Conserved and Distinct Modes of CREB/ATF Transcription Factor Regulation by PP2A/B56γ and Genotoxic Stress

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 B56γ, 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

Identification of Genetic Modifiers of TDP-43 Neurotoxicity in Drosophila

<div><p>Cytosolic aggregation of the nuclear RNA-binding protein TDP-43 is a histopathologic signature of degenerating neurons in amyotrophic lateral sclerosis (ALS), and mutations in the <i>TARDBP</i> gene encoding TDP-43 cause dominantly inherited forms of this condition. To understand the relationship between TDP-43 misregulation and neurotoxicity, we and others have used Drosophila as a model system, in which overexpression of either wild-type TDP-43 or its ALS-associated mutants in neurons is sufficient to induce neurotoxicity, paralysis, and early death. Using microarrays, we have examined gene expression patterns that accompany TDP-43-induced neurotoxicity in the fly system. Constitutive expression of TDP-43 in the Drosophila compound eye elicited widespread gene expression changes, with strong upregulation of cell cycle regulatory genes and genes functioning in the Notch intercellular communication pathway. Inducible expression of TDP-43 specifically in neurons elicited significant expression differences in a more restricted set of genes. Genes that were upregulated in both paradigms included <i>SpindleB</i> and the Notch target <i>Hey</i>, which appeared to be a direct TDP-43 target. Mutations that diminished activity of Notch or disrupted the function of downstream Notch target genes extended the lifespan of TDP-43 transgenic flies, suggesting that Notch activation was deleterious in this model. Finally, we showed that mutation of the nucleoporin <i>Nup50</i> increased the lifespan of TDP-43 transgenic flies, suggesting that nuclear events contribute to TDP-43-dependent neurotoxicity. The combined findings identified pathways whose deregulation might contribute to TDP-43-induced neurotoxicity in Drosophila.</p> </div

Effects of cell cycle gene mutations on D42-Gal4/UAS-TDP-43 fly lifespan.

<p>(<b>A</b>) Survival curves of D42-Gal4/UAS-TDP-43 flies in <i>string</i> and <i>greatwall</i> mutant backgrounds. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 26, N = 118; D42-Gal4/UAS-TDP-43, <i>string</i>^EY12388 = 27, N = 106, p<0.0001; D42-Gal4/UAS-TDP-43, <i>greatwall</i>^EP515 = 23, N = 67, p<0.0001. (<b>B</b>) Effects of the <i>string</i>^EY12388 allele in males. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 23.5, N = 60; D42-Gal4/UAS-TDP-43, <i>string</i>^EY12388 = 23, N = 52, p = 0.1831. (C) Effects of the <i>string</i>^EY12388 allele in females. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 28, N = 58; D42-Gal4/UAS-TDP-43, <i>string</i>^EY12388 = 32, N = 54, p<0.0001. (<b>D</b>) Survival curves of D42-Gal4 control flies with the <i>string</i>^EY12388 mutant allele, divided by gender. Median survival (days) of males: D42-Gal4/+ = 58, N = 49; D42-Gal4/<i>string</i>^EY12388 = 53.5, N = 60, p = 0.08. Median survival (days) of females: D42-Gal4/+ = 63, N = 53; D42-Gal4/<i>string</i>^EY12388 = 60, N = 60, p = 0.43.</p

Analysis of <i>Nup50</i> and other candidate genes.

<p>(<b>A</b>) Survival curves of D42-Gal4/UAS-TDP-43 crossed to <i>Pabp2</i> (<i>Pabp2</i>⁰¹or <i>Pabp2</i>^kg02359) and the <i>Rs1</i>^k09514 mutant lines. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 21, N = 110; D42-Gal4/UAS-TDP-43, <i>PABP</i>⁰¹ = 24, N = 63, p<0.0001; D42-Gal4/UAS-TDP-43, <i>PABP</i>^KG02359 = 22, N = 110, p<0.01. D42-Gal4/UAS-TDP-43, <i>Rs1</i>^K09514 = 21, N = 30, p = 0.8. (<b>B</b>) Survival curves of D42-Gal4/UAS-TDP-43 flies with the <i>Nup50</i>^kg09557 mutation. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 21, N = 110; D42-Gal4/UAS-TDP-43, <i>Nup50</i>^KG09557 = 26, N = 111; <i>p</i><0.0001. (<b>C</b>) Survival curves of D42-Gal4 control flies with <i>Nup50</i>^kg09557 mutation. Median survival (days) of: D42-Gal4/+ = 67.5, N = 110; D42-Gal4/<i>Nup50</i>^KG09557 = 75, N = 105, p<0.0001.</p

Expanded gene expression changes in inducible TDP-43 flies.

<p>Genes are from same experiment and analysis as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057214#pone-0057214-t003" target="_blank">Table 3</a>, with less stringent criteria to identify potentially important genes. nACR, nicotinic acetycholine receptor.</p

Effects of caspase inhibition and <i>TBPH</i> knockdown on D42-Gal4/UAS-TDP-43 fly lifespan.

<p>(<b>A</b>) Survival curves of D42-Gal4/UAS-TDP-43 flies crossed to UAS-P35 and UAS-dIAP lines. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 26, N = 118; D42-Gal4/UAS-TDP-43, UAS-P35 = 24, N = 111, p<0.05; D42-Gal4/UAS-TDP-43, UAS-dIAP = 26, N = 109, p = 0.47. (B) Survival curves of D42-Gal4/UAS-TDP-43 flies crossed to two <i>TBPH</i> null lines. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 22, N = 81; D42-Gal4/UAS-TDP-43, <i>TBPH</i>^Δ23 = 26, N = 122, p = 0.12; D42-Gal4/UAS-TDP-43, <i>TBPH</i>^Δ142 = 27.5, N = 118, p<0.0001.</p