pubmed.ncbi.nlm.nih.gov

Sam68 relocalization into stress granules in response to oxidative stress through complexing with TIA-1 - PubMed

  • ️Thu Jan 01 2009

Sam68 relocalization into stress granules in response to oxidative stress through complexing with TIA-1

Jorge Henao-Mejia et al. Exp Cell Res. 2009.

Abstract

Sam68 has been implicated in a variety of important cellular processes such as RNA metabolism and intracellular signaling. We have recently shown that Sam68 cytoplasmic mutants induce stress granules (SG) and inhibit HIV-1 nef mRNA translation [J. Henao-Mejia, Y. Liu, I.W. Park, J. Zhang, J. Sanford, J.J. He, Suppression of HIV-1 Nef translation by Sam68 mutant-induced stress granules and nef mRNA sequestration, Mol. Cell 33 (2009) 87-96]. These findings prompted us to investigate the possibility and the underlying mechanisms of the wild-type counterpart Sam68 SG recruitment. Herein, we revealed that Sam68 was significantly recruited into cytoplasmic SG under oxidative stress. We then demonstrated that domain aa269-321 and KH domain were both essential for this recruitment. Nevertheless, Sam68 knockdown had no effects on SG assembly, indicating that Sam68 is not a constitutive component of the SG. Moreover, we showed that Sam68 cytoplasmic mutant-induced SG formation was independent of eIF2alpha phosphorylation. Lastly, we demonstrated that Sam68 was complexed with T-cell intracellular antigen-1 (TIA-1), a core SG component, and that the complex formation was correlated with Sam68 SG recruitment. Taken together, these results provide direct evidence for the first time that Sam68 is recruited into SG through complexing with TIA-1 in response to oxidative stress and suggest that cytoplasmic SG recruitment of Sam68 and ensuing changes in Sam68 physiological functions are part of the host response to external stressful conditions.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Localization of exogenous Sam68 into SG upon oxidative stress

293T cells were transfected with RFP-Sam68 and GFP.TIA-1 (A), GFP.G3BP (B), GFP.hnRNP A1 (C), GFP.PABP (D), GFP.hdcp1α (E). Cells were cultured for 48 hr and then treated with 0.5 mM ARS (+ ARS) or without ARS (− ARS) for 1 hr prior to fixation and microscopic imaging. The images were representative of each transfection from at least three independent experiments. Co-localization of each marker with exogenous Sam68 was shown in the column marked as “Merge”.

Figure 1
Figure 1. Localization of exogenous Sam68 into SG upon oxidative stress

293T cells were transfected with RFP-Sam68 and GFP.TIA-1 (A), GFP.G3BP (B), GFP.hnRNP A1 (C), GFP.PABP (D), GFP.hdcp1α (E). Cells were cultured for 48 hr and then treated with 0.5 mM ARS (+ ARS) or without ARS (− ARS) for 1 hr prior to fixation and microscopic imaging. The images were representative of each transfection from at least three independent experiments. Co-localization of each marker with exogenous Sam68 was shown in the column marked as “Merge”.

Figure 2
Figure 2. Localization of endogenous Sam68 into stress granules upon oxidative stress

293T cells were plated and cultured for 24 hr to reach 60–70% confluence. The cells were treated with 0.5 mM ARS (+ ARS) or without ARS (− ARS) for 1 hr prior to fixation, and then processed for double immunofluorescence staining using pairs of primary and secondary antibodies as specified below: A. rabbit α-Sam68/Rh-conjugated goat α-rabbit; B. rabbit α-Sam68/Rh-conjugated goat α-rabbit, mouse α-G3BP/FITC-conjugated goat α-mouse; C. rabbit α-Sam68/Rh-conjugated bovine α-rabbit, goat α-eIF3η/FITC-conjugated donkey α-goat; D. rabbit α-Sam68/Rh-conjugated bovine α-rabbit, goat α-TIA-1/FITC-conjugated donkey α-goat. The images were representative of each staining from at least three independent experiments. Co-localization of each SG marker with endogenous Sam68 was shown in the column marked as “Merge”.

Figure 3
Figure 3. Requirement of specific Sam68 domain for its SG recruitment

A. P: proline-rich domain; KH: KH domain; RG: arginine-glycine rich domain; and NLS: nuclear localization signal. The single lines in GFP. 269–321 and GFP.269–321 represent deleted regions. B–G. 293T cells were transfected with GFP-tagged Sam68 (B), deletion mutants containing various lengths of C-terminal deletion, i.e. Δ410 (C), Δ321 (D) or Δ 269 (E), Δ269–321 lacking the domain of aa269–321 (F), or 269–321 expressing the domain of aa269–321 (G). Cells were cultured for 48 hr and then treated with 0.5 mM ARS (+ ARS) or without ARS (− ARS) for 1 hr prior to fixation. The cells were then stained using a mouse α-G3BP antibody followed by PE-conjugated goat α-mouse secondary antibody. The images were representative of each transfection. Co-localization of G3BP with Sam68 or each mutant was shown in the column marked as “Merge”. H. Quantitation of GFP+G3BP+ cells in each transfection in B–G, which was expressed as a percentage of the total number of GFP+ cells. The data were mean ± SEM of at least three independent experiments. The comparison was made between ARS-treated and untreated cells. *: p < 0.05; **: p < 0.05. I. 293T cells were transfected with GFP-tagged Sam68 or each of its mutants as indicated, followed by Western blot analysis using α-eIF2α, α-phosphorylated eIF2α (α-eIF2α-P), or α-GFP antibodies. Cells that were transfected with GFP and treated with 0.5 mM ARS or without ARS were included as positive and negative controls for eIF2α phosphorylation, respectively, in the Western blot analysis. *: GFP-tagged proteins.

Figure 3
Figure 3. Requirement of specific Sam68 domain for its SG recruitment

A. P: proline-rich domain; KH: KH domain; RG: arginine-glycine rich domain; and NLS: nuclear localization signal. The single lines in GFP. 269–321 and GFP.269–321 represent deleted regions. B–G. 293T cells were transfected with GFP-tagged Sam68 (B), deletion mutants containing various lengths of C-terminal deletion, i.e. Δ410 (C), Δ321 (D) or Δ 269 (E), Δ269–321 lacking the domain of aa269–321 (F), or 269–321 expressing the domain of aa269–321 (G). Cells were cultured for 48 hr and then treated with 0.5 mM ARS (+ ARS) or without ARS (− ARS) for 1 hr prior to fixation. The cells were then stained using a mouse α-G3BP antibody followed by PE-conjugated goat α-mouse secondary antibody. The images were representative of each transfection. Co-localization of G3BP with Sam68 or each mutant was shown in the column marked as “Merge”. H. Quantitation of GFP+G3BP+ cells in each transfection in B–G, which was expressed as a percentage of the total number of GFP+ cells. The data were mean ± SEM of at least three independent experiments. The comparison was made between ARS-treated and untreated cells. *: p < 0.05; **: p < 0.05. I. 293T cells were transfected with GFP-tagged Sam68 or each of its mutants as indicated, followed by Western blot analysis using α-eIF2α, α-phosphorylated eIF2α (α-eIF2α-P), or α-GFP antibodies. Cells that were transfected with GFP and treated with 0.5 mM ARS or without ARS were included as positive and negative controls for eIF2α phosphorylation, respectively, in the Western blot analysis. *: GFP-tagged proteins.

Figure 3
Figure 3. Requirement of specific Sam68 domain for its SG recruitment

A. P: proline-rich domain; KH: KH domain; RG: arginine-glycine rich domain; and NLS: nuclear localization signal. The single lines in GFP. 269–321 and GFP.269–321 represent deleted regions. B–G. 293T cells were transfected with GFP-tagged Sam68 (B), deletion mutants containing various lengths of C-terminal deletion, i.e. Δ410 (C), Δ321 (D) or Δ 269 (E), Δ269–321 lacking the domain of aa269–321 (F), or 269–321 expressing the domain of aa269–321 (G). Cells were cultured for 48 hr and then treated with 0.5 mM ARS (+ ARS) or without ARS (− ARS) for 1 hr prior to fixation. The cells were then stained using a mouse α-G3BP antibody followed by PE-conjugated goat α-mouse secondary antibody. The images were representative of each transfection. Co-localization of G3BP with Sam68 or each mutant was shown in the column marked as “Merge”. H. Quantitation of GFP+G3BP+ cells in each transfection in B–G, which was expressed as a percentage of the total number of GFP+ cells. The data were mean ± SEM of at least three independent experiments. The comparison was made between ARS-treated and untreated cells. *: p < 0.05; **: p < 0.05. I. 293T cells were transfected with GFP-tagged Sam68 or each of its mutants as indicated, followed by Western blot analysis using α-eIF2α, α-phosphorylated eIF2α (α-eIF2α-P), or α-GFP antibodies. Cells that were transfected with GFP and treated with 0.5 mM ARS or without ARS were included as positive and negative controls for eIF2α phosphorylation, respectively, in the Western blot analysis. *: GFP-tagged proteins.

Figure 4
Figure 4. Role of Sam68 KH domain in SG recruitment, Sam68 solubility and HIV-1 Nef mRNA translation

A. KH domain deletion mutants: P: proline-rich domain; KH: KH domain; and NLS: nuclear localization signal. The dotted lines represent deleted regions. B–E. 293T cells were transfected with GFP-tagged GFPSam68 (B), GFP.ΔKH (C), GFP.Δ410 (D), and GFP.Δ410ΔKH (E). Cells were cultured for 48 hr and prior to fixation, the cells were treated with 0.5 mM ARS (+ ARS) or without ARS (− ARS) for 1 hr. The cells were then stained using a mouse α-G3BP antibody followed by phycoerythrin (PE)-conjugated goat anti-mouse antibody. The images were representative of each transfection from at least three independent experiments. Co-localization of G3BP and GFP-tagged Sam68 or its mutants was shown in the column marked as “Merge”. F. 293T cells were transfected with GFP-tagged Sam68 or each of its mutants as indicated, then cell lysates were separated into soluble and insoluble fractions as described in Materials and Methods, followed by Western blot analysis using α-GFP or α-β-actin antibody. G. 293T cells were transfected with HIV-1 isolate NL4-3 and GFP-tagged Sam68 or each of its mutants as indicated, followed by Western blot analysis using α-Nef or α-β-actin antibody.

Figure 4
Figure 4. Role of Sam68 KH domain in SG recruitment, Sam68 solubility and HIV-1 Nef mRNA translation

A. KH domain deletion mutants: P: proline-rich domain; KH: KH domain; and NLS: nuclear localization signal. The dotted lines represent deleted regions. B–E. 293T cells were transfected with GFP-tagged GFPSam68 (B), GFP.ΔKH (C), GFP.Δ410 (D), and GFP.Δ410ΔKH (E). Cells were cultured for 48 hr and prior to fixation, the cells were treated with 0.5 mM ARS (+ ARS) or without ARS (− ARS) for 1 hr. The cells were then stained using a mouse α-G3BP antibody followed by phycoerythrin (PE)-conjugated goat anti-mouse antibody. The images were representative of each transfection from at least three independent experiments. Co-localization of G3BP and GFP-tagged Sam68 or its mutants was shown in the column marked as “Merge”. F. 293T cells were transfected with GFP-tagged Sam68 or each of its mutants as indicated, then cell lysates were separated into soluble and insoluble fractions as described in Materials and Methods, followed by Western blot analysis using α-GFP or α-β-actin antibody. G. 293T cells were transfected with HIV-1 isolate NL4-3 and GFP-tagged Sam68 or each of its mutants as indicated, followed by Western blot analysis using α-Nef or α-β-actin antibody.

Figure 4
Figure 4. Role of Sam68 KH domain in SG recruitment, Sam68 solubility and HIV-1 Nef mRNA translation

A. KH domain deletion mutants: P: proline-rich domain; KH: KH domain; and NLS: nuclear localization signal. The dotted lines represent deleted regions. B–E. 293T cells were transfected with GFP-tagged GFPSam68 (B), GFP.ΔKH (C), GFP.Δ410 (D), and GFP.Δ410ΔKH (E). Cells were cultured for 48 hr and prior to fixation, the cells were treated with 0.5 mM ARS (+ ARS) or without ARS (− ARS) for 1 hr. The cells were then stained using a mouse α-G3BP antibody followed by phycoerythrin (PE)-conjugated goat anti-mouse antibody. The images were representative of each transfection from at least three independent experiments. Co-localization of G3BP and GFP-tagged Sam68 or its mutants was shown in the column marked as “Merge”. F. 293T cells were transfected with GFP-tagged Sam68 or each of its mutants as indicated, then cell lysates were separated into soluble and insoluble fractions as described in Materials and Methods, followed by Western blot analysis using α-GFP or α-β-actin antibody. G. 293T cells were transfected with HIV-1 isolate NL4-3 and GFP-tagged Sam68 or each of its mutants as indicated, followed by Western blot analysis using α-Nef or α-β-actin antibody.

Figure 5
Figure 5. Effects of Sam68 knockdown on SG assembly

A. 293T cells were plated at a density of 1.75×105 per well and transfected with 75 nM of control siRNA or Sam68 siRNA. The cells were harvested 24 hr after transfection for Western Blot analysis using anti-Sam68 or anti-β-actin antibodies. B. 293T cells were plated and transfected with control siRNA or Sam68 siRNA as above. The cells were treated with 0.5 mM ARS (+ ARS) or without ARS (− ARS) for 1 hr prior to fixation. The cells were then stained using a mouse α-G3BP antibody followed by FITC-conjugated α-mouse antibody. Transfected cells were also stained with 500 ng/ml of DAPI for nuclei. The images were representative of each transfection from at least three independent experiments. C. Quantitation of G3BP+ SG-containing cells in B, which was expressed as percentage of the total number of G3BP+ cells. The data were mean ± SEM of at least three independent experiments.

Figure 6
Figure 6. Complex formation between Sam68 and TIA-1 and Sam68 SG recruitment

A. 293T cells were transfected with HA.Sam68, GFP-TIA-1, or both and harvested 48 hr after transfection for cell lysates. Cell lysates were then immunoprecipitated with normal IgG, anti-HA, or anti-TIA-1 antibody, followed by Western blotting using α-HA or α-GFP antibody. Input lysates were directly blotted to ensure comparable Sam68 and TIA-1 expression among transfections. *: reactive IgG bands. B. Cell lysates were prepared from 293T cells and blotted for endogenous Sam68 and TIA-1 expression (Input lysates), or immunoprecipitated with normal IgG, α-Sam68, or α-TIA-1, followed by Western blot analysis using α-TIA-1 or α-Sam68 antibody. C. 293T cells were transfected with GFP-TIA-1, in combination with HA.Sam68, or each of its mutants. Input lysates were directly blotted with α-GFP or α-HA antibody to ensure comparable protein expression, or immunoprecipitated with α-HA antibody, followed by Western blot analysis using α-GFP or α-HA antibody. D. 293T cells were transfected with GFP-TIA-1 in combination with HA.Δ410 or HA.Δ269–321. The cells were then stained using an anti-HA antibody followed by PE-conjugated goat anti-mouse antibody. Transfected cells were also stained with 500 ng/ml of DAPI for nuclei. E. 293T cells were transfected with GFP.Δ410 or GFP.Δ269–321, cultured for 48 hr and then treated with 0.5 mM ARS for 1 hr prior to fixation. The cells were then stained using a mouse α-TIA-1 antibody followed by PE-conjugated donkey α-goat secondary antibody. The images were representative of each transfection from at least three independent experiments. F. 293T cells were transfected with HA.Sam68, GFP-G3BP, or both and harvested 48 hr after transfection for cell lysates. Cell lysates were then immunoprecipitated with normal IgG, anti-HA, or anti-G3BP antibody, followed by Western blotting using α-HA or α-G3BP antibody. Input lysates were directly blotted to ensure comparable Sam68 and G3BP expression among transfections. G. Cell lysates were prepared from 293T cells and blotted for endogenous Sam68 and G3BP expression (Input lysates), or immunoprecipitated with normal IgG or α-Sam68, followed by Western blot analysis using α-G3BP or α-Sam68 antibody.*: reactive IgG bands.

Figure 6
Figure 6. Complex formation between Sam68 and TIA-1 and Sam68 SG recruitment

A. 293T cells were transfected with HA.Sam68, GFP-TIA-1, or both and harvested 48 hr after transfection for cell lysates. Cell lysates were then immunoprecipitated with normal IgG, anti-HA, or anti-TIA-1 antibody, followed by Western blotting using α-HA or α-GFP antibody. Input lysates were directly blotted to ensure comparable Sam68 and TIA-1 expression among transfections. *: reactive IgG bands. B. Cell lysates were prepared from 293T cells and blotted for endogenous Sam68 and TIA-1 expression (Input lysates), or immunoprecipitated with normal IgG, α-Sam68, or α-TIA-1, followed by Western blot analysis using α-TIA-1 or α-Sam68 antibody. C. 293T cells were transfected with GFP-TIA-1, in combination with HA.Sam68, or each of its mutants. Input lysates were directly blotted with α-GFP or α-HA antibody to ensure comparable protein expression, or immunoprecipitated with α-HA antibody, followed by Western blot analysis using α-GFP or α-HA antibody. D. 293T cells were transfected with GFP-TIA-1 in combination with HA.Δ410 or HA.Δ269–321. The cells were then stained using an anti-HA antibody followed by PE-conjugated goat anti-mouse antibody. Transfected cells were also stained with 500 ng/ml of DAPI for nuclei. E. 293T cells were transfected with GFP.Δ410 or GFP.Δ269–321, cultured for 48 hr and then treated with 0.5 mM ARS for 1 hr prior to fixation. The cells were then stained using a mouse α-TIA-1 antibody followed by PE-conjugated donkey α-goat secondary antibody. The images were representative of each transfection from at least three independent experiments. F. 293T cells were transfected with HA.Sam68, GFP-G3BP, or both and harvested 48 hr after transfection for cell lysates. Cell lysates were then immunoprecipitated with normal IgG, anti-HA, or anti-G3BP antibody, followed by Western blotting using α-HA or α-G3BP antibody. Input lysates were directly blotted to ensure comparable Sam68 and G3BP expression among transfections. G. Cell lysates were prepared from 293T cells and blotted for endogenous Sam68 and G3BP expression (Input lysates), or immunoprecipitated with normal IgG or α-Sam68, followed by Western blot analysis using α-G3BP or α-Sam68 antibody.*: reactive IgG bands.

Figure 6
Figure 6. Complex formation between Sam68 and TIA-1 and Sam68 SG recruitment

A. 293T cells were transfected with HA.Sam68, GFP-TIA-1, or both and harvested 48 hr after transfection for cell lysates. Cell lysates were then immunoprecipitated with normal IgG, anti-HA, or anti-TIA-1 antibody, followed by Western blotting using α-HA or α-GFP antibody. Input lysates were directly blotted to ensure comparable Sam68 and TIA-1 expression among transfections. *: reactive IgG bands. B. Cell lysates were prepared from 293T cells and blotted for endogenous Sam68 and TIA-1 expression (Input lysates), or immunoprecipitated with normal IgG, α-Sam68, or α-TIA-1, followed by Western blot analysis using α-TIA-1 or α-Sam68 antibody. C. 293T cells were transfected with GFP-TIA-1, in combination with HA.Sam68, or each of its mutants. Input lysates were directly blotted with α-GFP or α-HA antibody to ensure comparable protein expression, or immunoprecipitated with α-HA antibody, followed by Western blot analysis using α-GFP or α-HA antibody. D. 293T cells were transfected with GFP-TIA-1 in combination with HA.Δ410 or HA.Δ269–321. The cells were then stained using an anti-HA antibody followed by PE-conjugated goat anti-mouse antibody. Transfected cells were also stained with 500 ng/ml of DAPI for nuclei. E. 293T cells were transfected with GFP.Δ410 or GFP.Δ269–321, cultured for 48 hr and then treated with 0.5 mM ARS for 1 hr prior to fixation. The cells were then stained using a mouse α-TIA-1 antibody followed by PE-conjugated donkey α-goat secondary antibody. The images were representative of each transfection from at least three independent experiments. F. 293T cells were transfected with HA.Sam68, GFP-G3BP, or both and harvested 48 hr after transfection for cell lysates. Cell lysates were then immunoprecipitated with normal IgG, anti-HA, or anti-G3BP antibody, followed by Western blotting using α-HA or α-G3BP antibody. Input lysates were directly blotted to ensure comparable Sam68 and G3BP expression among transfections. G. Cell lysates were prepared from 293T cells and blotted for endogenous Sam68 and G3BP expression (Input lysates), or immunoprecipitated with normal IgG or α-Sam68, followed by Western blot analysis using α-G3BP or α-Sam68 antibody.*: reactive IgG bands.

Figure 7
Figure 7. Effects of oxidative stress on Sam68 phosphorylation

A. 293T cells were treated with 0.5 mM ARS (+ ARS) or without ARS (− ARS) for 1 hr prior to harvesting the cells. Cell lysates were then immunoprecipitated with normal anti-Sam68 antibody, followed by Western blotting using α-pTyr, α-pThr, α-pSer or α-Sam68 antibody. Input lysates were directly blotted to ensure comparable Sam68 and actin expression among samples. B. 293T cells were treated with 0.5 mM ARS for 0, 10, 20, 30, 40, 50, and 60 min, followed by Western blot analysis using anti-ERK1, anti-phosphorylated ERK1/2 (p-ERK1/2), or anti-β-actin.

Similar articles

Cited by

References

    1. Henao-Mejia J, Liu Y, Park IW, Zhang J, Sanford J, He JJ. Suppression of HIV-1 Nef Translation by Sam68 Mutant-Induced Stress Granules and nef mRNA Sequestration. Mol Cell. 2009;33:87–96. - PMC - PubMed
    1. Ellis C, Moran M, McCormick F, Pawson T. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature. 1990;343:377–381. - PubMed
    1. Moran MF, Koch CA, Anderson D, Ellis C, England L, Martin GS, Pawson T. Src homology region 2 domains direct protein-protein interactions in signal transduction. Proc Natl Acad Sci U S A. 1990;87:8622–8626. - PMC - PubMed
    1. Wong G, Muller O, Clark R, Conroy L, Moran MF, Polakis P, McCormick F. Molecular cloning and nucleic acid binding properties of the GAP-associated tyrosine phosphoprotein p62. Cell. 1992;69:551–558. - PubMed
    1. Fumagalli S, Totty NF, Hsuan JJ, Courtneidge SA. A target for Src in mitosis. Nature. 1994;368:871–874. - PubMed

Publication types

MeSH terms

Substances