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.
PLoS One. 2010 Aug 13;5(8):e12173. doi: 10.1371/journal.pone.0012173.
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.
激活转录因子 1（ATF1）和密切相关的蛋白 CREB（环 AMP 反应元件结合蛋白）和 CREM（环 AMP 反应元件调节剂）构成了 bZIP 转录因子的一个亚家族，它们在调节细胞生长、代谢和存活方面起着至关重要的作用。先前的研究表明，CREB 在 DNA 损伤后，通过共济失调毛细血管扩张症突变（ATM）蛋白激酶和酪蛋白激酶 1 和 2（CK1/2）的协调作用，在一组保守的丝氨酸残基（包括 Ser-111 和 Ser-121）上磷酸化。在这里，我们表明 ATM 诱导的 DNA 损伤诱导的磷酸化是 CREB 和 ATF1 的一个普遍特征。ATF1 具有一个保守的 ATM/CK 簇，该簇在非同步生长的细胞中被 CK1 和 CK2 组成型和化学计量地磷酸化。暴露于 DNA 损伤进一步通过 ATM 诱导 ATF1 在 Ser-51 上磷酸化，这需要上游 CK 残基的磷酸化。高度磷酸化的 ATF1 对 CREB 结合蛋白的亲和力降低了 4 倍。我们进一步表明，PP2A 与其靶向亚基 B56gamma 一起，拮抗了 ATM 和 CK1/2 依赖性的 CREB 和 ATF1 的磷酸化。最后，我们表明在细胞生长过程中 CREB 的 CK 位点被磷酸化，并且这些残基的磷酸化降低了 ATM 依赖性磷酸化抑制性 Ser-121 残基所需的 DNA 损伤阈值。这些研究定义了 CREB 和 ATF1 磷酸化调节的重叠和不同模式，这可能确保了这些因子介导的基因表达的协同变化。
