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. 2008 Feb 8;283(6):3465-3475.
doi: 10.1074/jbc.M706643200. Epub 2007 Dec 10.

Rapid turnover of the mTOR complex 1 (mTORC1) repressor REDD1 and activation of mTORC1 signaling following inhibition of protein synthesis

Scot R Kimball  1 , A N Dang Do  2 , Lydia Kutzler  2 , Douglas R Cavener  3 , Leonard S Jefferson  2
Affiliations

Rapid turnover of the mTOR complex 1 (mTORC1) repressor REDD1 and activation of mTORC1 signaling following inhibition of protein synthesis

Scot R Kimball et al. J Biol Chem. .

Abstract

mTORC1 is a complex of proteins that includes the mammalian target of rapamycin (mTOR) and several regulatory proteins. It is activated by a variety of hormones (e.g. insulin) and nutrients (e.g. amino acids) that act to stimulate cell growth and proliferation and repressed by hormones (e.g. glucocorticoids) that act to reduce cell growth. Curiously, mTORC1 signaling is reported to be rapidly (e.g. within 1-2 h) activated by inhibitors of protein synthesis that act on either mRNA translation elongation or gene transcription. However, the basis for the mTORC1 activation has not been satisfactorily delineated. In the present study, mTORC1 signaling was found to be activated in response to inhibition of either the initiation or elongation phases of mRNA translation. Changes in mTORC1 signaling were inversely proportional to alterations in the expression of the mTORC1 repressor, REDD1, but not the expression of TRB3 or TSC2. Moreover the cycloheximide-induced increase in mTORC1 signaling was significantly attenuated in cells lacking REDD1, showing that REDD1 plays an integral role in the response. Finally, the half-life of REDD1 was estimated to be 5 min or less. Overall, the results are consistent with a model in which inhibition of protein synthesis leads to a loss of REDD1 protein because of its rapid degradation, and in part reduced REDD1 expression subsequently leads to de-repression of mTORC1 activity.

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Figures

Figure 1
Figure 1
Regulation of protein synthesis and eIF2α phosphorylation in wildtype and GCN2-/- MEFs by cycloheximide, His deprivation, and/or histidinol treatment. (A and C) Wildtype and (B and D) GCN2-/- MEFs were treated with 1 μM cycloheximide or 5 mM histidinol or were deprived of His in the presence or absence of 5 mM histidinol for 2 h prior to harvest. Global rates of protein synthesis were measured as the incorporation of [35S]methionine and [35S]cysteine into protein as described under “Experimental Procedures”. The results are presented as CPM of [35S]methionine and [35S]cysteine incorporated into protein corrected for the amount of protein in cell homogenates and represent the mean ± SE of 9 dishes of cells per condition. The phosphorylation state of eIF2α in (C) wildtype and (D) GCN2-/- MEFs was assessed by Western blot analysis using an antibody specific for eIF2α phosphorylated on Ser51 as described under “Experimental Procedures”. Values for phosphorylated eIF2α were corrected for the total amount of the protein present in the sample. Representative blots are shown as inserts to panels C and D. Lane 1, control cells; lane 2, cells treated with cycloheximide; lane 3, cells treated with 5 mM histidinol; lane 4, cells deprived of His; lane 5, cells deprived of His and treated with 5 mM histidinol. The results represent the mean ± SE of 3 dishes of cells per condition. Means not sharing a superscript are significantly different (p<0.05).
Figure 2
Figure 2
Analysis of polysomal aggregation by sucrose density gradient centrifugation. (A, C, E, G, and I) Wild type and (B, D, F, H, and J) GCN2-/- cells were treated with (C and D) cycloheximide or (E and F) histidinol or were deprived of His (I and J) with or (G and H) without histidinol treatment as described in the legend to Fig. 1. Cell supernatants were subjected to sucrose density gradient centrifugation as described under “Experimental Procedures”. The black arrow in each panel denotes the location of 80S monomers. The ratio of the ribosomal content of the non-polysomal to the polysomal fraction (NP/P) is shown as an inset to each panel. The dashed vertical line in each panel denotes the separation between the two fractions. The results are representative of two studies that were individually analyzed per condition.
Figure 3
Figure 3
Effect of inhibition of translation initiation or elongation on mTOR signaling in wildtype and GCN2-/- MEFs. (A and C) Wildtype and (B and D) GCN2-/- MEFs were treated with cycloheximide or histidinol or were deprived of His with or without histidinol treatment as described in the legend to Fig. 1. Phosphorylation of (A and B) 4E-BP1 and (C and D) S6K1 was assessed by changes in migration during SDS-polyacrylamide gel electrophoresis as described under “Experimental Procedures”. Phosphorylation of 4E-BP1 was calculated as the proportion of the protein present in the hyperphosphorylated γ-form and phosphorylation of S6K1 was calculated as the proportion of the protein present in the hyperphosphorylated β, γ, and δ-forms. Representative blots are shown as inserts to the panels. Lane 1, control cells; lane 2, cells treated with cycloheximide; lane 3, cells treated with 5 mM histidinol; lane 4, cells deprived of His; lane 5, cells deprived of His and treated with 5 mM histidinol. The results represent the mean ± SE of 8-9 dishes of cells per condition. Means not sharing a superscript are significantly different (p<0.05).
Figure 4
Figure 4
Inhibition of mRNA translation results in reduced expression of REDD1, but not TSC2 or TRB3. (A, C, and E) Wildtype and (B, D, and F) GCN2-/- cells were treated with cycloheximide or histidinol or were deprived of His with or without histidinol treatment as described in the legend to Fig. 1. The expression of (A and B) TSC2, (C and D) TRB3, and (E and F) REDD1 was measured by Western blot analysis as described under “Experimental Procedures”. Representative blots are shown as inserts to the panels. Lane 1, control cells; lane 2, cells treated with cycloheximide; lane 3, cells treated with 5 mM histidinol; lane 4, cells deprived of His; lane 5, cells deprived of His and treated with 5 mM histidinol. The results are presented as mean and SE of 7 (TSC2) 6 (TRB3) or 9 (REDD1) dishes of cells. Means not sharing a superscript are significantly different, p<0.05.
Figure 5
Figure 5
Time course of cycloheximide-induced changes in REDD1 expression and mTORC1 signaling. Cycloheximide was added to the culture medium of (A, C, and E) wildtype and (B, D, and F) GCN2-/- MEFs and at the times indicated in the figure, the cells were quickly harvested as described under “Experimental Procedures”. The expression of (A and B) REDD1 was measured as described in the legend to Fig. 4 and (C and D) 4E-BP1 and (E and F) S6K1 phosphorylation was assessed as described in the legend to Fig. 3. Representative blots are shown as inserts to the panels. The time of exposure to cycloheximide in min is depicted below each blot. The results represent the mean ± SE of 6 dishes of cells per condition. Means not sharing a superscript are significantly different, p<0.05.
Figure 6
Figure 6
Time course of cycloheximide-induced changes in REDD1 mRNA expression. Cycloheximide was added to the culture medium of (A) wildtype and (B) REDD1-/- MEFs and at the times indicated in the figure, the cells were quickly harvested as described under “Experimental Procedures”. RNA was isolated from cells and analyzed by quantitative real-time PCR as described under “Experimental Procedures”. REDD1 mRNA content was normalized for the expression of GAPDH mRNA. The results represent the mean ± SE of 6 dishes of cells per condition. Means not sharing a superscript are significantly different, p<0.05.
Figure 7
Figure 7
Activation of mTORC1 signaling by cycloheximide is attenuated in REDD1-/- compared to wildtype MEFs. Cycloheximide was added to the culture medium of (A, C, and E) wildtype and (B, D, and F) REDD1-/- MEFs and 15 min later, the cells were quickly harvested as described under “Experimental Procedures”. The expression of (A and B) 4E-BP1 phosphorylation, (C and D) S6K1 phosphorylation, and (E and F) REDD1 expression was assessed as described in the legend to Fig. 5. Representative blots are shown as inserts to the panels. The time of exposure to cycloheximide in min is depicted below each blot. The results represent the mean ± SE of 6 dishes of cells per condition. Means not sharing a superscript are significantly different, p<0.05. † p<0.01 vs. REDD1+/+ cells at time=0.
Figure 8
Figure 8
REDD1 expression is inversely proportional to mTORC1 signaling. Wildtype and GCN2-/- cells were treated with cycloheximide or histidinol or were deprived of His with or without histidinol treatment as described in the legend to Fig. 1. Values for REDD1 expression are from Fig. 4 and values for 4E-BP1 and S6K1 phosphorylation are from Fig. 3. Note that the same samples were analyzed for all three proteins, so that a direct comparison could be made. The results represent the mean ± SE of 8-9 dishes of cells per condition. The results of a least squares analysis are presented in the figure.
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