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. 2012 Apr;4(4):290-304.
doi: 10.18632/aging.100452.

ATM-dependent phosphorylation of SNEVhPrp19/hPso4 is involved in extending cellular life span and suppression of apoptosis

Hanna Dellago  1 , Abdulhameed Khan, Monika Nussbacher, Anna Gstraunthaler, Ingo Lämmermann, Markus Schosserer, Christoph Mück, Dorothea Anrather, Annika Scheffold, Gustav Ammerer, Pidder Jansen-Dürr, Karl Lenhard Rudolph, Regina Voglauer-Grillari, Johannes Grillari
Affiliations

ATM-dependent phosphorylation of SNEVhPrp19/hPso4 is involved in extending cellular life span and suppression of apoptosis

Hanna Dellago et al. Aging (Albany NY). 2012 Apr.

Abstract

Defective DNA repair is widely acknowledged to negatively impact on healthy aging, since mutations in DNA repair factors lead to accelerated and premature aging. However, the opposite, namely if improved DNA repair will also increase the life or health span is less clear, and only few studies have tested if overexpression of DNA repair factors modulates life and health span in cells or organisms. Recently, we identified and characterized SNEVhPrp19/hPso4, a protein that plays a role in DNA repair and pre-mRNA splicing, and observed a doubling of the replicative life span upon ectopic overexpression, accompanied by lower basal DNA damage and apoptosis levels as well as an increased resistance to oxidative stress. Here we find that SNEVhPrp19/hPso4 is phosphorylated at S149 in an ataxia telangiectasia mutated protein (ATM)-dependent manner in response to oxidative stress and DNA double strand break inducing agents. By overexpressing wild-type SNEVhPrp19/hPso4 and a phosphorylation-deficient point-mutant, we found that S149 phosphorylation is necessary for mediating the resistance to apoptosis upon oxidative stress and is partially necessary for elongating the cellular life span. Therefore, ATM dependent phosphorylation of SNEVhPrp19/hPso4 upon DNA damage or oxidative stress might represent a novel axis capable of modulating cellular life span.

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Conflict of interest statement

JG and RGV are co-founders of Evercyte GmbH.

Figures

Figure 1
Figure 1. Upon oxidative stress, SNEV is detected as double band, probably representing a phosphorylated species
(A) Upon treatment with hydrogen peroxide, additional bands of higher molecular weight were detected with anti-SNEV antibody. These bands disappear upon incubation with phosphatase. (B) Similarly, upon bleomycin treatment of fibroblasts, we detected an additional band with anti-SNEV antibody, which increased in a dose-and time-dpendent manner. Fibrolblasts were incubated with 0, 5, 10, 25, 50 or 100μg/ml bleomycin for 1hour (left panels) or with 25μg/ml bleomycin for 0, 0.5, 1, 2, 4 hours (right panels), scraped on ice in 2x SDS loading dye and subjected to Western Blotting with anti-SNEV antibody. Anti-β-actin was used to ensure equal loading. Anti-γH2AX antibody was used to confirm that the treatment induces DNA damage. (C)Collision-induced dissociation spectrum of the SNEV peptide AVPSS(ph)QPSVVGAGEPM(ox)DLG Indeed, a phosphorylation was detected and assigned to S149 with a probability of 76.5% (Fig. 1C). While the consistence of three different spectra with different m/z ratios underscores the correct assignment of the phosphorylation to this site, the remaining uncertainty comes from the presence of 3 serine residues within a quite long peptide. Together with the problem of lack of trypsin or chymotrypsin proteolytic sites near these serines, a better proof of S149 as the really phosphorylated serine by mass spectrometry is hampered. (D) Sequence comparison of SNEV homologues in the putative ATM target site. The consensus sequence surrounding the phospho-SQ site on ATM substrates that are regulated by DNA damage is conserved in the SNEVamino acid sequences across different vertebrates, but not in non-vertebrates.
Figure 2
Figure 2. Phosphorylation of SNEV at serine 149 is induced by oxidative stress in a time- and dose dependent manner
(A) Upon oxidative stress treatment, the band detected by a phospho-SNEVspecific antibody (anti-pSNEV(S149)) increases, and diminishes upon phosphatase treatment. HeLa cells were treated with 1mM H2O2 for 1h or left untreated. Lysates were incubated with CIP and subjected to Western Blotting with a specific anti-phosphoSNEV(S149) antibody, that was generated by immunizing rabbits with an artificial SNEV-phosphopeptide. (B) Human diploid fibroblasts (HDF) were treated with 0, 100, 200 or 400 μM H2O2 for 1h, scraped on ice in 2x SDS loading dye and subjected to SDS PAGE. Western Blot was detected with anti-pSNEV(S149) and anti-SNEV to compare pSNEV to total SNEV levels. anti-γH2AX antibody was used as positive control for stress-dependent phosphorylation. anti-?-Actin was used as loading control. (C) HDF were treated with 100 μM H2O2 for 0, 5, 15, 30, 60 or 120 min. Lysis and Western Blot as in B.
Figure 3
Figure 3. Upon oxidative stress, SNEV is phosphorylated and localizes mainly to the nucleus
(A) Hela cells were seeded on coverslipsand treated with 100 μMH2O2 for 1 hour. Cells were stained with anti-pSNEV(S149) and DAPI as described in the Materials and Methods section and subjected to fluorescence microscopy. (B) HeLa cells were treated with 25 μg/ml MMC for 24 hours prior to indirect immunofluorescence staining with anti-pSNEV(S149) antibody.
Figure 4
Figure 4. Phosphorylation of SNEV at S149A is ATM-dependent
(A) ATM inhibition alleviates phosphorylation of SNEV in response to oxidative stress. HDFs were treated with 100 μM H2O2 and the specific ATM inhibitor KU-55933. DMSO, the solvent for Ku-99533, was used as negative control. Phosphorylation of SNEV and known ATM target γH2AX was reduced by inhibition of ATM. (B) Phopshorylation of SNEV at S149 is reduced in ATM conditional knockout MEF. Upper panel: wt and ATM -/- MEF were treated with 200 μM H2O2 for 1h, harvested, resuspended in 2x SDS loading dye and subjected to SDS PAGE. Western Blot was detected with anti-pSNEV(S149) and anti-βActin to ensure equael loading. Lower panel: Genomic DNA was isolated from wt and ATM lox/lox MEF before and after transfection with Cre and used in a genotyping PCR. The band for deleted ATM is present only after transfection with Cre, nonetheless the deletion is not quantitative and a small portion of ATM is maintained.
Figure 5
Figure 5. Phosphorylation at S149 is necessary for apoptosis resistance and partially for life span extension conferred by SNEV
(A) Normal HUVEC as well as stable HUVEC overexpressing SNEV wt, SNEV S149A or empty vector control cells were lysed and submitted to SDS PAGE. Western Blots were probed with anti-SNEV and anti-?Actin antibodies. SNEV protein expression levels are increased five-fold in SNEV wt and ten-fold in SNEV S149A HUVEC as compared to untransfected HUVEC. (B) Growth curves of HUVEC overexpressing SNEV wt, SNEV S149A and empty vector. (C) SNEV wt as well as SNEV S149A overexpression reduces the basal level of DNA damage. Upper panel: Representative pictures of Comet assay performed with empty vector, SNEV wt and S149A point mutant overexpressing HUVEC. Lower panel: Comets were classified into 5 categories depending on the percentage of the DNA content in the tail. DNA damage levels of SNEV S149A. (D) Cells were pre-incubated with 1 mM BSO for 48h, followed by treatment with 100 μg/ml Bleomycin for 24h. Apoptosis was measured by Annexin-FITC and PI staining and subsequent flow cytometric analysis. (E) Cells were incubated with 100μg/ml Cisplatin for 24h. Apoptosis was measured as in D.
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