An intrinsic ATPase activity of phospho-MEK-1 uncoupled from downstream ERK phosphorylation.
We have developed a highly sensitive assay of MEK-mediated ATP hydrolysis by coupling the formation of ADP to NADH oxidation through the enzymes pyruvate kinase and lactate dehydrogenase. Robust ATP hydrolysis is catalyzed by phosphorylated MEK in the absence of the protein substrate ERK. This ERK-uncoupled ATPase activity is dependent on the phosphorylation status of MEK and is abrogated by the selective MEK kinase inhibitor U0126. ADP production is concomitant with Raf-mediated phosphorylation of MEK. Based on this finding, a coupled Raf/MEK assay is developed for measuring the Raf activity. A kinetic treatment derived under steady-state assumptions is presented for the analysis of the reaction progress curve generated by this coupled assay. We have shown that inhibitory potency of selective Raf inhibitors can be determined accurately by this assay.
Rominger
Cynthia M
CM
Department of Enzymology and Mechanistic Pharmacology, GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Road, Collegeville, PA 19426, USA.
Schaber
Michael D
MD
Yang
Jingsong
J
Gontarek
Richard R
RR
Weaver
Kurt L
KL
Broderick
Timothy
T
Carter
Luke
L
Copeland
Robert A
RA
May
Earl W
EW
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D048369:MAP Kinase Kinase 1 / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D048493:Proto-Oncogene Proteins B-raf / Q000378:metabolism; D011817:Rabbits; D015398:Signal Transduction; D013997:Time Factors
D002070:Butadienes; D009570:Nitriles; D010710:Phosphates; C113580:U 0126; D000255:Adenosine Triphosphate; D048493:Proto-Oncogene Proteins B-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D048369:MAP Kinase Kinase 1; D000251:Adenosine Triphosphatases