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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

Mutation of K226 results in loss of sumoylation of eIF4A2 in vivo and a reduction in stress granule size. (A) His–SUMO1 stably transfected cells were reverse transfected with eIF4A2 siRNA (lanes 3–5). After 48 h, cells were mock treated (lane 2) or transfected with FLAG–myc–eIF4A2 wt or FLAG–myc–eIF4A2-K226R mutant as indicated. His-SUMO1 was purified from non-transfected cells (NT, lane 1) or His–SUMO1 stably transfected cells (S1, lanes 2–5) using Ni2+ agarose under denaturing conditions as in Figs 1 and 2. Proteins were analysed by SDS-PAGE and western blotted with anti-eIF4A2 antisera. (B) HeLa cells were depleted of endogenous eIF4A2 and transfected with either FLAG–myc–eIF4A2 wt or FLAG–myc–eIF4A2-K226 and either left untreated (UT) or treated with 1 mM arsenite (AR) for 30 min. Cells were immunostained with an anti-FLAG antibody and anti-SUMO. High-resolution images (lower panels) were taken over a z-plane of 4 μm at 0.05 μm slices. Scale bars: 10 μm. (C) HeLa cells were prepared as in B and immunostained for FLAG (eIF4A2) and TIA-1. Scale bars: 10 μm. (D) High-resolution z-stack images were taken and deconvolved using Huygens deconvolution software. Images were three-dimensionally rendered using the IMARIS software suite. 3D images were used to calculate the volumes of stress granules for both TIA-1 and FLAG (eIF4A2). Box-and-whisker plots were used to show the distribution of stress granule volumes. The box represents the 25–75th percentiles, and the median is indicated. The whiskers show the 10–90th percentiles. n=2300 for TIA-1 and 800 for FLAG. (E) Cells processed as in C were analysed for colocalisation of FLAG and TIA-1 signals. The chart shows the percentage of FLAG (eIF4A2) signal that overlaps with TIA-1 in cells transfected with either FLAG-eIF4A2 WT or the K226R mutant (mean±s.d., n=120).
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JCS184614F6: Mutation of K226 results in loss of sumoylation of eIF4A2 in vivo and a reduction in stress granule size. (A) His–SUMO1 stably transfected cells were reverse transfected with eIF4A2 siRNA (lanes 3–5). After 48 h, cells were mock treated (lane 2) or transfected with FLAG–myc–eIF4A2 wt or FLAG–myc–eIF4A2-K226R mutant as indicated. His-SUMO1 was purified from non-transfected cells (NT, lane 1) or His–SUMO1 stably transfected cells (S1, lanes 2–5) using Ni2+ agarose under denaturing conditions as in Figs 1 and 2. Proteins were analysed by SDS-PAGE and western blotted with anti-eIF4A2 antisera. (B) HeLa cells were depleted of endogenous eIF4A2 and transfected with either FLAG–myc–eIF4A2 wt or FLAG–myc–eIF4A2-K226 and either left untreated (UT) or treated with 1 mM arsenite (AR) for 30 min. Cells were immunostained with an anti-FLAG antibody and anti-SUMO. High-resolution images (lower panels) were taken over a z-plane of 4 μm at 0.05 μm slices. Scale bars: 10 μm. (C) HeLa cells were prepared as in B and immunostained for FLAG (eIF4A2) and TIA-1. Scale bars: 10 μm. (D) High-resolution z-stack images were taken and deconvolved using Huygens deconvolution software. Images were three-dimensionally rendered using the IMARIS software suite. 3D images were used to calculate the volumes of stress granules for both TIA-1 and FLAG (eIF4A2). Box-and-whisker plots were used to show the distribution of stress granule volumes. The box represents the 25–75th percentiles, and the median is indicated. The whiskers show the 10–90th percentiles. n=2300 for TIA-1 and 800 for FLAG. (E) Cells processed as in C were analysed for colocalisation of FLAG and TIA-1 signals. The chart shows the percentage of FLAG (eIF4A2) signal that overlaps with TIA-1 in cells transfected with either FLAG-eIF4A2 WT or the K226R mutant (mean±s.d., n=120).

Mentions: Given that eIF4A2 is more highly sumoylated in vivo than is eIF4A1 (Fig. 2A), we concentrated our studies on the eIF4A2 isoform, focusing on SUMO1-containing species, as this would cover singly sumoylated species as well as those containing SUMO chains terminating with SUMO1. Having shown that eIF4A2 is sumoylated in vitro on K226, we wished to confirm that this residue is used for sumoylation in vivo. To do this, expression of endogenous eIF4A2 was knocked down using small interfering RNA (siRNA), and cells were then transfected with wild-type (wt) or mutant eIF4A2 (Fig. 6A). Despite the fact that the knockdown was only partial (eIF4A2 is a very abundant protein making it difficult to achieve 100% knockdown), introduction of siRNA-resistant wild-type eIF4A2 into cells clearly results in new high-molecular-mass species being observed following affinity-purification of His-SUMO1 (lane 4). These species are of similar molecular mass to those observed in Figs 1B and 2B, and are not observed in the untransfected controls (lanes 1–3). In contrast, although it is clear that the eIF4A2-K226 protein was expressed at similar levels to the wt protein (lanes 4 and 5, lower panel), such species were not observed when the eIF4A2-K226R mutant is introduced (lane 5). This indicates that K226 is the main sumoylation site in eIF4A2 that is used in vivo.Fig. 6.


Sumoylation of eIF4A2 affects stress granule formation.

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

Mutation of K226 results in loss of sumoylation of eIF4A2 in vivo and a reduction in stress granule size. (A) His–SUMO1 stably transfected cells were reverse transfected with eIF4A2 siRNA (lanes 3–5). After 48 h, cells were mock treated (lane 2) or transfected with FLAG–myc–eIF4A2 wt or FLAG–myc–eIF4A2-K226R mutant as indicated. His-SUMO1 was purified from non-transfected cells (NT, lane 1) or His–SUMO1 stably transfected cells (S1, lanes 2–5) using Ni2+ agarose under denaturing conditions as in Figs 1 and 2. Proteins were analysed by SDS-PAGE and western blotted with anti-eIF4A2 antisera. (B) HeLa cells were depleted of endogenous eIF4A2 and transfected with either FLAG–myc–eIF4A2 wt or FLAG–myc–eIF4A2-K226 and either left untreated (UT) or treated with 1 mM arsenite (AR) for 30 min. Cells were immunostained with an anti-FLAG antibody and anti-SUMO. High-resolution images (lower panels) were taken over a z-plane of 4 μm at 0.05 μm slices. Scale bars: 10 μm. (C) HeLa cells were prepared as in B and immunostained for FLAG (eIF4A2) and TIA-1. Scale bars: 10 μm. (D) High-resolution z-stack images were taken and deconvolved using Huygens deconvolution software. Images were three-dimensionally rendered using the IMARIS software suite. 3D images were used to calculate the volumes of stress granules for both TIA-1 and FLAG (eIF4A2). Box-and-whisker plots were used to show the distribution of stress granule volumes. The box represents the 25–75th percentiles, and the median is indicated. The whiskers show the 10–90th percentiles. n=2300 for TIA-1 and 800 for FLAG. (E) Cells processed as in C were analysed for colocalisation of FLAG and TIA-1 signals. The chart shows the percentage of FLAG (eIF4A2) signal that overlaps with TIA-1 in cells transfected with either FLAG-eIF4A2 WT or the K226R mutant (mean±s.d., n=120).
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JCS184614F6: Mutation of K226 results in loss of sumoylation of eIF4A2 in vivo and a reduction in stress granule size. (A) His–SUMO1 stably transfected cells were reverse transfected with eIF4A2 siRNA (lanes 3–5). After 48 h, cells were mock treated (lane 2) or transfected with FLAG–myc–eIF4A2 wt or FLAG–myc–eIF4A2-K226R mutant as indicated. His-SUMO1 was purified from non-transfected cells (NT, lane 1) or His–SUMO1 stably transfected cells (S1, lanes 2–5) using Ni2+ agarose under denaturing conditions as in Figs 1 and 2. Proteins were analysed by SDS-PAGE and western blotted with anti-eIF4A2 antisera. (B) HeLa cells were depleted of endogenous eIF4A2 and transfected with either FLAG–myc–eIF4A2 wt or FLAG–myc–eIF4A2-K226 and either left untreated (UT) or treated with 1 mM arsenite (AR) for 30 min. Cells were immunostained with an anti-FLAG antibody and anti-SUMO. High-resolution images (lower panels) were taken over a z-plane of 4 μm at 0.05 μm slices. Scale bars: 10 μm. (C) HeLa cells were prepared as in B and immunostained for FLAG (eIF4A2) and TIA-1. Scale bars: 10 μm. (D) High-resolution z-stack images were taken and deconvolved using Huygens deconvolution software. Images were three-dimensionally rendered using the IMARIS software suite. 3D images were used to calculate the volumes of stress granules for both TIA-1 and FLAG (eIF4A2). Box-and-whisker plots were used to show the distribution of stress granule volumes. The box represents the 25–75th percentiles, and the median is indicated. The whiskers show the 10–90th percentiles. n=2300 for TIA-1 and 800 for FLAG. (E) Cells processed as in C were analysed for colocalisation of FLAG and TIA-1 signals. The chart shows the percentage of FLAG (eIF4A2) signal that overlaps with TIA-1 in cells transfected with either FLAG-eIF4A2 WT or the K226R mutant (mean±s.d., n=120).
Mentions: Given that eIF4A2 is more highly sumoylated in vivo than is eIF4A1 (Fig. 2A), we concentrated our studies on the eIF4A2 isoform, focusing on SUMO1-containing species, as this would cover singly sumoylated species as well as those containing SUMO chains terminating with SUMO1. Having shown that eIF4A2 is sumoylated in vitro on K226, we wished to confirm that this residue is used for sumoylation in vivo. To do this, expression of endogenous eIF4A2 was knocked down using small interfering RNA (siRNA), and cells were then transfected with wild-type (wt) or mutant eIF4A2 (Fig. 6A). Despite the fact that the knockdown was only partial (eIF4A2 is a very abundant protein making it difficult to achieve 100% knockdown), introduction of siRNA-resistant wild-type eIF4A2 into cells clearly results in new high-molecular-mass species being observed following affinity-purification of His-SUMO1 (lane 4). These species are of similar molecular mass to those observed in Figs 1B and 2B, and are not observed in the untransfected controls (lanes 1–3). In contrast, although it is clear that the eIF4A2-K226 protein was expressed at similar levels to the wt protein (lanes 4 and 5, lower panel), such species were not observed when the eIF4A2-K226R mutant is introduced (lane 5). This indicates that K226 is the main sumoylation site in eIF4A2 that is used in vivo.Fig. 6.

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