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.
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.
Jia
Yong
Y
Abbott Bioresearch Center, Department of Biochemistry, Worcester, MA 01605, USA. yong.jia@abbott.com
Quinn
Christopher M
CM
Bump
Nancy J
NJ
Clark
Kevin M
KM
Clabbers
Anca
A
Hardman
Jennifer
J
Gagnon
Andrew
A
Kamens
Joanne
J
Tomlinson
Medha J
MJ
Wishart
Neil
N
Allen
Hamish
H
D001665:Binding Sites; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D020930:MAP Kinase Kinase Kinases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D016328:NF-kappa B / Q000737:chemistry*; D052002:NF-kappa B p50 Subunit; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D011498:Protein Precursors / Q000737:chemistry*; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013329:Structure-Activity Relationship
D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D011498:Protein Precursors; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D020930:MAP Kinase Kinase Kinases; C069937:MAP3K8 protein, human