An intrinsic ATPase activity of phospho-MEK-1 uncoupled from downstream ERK phosphorylation
| Autor: | Timothy Broderick, Luke H. Carter, Michael D. Schaber, Robert A. Copeland, Earl May, Richard R. Gontarek, Cynthia M. Rominger, Kurt Weaver, Jingsong Yang |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Proto-Oncogene Proteins B-raf
MAPK/ERK pathway Time Factors MAP Kinase Kinase 1 Biophysics Biochemistry Phosphates chemistry.chemical_compound Adenosine Triphosphate ATP hydrolysis Lactate dehydrogenase Nitriles Butadienes Escherichia coli Animals Humans Phosphorylation Extracellular Signal-Regulated MAP Kinases Molecular Biology Adenosine Triphosphatases chemistry.chemical_classification Kinase Hydrolysis Cell biology Kinetics Enzyme chemistry Rabbits Signal transduction Pyruvate kinase Signal Transduction |
| Zdroj: | Archives of Biochemistry and Biophysics. 464:130-137 |
| ISSN: | 0003-9861 |
| DOI: | 10.1016/j.abb.2007.04.004 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect