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Conserved and distinct modes of CREB/ATF transcription factor regulation by PP2A/B56gamma and genotoxic stress.

Shanware NP, Zhan L, Hutchinson JA, Kim SH, Williams LM, Tibbetts RS - PLoS ONE (2010)

Bottom Line: Hyperphosphorylated ATF1 showed a 4-fold reduced affinity for CREB-binding protein.We further show that PP2A, in conjunction with its targeting subunit B56gamma, antagonized ATM and CK1/2-dependent phosphorylation of CREB and ATF1 in cellulo.These studies define overlapping and distinct modes of CREB and ATF1 regulation by phosphorylation that may ensure concerted changes in gene expression mediated by these factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Program in Molecular and Cellular Pharmacology and Molecular and Environmental and Toxicology Center, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.

ABSTRACT
Activating transcription factor 1 (ATF1) and the closely related proteins CREB (cyclic AMP resonse element binding protein) and CREM (cyclic AMP response element modulator) constitute a subfamily of bZIP transcription factors that play critical roles in the regulation of cellular growth, metabolism, and survival. Previous studies demonstrated that CREB is phosphorylated on a cluster of conserved Ser residues, including Ser-111 and Ser-121, in response to DNA damage through the coordinated actions of the ataxia-telangiectasia-mutated (ATM) protein kinase and casein kinases 1 and 2 (CK1/2). Here, we show that DNA damage-induced phosphorylation by ATM is a general feature of CREB and ATF1. ATF1 harbors a conserved ATM/CK cluster that is constitutively and stoichiometrically phosphorylated by CK1 and CK2 in asynchronously growing cells. Exposure to DNA damage further induced ATF1 phosphorylation on Ser-51 by ATM in a manner that required prior phosphorylation of the upstream CK residues. Hyperphosphorylated ATF1 showed a 4-fold reduced affinity for CREB-binding protein. We further show that PP2A, in conjunction with its targeting subunit B56gamma, antagonized ATM and CK1/2-dependent phosphorylation of CREB and ATF1 in cellulo. Finally, we show that CK sites in CREB are phosphorylated during cellular growth and that phosphorylation of these residues reduces the threshold of DNA damage required for ATM-dependent phosphorylation of the inhibitory Ser-121 residue. These studies define overlapping and distinct modes of CREB and ATF1 regulation by phosphorylation that may ensure concerted changes in gene expression mediated by these factors.

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DNA damage induces ATM dependent phosphorylation of ATF1 on Ser-51.(A) Sequence of peptide antigen used to generate α-pATF1-47/50/51 antibody. (B) Phosphatase sensitivity of α-pATF1-47/50/51 antibody. HEK 293T cells were transfected with Myc-ATF1WT plasmid. Cell extracts were prepared and treated with λ phosphatase prior to analysis by immunoblotting using α-Myc and α-pATF1-47/50/51 antibodies. (C) Phosphorylation site requirements and IR inducibility. HEK 293T cells were transfected with vector DNA (−), Myc-ATF1WT, Myc-ATF1S50A or Myc-ATF1S51A plasmids and exposed to IR (10 Gy, 2 h). Cell extracts were prepared and analyzed by immunoblotting using α-Myc and α-pATF1-47/50/51 antibodies. (D) IR dependent ATF1 phosphorylation is ATM dependent. HEK 293T cells either left untreated or treated with IR in the presence of 10 µM ATM inhibitor (KU-55933). Immunoprecipitation reactions were performed using a mock antibody or α-ATF1 antibody and immunoprecipitates were analyzed by immunoblotting using α-Myc and α-pATF1-47/50/51 antibodies. (E) The ATF1S36/41A mutant is defective for IR induced pATF1-47/50/51 phosphorylation. HEK 293T cells were transfected with plasmid DNA encoding Myc-ATF1WT or the Myc-ATF1S36/41A mutant and either left untreated or subjected to 10 Gy IR for 2 h. Cell extracts were prepared and analyzed by immunoblotting using α-Myc and α-pATF1-47/50/51 antibodies. (F) Hyperphosphorylated ATF1 shows reduced binding to the KIX domain of CBP. HEK 293T cells were transfected with plasmids encoding Myc-ATF1WT or the Myc-ATF1S36/41A mutant and either left untreated or subjected to 10 Gy IR. Cell extracts were prepared 2 h later, incubated with GST-KIX-loaded beads, and bound protein analyzed by immunoblotting using α-Myc antibodies. Numbers under GST-KIX result demote fold changes in ATF1 levels.
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pone-0012173-g002: DNA damage induces ATM dependent phosphorylation of ATF1 on Ser-51.(A) Sequence of peptide antigen used to generate α-pATF1-47/50/51 antibody. (B) Phosphatase sensitivity of α-pATF1-47/50/51 antibody. HEK 293T cells were transfected with Myc-ATF1WT plasmid. Cell extracts were prepared and treated with λ phosphatase prior to analysis by immunoblotting using α-Myc and α-pATF1-47/50/51 antibodies. (C) Phosphorylation site requirements and IR inducibility. HEK 293T cells were transfected with vector DNA (−), Myc-ATF1WT, Myc-ATF1S50A or Myc-ATF1S51A plasmids and exposed to IR (10 Gy, 2 h). Cell extracts were prepared and analyzed by immunoblotting using α-Myc and α-pATF1-47/50/51 antibodies. (D) IR dependent ATF1 phosphorylation is ATM dependent. HEK 293T cells either left untreated or treated with IR in the presence of 10 µM ATM inhibitor (KU-55933). Immunoprecipitation reactions were performed using a mock antibody or α-ATF1 antibody and immunoprecipitates were analyzed by immunoblotting using α-Myc and α-pATF1-47/50/51 antibodies. (E) The ATF1S36/41A mutant is defective for IR induced pATF1-47/50/51 phosphorylation. HEK 293T cells were transfected with plasmid DNA encoding Myc-ATF1WT or the Myc-ATF1S36/41A mutant and either left untreated or subjected to 10 Gy IR for 2 h. Cell extracts were prepared and analyzed by immunoblotting using α-Myc and α-pATF1-47/50/51 antibodies. (F) Hyperphosphorylated ATF1 shows reduced binding to the KIX domain of CBP. HEK 293T cells were transfected with plasmids encoding Myc-ATF1WT or the Myc-ATF1S36/41A mutant and either left untreated or subjected to 10 Gy IR. Cell extracts were prepared 2 h later, incubated with GST-KIX-loaded beads, and bound protein analyzed by immunoblotting using α-Myc antibodies. Numbers under GST-KIX result demote fold changes in ATF1 levels.

Mentions: While our previous data showed that ATF1 was stoichiometrically phosphorylated on CK sites in vivo, it was still not clear if ATF1 is a target of the DNA damage response. Ser-51 is positionally analogous to Ser-121 in CREB, which undergoes ATM-dependent phosphorylation in response to DNA damage (Fig. 1A). To test if Ser-51 is a target of the DNA damage in vivo, we generated an antibody against a peptide containing phosphorylated Ser-47, Ser-50 and Ser-51, using the rationale that Ser-47 and Ser-50 are likely phosphorylated by CK1 and CK2 (Fig. 2A). The α-pATF1-47/50/51 antibody was first tested against HEK 293T cell extract overexpressing Myc-tagged ATF1. The antibody displayed strong phosphatase-sensitive reactivity with Myc-ATF1 in HEK 293T cells (Fig. 2B). We further tested the specificity and IR-inducibility of the α-pATF1-47/50/51 antibody by assessing the effects of single Ser→Ala substitutions at Ser-50 and Ser-51 on basal and IR-induced immunoreactivity. Whereas wild-type Myc-ATF1 exhibited modest IR-induced phosphorylation, Myc-ATF1 harboring Ala substitutions at Ser-50- or Ser-51- failed to react with α-pATF1-47/50/51, indicating that the integrity of these sites was required for antibody recognition (Fig. 2C).


Conserved and distinct modes of CREB/ATF transcription factor regulation by PP2A/B56gamma and genotoxic stress.

Shanware NP, Zhan L, Hutchinson JA, Kim SH, Williams LM, Tibbetts RS - PLoS ONE (2010)

DNA damage induces ATM dependent phosphorylation of ATF1 on Ser-51.(A) Sequence of peptide antigen used to generate α-pATF1-47/50/51 antibody. (B) Phosphatase sensitivity of α-pATF1-47/50/51 antibody. HEK 293T cells were transfected with Myc-ATF1WT plasmid. Cell extracts were prepared and treated with λ phosphatase prior to analysis by immunoblotting using α-Myc and α-pATF1-47/50/51 antibodies. (C) Phosphorylation site requirements and IR inducibility. HEK 293T cells were transfected with vector DNA (−), Myc-ATF1WT, Myc-ATF1S50A or Myc-ATF1S51A plasmids and exposed to IR (10 Gy, 2 h). Cell extracts were prepared and analyzed by immunoblotting using α-Myc and α-pATF1-47/50/51 antibodies. (D) IR dependent ATF1 phosphorylation is ATM dependent. HEK 293T cells either left untreated or treated with IR in the presence of 10 µM ATM inhibitor (KU-55933). Immunoprecipitation reactions were performed using a mock antibody or α-ATF1 antibody and immunoprecipitates were analyzed by immunoblotting using α-Myc and α-pATF1-47/50/51 antibodies. (E) The ATF1S36/41A mutant is defective for IR induced pATF1-47/50/51 phosphorylation. HEK 293T cells were transfected with plasmid DNA encoding Myc-ATF1WT or the Myc-ATF1S36/41A mutant and either left untreated or subjected to 10 Gy IR for 2 h. Cell extracts were prepared and analyzed by immunoblotting using α-Myc and α-pATF1-47/50/51 antibodies. (F) Hyperphosphorylated ATF1 shows reduced binding to the KIX domain of CBP. HEK 293T cells were transfected with plasmids encoding Myc-ATF1WT or the Myc-ATF1S36/41A mutant and either left untreated or subjected to 10 Gy IR. Cell extracts were prepared 2 h later, incubated with GST-KIX-loaded beads, and bound protein analyzed by immunoblotting using α-Myc antibodies. Numbers under GST-KIX result demote fold changes in ATF1 levels.
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pone-0012173-g002: DNA damage induces ATM dependent phosphorylation of ATF1 on Ser-51.(A) Sequence of peptide antigen used to generate α-pATF1-47/50/51 antibody. (B) Phosphatase sensitivity of α-pATF1-47/50/51 antibody. HEK 293T cells were transfected with Myc-ATF1WT plasmid. Cell extracts were prepared and treated with λ phosphatase prior to analysis by immunoblotting using α-Myc and α-pATF1-47/50/51 antibodies. (C) Phosphorylation site requirements and IR inducibility. HEK 293T cells were transfected with vector DNA (−), Myc-ATF1WT, Myc-ATF1S50A or Myc-ATF1S51A plasmids and exposed to IR (10 Gy, 2 h). Cell extracts were prepared and analyzed by immunoblotting using α-Myc and α-pATF1-47/50/51 antibodies. (D) IR dependent ATF1 phosphorylation is ATM dependent. HEK 293T cells either left untreated or treated with IR in the presence of 10 µM ATM inhibitor (KU-55933). Immunoprecipitation reactions were performed using a mock antibody or α-ATF1 antibody and immunoprecipitates were analyzed by immunoblotting using α-Myc and α-pATF1-47/50/51 antibodies. (E) The ATF1S36/41A mutant is defective for IR induced pATF1-47/50/51 phosphorylation. HEK 293T cells were transfected with plasmid DNA encoding Myc-ATF1WT or the Myc-ATF1S36/41A mutant and either left untreated or subjected to 10 Gy IR for 2 h. Cell extracts were prepared and analyzed by immunoblotting using α-Myc and α-pATF1-47/50/51 antibodies. (F) Hyperphosphorylated ATF1 shows reduced binding to the KIX domain of CBP. HEK 293T cells were transfected with plasmids encoding Myc-ATF1WT or the Myc-ATF1S36/41A mutant and either left untreated or subjected to 10 Gy IR. Cell extracts were prepared 2 h later, incubated with GST-KIX-loaded beads, and bound protein analyzed by immunoblotting using α-Myc antibodies. Numbers under GST-KIX result demote fold changes in ATF1 levels.
Mentions: While our previous data showed that ATF1 was stoichiometrically phosphorylated on CK sites in vivo, it was still not clear if ATF1 is a target of the DNA damage response. Ser-51 is positionally analogous to Ser-121 in CREB, which undergoes ATM-dependent phosphorylation in response to DNA damage (Fig. 1A). To test if Ser-51 is a target of the DNA damage in vivo, we generated an antibody against a peptide containing phosphorylated Ser-47, Ser-50 and Ser-51, using the rationale that Ser-47 and Ser-50 are likely phosphorylated by CK1 and CK2 (Fig. 2A). The α-pATF1-47/50/51 antibody was first tested against HEK 293T cell extract overexpressing Myc-tagged ATF1. The antibody displayed strong phosphatase-sensitive reactivity with Myc-ATF1 in HEK 293T cells (Fig. 2B). We further tested the specificity and IR-inducibility of the α-pATF1-47/50/51 antibody by assessing the effects of single Ser→Ala substitutions at Ser-50 and Ser-51 on basal and IR-induced immunoreactivity. Whereas wild-type Myc-ATF1 exhibited modest IR-induced phosphorylation, Myc-ATF1 harboring Ala substitutions at Ser-50- or Ser-51- failed to react with α-pATF1-47/50/51, indicating that the integrity of these sites was required for antibody recognition (Fig. 2C).

Bottom Line: Hyperphosphorylated ATF1 showed a 4-fold reduced affinity for CREB-binding protein.We further show that PP2A, in conjunction with its targeting subunit B56gamma, antagonized ATM and CK1/2-dependent phosphorylation of CREB and ATF1 in cellulo.These studies define overlapping and distinct modes of CREB and ATF1 regulation by phosphorylation that may ensure concerted changes in gene expression mediated by these factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Program in Molecular and Cellular Pharmacology and Molecular and Environmental and Toxicology Center, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.

ABSTRACT
Activating transcription factor 1 (ATF1) and the closely related proteins CREB (cyclic AMP resonse element binding protein) and CREM (cyclic AMP response element modulator) constitute a subfamily of bZIP transcription factors that play critical roles in the regulation of cellular growth, metabolism, and survival. Previous studies demonstrated that CREB is phosphorylated on a cluster of conserved Ser residues, including Ser-111 and Ser-121, in response to DNA damage through the coordinated actions of the ataxia-telangiectasia-mutated (ATM) protein kinase and casein kinases 1 and 2 (CK1/2). Here, we show that DNA damage-induced phosphorylation by ATM is a general feature of CREB and ATF1. ATF1 harbors a conserved ATM/CK cluster that is constitutively and stoichiometrically phosphorylated by CK1 and CK2 in asynchronously growing cells. Exposure to DNA damage further induced ATF1 phosphorylation on Ser-51 by ATM in a manner that required prior phosphorylation of the upstream CK residues. Hyperphosphorylated ATF1 showed a 4-fold reduced affinity for CREB-binding protein. We further show that PP2A, in conjunction with its targeting subunit B56gamma, antagonized ATM and CK1/2-dependent phosphorylation of CREB and ATF1 in cellulo. Finally, we show that CK sites in CREB are phosphorylated during cellular growth and that phosphorylation of these residues reduces the threshold of DNA damage required for ATM-dependent phosphorylation of the inhibitory Ser-121 residue. These studies define overlapping and distinct modes of CREB and ATF1 regulation by phosphorylation that may ensure concerted changes in gene expression mediated by these factors.

Show MeSH
Related in: MedlinePlus