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Updated: Jul 15, 2026

Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
An intrinsic ATPase activity of phospho-MEK-1 uncoupled from downstream ERK phosphorylation
Cynthia M Rominger1, Michael D Schaber, Jingsong Yang
1Department of Enzymology and Mechanistic Pharmacology, GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Road, Collegeville, PA 19426, USA.
Abstract:
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.
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Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
Protein Kinases and Phosphatases
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...

