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Purification and kinetic characterization of recombinant human MAP3K COT and the complexes with its cellular partner NF-kB1 p105

Literature Life Science

Development of an HTRF assay using labeled anti phospho-MEK antibody and anti-tag reagent. RIA compared to non radioactive homogeneous HTRF method

Abstract

Cancer osaka thyroid (COT), a human MAP3K, is essential for lipopolysaccharide activation of the Erk MAPK cascade in macrophages. COT30-467 is insoluble, whereas low levels of COT30-397 can be expressed, but this protein is unstable. However, both COT30-467 and COT30-397 are expressed in a soluble and stable form when produced in complex with the C-terminal half of p105. The kcat of COT30-397 is reduced 47-fold in the COT30-467/p105DN complex. COT prefers Mn2+ to Mg2+ as the ATP metal cofactor, exhibiting an unusually high ATP Km in the presence of Mg2+. When using Mn2+ as the cofactor, the ATP Km 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.

Details

Arch Biochem Biophys. 2005;441(1):64-74.

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