| Abstract
| - Activation of mitogen-activated protein kinases (MAPKs), alsoknown as extracellular-signal-regulated kinases (ERKs), by MAPK/extracellular protein kinase kinases(MEKs) requires phosphorylationat two sites. The first step in MAPK activation by MEK must be theformation of a MEK·MAPK enzyme−substrate complex, followed by phosphorylation producingmonophosphorylated MAPK (pMAPK).Subsequently, one of two events may occur. (1) MEK catalyzesthe second and fully activatingphosphorylation of MAPK, producing ppMAPK (a processive mechanism).(2) The complex ofMEK·pMAPK dissociates before the second phosphorylation occurs, fullactivation requiring a reassociationof pMAPK with MEK (a nonprocessive or distributive mechanism).Simulations indicate that these twomechanisms predict different kinetics of MAPK activation.Specifically, the nonprocessive mechanismpredicts that there will be a paradoxical decrease in the rate of MAPKactivation as the MAPK concentrationis increased. The present study uses p42 MAPK, also known as ERK2,and MEK1 as representatives oftheir respective classes of enzymes. We find that increasing theERK2 concentration decreases the rateof activation by a mechanism which does not involve inhibition of MEK1function. The accumulationof the active, doubly phosphorylated ERK2 (ppERK2) was directlyassessed using a phosphorylation-state-specific antibody. The rate of accumulation of ppERK2 isdecreased by increasing the ERK2concentration. Therefore, the mechanism of ERK2 activation by MEK1in vitro is nonprocessive.
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