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

The mitogen-activated protein kinases (MAPKs) are a family of enzymes conserved among eukaryotes that regulate cellular activities in response to numerous external signals. They are the terminal component of a three-kinase cascade that is evolutionarily conserved and whose arrangement appears to offer considerable flexibility in encompassing the diverse biological situations for which they are employed. Although multistep protein phosphorylation within mitogen-activated protein kinase (MAPK) cascades can dramatically influence the sensitivity of signal propagation, an investigation of the mechanism 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 of the glutathione S-transferase fusion protein of the trans-activation domain of activating transcription factor-a (GST-ATFB-(1-115)) by p38 MAPK alpha (p38 alpha) as a model system for the phosphorylation of ATF2 by p38 alpha. Our experiments demonstrated that GST-ATF2-(1-115) is phosphorylated in a two-step distributive mechanism, where p38 alpha dissociates from GST-ATFB-(1-115) after the initial phosphorylation of either Thr-69 or Thr-71, Whereas p38 alpha showed similar specificity for Thr-71 and Thr-69 in the unphosphorylated protein, it displayed a marked difference in specificity toward the mono-phosphoisomers. Phosphorylation of Thr-71 had no significant effect on the rate of Thr-69 phosphorylation, but Thr-69 phosphorylation reduced 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 into how the kinetics of a two-step distributive mechanism can be adapted to modulate effectively the sensitivity of a signal transduction pathway. This work also suggests that whereas MAPKs utilize docking interactions to bind substrates, they can be weak and transient in nature, providing just enough binding energy to promote the phosphorylation of a specific substrate.