Overexpression of PTEN or dominant-negative p85 or PKB mutants suppresses the translational activation by amino acid refeeding. 293 cells were cotransfected with 2 μg of a vector encoding L32-GFP or Act-GFP and 16 μg of an empty vector (pSG5) or the expression vector indicated at the left. Twenty-four hours later cells were amino acid starved for 2 h and then refed for 30 min. The polysomal distribution of mRNAs encoding L32-GFP and Act-GFP was analyzed and is presented as described in the legend to Fig. 2.

Schematic representation of signal transduction pathways involved in activation of rpS6 phosphorylation and translational control of TOP mRNAs in amino acid-stimulated cells. Arrows delineate the flow of information. Open arrowhead, partial and delayed effect of rapamycin (through mTOR?) on TOP mRNA translation. The site of convergence of this effect with that of other signals is purely speculative. Circled and boxed question marks, putative links and unknown targets, respectively. See text for details.

LY-294002 inhibits both the phosphorylation of rpS6 and the translational activation of TOP mRNAs upon amino acid refeeding. (a) 293 cells were untreated (C), amino acid starved for 2 h (0′), or refed with amino acids without (−) or with (+) LY-294002 (50 μM) for the indicated times, after which cells were harvested. The cytoplasmic proteins were subjected to Western blot analysis using the indicated antibodies. (b) 293 cells were untreated or amino acid starved for 2 h and then refed for 30 min without (−) or with (+) LY294002 (50 μM). The polysomal distribution of rpL32 mRNA was analyzed and is presented as described in the legend to Fig. 2.

Calyculin A induces phosphorylation of rpS6 but fails to relieve the translational repression of TOP mRNAs. (a) 293 cells were either untreated (C) or amino acid starved without (−) or with (+) calyculin A (20 nM) for the indicated time, after which cells were harvested. The cytoplasmic proteins were subjected to Western blot analysis using anti-rpS6. α and β, unphosphorylated and hyperphosphorylated forms of rpS6, respectively. (b) 293 cells were untreated or amino acid starved for 1 h without (−) or with (+) calyculin A (20 nM), after which cells were harvested. The cytoplasmic proteins were subjected to Western blot analysis using the indicated antibodies. (c) 293 cells were amino acid starved for 1 h without (−) or with (+) calyculin A (20 nM) and then harvested, and the polysomal distribution of the mRNAs encoding rpL32 and actin was analyzed and presented as described in the legend to Fig. 2.

Rapamycin inhibits phosphorylation of rpS6, but does not prevent translational activation of rpL32. (a) 293 cells were untreated (C), amino acid starved for 2 h (0′), or refed with amino acids for the indicated times without (−) or with (+) rapamycin (20 nM) for the indicated times, after which cells were harvested. The cytoplasmic proteins were subjected to Western blot analysis using the indicated antibodies. (b) 293 cells were amino acid starved for 2 h and then refed without (left) or with (right) rapamycin (20 nM) and then harvested. The distribution of the mRNAs encoding rpL32 and SOD among 12 sucrose gradient fractions was analyzed and is presented as described in the legend to Fig. 1a. The shaded areas depict the profiles of optical density at 260 nm of polysomal and subpolysomal fractions.

TOP mRNAs are translationally regulated by amino acid sufficiency in ES cells lacking S6K activity. (a) Wild type ES cells (+/+) and p70^S6K−/− cells (−/−) were harvested untreated (C) or after being amino acid starved for 2 h (−aa) or starved and then refed for 1 h (ref). The cytoplasmic proteins were subjected to Western blot analysis using the indicated antibodies (the results represent two independent experiments with identical results). (b to d) p70^S6K−/− cells were treated as described in for panel a and then harvested, and the profiles of optical density at 260 nm are presented (dashed lines separate the polysomal fractions [left] and the subpolysomal fractions [right]). (e) The polysomal distribution of mRNAs encoding rpL32 and actin was analyzed and presented as described in the legend to Fig. 2.

Overexpression of RSK2 led to phosphorylation of rpS6 but failed to relieve the translational repression of L32-GFP mRNA in amino acid-starved cells. (a) 293 cells were transiently transfected with 16 μg of pHA-K3H.RSK2(Y707A) (lane 1) or its inactive counterpart, pHA-K3H.RSK2(K100A) (lane 2). Twenty-four hours later transfectants were starved of amino acids for 1 h and harvested, and their cytoplasmic proteins were used either for Western blot analysis with anti-HA or for assaying the activity of RSK2 following immunoprecipitation with an anti-HA antibody. In the latter case the reaction mixture was separated by SDS-PAGE, transferred onto nitrocellulose membrane, and subjected to autoradiography (³²P-S6). (b) In a parallel experiment, 293 cells were similarly transfected with 16 μg of one of the expression vectors encoding HA-K3H.RSK2(Y707A) (lane 1) or HA-K3H.RSK2(K100A) (lane 2). Twenty-four hours later cells were starved for 1 h and harvested, and their cytoplasmic proteins were subjected to Western blot analysis using anti-rpS6 and anti-phospho-Ser235/236 or anti-phospho-Ser240/244. α and β, hypophosphorylated and hyperphosphorylated forms of rpS6, respectively. (c) 293 cells were cotransfected with 2 μg of a vector encoding L32-GFP and 16 μg of the expression vector indicated at the left. Twenty-four hours later cells were starved for 1 h and harvested, and the polysomal distribution of L32-GFP mRNA was analyzed and presented as described in the legend to Fig. 2.

Amino acid deficiency induces reversible translational repression of TOP mRNAs. (a) Untreated 293 cells (Control) and 293 cells starved of amino acids for 1 h [−AA (1 h)] were harvested, and cytoplasmic extracts were prepared. These extracts were centrifuged through sucrose gradients and separated into 12 fractions. RNA isolated from these fractions was applied to Northern blot analysis and hybridized with labeled cDNAs encoding SOD and rpL32. The radioactive signals were quantified by phosphorimager, and the results for each fraction are presented as the percentage of total mRNA (dashed lines separate the polysomal fractions [left] and the subpolysomal fractions [right]). For each treatment the same RNA preparations were hybridized with the different probes. The shaded areas depict the profiles of optical density at 260 nm for polysomal and subpolysomal fractions. (b) Untreated 293 cells and cells starved of amino acids for the indicated times or starved and then refed for 0.5 h were harvested, and cytoplasmic extracts were prepared. These extracts were centrifuged through sucrose gradients and separated into two fractions: polysomal and subpolysomal. RNA isolated from these fractions was subjected to Northern blot analysis and hybridized with labeled cDNAs encoding SOD and rpL32. The radioactive signals were quantified by phosphorimager, and the relative amounts of the mRNAs in polysomes are shown. Dashed lines, changes in polysomal association following 0.5 h of refeeding. Vertical bars, standard errors of the means.

The 5′TOP motif plays a critical role in the translational repression of TOP mRNAs upon amino acid starvation. 293 cells were transfected with 2 μg of the indicated chimeric GH or GFP constructs together with 16 μg of an empty vector. Cytoplasmic extracts were prepared about 24 h later from untreated (CON) or cells starved of amino acids for 1 h (−AA). These extracts were centrifuged through sucrose gradients and separated into polysomal (P) and subpolysomal (S) fractions. RNA from equivalent aliquots of these fractions was analyzed by Northern blot hybridization with hGH or GFP cDNAs for detection of the chimeric transcripts and cDNAs for L32 and actin for detection of the corresponding endogenous mRNAs. The radioactive signals were quantified by phosphorimager, and the relative translational efficiency of each mRNA is numerically presented at the right as a percentage of the mRNA engaged in polysomes. These figures are averages ± standard errors of the means of the number of determinations in parentheses or are the averages with the individual values in parentheses if only two determinations are available. The first 10 nt in the respective chimeric construct are indicated at the left, and the underlined letters represent nucleotides mutated relative to the wild-type sequence.

Overexpression of kinase-dead S6K1 mutants inhibited S6K activity but failed to suppress the translational activation of L32-GFP mRNA. (a) 293 cells were transiently cotransfected with 1 μg of HA-p70S6K1 together with 1 μg of pEGFP-C1 and 16 μg of either a vector encoding the HA epitope (EMPTY) or a vector encoding HA-tagged p70S6K,K123M. Twenty-four hours later, highly fluorescent cells were sorted by fluorescence-activated cell sorter and reseeded. Forty-eight hours posttransfection cells were harvested, and cytoplasmic proteins were subjected to Western blot analysis using anti-rpS6 and anti-phospho-Ser240/244 antibodies. (b) 293 cells were transiently transfected with 1 μg of HA-S6K1, 1 μg of pEGFP-C1, and 16 μg of either a vector encoding HA (EMPTY) or a vector encoding HA-tagged p70Δ2-46/ΔCT104, K123M/T412E (Kinase-dead). Twenty-four hours later cells were starved for 2 h and then refed for 0.5 h and harvested. Cell extracts were subjected to immunoprecipitation with an anti-HA antibody, and a portion was assayed for S6 kinase activity. The reaction mixture was separated by SDS-PAGE and transferred onto a nitrocellulose membrane, and the radioactive signals of ³²P-S6 were autoradiographed (HA-S6K1 activity). Immunoblotting of the membrane with an anti-HA antibody enabled the detection of the wild-type S6K1 (HA-S6K1) and of the shorter kinase-dead (p70Δ2-46/ΔCT104, K123M/T412E) variant (Kinase-dead). (c) 293 cells were transfected with 2 μg of a vector encoding L32-GFP and 16 μg of a vector encoding the HA tag, p70S6K,K123M, and p70Δ2-46/ΔCT104, K123M/ T412E (Kinase-dead). Twenty-four to 36 h later cells were harvested untreated (Con) or after 2 h of amino acid starvation followed by 0.5 h of refeeding (aa refed). The translational efficiency of L32-GFP mRNA was analyzed and is presented as described in the legend to Fig. 2.

Translational activation of rpL32 mRNA is far more resistant to rapamycin than rpS6 phosphorylation. (a) 293 cells were amino acid starved for 2 h and then refed for 30 min in the absence or presence of rapamycin at the indicated concentrations. Subsequently, the cytoplasmic proteins were subjected to Western blot analysis using an anti-rpS6 antibody. (b) 293 cells were untreated (C), amino acid starved for 2 h (−AA), or amino acid starved for 2 h and then refed for 30 min without rapamycin (refed) or with the indicated concentrations of rapamycin. The polysomal distribution of rpL32 mRNA was analyzed and presented as described in the legend to Fig. 2. (c) Dose-response curves of the effects of rapamycin on rpS6 phosphorylation and on translational efficiency of rpL32 mRNA. The chemiluminescence signals of the hypophosphorylated and phosphorylated bands, α and β, respectively, whose images appear in panel a, were quantified by the Chemi Doc system (Bio-Rad). The relative phosphorylation of rpS6 is presented numerically as the β/α ratio. The translational efficiency of rpL32 mRNA is presented as the percentage of mRNA associated in polysomes. (d) Kinetics of the effect of rapamycin on the translational efficiency of rpL32 mRNA. 293 cells were amino acid starved for 2 h (time zero) and then refed in the absence (white bars) or presence (gray bars) of rapamycin for the indicated times. Rapamycin was simultaneously added with amino acids when cells were refed for 0.5 and 2 h or for 15 min prior to addition of the amino acids when the latter were added for 1 h. The polysomal distribution of rpL32 mRNA was analyzed as described in the legend to Fig. 2. The percentage of mRNA in polysomes is presented as an averages ± standard errors of the means for three experiments.