Probing kinase activation and substrate specificity with an engineered monomeric IKK2
| Autor: | W. Eric Rogers, De-Bin Huang, Tom Huxford, Jacob D. Shaul, Gourisankar Ghosh, Arthur V. Hauenstein |
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| Rok vydání: | 2014 |
| Předmět: |
Biochemistry & Molecular Biology
Protein subunit 1.1 Normal biological development and functioning Plasma protein binding IκB kinase Biology Medical Biochemistry and Metabolomics Crystallography X-Ray Protein Engineering Biochemistry Article Substrate Specificity Enzyme activator Medicinal and Biomolecular Chemistry Underpinning research Humans Phosphorylation CHUK Crystallography Kinase I-Kappa-B Kinase Substrate (chemistry) I-kappa B Kinase Enzyme Activation Biophysics X-Ray Generic health relevance Biochemistry and Cell Biology Protein Multimerization Protein Binding |
| Zdroj: | Biochemistry, vol 53, iss 12 Biochemistry |
| Popis: | Catalytic subunits of the IκB kinase (IKK), IKK1/IKKα, and IKK2/IKKβ function in vivo as dimers in association with the necessary scaffolding subunit NEMO/IKKγ. Recent X-ray crystal structures of IKK2 suggested that dimerization might be mediated by a smaller protein-protein interaction than previously thought. Here, we report that removal of a portion of the scaffold dimerization domain (SDD) of human IKK2 yields a kinase subunit that remains monomeric in solution. Expression in baculovirus-infected Sf9 insect cells and purification of this engineered monomeric human IKK2 enzyme allows for in vitro analysis of its substrate specificity and mechanism of activation. We find that the monomeric enzyme, which contains all of the amino-terminal kinase and ubiquitin-like domains as well as the more proximal portions of the SDD, functions in vitro to direct phosphorylation exclusively to residues S32 and S36 of its IκBα substrate. Thus, the NF-κB-inducing potential of IKK2 is preserved in the engineered monomer. Furthermore, we observe that our engineered IKK2 monomer readily autophosphorylates activation loop serines 177 and 181 in trans. However, when residues that were previously observed to interfere with IKK2 trans autophosphorylation in transfected cells are mutated within the context of the monomer, the resulting Sf9 cell expressed and purified proteins were significantly impaired in their trans autophosphorylation activity in vitro. This study further defines the determinants of substrate specificity and provides additional evidence in support of a model in which activation via trans autophosphorylation of activation loop serines in IKK2 requires transient assembly of higher-order oligomers. |
| Databáze: | OpenAIRE |
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