T813A, T838A, and T842A on ASK1 Activity(A) 293T cells were transfected with various ASK1 mutants in the presence of an ASK1-JNK-dependent reporter gene. A renilla construct was cotransfected as an internal control. HIPK1-WT and T838A were used as controls. Both luciferase and renilla units were measured. Relative luciferase activities are presented from mean of duplicate samples by taking vector control as 1. Similar results were obtained from two additional experiments. Data are presented as mean of duplicates from two independent experiments. ASK1 protein expression was determined by Western blot (lower panel) with anti-HA-POD (anti-HA-conjugated peroxidase; Roche).(B) The ASK signaling pathway was reconstituted in vitro with recombinant ASK1 and its phosphorylation site mutants, MKK6 as well as p38. p38 phosphorylation was detected with an antibody specific phosphorylated p38. Corresponding Coomassie gels as well as a his-tag-specific antibody has been used to demonstrate identical loading concentrations of the samples.
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fig6: Effects of Mutation at T813A, T838A, and T842A on ASK1 Activity(A) 293T cells were transfected with various ASK1 mutants in the presence of an ASK1-JNK-dependent reporter gene. A renilla construct was cotransfected as an internal control. HIPK1-WT and T838A were used as controls. Both luciferase and renilla units were measured. Relative luciferase activities are presented from mean of duplicate samples by taking vector control as 1. Similar results were obtained from two additional experiments. Data are presented as mean of duplicates from two independent experiments. ASK1 protein expression was determined by Western blot (lower panel) with anti-HA-POD (anti-HA-conjugated peroxidase; Roche).(B) The ASK signaling pathway was reconstituted in vitro with recombinant ASK1 and its phosphorylation site mutants, MKK6 as well as p38. p38 phosphorylation was detected with an antibody specific phosphorylated p38. Corresponding Coomassie gels as well as a his-tag-specific antibody has been used to demonstrate identical loading concentrations of the samples.

Mentions: To study the role of each of the identified autophosphorylation sites, we cloned site-directed alanine mutants into an expression vector and studied the effects of the mutations in transient transfection assays, monitoring JNK/p38 reporter gene activity. As expected from earlier studies, mutation of Thr838 drastically reduced reporter gene activity when compared to unstimulated control levels. Interestingly, mutation of the other two sites also provided a significant reduction in ASK1 function (Figure 6A), suggesting that autophosphorylation at the residues Thr842 and Thr813 regulates ASK1 signaling.


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)

Effects of Mutation at T813A, T838A, and T842A on ASK1 Activity(A) 293T cells were transfected with various ASK1 mutants in the presence of an ASK1-JNK-dependent reporter gene. A renilla construct was cotransfected as an internal control. HIPK1-WT and T838A were used as controls. Both luciferase and renilla units were measured. Relative luciferase activities are presented from mean of duplicate samples by taking vector control as 1. Similar results were obtained from two additional experiments. Data are presented as mean of duplicates from two independent experiments. ASK1 protein expression was determined by Western blot (lower panel) with anti-HA-POD (anti-HA-conjugated peroxidase; Roche).(B) The ASK signaling pathway was reconstituted in vitro with recombinant ASK1 and its phosphorylation site mutants, MKK6 as well as p38. p38 phosphorylation was detected with an antibody specific phosphorylated p38. Corresponding Coomassie gels as well as a his-tag-specific antibody has been used to demonstrate identical loading concentrations of the samples.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2100151&req=5

fig6: Effects of Mutation at T813A, T838A, and T842A on ASK1 Activity(A) 293T cells were transfected with various ASK1 mutants in the presence of an ASK1-JNK-dependent reporter gene. A renilla construct was cotransfected as an internal control. HIPK1-WT and T838A were used as controls. Both luciferase and renilla units were measured. Relative luciferase activities are presented from mean of duplicate samples by taking vector control as 1. Similar results were obtained from two additional experiments. Data are presented as mean of duplicates from two independent experiments. ASK1 protein expression was determined by Western blot (lower panel) with anti-HA-POD (anti-HA-conjugated peroxidase; Roche).(B) The ASK signaling pathway was reconstituted in vitro with recombinant ASK1 and its phosphorylation site mutants, MKK6 as well as p38. p38 phosphorylation was detected with an antibody specific phosphorylated p38. Corresponding Coomassie gels as well as a his-tag-specific antibody has been used to demonstrate identical loading concentrations of the samples.
Mentions: To study the role of each of the identified autophosphorylation sites, we cloned site-directed alanine mutants into an expression vector and studied the effects of the mutations in transient transfection assays, monitoring JNK/p38 reporter gene activity. As expected from earlier studies, mutation of Thr838 drastically reduced reporter gene activity when compared to unstimulated control levels. Interestingly, mutation of the other two sites also provided a significant reduction in ASK1 function (Figure 6A), suggesting that autophosphorylation at the residues Thr842 and Thr813 regulates ASK1 signaling.

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