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Sumoylation of eIF4A2 affects stress granule formation.

Jongjitwimol J, Baldock RA, Morley SJ, Watts FZ - J. Cell. Sci. (2016)

Bottom Line: We demonstrate that sumoylation of eIF4A2 is modestly increased in response to arsenite and ionising radiation, but decreases in response to heat shock or hippuristanol.In arsenite-treated cells, but not in hippuristanol-treated cells, eIF4A2 is recruited to stress granules, suggesting sumoylation of eIF4A2 correlates with its recruitment to stress granules.Furthermore, we demonstrate that the inability to sumoylate eIF4A2 results in impaired stress granule formation, indicating a new role for sumoylation in the stress response.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9RQ, UK.

No MeSH data available.


Related in: MedlinePlus

Exposure to arsenite and ionising radiation affects sumoylation of eIF4G, eIF4E, eIF4A1 and eIF4A2. Whole-cell extracts (WCE) and affinity purification under denaturing conditions as in Fig. 1 (AP) of His-tagged SUMO1 (S1) or SUMO2 (S2) from stably transfected HeLa cell lines or non-transfected cells (NT), untreated (UT) or subjected to 1 mM arsenite (AR) for 30 min or 3 Gy ionising radiation (IR) with 30 min recovery, analysed by SDS-PAGE (7.5%) and western blotted. (A) Western blots probed with antisera against eIF4G and eIF4E under conditions used in Fig. 1B, as indicated, and with affinity-purified antibodies against eIF4A1 and eIF4A2 as indicated. (B) Whole-cell extracts (WCE) and affinity purification (AP) as in Fig. 1A from cells that were exposed to 1 mM arsenite, heat shocked at 42°C for 30 min (HS) or treated with 1 μM hippuristanol (HP) for 60 min. Samples were analysed by western blotting as in A.
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JCS184614F2: Exposure to arsenite and ionising radiation affects sumoylation of eIF4G, eIF4E, eIF4A1 and eIF4A2. Whole-cell extracts (WCE) and affinity purification under denaturing conditions as in Fig. 1 (AP) of His-tagged SUMO1 (S1) or SUMO2 (S2) from stably transfected HeLa cell lines or non-transfected cells (NT), untreated (UT) or subjected to 1 mM arsenite (AR) for 30 min or 3 Gy ionising radiation (IR) with 30 min recovery, analysed by SDS-PAGE (7.5%) and western blotted. (A) Western blots probed with antisera against eIF4G and eIF4E under conditions used in Fig. 1B, as indicated, and with affinity-purified antibodies against eIF4A1 and eIF4A2 as indicated. (B) Whole-cell extracts (WCE) and affinity purification (AP) as in Fig. 1A from cells that were exposed to 1 mM arsenite, heat shocked at 42°C for 30 min (HS) or treated with 1 μM hippuristanol (HP) for 60 min. Samples were analysed by western blotting as in A.

Mentions: We have previously demonstrated that Schizosaccharomycespombe eIF4G is sumoylated in response to 1 M KCl (a condition that induces stress granules in yeast) (Jongjitwimol et al., 2014). We therefore investigated the effect of stress on sumoylation of eIF4G, eIF4E or eIF4A2 in mammalian cells. We began by investigating the effect of arsenite, which induces the formation of stress granules, and ionising radiation, which causes genotoxic stress. Analysis of global levels of sumoylation in response to arsenite (1 mM) and ionising radiation (3 Gy) (Fig. S2A), indicated that exposure to arsenite had a minimal effect on the levels of sumoylation by either SUMO1 or SUMO2. In contrast, exposure to ionising radiation reduced the level of global sumoylation by ∼50%. To analyse sumoylation of the individual eIFs, His–SUMO1 and His–SUMO2 were affinity-purified from untreated cells and cells exposed to arsenite or ionising radiation. Fig. 2A shows that sumoylation of eIF4G by SUMO1 was somewhat reduced following exposure of cells to arsenite. In contrast, sumoylation by SUMO1 was reduced in response to ionising radiation. In comparison to sumoylation by SUMO1, only low levels of SUMO2-containing species were observed. In the case of eIF4E, sumoylation by both SUMO1 and SUMO2 increased in response to both arsenite and ionising radiation (Fig. 2A). All four species seen represent sumoylated forms of eIF4E; because the significance of the four different forms is unknown, we have compared the levels of the total amount of sumoylated species here.Fig. 2.


Sumoylation of eIF4A2 affects stress granule formation.

Jongjitwimol J, Baldock RA, Morley SJ, Watts FZ - J. Cell. Sci. (2016)

Exposure to arsenite and ionising radiation affects sumoylation of eIF4G, eIF4E, eIF4A1 and eIF4A2. Whole-cell extracts (WCE) and affinity purification under denaturing conditions as in Fig. 1 (AP) of His-tagged SUMO1 (S1) or SUMO2 (S2) from stably transfected HeLa cell lines or non-transfected cells (NT), untreated (UT) or subjected to 1 mM arsenite (AR) for 30 min or 3 Gy ionising radiation (IR) with 30 min recovery, analysed by SDS-PAGE (7.5%) and western blotted. (A) Western blots probed with antisera against eIF4G and eIF4E under conditions used in Fig. 1B, as indicated, and with affinity-purified antibodies against eIF4A1 and eIF4A2 as indicated. (B) Whole-cell extracts (WCE) and affinity purification (AP) as in Fig. 1A from cells that were exposed to 1 mM arsenite, heat shocked at 42°C for 30 min (HS) or treated with 1 μM hippuristanol (HP) for 60 min. Samples were analysed by western blotting as in A.
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Related In: Results  -  Collection

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JCS184614F2: Exposure to arsenite and ionising radiation affects sumoylation of eIF4G, eIF4E, eIF4A1 and eIF4A2. Whole-cell extracts (WCE) and affinity purification under denaturing conditions as in Fig. 1 (AP) of His-tagged SUMO1 (S1) or SUMO2 (S2) from stably transfected HeLa cell lines or non-transfected cells (NT), untreated (UT) or subjected to 1 mM arsenite (AR) for 30 min or 3 Gy ionising radiation (IR) with 30 min recovery, analysed by SDS-PAGE (7.5%) and western blotted. (A) Western blots probed with antisera against eIF4G and eIF4E under conditions used in Fig. 1B, as indicated, and with affinity-purified antibodies against eIF4A1 and eIF4A2 as indicated. (B) Whole-cell extracts (WCE) and affinity purification (AP) as in Fig. 1A from cells that were exposed to 1 mM arsenite, heat shocked at 42°C for 30 min (HS) or treated with 1 μM hippuristanol (HP) for 60 min. Samples were analysed by western blotting as in A.
Mentions: We have previously demonstrated that Schizosaccharomycespombe eIF4G is sumoylated in response to 1 M KCl (a condition that induces stress granules in yeast) (Jongjitwimol et al., 2014). We therefore investigated the effect of stress on sumoylation of eIF4G, eIF4E or eIF4A2 in mammalian cells. We began by investigating the effect of arsenite, which induces the formation of stress granules, and ionising radiation, which causes genotoxic stress. Analysis of global levels of sumoylation in response to arsenite (1 mM) and ionising radiation (3 Gy) (Fig. S2A), indicated that exposure to arsenite had a minimal effect on the levels of sumoylation by either SUMO1 or SUMO2. In contrast, exposure to ionising radiation reduced the level of global sumoylation by ∼50%. To analyse sumoylation of the individual eIFs, His–SUMO1 and His–SUMO2 were affinity-purified from untreated cells and cells exposed to arsenite or ionising radiation. Fig. 2A shows that sumoylation of eIF4G by SUMO1 was somewhat reduced following exposure of cells to arsenite. In contrast, sumoylation by SUMO1 was reduced in response to ionising radiation. In comparison to sumoylation by SUMO1, only low levels of SUMO2-containing species were observed. In the case of eIF4E, sumoylation by both SUMO1 and SUMO2 increased in response to both arsenite and ionising radiation (Fig. 2A). All four species seen represent sumoylated forms of eIF4E; because the significance of the four different forms is unknown, we have compared the levels of the total amount of sumoylated species here.Fig. 2.

Bottom Line: We demonstrate that sumoylation of eIF4A2 is modestly increased in response to arsenite and ionising radiation, but decreases in response to heat shock or hippuristanol.In arsenite-treated cells, but not in hippuristanol-treated cells, eIF4A2 is recruited to stress granules, suggesting sumoylation of eIF4A2 correlates with its recruitment to stress granules.Furthermore, we demonstrate that the inability to sumoylate eIF4A2 results in impaired stress granule formation, indicating a new role for sumoylation in the stress response.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9RQ, UK.

No MeSH data available.


Related in: MedlinePlus