The Kinetic Mechanism of the Dual Phosphorylation of the ATF2 Transcription Factor by p38 Mitogen-activated Protein (MAP) Kinase α

Abstract

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-2 (GST-ATF2-(1-115)) by p38 MAPK␣ (p38␣) as a model system for the phosphorylation of ATF2 by p38␣. Our experiments demonstrated that GST-ATF2-(1-115) is phosphorylated in a two-step distributive mechanism, where p38␣ dissociates from GST-ATF2-(1-115) after the initial phosphorylation of either Thr-69 or Thr-71. Whereas p38␣ 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␣ for Thr-71 by approximately 40-fold. Computer simulation of the mechanism suggests that the activation of ATF2 by p38␣ 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.

The Serine Protease Plasmin Triggers Expression of MCP-1 and CD40 in Human Primary Monocytes via Activation of p38 MAPK and Janus Kinase (JAK)/STAT Signaling Pathways

“…Indeed, activated p38 MAPK can phosphorylate various transcription factors including cAMP-response element-binding protein (CREB) and ATF2, which are known to bind to the positive regulatory element in the promoter region of the c-fos gene and activate its transcription. Besides, ATF2 and c-Fos can heterodimerize with c-Jun, forming AP-1 transcription factor complexes that bind to AP-1 binding sites, which activate transcription of numerous proinflammatory genes (12,39,54). In line with such a signaling cascade and complementing our previous findings of nuclear translocation of AP-1 in plasminstimulated monocytes (8), both phosphorylated ATF2 and cJun were identified as components of the AP-1 complex after plasmin stimulation.…”

Activating Transcription Factor 2 Mediates Matrix Metalloproteinase-2 Transcriptional Activation Induced by p38 in Breast Epithelial Cells

“…Because the transcription factor ATF2 has been known to be phosphorylated and activated by p38 in several cell types (16,23,24), it is probable that ATF2 is activated by MKK6/p38 and H-Ras in MCF10A cells. To test this issue, we detected phosphorylated ATF2 in the nuclei of MKK6-and H-Ras-activated cells.…”

MAPK Kinase 3 Is a Tumor Suppressor with Reduced Copy Number in Breast Cancer

“…It is well established that MKK3 signaling through the p38 MAPK protein family culminates in the activation of downstream transcription factors, including activating transcription factor 2 (ATF2; ref. 19). Monitoring of ATF2 phosphorylation confirmed constitutive activity in MKK3ca cells and impaired signaling in MKK3dn cells, detectable in cells stimulated for activation of the MAPK pathway through the SCF receptor ckit (Fig.…”