Fully activated MEK1 exhibits compromised affinity for binding of allosteric inhibitors U0126 and PD0325901.

Kinases catalyze the transfer of γ-phosphate from ATP to substrate protein residues triggering signaling pathways responsible for a plethora of cellular events. Isolation and production of homogeneous preparations of kinases in their fully active forms is important for accurate in vitro measurements of activity, stability, and ligand binding properties of these proteins. Previous studies have shown that MEK1 can be produced in its active phosphorylated form by coexpression with RAF1 in insect cells. In this study, using activated MEK1 produced by in vitro activation by RAF1 (pMEK1in vitro), we demonstrate that the simultaneous expression of RAF1 for production of activated MEK1 does not result in stoichiometric phosphorylation of MEK1. The pMEK1in vitro showed higher specific activity toward ERK2 protein substrate compared to the pMEK1 that was activated via coexpression with RAF1 (pMEK1in situ). The two pMEK1 preparations showed quantitative differences in the phosphorylation of T-loop residue serine 222...