Single-well monitoring of protein-protein interaction and phosphorylation-dephosphorylation events.
We combined oxygen channeling assays with two distinct chemiluminescent beads to detect simultaneously protein phosphorylation and interaction events that are usually monitored separately. This novel method was tested in the ERK1/2 MAP kinase pathway. It was first used to directly monitor dissociation of MAP kinase ERK2 from MEK1 upon phosphorylation and to evaluate MAP kinase phosphatase (MKP) selectivity and mechanism of action. In addition, MEK1 and ERK2 were probed with an ATP competitor and an allosteric MEK1 inhibitor, which generated distinct phosphorylation-interaction patterns. Simultaneous monitoring of protein-protein interactions and substrate phosphorylation can provide significant mechanistic insight into enzyme activity and small molecule action.
Arcand
Mathieu
M
PerkinElmer BioSignal Inc., 1744 William Street, suite 600, Montréal, Québec, Canada H3J 1R4. mathieu.arcand@perkinelmer.com
Roby
Philippe
P
Bossé
Roger
R
Lipari
Francesco
F
Padrós
Jaime
J
Beaudet
Lucille
L
Marcil
Alexandre
A
Dahan
Sophie
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D001667:Binding, Competitive; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010101:Oxygen Consumption; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*
D011506:Proteins; D000255:Adenosine Triphosphate; D048049:Extracellular Signal-Regulated MAP Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases