Stability of the ATF2 transcription factor is regulated by phosphorylation and dephosphorylation

Trans-activation of the activating transcription factor-2 (ATF2) in response to cellular stress requires the N-terminal phosphorylation of ATF2 by stress-activated protein kinases (SAPK). In this study, we investigated the role of ATF2 phosphorylation in the maintenance of ATF2 stability. Activation of SAPK by forced expression of Delta MEKK1 increased overall ATF2 ubiquitination, presumably because of the enhanced dimerization of ATF2. Treatment of Delta MEKK1-expressing cells with okadaic acid led to the increase in N-terminal phosphorylation, protection from ubiquitination, and accumulation of exogenously expressed ATF2, indicating the role of protein phosphatases in balancing the effects of stress kinases. Analysis of ubiquitination and degradation of the constitutively dimerized ATF2 mutant (ATF2(Delta 150-248)) showed that activation of JNK or p38 kinase renders ATF2 resistant to ubiquitination and degradation. This effect is mediated by JNKp38-dependent phosphorylation of ATF2 at Thr-69 and Thr-71, because the phosphorylation-deficient mutant (ATF2(Delta 150-248-T69A,T71A)) was not protected from ubiquitination and degradation by the activation of SAPK, Treatment of cells with okadaic acid elevated the tumor necrosis factor alpha-induced ATF2 level and the extent of its specific N-terminal phosphorylation. Cycloheximide, which activates SAPK, while inhibiting protein synthesis, stabilized endogenous ATF2. However, treatment of cells with the high dose of SB203580, which inhibits JNK and p38 kinase, resulted in efficient degradation of ATF2 in cells exposed to cycloheximide. This degradation was abrogated by cotreatment with the proteasome inhibitor MG132. Our findings suggest that N-terminal phosphorylation of ATF2 dimers protect ATF2 from ubiquitination and degradation. We propose the hypothesis that the balance between SAPK and protein phosphatases affects the duration and magnitude of ATF2 transcriptional output because of the effect on substrate recognition for ubiquitination and degradation.