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 ΔMEKK1 increased overall ATF2 ubiquitination, presumably because of the enhanced dimerization of ATF2. Treatment of ΔMEKK1-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 (ATF2Δ150–248) showed that activation of JNK or p38 kinase renders ATF2 resistant to ubiquitination and degradation. This effect is mediated by JNK/p38-dependent phosphorylation of ATF2 at Thr-69 and Thr-71, because the phosphorylation-deficient mutant (ATF2Δ150–248-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 α-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 co-treatment 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.

[1]  T. Ito,et al.  Ceramide Induces Bcl2 Dephosphorylation via a Mechanism Involving Mitochondrial PP2A* , 1999, The Journal of Biological Chemistry.

[2]  A. Zeiher,et al.  Dephosphorylation Targets Bcl-2 for Ubiquitin-dependent Degradation: A Link between the Apoptosome and the Proteasome Pathway , 1999, The Journal of experimental medicine.

[3]  Z. Ronai,et al.  Ubiquitination and Degradation of ATF2 Are Dimerization Dependent , 1999, Molecular and Cellular Biology.

[4]  P. Dent,et al.  Inhibition of the MAPK pathway abrogates BCL2-mediated survival of leukemia cells after exposure to low-dose ionizing radiation. , 1999, Radiation research.

[5]  C. Prives,et al.  The p53 pathway , 1999, The Journal of pathology.

[6]  V. Adler,et al.  JNK targets p53 ubiquitination and degradation in nonstressed cells. , 1998, Genes & development.

[7]  M. Karin,et al.  Stabilization of interleukin-2 mRNA by the c-Jun NH2-terminal kinase pathway. , 1998, Science.

[8]  Kohei Miyazono,et al.  Mammalian thioredoxin is a direct inhibitor of apoptosis signal‐regulating kinase (ASK) 1 , 1998, The EMBO journal.

[9]  R. Firestein,et al.  Association of Activating Transcription Factor 2 (ATF2) with the Ubiquitin-conjugating Enzyme hUBC9 , 1998, The Journal of Biological Chemistry.

[10]  T. Nakamura,et al.  Activation of the rat cyclin A promoter by ATF2 and Jun family members and its suppression by ATF4. , 1998, Experimental cell research.

[11]  V. Fried,et al.  c-Jun NH2-terminal Kinases Target the Ubiquitination of Their Associated Transcription Factors* , 1997, The Journal of Biological Chemistry.

[12]  T. Collins,et al.  Tumor Necrosis Factor α-Induced E-selectin Expression Is Activated by the Nuclear Factor-κB and c-JUN N-terminal Kinase/p38 Mitogen-activated Protein Kinase Pathways* , 1997, The Journal of Biological Chemistry.

[13]  Dirk Bohmann,et al.  Reduced Ubiquitin-Dependent Degradation of c-Jun After Phosphorylation by MAP Kinases , 1997, Science.

[14]  Z. Ronai,et al.  Phosphorylation-dependent targeting of c-Jun ubiquitination by Jun N-kinase. , 1996, Oncogene.

[15]  X. Y. Li,et al.  Intramolecular inhibition of activating transcription factor-2 function by its DNA-binding domain. , 1996, Genes & development.

[16]  R. Davis,et al.  MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway , 1996, Molecular and cellular biology.

[17]  A. Rao,et al.  Tumor necrosis factor alpha gene regulation in activated T cells involves ATF-2/Jun and NFATp , 1996, Molecular and cellular biology.

[18]  R. Hipskind,et al.  Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1 , 1995, Molecular and cellular biology.

[19]  I. Herr,et al.  ATF‐2 is preferentially activated by stress‐activated protein kinases to mediate c‐jun induction in response to genotoxic agents. , 1995, The EMBO journal.

[20]  N. Jones,et al.  ATF‐2 contains a phosphorylation‐dependent transcriptional activation domain. , 1995, The EMBO journal.

[21]  B. Dérijard,et al.  Transcription factor ATF2 regulation by the JNK signal transduction pathway , 1995, Science.

[22]  G L Johnson,et al.  Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK. , 1994, Science.

[23]  L. Staszewski,et al.  Ubiquitin-dependent c-Jun degradation in vivo is mediated by the δ domain , 1994, Cell.

[24]  W. Beard,et al.  RNA polymerase II transcription. Rate of promoter clearance is enhanced by a purified activating transcription factor/cAMP response element-binding protein. , 1994, The Journal of biological chemistry.

[25]  M. Green,et al.  HTLV-I Tax protein stimulation of DNA binding of bZIP proteins by enhancing dimerization. , 1993, Science.

[26]  P. Herrlich,et al.  Heterodimer formation of cJun and ATF‐2 is responsible for induction of c‐jun by the 243 amino acid adenovirus E1A protein. , 1993, The EMBO journal.

[27]  Michael R. Green,et al.  Retinoblastoma gene product activates expression of the human TGF-β2 gene through transcription factor ATF-2 , 1992, Nature.

[28]  A. Siddiqui,et al.  HBV X protein alters the DNA binding specificity of CREB and ATF-2 by protein-protein interactions , 1991, Science.

[29]  Michael R. Green,et al.  A specific member of the ATF transcription factor family can mediate transcription activation by the adenovirus E1a protein , 1990, Cell.

[30]  Tsonwin Hai,et al.  Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. , 1989, Genes & development.

[31]  M. Yoshida,et al.  Leucine zipper structure of the protein CRE‐BP1 binding to the cyclic AMP response element in brain. , 1989, The EMBO journal.

[32]  P. Cohen The structure and regulation of protein phosphatases. , 1989, Annual review of biochemistry.