Holden J et al. (2017), Cip29 is phosphorylated following activation of...

XB-ART-53848

PLoS One 2017 Jul 10;127:e0181131. doi: 10.1371/journal.pone.0181131.

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Cip29 is phosphorylated following activation of the DNA damage response in Xenopus egg extracts.

Holden J , Taylor EM , Lindsay HD .

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Acting through a complex signalling network, DNA lesions trigger a range of cellular responses including DNA repair, cell cycle arrest, altered gene expression and cell death, which help to limit the mutagenic effects of such DNA damage. RNA processing factors are increasingly being recognised as important targets of DNA damage signalling, with roles in the regulation of gene expression and also more directly in the promotion of DNA repair. In this study, we have used a Xenopus laevis egg extract system to analyse the DNA damage-dependent phosphorylation of a putative RNA export factor, Cip29. We have found that Cip29 is rapidly phosphorylated in response to DNA double-strand breaks in this experimental system. We show that the DNA damage-inducible modification of Cip29 is dependent on the activity of the key double-strand break response kinase, ATM, and we have identified a conserved serine residue as a damage-dependent phosphorylation site. Finally, we have determined that Cip29 is not required for efficient DNA end-joining in egg extracts. Taken together, these data identify Cip29 as a novel target of the DNA damage response and suggest that the damage-dependent modification of Cip29 may relate to a role in the regulation of gene expression after DNA damage.

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Species referenced: Xenopus laevis
Genes referenced: atm chek1 chek2 mre11 sarnp uhrf1

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	Fig 1. Detection of Cip29 and phosphorylated Cip29 proteins in X. laevis egg extract.(A) Immunodetection of Cip29 on a Western blot of X. laevis egg extract (E), in vitro translated Cip29 (T1) and in vitro translated Cip29b (T2). A non-specific band detected by the α-Cip29 antibodies in X. laevis egg extract is indicated with an asterisk. Molecular weight markers (kDa) are indicated on the left. (B) Western blot of Cip29 in X. laevis egg extract, undepleted or immunodepleted with either non-specific rabbit IgGs (mock) or α-Cip29 antibodies (left panel), and in the immunoprecipitated samples (right panel). The non-specific band (*) serves as a loading control for extract samples. (C) Amino acid sequence alignment of X. laevis Cip29 and Cip29b. (D) Western blots of egg extract incubated with or without 50ng/μl AT70 (21°C, 1h). Samples were resolved by electrophoresis on a standard 12% SDS-PAGE gel (left panel) and an 8% SDS-PAGE gel containing 15μM Phos-tag (right panel) before immunoblotting with the indicated antibodies. Modified Cip29 protein is denoted by Cip29-P (E) Cip29 was immunoprecipitated from egg extract supplemented with 50ng/μl AT70 (21°C, 1h). The immunoprecipitation reactions were treated with either 1 x NEBuffer 3 (-CIP), or NEBuffer 3 containing 10 units of CIP, or 10 units of heat-inactivated CIP (21°C, 1h) before glycine elution of the immunoprecipitated proteins. Eluted protein was supplemented with 2μg of Cip29-depleted extract before separation on Phos-tag SDS-PAGE, to optimise resolution of the modified form of Cip29.
	Fig 2. Cip29 is phosphorylated in a DNA damage- and ATM-dependent manner in X. laevis egg extract.(A) Egg extract was supplemented with 50ng/μl AT70 and incubated at 21°C for the indicated times before reactions were stopped by addition of 5 x SDS-PAGE gel loading buffer. Samples were analysed by Phos-tag SDS-PAGE and western blotting with the indicated antibodies. (B) Egg extract was incubated with either AT70, cut plasmid or uncut plasmid (50ng/μl), in the absence or presence of caffeine (5mM) (21°C, 1h) before analysis by Phos-tag SDS-PAGE and western blotting. Dotted line indicates removal of intervening lanes on the same gel for the sake of clarity. (C) Egg extract was incubated in the absence or presence of 50ng/μl AT70 supplemented with 5mm caffeine, 10μM DNA-PKi (NU7441), 20μM and 40μM ATMi (KU55933) or 20μM and 40μM ATRi (VE821) as indicated (21°C, 1h), and samples were analysed by Phos-tag SDS-PAGE and western blotting. (D) Egg extract, immunodepleted with non-specific rabbit IgGs (mock), α-Chk1 or α-Chk2 antibodies, was incubated with 50ng/μl AT70 (21°C, 30min) and analysed by Phos-tag SDS-PAGE and western blotting. (E) Western blot of immunodepleted extracts from (D) indicating depletion efficiency for Chk1 and Chk2. Molecular weight markers (kDa) are indicated on the right.
	Fig 3. Phosphorylation of GST-Cip29 protein fragments in X. laevis egg extract.(A) X. laevis Cip29 amino acid sequence indicating potential phosphorylation sites as predicted using the protein phosphorylation prediction tools, NetPhos 2.0, PHOSIDA, KinasePhos 2.0 and Scansite. The number of times each residue is predicted as a candidate phospho-site is indicated by shading, as described in the key. Ser/Thr residues that are conserved between X. laevis Cip29 and other vertebrates are underlined. (B) Schematic representation of GST-Cip29 fragments. Five Cip29 protein fragments (F1-F5), encompassing the entire Cip29 protein sequence, were each expressed with an N-terminal GST tag to facilitate purification. (C) Purified GST-Cip29 fusion proteins, F1-F5, (5μg each) were incubated in egg extract supplemented with 32P-ATP, in the absence or presence of 50ng/μl AT70, (21°C, 1h), then the recombinant proteins were recovered and subject to SDS-PAGE. Proteins were visualised by Sypro Ruby staining. Premature termination products or degradation products are denoted with asterisks. (D) After staining and protein quantification, the SDS-PAGE gel from (C) was dried and exposed to a phosphorimager screen for 48h to determine 32P-incorporation. (E) 32P-incorporation is presented for each fragment, in the absence or presence of AT70, relative to the amount of protein per band. The data shown in the graphs correspond to the mean of three independent experiments, and error bars indicate the SD. Note the axis break and scale change on the y axis to accommodate the high incorporation level in F5, relative to F1-F4.
	Fig 4. Damage-dependent phosphorylation of Cip29 occurs on a conserved Ser residue of fragment 3.(A) F3 (5μg) was incubated in egg extract with 32P-ATP in the absence or presence of 50ng/μl AT70 and 5mM caffeine (21°C, 1h). F3 was recovered from the extract and 32P-incorporation was determined for each condition. (B) 5μg each of F3 (WT), F3 containing the double mutation S98A/T100A or the single mutation S95, were incubated as in (A) and 32P-incorporation determined for each fragment. (C) 5μg of full length His10-Cip29 was incubated as in (A) and 32P-incorporation was determined. In each case, the data represent an average of three independent experiments where error bars indicate the SD. Significance of the observed differences was evaluated using Student’s t test (P 0.01–0.05; *P 0.001–0.01).
	Fig 5. Damage-dependent phosphorylation of recombinant full length Cip29.(A) Western blot of full length His10-Cip29 (WT) (lane 1) alongside His10-Cip29 (WT and S95A) incubated in egg extract with or without 50ng/μl AT70 (21°C, 1h) and recovered on nickel NTA agarose. Samples were resolved by electrophoresis on an 8% SDS-PAGE gel containing 15μM Phos-tag before immunoblotting with the anti-His antibodies. A modified Cip29 isoform is denoted by . (B) Quantification of signal intensity of the modified Cip29 isoform () relative to the total Cip29 signal in each lane, expressed as fold-increase for AT70-treated extract relative to untreated extract. The data represent an average of three independent experiments where error bars indicate the SD. Significance of the observed differences was evaluated using Student’s t test (*P 0.01–0.05).
	Fig 6. Analysis of DNA end joining in Cip29-depleted egg extract.(A) Sequencing gel analysis of DNA end joining in X. laevis egg extracts using a defined repair substrate. Linearized repair substrate was incubated at 1ng/μl in mock-depleted, Mre11-depleted or Cip29-depleted extract (21°C, 6h). Where indicated, DMSO was added to a final concentration of 0.4% and DNA-PKi (NU7441), dissolved in DMSO, was added to a final concentration of 8μM. Cip29-depletion was performed with two different α-Cip29 antibodies (ΔCip29-1 and ΔCip29-2). Following incubation, plasmid DNA was recovered, digested with TaqαI and BstXI, resolved on a 20% denaturing polyacrylamide gel and exposed to a phosphorimager screen. Linearized substrate added to mock-depleted extract and processed immediately serves as an unrepaired control (0h). (B) Western blot of immunodepleted extracts from (A) indicating the depletion efficiency for Cip29 with both α-Cip29 antibodies. Uhrf1 serves as a loading control. Molecular weight markers (kDa) are indicated on the right.

References [+] :

Adamson, A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response. 2012, Pubmed