mTOR-dependent regulation of ribosomal gene transcription requires S6K1 and is mediated by phosphorylation of the carboxy-terminal activation domain of the nucleolar transcription factor UBF

Abstract

Mammalian target of rapamycin (mTOR) is a key regulator of cell growth acting via two independent targets, ribosomal protein S6 kinase 1 (S6K1) and 4EBP1. While each is known to regulate translational efficiency, the mechanism by which they control cell growth remains unclear. In addition to increased initiation of translation, the accelerated synthesis and accumulation of ribosomes are fundamental for efficient cell growth and proliferation. Using the mTOR inhibitor rapamycin, we show that mTOR is required for the rapid and sustained serum-induced activation of 45S ribosomal gene transcription (rDNA transcription), a major rate-limiting step in ribosome biogenesis and cellular growth. Expression of a constitutively active, rapamycin-insensitive mutant of S6K1 stimulated rDNA transcription in the absence of serum and rescued rapamycin repression of rDNA transcription. Moreover, overexpression of a dominant-negative S6K1 mutant repressed transcription in exponentially growing NIH 3T3 cells. Rapamycin treatment led to a rapid dephosphorylation of the carboxy-terminal activation domain of the rDNA transcription factor, UBF, which significantly reduced its ability to associate with the basal rDNA transcription factor SL-1. Rapamycin-mediated repression of rDNA transcription was rescued by purified recombinant phosphorylated UBF and endogenous UBF from exponentially growing NIH 3T3 cells but not by hypophosphorylated UBF from cells treated with rapamycin or dephosphorylated recombinant UBF. Thus, mTOR plays a critical role in the regulation of ribosome biogenesis via a mechanism that requires S6K1 activation and phosphorylation of UBF.

Figures

FIG. 1.

Regulation of NIH 3T3 cell growth correlates with rapid and sustained activation of rDNA transcription by mTOR. NIH 3T3 cells were plated in DMEM with 10% FBS, allowed to grow overnight, and made quiescent by incubation for 24 h in DMEM with 0.5% BSA (serum starved as a control). After serum starvation, cells were pretreated with rapamycin (20 nM) or vehicle (ethanol) for 30 min before stimulation with DMEM plus 10% FBS for 30 min, 3 h, or 24 h. Cells harvested at 24 h were assayed for S6K1 activity, protein content per cell, and cell volume (A) as described in Materials and Methods. Cells harvested at 30 min, 3 h, and 24 h were assayed for endogenous rDNA transcription rates by nuclear run-on analysis (B) as described in Materials and Methods. Alternatively, quiescent cells were stimulated with 10% FBS for 24 h before treatment with rapamycin (20 nM) for 30 min and 3 h as illustrated in the schematic (C), then harvested and assayed for S6K1 activity (C), endogenous rDNA transcription (D), and for their ability to transcribe 0.1 μg of 45S template gene in vitro (E) as described in Materials and Methods. Lane 1, markers; lanes 2 to 5, 45S transcripts (45S Transc.); lane 6, internal standard (Int. Std.); lane 7, 45S transcript. Values that are significantly different (P < 0.05) from the control values (*) and values that are significantly different (P < 0.05 [#] and P < 0.005 [##]) from the values for serum-treated cells (n = 4 to 7) are indicated. Abbreviations: Ctl, control; S, 10% FBS; S+ Rapa, 10% FBS plus 20 nM rapamycin.

FIG. 2.

S6K1 is necessary and sufficient to regulate rDNA transcription in mammalian cells. (A) NIH 3T3 cells (0.18 × 106 cells/well) were transfected 24 h after plating with the following plasmid constructs: a reporter construct for rDNA transcription (pSMECAT) (0.45 μg), a control vector in which an essential G at position −7 in the 45S promoter was mutated in the control reporter (pSMECAT-7), empty vector PRK5 (0.45 μg), or the indicated S6K1 mutants, dED3E or D3E (0.45 μg). The cells were then transferred into DMEM containing 0.5% BSA. After 24 h of serum starvation, the cells were pretreated with rapamycin (20 nM) or vehicle (ethanol) for 30 min and then stimulated with DMEM plus 10% FBS for a further 24 h before being assayed for CAT activity. Samples were analyzed in parallel for endogenous and recombinant S6K1 expression with an antibody directed to the carboxy-terminal region of S6K1 or the myc tag. (B) Cells were plated and transfected as described above with pSMECAT (0.45 μg) and increasing amounts of the dominant-negative construct E2BQ (0.45, 0.9, 1.8, and 3.6 μg) or dED3E (0.45, 0.9, 1.8, and 3.6 μg) and then transferred into DMEM containing 10% BSA. Total plasmid DNA per transfection was equalized with the empty vector, PRK5. After 48 h, the cells were assayed for CAT activity. Samples were analyzed in parallel for recombinant S6K1 expression (myc) and tubulin as a control for loading (see insert). Values that are significantly different (P < 0.05) from the control values (serum-starved cells) (n = 5) are indicated (*). Abbreviations: Ctl, control; Rapa, 20 nM rapamycin; S, 10% FBS; S+Rapa, 10% FBS plus 20 nM rapamycin; α-S6K1, anti-S6K1; Unt., untransfected.

FIG. 3.

Rapamycin inhibits rDNA transcription independent of its effect on proliferation. Cultures of primary neonatal cardiomyocytes maintained in defined serum-free medium were stimulated with vehicle (ethanol) or the hypertrophic agent phenylephrine (25 μM) in the presence or absence of rapamycin (20 nM). After 48 h, the cells were harvested and assayed for S6K1 activity (A), protein content (B), DNA content (C), or rDNA transcription (D) as described in Materials and Methods. Alternatively, cultures of primary neonatal cardiomyocytes were transfected with pSMECAT (0.45 μg), S6K1 mutant dED3E (0.45 μg), or empty vector PRK5 (0.45 μg) and then transferred to defined serum-free medium. After 24 h, the cells were stimulated as described above, harvested 24 h later, and then assayed for CAT activity (E). Expression of endogenous and recombinant S6K1 was determined by Western blotting with an antibody directed to the carboxy-terminal region of S6K1 (α-S6K1) or the myc tag, respectively. Values that are significantly different from the control values (n = 5 to 7) are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.005. Abbreviations: Ctl, control; PE, 25 μM phenylephrine; PE + Rapa, PE plus 20 nM rapamycin.

FIG. 4.

mTOR regulates UBF expression. (A) NIH 3T3 cells were made quiescent, pretreated with 20 nM rapamycin or vehicle (ethanol) for 30 min, stimulated with serum (by growing in DMEM containing 10% FBS for 24 h), and harvested. Equal amounts of protein were analyzed for UBF expression by Western blotting using polyclonal antibodies that recognize both forms of UBF (UBF1 and UBF2) (α-UBF) and an anti-actin antibody (α-Actin) to verify loading. Samples from duplicate cultures are shown. (B) NIH 3T3 cells were transfected with pSMECAT (0.45 μg) and where indicated with increasing amounts (0.9 and 1.8 μg) of a vector driving expression of UBF antisense RNA (pCMV5-UBFas) or the empty vector (pCMV5) and then transferred to DMEM containing 0.5% BSA. After 24 h of serum starvation, cells were pretreated with rapamycin (20 nM) or vehicle (ethanol) for 30 min before stimulation with DMEM containing 10% FBS for a further 24 h. The cells were harvested and assayed for CAT activity. (C) NIH 3T3 cells growing exponentially in the presence of DMEM containing 10% FBS were treated with rapamycin (20 nM) for 30 min or 3 h, harvested, and assayed by Western blotting for UBF expression as described above for panel A. (D and E) NIH 3T3 cells were transfected with pSMECAT (0.45 μg) and where indicated, with increasing amounts of a construct driving the expression of an epitope-tagged version of UBF1 (FLAG-tagged UBF-1 [FLAG-UBF1]) (0.9 and 1.8 μg) or the empty vector (pCDNA3) and then transferred to DMEM containing 0.5% BSA. After 24 h of serum starvation, cells were pretreated with rapamycin (20 nM) or vehicle (ethanol) for 30 min before stimulation with DMEM containing 10% FBS for a further 24 h as indicated. The cells were then harvested and assayed for CAT activity (D) or expression of recombinant UBF using an anti-FLAG antibody; actin was used as a loading control (E). Values that are significantly different from the control values (*, P < 0.05; **, P < 0.01) and values that are significantly different (P < 0.05) from the values for serum-treated cells (n = 5) (#) are indicated. Abbreviations: Ctl, control (serum starved); S, 10% FBS; S+Rapa, 10% FBS plus 20 nM rapamycin.

FIG. 5.

mTOR regulates phosphorylation of the UBF carboxy-terminal activation domain. NIH 3T3 cells were made quiescent (serum starved as a control), stimulated with serum (grown in DMEM containing 10% FBS for 24 h), and then treated with rapamycin (20 nM) or vehicle (ethanol) for 30 min or 3 h. The cells were also labeled with 1 mCi of 32Pi per plate 12 h after serum was added. 32P-labeled UBF was immunoprecipitated, separated by SDS-PAGE, and transferred to Immobilon P membranes. The membranes were then subjected to Western blot analysis using anti-UBF antibodies and phosphorimager analysis (A). Alternatively, 32P-labeled UBF was excised and digested with trypsin, and the resultant peptides were separated by electrophoresis and chromatography as described in Materials and Methods. Radiolabeled phosphopeptides were visualized after 24-h exposure using a PhosphorImager (B). Panels A and B show typical results for a 3-h rapamycin treatment. (C) The results from two or three separate experiments incorporating 30-min and 3-h treatment with rapamycin were quantitated using ImageQuant software (Molecular Dynamics), and the level of carboxy-terminal domain (CT) phosphorylation was expressed relative to the phosphorylation of peptides 1 and 2 (circled 1 and 2 in panel B). Abbreviations: Ctl, control; S, 10% FBS; S+ Rapa, 10% FBS plus 20 nM rapamycin.

FIG. 6.

UBF from serum-stimulated cells and recombinant baculovirus UBF rescues rapamycin-inhibited RPI activity. NIH 3T3 cells were made quiescent, stimulated with serum (in DMEM plus 10% FBS) for 24 h, and treated with rapamycin (20 nM) or vehicle (ethanol) for 3 h. Nuclear extracts were prepared and fractionated as described in Materials and Methods. (A) Fractions containing RPI/SL-1 (MQ340) and UBF (MQ570) were pooled and analyzed by Western blotting for expression of RPI (anti-A127), SL-1 (anti-TBP, anti-TAFI p95, and anti-TAFI p68), Rrn3 (anti-Rrn3), or UBF (anti-UBF). (B) The RPI/SL-1 and UBF fractions were concentrated, and the volume was adjusted to ensure that an equal amount of each factor purified could be added to the transcription reaction mixtures. In addition, the level of Rrn3 was determined asillustrated by Western blotting. (C) Nuclear extracts (50 μg) were assayed for their ability to transcribe 0.1 μg of the 45S template gene in vitro and respond to partially purified UBF (MQ570). Lanes 3 to 5 and 6 to 8 contain 1, 3, and 10 μl of UBF (MQ570) from rapamycin- or serum-treated extracts, respectively. In lanes 9 and 10, the UBF fraction was precleared with anti-UBF or preimmune serum before addition to the transcription reaction mixture. Lane 11 contains molecular size markers. (D) Western blot illustrates effective immunodepletion of UBF from MQ570 fraction. (E) Nuclear extracts (50 μg) were assayed for their ability to transcribe 0.1 μg of the 45S template gene in vitro and respond to purified baculovirus-expressed UBF. Lanes 1 to 3 contain 10, 25, and 50 ng of UBF added to a rapamycin-treated nuclear extract. Lane 4 contains serum-treated nuclear extract. Lanes 5 to 6 illustrates the effect of dephosphorylating the baculovirus UBF on transcription in a rapamycin-treated nuclear extract. The purity of the baculovirus-expressed UBF is illustrated by the Coomassie blue and Western blots. (F) Nuclear extracts (200 μg) from serum- or rapamycin treated cells were incubated with anti-TAF1 p95 antibodies (2 μg) in C-10 buffer containing 0.5% NP-40 at 4°C for 3 h, and then washed protein A Sepharose beads (25 μl) were added. The sample was tumbled for 30 min, and the beads were washed three times with C-10 buffer containing 0.5% NP-40. Sample loading buffer was added, and the samples were boiled and separated on SDS-8% polyacrylamide gels, transferred, and Western blotted with anti-UBF or anti-TAF1 p95 antibodies. Abbreviations: α-UBF, anti-UBF; UBF1/2, UBF1 and UBF2; S, 10% FBS; S+ Rapa, 10% FBS plus 20 nM rapamycin; 45S Transc., 45S transcripts; Int. Std., internal standard; Std., standards; dephos, dephosphorylated; I.P., immunoprecipitation.

FIG. 7.

Rrn3, RPI, and SL-1 activities are not inhibited by rapamycin. (A) The ability of purified Rrn3 to complement in vitro transcription of 0.1 μg of the 45S template gene in S100 extracts from NIH 3T3 cells (40 μg) treated with serum or rapamycin (3 h) was assayed. Lanes 1 to 4 contain S100 extracts from serum-treated cells complemented with equal amounts (50 ng) of affinity-purified FLAG-tagged Rrn3 from cells treated with serum or rapamycin (30 min or 3 h). Lanes 5 to 8 contain S100 extracts from cells treated with rapamycin (3 h) complemented with purified Rrn3 from cells treated with serum or rapamycin (30 min or 3 h). The amount of affinity-purified Rrn3 was determined by silver-stained polyacrylamide gels and Western blotting against recombinant Rrn3 from Sf9 cells (, ; data not shown). (B) The ability of purified Rrn3 to complement in vitro transcription of 0.1 μg of the 45S template gene in S100 extracts from CHX-treated NISI cells (40 μg) was assayed. Lane 2 contains purified baculovirus-expressed Rrn3, and lanes 3 and 4 contain active FLAG-tagged Rrn3 complementing transcription from the CHX-treated S100 cell extracts. Lanes 5 to 12 contain increasing doses (10 and 25 ng) of FLAG-tagged Rrn3 purified from serum-starved cells (lanes 5 and 6), serum-treated cells (lanes 7 and 8), and cells treated with rapamycin for 30 min (lanes 9 and 10) or 3 h (lanes 11 and 12). (C) UBF (MQ570 [lanes 1 and 2]) and RPI/SL-1 (MQ340 [lanes 3 and 4]) fractions from cells treated with serum or rapamycin (3 h) were assayed for their ability to transcribe 0.1 μg of the 45S template gene in vitro. Alternatively, RPI/SL-1 fractions precleared with anti-UBF or preimmune sera were assayed in the presence (+) of UBF from cells treated with serum (lanes 5 to 8) or rapamycin (lanes 9 to 12). The amount of affinity-purified FLAG-tagged Rrn3 was determined by silver-stained polyacrylamide gels and Western blotting against recombinant Rrn3 from Sf9 cells (, ; data not shown). Abbreviations: S, 10% FBS; S + Rapa, 10% FBS plus 20 nM rapamycin; FLAG-Rrn3, FLAG-tagged Rrn3, Ctl, control; 45S Transc., 45S transcripts; Int. Std., internal standard; α-UBF, anti-UBF.