An RNA interference screen identifies a novel regulator of target of rapamycin that mediates hypoxia suppression of translation in Drosophila S2 cells

Abstract

In addition to its central role in energy production, oxygen has pervasive regulatory actions. Hypoxia (oxygen limitation) triggers the shutdown of major cellular processes, including gene expression. We carried out a genome-wide RNA interference (RNAi) screen in Drosophila S2 cells for functions required to down-regulate translation during hypoxia. RNAi knockdown of specific genes allowed induction of a green fluorescent protein (GFP) reporter gene and continued protein synthesis during hypoxia. Among the identified genes, Tsc1 and Tsc2, which together form the tuberose sclerosis complex that negatively regulates target of rapamycin (TOR) kinase, gave an especially strong effect. This finding is consistent with the involvement of TOR in promoting translation. Another gene required for efficient inhibition of protein translation during hypoxia, the protein tyrosine phosphatase 61F (Ptp61F), down-regulates TOR activity under hypoxia. Lack of Ptp61F or Tsc2 improves cell survival under prolonged hypoxia in a TOR-dependent manner. Our results identify Ptp61F as a novel modulator of TOR activity and suggest that its function during hypoxia contributes to the down-regulation of protein synthesis.

Figure 1.

Hypoxia induces inhibition of protein translation in Drosophila S2 cells. (A) Protein translation is inhibited in an oxygen concentration-dependent manner. Cells were exposed to the indicated concentration of O₂ for 1 h, [³⁵S]-methionine was then added, and incorporation assessed after an additional hour incubation. Counts per minute of [³⁵S]-methionine incorporation were normalized to that in normoxia (100%). (B) Protein translation is rapidly inhibited under hypoxia. Cells were exposed to hypoxia (0.5% O₂) for the indicated time. [³⁵S]-Methionine was added during the last hour of the hypoxia treatment.

Figure 2.

Genome wide RNAi screening in S2 cells identifies sets of genes required for efficient suppression of translation in response to hypoxia. (A) Cu²⁺-inducible GFP reporter. (B) The expression of a GFP reporter gene is inhibited under hypoxia. CuSO₄ (400 μM final concentration) was added to S2 cells harboring the Cu²⁺-inducible GFP reporter. Cells were incubated in either normoxia or hypoxia for 16 h. GFP expression levels were measured by Western blotting of cell lysates with an anti-GFP antibody. (C) Scheme of RNAi screening. S2 cells harboring the GFP reporter were treated with dsRNA in a 96-well format and incubated for 4 d. CuSO₄ was added to dsRNA-treated cells. Cells were placed under hypoxia (∼0.5% O₂) overnight, fixed with 2% formaldehyde, and screened for GFP signal. (D) Western analysis of reporter gene induction shows that RNAi against genes identified in the screen diminishes translational suppression during hypoxia. Cells were treated for 4 d with dsRNA against genes identified in our screen. CuSO₄ was added to dsRNA-treated cells. Cells were placed under hypoxia (∼0.5% O₂) overnight. GFP expression level was measured by Western blotting of cell lysates with an anti-GFP antibody. Tubulin was detected with anti-tubulin antibody (Sigma-Aldrich) as a loading control. (E) Transcript levels from the GFP reporter were not affected by hypoxia. Cells were treated as described above, and the induction of the GFP reporter under hypoxia and normoxia was measured by RT-PCR.

Figure 3.

Ptp61F is required for efficient inhibition of protein translation under hypoxia. (A) The GFP reporter is induced in cells treated with RNAi against Tsc1, Tsc2, or Ptp61F. Experiments were performed as described in Figure 2D. Rapamycin was added to 50 nM (final concentration) where indicated. (B) The effectiveness of dsRNA against Ptp61F is confirmed by RT-PCR. Cells were treated with indicated dsRNA for 4 d, and depletion of mRNA of indicated genes was examined by RT-PCR. (C) [³⁵S]-Methionine incorporation is decreased by exposure to hypoxia and incorporation in hypoxia is increased by depletion of Tsc2 or Ptp61F. Cells were treated with dsRNA for 4 d, and exposed to hypoxia for 16 h. [³⁵S]-Methionine incorporation was measured as described in Figure 1A. Counts per minute of [³⁵S]-methionine incorporation was normalized to that of control RNAi-treated cells in normoxia (100%). *p < 0.05.

Figure 4.

Ptp61F contributes to inhibition of protein translation under hypoxia by down-regulating TOR activity. (A) Ptp61F is required for down-regulation of TOR activity. Cells were treated with dsRNA against Tsc2 or Ptp61F, exposed to hypoxia (0.5% O₂, 16 h), and lysed. Phosphorylated p70^S6K (phospho-S6K) and phosphorylated 4E-BP (phospho-4E-BP) were detected by Western blotting with antibodies recognizing phosphorylated forms of these proteins (Cell Signaling Technology). N, normoxia; H, hypoxia. (B) Expression of Ptp61F-A with C-terminal Myc tag restores a decrease in phosphorylation of p70^S6K (phospho-S6K) in hypoxia to Ptp61F-A/D–depleted cells. Cells harboring a copper inducible Ptp61F-A-myc gene were treated for 4 d with dsRNA specifically targeting the 3′ UTR of Ptp61F-A/D. Ptp61F-A was induced with copper sulfate (100 μM) for the same period. (C) Inhibition of TOR activity by rapamycin suppresses the effect of Ptp61F depletion on inhibition of protein translation under hypoxia. Cells were treated with dsRNA against Ptp61F for 4 d. Rapamycin was added where indicated, and cells were exposed to hypoxia (0.5% O₂ for 16 h). [³⁵S]-Methionine incorporation was measured as described in Figure 1A. Rapa, rapamycin. *p < 0.05.

Figure 5.

Ptp61F down-regulates phosphorylation of a TOR substrate in hypoxia by suppressing an Akt-dependent signal. (A) Ptp61F is required for efficient down-regulation of insulin signaling under hypoxia. Cells treated with dsRNA against Ptp61F were stimulated with 10 μg/ml bovine insulin for an hour, exposed to normoxia or hypoxia (0.5% O₂) for the indicated time, and lysed. Antibodies against phospho-tyrosine (4G10; Millipore), insulin receptor (from O. Puig), phosphorylated Akt, Akt (Cell Signaling Technology), and tubulin (Sigma-Aldrich) were used in Western blot analysis to detect activation of insulin signaling pathway. The star-like symbol indicates a band that stains for phospho-tyrosine that is unrelated to but is close to the insulin receptor band. (B) dsRNAi targeting the insulin receptor, JAK, or STAT does not compromise Ptp61F modulation of phosphorylation of p70^S6K (phospho-S6K) under hypoxia. (C) Depletion of Akt inhibits the effect of Ptp61F RNAi on phosphorylated p70^S6K under hypoxia. Cells treated with dsRNA against Ptp61F, Akt, or Akt in combination with Ptp61F were exposed to hypoxia (<0.1% O₂ for 16 h) or normoxia and phosphorylated p70^S6K was detected as described in Figure 4A.

Figure 6.

Lack of Ptp61F improves cell survival under hypoxia. (A) Cells deficient in Ptp61F displayed enhanced survival under hypoxia compared with control cells (LacI, GFP, or luciferase RNAi). *p < 0.05. (B and C) Inhibition of TORC1 activity abolishes the effect of depletion of Ptp61F (B) or Tsc2 (C) on cell survival under hypoxia. In Figure 6, cells were treated with various dsRNAs for 4 d and exposed to hypoxia treatment for indicated days. Cell death was measured by staining cells with Sytox Green, which only stains dead cells, followed by FACS analysis.