Purification and kinetic characterization of recombinant human mitogen-activated protein kinase kinase kinase COT and the complexes with its cellular partner NF-kappa B1 p105
Autor: | Andrew I. Gagnon, Joanne Kamens, Hamish Allen, Jennifer Hardman, Kevin M. Clark, Neil Wishart, Nancy J. Bump, Anca Clabbers, Medha J. Tomlinson, Yong Jia, Christopher M. Quinn |
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Rok vydání: | 2005 |
Předmět: |
Biophysics
Mitogen-activated protein kinase kinase Protein Engineering Biochemistry Cofactor law.invention Jurkat Cells Structure-Activity Relationship law Proto-Oncogene Proteins Enzyme Stability Humans Enzyme kinetics Kinase activity Protein Precursors Molecular Biology Binding Sites MAP kinase kinase kinase biology Chemistry Kinase NF-kappa B NF-kappa B p50 Subunit MAP Kinase Kinase Kinases Molecular biology Recombinant Proteins Enzyme Activation Kinetics Mitogen-activated protein kinase Recombinant DNA biology.protein Protein Binding |
Zdroj: | Archives of biochemistry and biophysics. 441(1) |
ISSN: | 0003-9861 |
Popis: | Cancer osaka thyroid (COT), a human MAP 3 K, is essential for lipopolysaccharide activation of the Erk MAPK cascade in macrophages. COT 30--467 is insoluble, whereas low levels of COT 30--397 can be expressed, but this protein is unstable. However, both COT 30--467 and COT 30--397 are expressed in a soluble and stable form when produced in complex with the C-terminal half of p105. The k(cat) of COT 30--397 is reduced approximately 47--fold in the COT 30--467/p105 Delta N complex. COT prefers Mn(2+) to Mg(2+) as the ATP metal cofactor, exhibiting an unusually high ATP K(m) in the presence of Mg(2+). When using Mn(2+) as the cofactor, the ATP K(m) is reduced to a level typical of most kinases. In contrast, the binding affinity of COT for its other substrate MEK is cofactor independent. Our results using purified proteins indicate that p105 binding improves COT solubility and stability while down-regulating kinase activity, consistent with cellular data showing that p105 functions as an inhibitor of COT. |
Databáze: | OpenAIRE |
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