The kinetic mechanism of the dual phosphorylation of the ATF2 transcription factor by p38 mitogen-activated protein (MAP) kinase alpha - Implicationsfor signal/response profiles of map kinase pathways

The mitogen-activated protein kinases (MAPKs) are a family of enzymes conse rved among eukaryotes that regulate cellular activities in response to nume rous external signals. They are the terminal component of a three-kinase ca scade that is evolutionarily conserved and whose arrangement appears to off er considerable flexibility in encompassing the diverse biological situatio ns for which they are employed. Although multistep protein phosphorylation within mitogen-activated protein kinase (MAPK) cascades can dramatically in fluence the sensitivity of signal propagation, an investigation of the mech anism of multisite phosphorylation by a MAPK has not been reported. Here we report a kinetic examination of the phosphorylation of Thr-69 and Thr-71 o f the glutathione S-transferase fusion protein of the trans-activation doma in of activating transcription factor-a (GST-ATFB-(1-115)) by p38 MAPK alph a (p38 alpha) as a model system for the phosphorylation of ATF2 by p38 alph a. Our experiments demonstrated that GST-ATF2-(1-115) is phosphorylated in a two-step distributive mechanism, where p38 alpha dissociates from GST-ATF B-(1-115) after the initial phosphorylation of either Thr-69 or Thr-71, Whe reas p38 alpha showed similar specificity for Thr-71 and Thr-69 in the unph osphorylated protein, it displayed a marked difference in specificity towar d the mono-phosphoisomers. Phosphorylation of Thr-71 had no significant eff ect on the rate of Thr-69 phosphorylation, but Thr-69 phosphorylation reduc ed the specificity, k(cat)/K-M, of p38 alpha for Thr-71 by approximately 40 -fold. Computer simulation of the mechanism suggests that the activation of ATF2 by p38 alpha in vivo is essentially Michaelian and provides insight i nto how the kinetics of a two-step distributive mechanism can be adapted to modulate effectively the sensitivity of a signal transduction pathway. Thi s work also suggests that whereas MAPKs utilize docking interactions to bin d substrates, they can be weak and transient in nature, providing just enou gh binding energy to promote the phosphorylation of a specific substrate.