Abstract
Apoptosis signal-regulating kinase 1 (ASK1, also known as MAP3K5), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, regulates diverse physiological processes. The activity of ASK1 is triggered by various stress stimuli and is involved in the pathogenesis of cancer, neurodegeneration, inflammation, and diabetes. ASK1 forms a high molecular mass complex whose activity is, under non-stress conditions, suppressed through interaction with thioredoxin and the scaffolding protein 14-3-3. The 14-3-3 protein binds to the phosphorylated Ser-966 motif downstream of the ASK1 kinase domain. The role of 14-3-3 in the inhibition of ASK1 has yet to be elucidated. In this study we performed structural analysis of the complex between the ASK1 kinase domain phosphorylated at Ser-966 (pASK1-CD) and the 14-3-3ζ protein. Small angle x-ray scattering (SAXS) measurements and chemical cross-linking revealed that the pASK1-CD·14-3-3ζ complex is dynamic and conformationally heterogeneous. In addition, structural analysis coupled with the results of phosphorus NMR and time-resolved tryptophan fluorescence measurements suggest that 14-3-3ζ interacts with the kinase domain of ASK1 in close proximity to its active site, thus indicating this interaction might block its accessibility and/or affect its conformation.
Highlights
ResultsBiophysical Characterization of the pASK1-CD1⁄714-3-3 Complex—The interaction between pASK1-CD and 14-3-3 was first studied using sedimentation velocity (SV) analytical ultracentrifugation (AUC) by analyzing their mixtures at various molar ratios (Fig. 1D)
Experiments were recorded at 23 °C on samples containing 100 –200 M pASK1-CD and 500 –900 M 14-3-3⌬C dissolved in 50 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 5 mM DTT, 10% (w/v) glycerol, and 10% (v/v) D2O
Samples were placed in a thermostatic holder, and all experiments were performed at 23 °C in buffer containing 50 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 5 mM DTT, 10% (w/v) glycerol, and 0.05% (w/v) Nonidet P-40
Summary
Biophysical Characterization of the pASK1-CD1⁄714-3-3 Complex—The interaction between pASK1-CD and 14-3-3 was first studied using sedimentation velocity (SV) analytical ultracentrifugation (AUC) by analyzing their mixtures at various molar ratios (Fig. 1D). 14-3-3⌬C showed significantly increased binding affinity for pASK1-CD with the best-fit KD value of 4 Ϯ 2 M (Fig. 1E). 14-3-3 Binding Suppresses the Kinase Activity of pASK1CD—The pASK1-CD used in this study is enzymatically active, allowing us to perform activity measurements to compare the properties of complexed and free pASK1-CD. The addition of 14-3-3⌬C suppressed the activity of pASK1-CD relative to the uncomplexed protein by ϳ40%, whereas no significant decrease in kinase activity was observed for the 14-3-3 binding-defective mutant pASK1-CD S966A (Fig. 1F, the specific activities are listed in supplemental Table S1). Characterization of the pASK1-CD1⁄714-3-3 Complex by SAXS—The scattering data for 14-3-3⌬C alone and ASK1-CD alone as well as the pASK1-CD1⁄714-3-3⌬C complex (mixed with 2:2 molar stoichiometry) were collected at various concentrations (Table 1). The Guinier plots and the concentration dependence of the forward scattering intensity I(0), the Porod volume Vp, and the radius of gyration Rg were used to assess the data quality
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