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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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CREB/ATF1 regulate ATM transcription.(A) Schematic of the human ATM with putative CREs is shown. CREs conserved between human and mouse ATM promoters are in boldface. The arrow indicates the transcription start site. (B) Knockdown of CREB and ATF1 in MeWo cells. MeWo cells were transfected with shRNA for ATF1, siRNA for CREB or both and compared to cells expressing a non-targeting construct for 48 h. Cell extracts were subjected to immunoblotting with α-CREB, α-ATF1 and α-β-Tubulin antibodies. (C) Real-time PCR analysis of ATM mRNA in CREB/ATF1-deficient MeWo cells. Relative fold-expression levels of ATM mRNA normalized for GAPDH is shown. Error bars denote standard deviation from the mean from three independent experiments. (D) Effects of CREB and ATF1 knockdown on ATM protein levels. MeWo cells were transfected with shRNA for ATF1, siRNA for CREB or both and compared to cells expressing a non-targeting construct for 48 h before IR exposure. Cell extracts were prepared and subjected to immunoblotting with α-CREB, α-ATF1, α-ATM and α-β-Tubulin antibodies.
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pone-0012173-g005: CREB/ATF1 regulate ATM transcription.(A) Schematic of the human ATM with putative CREs is shown. CREs conserved between human and mouse ATM promoters are in boldface. The arrow indicates the transcription start site. (B) Knockdown of CREB and ATF1 in MeWo cells. MeWo cells were transfected with shRNA for ATF1, siRNA for CREB or both and compared to cells expressing a non-targeting construct for 48 h. Cell extracts were subjected to immunoblotting with α-CREB, α-ATF1 and α-β-Tubulin antibodies. (C) Real-time PCR analysis of ATM mRNA in CREB/ATF1-deficient MeWo cells. Relative fold-expression levels of ATM mRNA normalized for GAPDH is shown. Error bars denote standard deviation from the mean from three independent experiments. (D) Effects of CREB and ATF1 knockdown on ATM protein levels. MeWo cells were transfected with shRNA for ATF1, siRNA for CREB or both and compared to cells expressing a non-targeting construct for 48 h before IR exposure. Cell extracts were prepared and subjected to immunoblotting with α-CREB, α-ATF1, α-ATM and α-β-Tubulin antibodies.

Mentions: The ATM promoter contains positionally conserved CRE elements suggesting the interesting possibility that ATM is regulated by its own substrates (Fig. 5A and Ref. [2], [33]. To test if ATM is a direct target of CREB/ATF1 we transfected human MeWo melanoma cells with siRNA directed against CREB and ATF1 either individually or in combination. While ATF1 knockdown and CREB knockdown significantly reduced ATM mRNA levels, a double knockdown caused an almost 10-fold reduction in ATM mRNA levels (Figs. 5B and C). ATM protein levels were also significantly reduced in CREB/ATF1 knockdown cells (Fig. 5D). However, in no case did we observe effects of DNA damage on ATM mRNA or protein levels, indicating that although CREB and ATF1 contribute to constitutive ATM expression, it is unlikely that ATM-mediated phosphorylation of these factors modulates ATM promoter activity during DNA damage.


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)

CREB/ATF1 regulate ATM transcription.(A) Schematic of the human ATM with putative CREs is shown. CREs conserved between human and mouse ATM promoters are in boldface. The arrow indicates the transcription start site. (B) Knockdown of CREB and ATF1 in MeWo cells. MeWo cells were transfected with shRNA for ATF1, siRNA for CREB or both and compared to cells expressing a non-targeting construct for 48 h. Cell extracts were subjected to immunoblotting with α-CREB, α-ATF1 and α-β-Tubulin antibodies. (C) Real-time PCR analysis of ATM mRNA in CREB/ATF1-deficient MeWo cells. Relative fold-expression levels of ATM mRNA normalized for GAPDH is shown. Error bars denote standard deviation from the mean from three independent experiments. (D) Effects of CREB and ATF1 knockdown on ATM protein levels. MeWo cells were transfected with shRNA for ATF1, siRNA for CREB or both and compared to cells expressing a non-targeting construct for 48 h before IR exposure. Cell extracts were prepared and subjected to immunoblotting with α-CREB, α-ATF1, α-ATM and α-β-Tubulin antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2921338&req=5

pone-0012173-g005: CREB/ATF1 regulate ATM transcription.(A) Schematic of the human ATM with putative CREs is shown. CREs conserved between human and mouse ATM promoters are in boldface. The arrow indicates the transcription start site. (B) Knockdown of CREB and ATF1 in MeWo cells. MeWo cells were transfected with shRNA for ATF1, siRNA for CREB or both and compared to cells expressing a non-targeting construct for 48 h. Cell extracts were subjected to immunoblotting with α-CREB, α-ATF1 and α-β-Tubulin antibodies. (C) Real-time PCR analysis of ATM mRNA in CREB/ATF1-deficient MeWo cells. Relative fold-expression levels of ATM mRNA normalized for GAPDH is shown. Error bars denote standard deviation from the mean from three independent experiments. (D) Effects of CREB and ATF1 knockdown on ATM protein levels. MeWo cells were transfected with shRNA for ATF1, siRNA for CREB or both and compared to cells expressing a non-targeting construct for 48 h before IR exposure. Cell extracts were prepared and subjected to immunoblotting with α-CREB, α-ATF1, α-ATM and α-β-Tubulin antibodies.
Mentions: The ATM promoter contains positionally conserved CRE elements suggesting the interesting possibility that ATM is regulated by its own substrates (Fig. 5A and Ref. [2], [33]. To test if ATM is a direct target of CREB/ATF1 we transfected human MeWo melanoma cells with siRNA directed against CREB and ATF1 either individually or in combination. While ATF1 knockdown and CREB knockdown significantly reduced ATM mRNA levels, a double knockdown caused an almost 10-fold reduction in ATM mRNA levels (Figs. 5B and C). ATM protein levels were also significantly reduced in CREB/ATF1 knockdown cells (Fig. 5D). However, in no case did we observe effects of DNA damage on ATM mRNA or protein levels, indicating that although CREB and ATF1 contribute to constitutive ATM expression, it is unlikely that ATM-mediated phosphorylation of these factors modulates ATM promoter activity during DNA damage.

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