Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E

Abstract

The coordinated action of cell cycle progression and cell growth (an increase in cell size and cell mass) is critical for sustained cellular proliferation, yet the biochemical signals that control cell growth are poorly defined, particularly in mammalian systems. We find that cell growth and cell cycle progression are separable processes in mammalian cells and that growth to appropriate cell size requires mTOR- and PI3K-dependent signals. Expression of a rapamycin-resistant mutant of mTOR rescues the reduced cell size phenotype induced by rapamycin in a kinase-dependent manner, showing the evolutionarily conserved role of mTOR in control of cell growth. Expression of S6K1 mutants that possess partial rapamycin-resistant activity or overexpression of eIF4E individually and additively partially rescues the rapamycin-induced decrease in cell size. In the absence of rapamycin, overexpression of S6K1 or eIF4E increases cell size, and, when coexpressed, they cooperate to increase cell size further. Expression of a phosphorylation site-defective mutant of 4EBP1 that constitutively binds the eIF4E-Cap complex to inhibit translation initiation reduces cell size and blocks eIF4E effects on cell size. These data show that mTOR signals downstream to at least two independent targets, S6K1 and 4EBP1/eIF4E, that function in translational control to regulate mammalian cell size.

Figure 1

mTOR- and PI3K-mediated cell growth continues when cell cycle progression is blocked. (A) Rat.1a fibroblasts cultured in DMEM/10% FBS were transiently cotransfected with the cell surface marker CD20 (2 μg) and plasmids encoding various pRb pathway cell cycle inhibitory proteins (p16, p21, pRb 328–392, and cdk2 dn; 20 μg). At 48 h posttransfection, cells were harvested for analysis by flow cytometry to determine DNA content and mean FSC-H of G₁-phase cells. (B) Rat.1a RT16.15 cells were cultured in the presence of tetracycline (+Tet; p16 Off). To induce p16 expression, tetracycline was removed from the RT16.15 cell line for 24–48 h (−Tet; p16 On). Cells were then either lysed and immunoblotted with anti-p16 antibodies (24 h) or harvested for analysis by flow cytometry to determine DNA content and mean FSC-H of G₁-phase cells (48 h). (C) Rat.1a RT16.15 cells were deprived of serum for 48 h in the presence of tetracycline (p16 Off) and then pretreated with the drug inhibitors rapamycin (50 ng/mL) or LY294002 (50 μM) for 30 min. Cells were then shifted to DMEM/10% FBS lacking tetracycline (p16 On) in the absence or presence of drug inhibitors. After 48 h, cells were harvested for analysis by flow cytometry to determine the mean FSC-H of G₁-phase cells. The mean FSC-H values for each histogram curve are indicated. Rapamycin and LY294002 inhibited the activity of S6K1 as determined by mobility on SDS-PAGE (data not shown).

Figure 1

mTOR- and PI3K-mediated cell growth continues when cell cycle progression is blocked. (A) Rat.1a fibroblasts cultured in DMEM/10% FBS were transiently cotransfected with the cell surface marker CD20 (2 μg) and plasmids encoding various pRb pathway cell cycle inhibitory proteins (p16, p21, pRb 328–392, and cdk2 dn; 20 μg). At 48 h posttransfection, cells were harvested for analysis by flow cytometry to determine DNA content and mean FSC-H of G₁-phase cells. (B) Rat.1a RT16.15 cells were cultured in the presence of tetracycline (+Tet; p16 Off). To induce p16 expression, tetracycline was removed from the RT16.15 cell line for 24–48 h (−Tet; p16 On). Cells were then either lysed and immunoblotted with anti-p16 antibodies (24 h) or harvested for analysis by flow cytometry to determine DNA content and mean FSC-H of G₁-phase cells (48 h). (C) Rat.1a RT16.15 cells were deprived of serum for 48 h in the presence of tetracycline (p16 Off) and then pretreated with the drug inhibitors rapamycin (50 ng/mL) or LY294002 (50 μM) for 30 min. Cells were then shifted to DMEM/10% FBS lacking tetracycline (p16 On) in the absence or presence of drug inhibitors. After 48 h, cells were harvested for analysis by flow cytometry to determine the mean FSC-H of G₁-phase cells. The mean FSC-H values for each histogram curve are indicated. Rapamycin and LY294002 inhibited the activity of S6K1 as determined by mobility on SDS-PAGE (data not shown).

Figure 2

Rapamycin and LY294002 treatment of cycling U2OS cells reduces cell size and inhibits proliferation and cell cycle progression. (A,B) U2OS cells cultured in DMEM/10% FBS were incubated in the absence (−Rapa; ethanol vehicle) or presence (+Rapa) of rapamycin (20 ng/mL) or in the absence (−LY; DMSO vehicle) or presence (+LY) of LY294002 (50 μM) for 72 h. Mean FSC-H histograms of G₁-phase cells are shown in A, whereas quantitation of triplicate samples is shown in B. *P < 0.002 compared with vehicle control. The ratio of mean FSC-H +Rapa/−Rapa is indicated above each +Rapa bar. (C) U2OS cells were plated at 1 × 10⁴ cells/well, cultured in the absence or presence of rapamycin (rapa) or LY294002 (LY), and after 2 and 4 d of drug treatment, counted in a hemocytometer. (D) DNA-content histograms of the cells shown in A. (E) U2OS cells were incubated in the absence (−) or presence (+) of rapamycin or LY294002 for 72 h, and the in vitro kinase activity of endogenous S6K1 toward GST-S6 was assayed and quantitated; equal protein amounts were assayed (upper). Equal total protein was incubated with m⁷GTP-sepharose beads or protein A-sepharose beads as a negative control, and the amount of eIF4E and 4EBP1 bound to the m⁷GTP–Cap complex after 72 h of ethanol vehicle (−) or rapamycin (+) treatment is shown (lower). The α-, β-, and γ-isoforms of 4EBP1 are indicated by arrows: The α-band represents a hypophosphorylated form, the γ-band represents a hyperphosphorylated form, but the β-band represents an intermediate phosphorylation state. (F) The mean FSC-H of S- and G₂/M-phase cells is shown (same cell population as in A). *P < 0.002, **P < 0.02, ***P = 0.08 versus −Rapa condition. (G) Cells cultured in DMEM/10% FBS in the absence (−Rapa; black bar) or presence (+Rapa; gray bar) of rapamycin 20 ng/mL) for 72 h were trypsinized and counted. Equal numbers of cells were lysed, and protein assays were performed to determine total cellular protein content +/− S.E. (H) Triplicate plates of U2OS, 293, or HeLa cells were incubated in the absence (−) or presence (+) of rapamycin for 72 h. The ratio of the mean FSC-H of cells treated +Rapa/−Rapa +/− S.E. is shown. The relative cell size of vehicle-treated cells is set to 1.0.

Figure 2

Rapamycin and LY294002 treatment of cycling U2OS cells reduces cell size and inhibits proliferation and cell cycle progression. (A,B) U2OS cells cultured in DMEM/10% FBS were incubated in the absence (−Rapa; ethanol vehicle) or presence (+Rapa) of rapamycin (20 ng/mL) or in the absence (−LY; DMSO vehicle) or presence (+LY) of LY294002 (50 μM) for 72 h. Mean FSC-H histograms of G₁-phase cells are shown in A, whereas quantitation of triplicate samples is shown in B. *P < 0.002 compared with vehicle control. The ratio of mean FSC-H +Rapa/−Rapa is indicated above each +Rapa bar. (C) U2OS cells were plated at 1 × 10⁴ cells/well, cultured in the absence or presence of rapamycin (rapa) or LY294002 (LY), and after 2 and 4 d of drug treatment, counted in a hemocytometer. (D) DNA-content histograms of the cells shown in A. (E) U2OS cells were incubated in the absence (−) or presence (+) of rapamycin or LY294002 for 72 h, and the in vitro kinase activity of endogenous S6K1 toward GST-S6 was assayed and quantitated; equal protein amounts were assayed (upper). Equal total protein was incubated with m⁷GTP-sepharose beads or protein A-sepharose beads as a negative control, and the amount of eIF4E and 4EBP1 bound to the m⁷GTP–Cap complex after 72 h of ethanol vehicle (−) or rapamycin (+) treatment is shown (lower). The α-, β-, and γ-isoforms of 4EBP1 are indicated by arrows: The α-band represents a hypophosphorylated form, the γ-band represents a hyperphosphorylated form, but the β-band represents an intermediate phosphorylation state. (F) The mean FSC-H of S- and G₂/M-phase cells is shown (same cell population as in A). *P < 0.002, **P < 0.02, ***P = 0.08 versus −Rapa condition. (G) Cells cultured in DMEM/10% FBS in the absence (−Rapa; black bar) or presence (+Rapa; gray bar) of rapamycin 20 ng/mL) for 72 h were trypsinized and counted. Equal numbers of cells were lysed, and protein assays were performed to determine total cellular protein content +/− S.E. (H) Triplicate plates of U2OS, 293, or HeLa cells were incubated in the absence (−) or presence (+) of rapamycin for 72 h. The ratio of the mean FSC-H of cells treated +Rapa/−Rapa +/− S.E. is shown. The relative cell size of vehicle-treated cells is set to 1.0.

Figure 3

Rapamycin-resistant (RR) mTOR rescues rapamycin-inhibited downstream biochemical signaling and rapamycin-induced decreased cell size. (A) U2OS cells cultured in DMEM/FBS were transiently transfected with pcDNA3 vector control or AU1-tagged mTOR constructs (5 μg) and HA-tagged 4EBP1 (0.5 μg). Transfected cells were incubated in the absence (−) or presence (+) of rapamycin (20 ng/mL) for 20 h, lysed, resolved on SDS-PAGE, and immunoblotted (IB) with anti-phospho-S6, anti-HA, anti-AU1, or anti-MAPK antibodies as loading control, as indicated. (B) Cells were transiently transfected with pcDNA3 vector control or a panel of mTOR plasmids (10 μg) plus CD20 (1 μg). Each transfected plate was split into two plates containing DMEM/FBS in the absence (−Rapa; black curve) or presence (+Rapa; gray curve) of rapamycin (20 ng/mL) for 72 h. Cells were harvested for analysis by flow cytometry, as described in Materials and Methods, and the mean FSC-H of the G₁-phase FITC+ cell population was determined. The mean FSC-H values corresponding to each histogram curve are shown. (C) As in B, except quantitation of triplicate transfections is shown. The ratio of the mean FSC-H +Rapa/−Rapa is shown over each +Rapa bar. (NS) Not significant. (D) Cells were cotransfected with 1 μg of HA-tagged S6K1 (left panel) or 1 μg of HA-tagged 4EBP1 (right panel), together with 10 μg of AU1-tagged mTOR constructs and cultured identically to the cell size experiments. Transfected cells were incubated in the absence (−) or presence (+) of rapamycin for 72 h and analyzed by anti-HA-S6K1 in vitro kinase assay or immunoblotting, as indicated.

Figure 3

Rapamycin-resistant (RR) mTOR rescues rapamycin-inhibited downstream biochemical signaling and rapamycin-induced decreased cell size. (A) U2OS cells cultured in DMEM/FBS were transiently transfected with pcDNA3 vector control or AU1-tagged mTOR constructs (5 μg) and HA-tagged 4EBP1 (0.5 μg). Transfected cells were incubated in the absence (−) or presence (+) of rapamycin (20 ng/mL) for 20 h, lysed, resolved on SDS-PAGE, and immunoblotted (IB) with anti-phospho-S6, anti-HA, anti-AU1, or anti-MAPK antibodies as loading control, as indicated. (B) Cells were transiently transfected with pcDNA3 vector control or a panel of mTOR plasmids (10 μg) plus CD20 (1 μg). Each transfected plate was split into two plates containing DMEM/FBS in the absence (−Rapa; black curve) or presence (+Rapa; gray curve) of rapamycin (20 ng/mL) for 72 h. Cells were harvested for analysis by flow cytometry, as described in Materials and Methods, and the mean FSC-H of the G₁-phase FITC+ cell population was determined. The mean FSC-H values corresponding to each histogram curve are shown. (C) As in B, except quantitation of triplicate transfections is shown. The ratio of the mean FSC-H +Rapa/−Rapa is shown over each +Rapa bar. (NS) Not significant. (D) Cells were cotransfected with 1 μg of HA-tagged S6K1 (left panel) or 1 μg of HA-tagged 4EBP1 (right panel), together with 10 μg of AU1-tagged mTOR constructs and cultured identically to the cell size experiments. Transfected cells were incubated in the absence (−) or presence (+) of rapamycin for 72 h and analyzed by anti-HA-S6K1 in vitro kinase assay or immunoblotting, as indicated.

Figure 3

Rapamycin-resistant (RR) mTOR rescues rapamycin-inhibited downstream biochemical signaling and rapamycin-induced decreased cell size. (A) U2OS cells cultured in DMEM/FBS were transiently transfected with pcDNA3 vector control or AU1-tagged mTOR constructs (5 μg) and HA-tagged 4EBP1 (0.5 μg). Transfected cells were incubated in the absence (−) or presence (+) of rapamycin (20 ng/mL) for 20 h, lysed, resolved on SDS-PAGE, and immunoblotted (IB) with anti-phospho-S6, anti-HA, anti-AU1, or anti-MAPK antibodies as loading control, as indicated. (B) Cells were transiently transfected with pcDNA3 vector control or a panel of mTOR plasmids (10 μg) plus CD20 (1 μg). Each transfected plate was split into two plates containing DMEM/FBS in the absence (−Rapa; black curve) or presence (+Rapa; gray curve) of rapamycin (20 ng/mL) for 72 h. Cells were harvested for analysis by flow cytometry, as described in Materials and Methods, and the mean FSC-H of the G₁-phase FITC+ cell population was determined. The mean FSC-H values corresponding to each histogram curve are shown. (C) As in B, except quantitation of triplicate transfections is shown. The ratio of the mean FSC-H +Rapa/−Rapa is shown over each +Rapa bar. (NS) Not significant. (D) Cells were cotransfected with 1 μg of HA-tagged S6K1 (left panel) or 1 μg of HA-tagged 4EBP1 (right panel), together with 10 μg of AU1-tagged mTOR constructs and cultured identically to the cell size experiments. Transfected cells were incubated in the absence (−) or presence (+) of rapamycin for 72 h and analyzed by anti-HA-S6K1 in vitro kinase assay or immunoblotting, as indicated.

Figure 4

Rapamycin-resistant (RR) S6K1s partially rescue rapamycin-inhibited downstream signaling and partially rescue the rapamycin-induced decrease in cell size. (A) U2OS cells were transiently transfected with pRK7 vector control or HA-tagged S6K1 constructs (5 μg), incubated in the absence (−) or presence (+) of rapamycin (20 ng/mL) for 20 h, lysed, resolved on SDS-PAGE, and immunoblotted (IB) with anti-HA, anti-phospho-S6, or anti-MAPK antibodies as loading control, as indicated. Equal protein amounts were immunoprecipitated with αHA antibodies, and the in vitro kinase activity of HA-S6K1 was determined. (B) Cells were cotransfected with pRK7 vector control or a panel of S6K1 plasmids (10 μg) plus CD20 (1 μg). Each transfected plate was split into two plates containing DMEM/FBS, incubated in the absence (−Rapa; white bar) or presence (+Rapa; black bar) of rapamycin (20 ng/mL) for 72 h, and harvested for analysis by flow cytometry. For the −Rapa treatment, mean FSC-H of the G₁-phase FITC+ cell population from a single transfection is shown; for the +Rapa treatment, the mean FSC-H of triplicate transfections +/− S.E. is shown. *P < 0.02 compared with vector control +Rapa. (C) Cells were transfected with HA-tagged S6K1 (10 μg) and cultured identically to the cell size experiments. Transfected cells were incubated in the absence (−) or presence (+) of rapamycin for 72 h and analyzed by anti-HA -S6K1 immunoblotting or in vitro kinase assay, or as indicated.

Figure 5

eIF4E overexpression partially rescues the rapamycin-induced decrease in cell size in a manner dependent on Cap-dependent translation, and coexpression of both eIF4E and RR-S6K1 cooperates to provide stronger rescue. (A) U2OS cells were cotransfected with the pMV7 vector control or eIF4E (10 μg) together with CD20 (1 μg), incubated in the absence (−) or presence (+) of rapamycin for 72 h, and analyzed by flow cytometry to determine cell size. The mean FSC-H +/− S.E. of the G₁-phase FITC+ cell population from quadruplicate transfections is shown. *P < 0.03 compared with vector control +Rapa. (B) Cells were transfected with the indicated combinations of plasmids (8 μg of pMV7 or pMV7/eIF4E + 2 μg of pACTAG2 or pACT/AA-4EBP1) plus CD20 (1 μg) and incubated in the presence of rapamycin for 72 h followed by flow cytometry to determine cell size, as above. (C) Cells were cotransfected with the indicated combinations of plasmids (5 μg + 5 μg) plus CD20 (1 μg) and incubated in the absence (−) or presence (+) of rapamycin for 72 h followed by flow cytometry to determine cell size, as above. For the −Rapa treatment, mean FSC-H of a single transfection is shown; for the +Rapa treatment, the mean FSC-H of triplicate transfections +/− S.E. is shown. Statistical comparisons between +Rapa-treated cells are shown with brackets.

Figure 6

Overexpression of S6K1 or eIF4E individually increases cell size, coexpression of both S6K1 and eIF4E cooperate to increase cell size further, and overexpression of a dominant mutant of 4EBP1 reduces cell size. Cap-dependent translation likely mediates the eIF4E and 4EBP1 effects on cell size. (A) U2OS cells were transiently cotransfected with CD20 (1 μg) plus pRK7 vector control, wild-type (WT), or kinase dead (KD) S6K1 constructs (10 μg), cultured for 72 h in DMEM/FBS, and assayed by flow cytometry to determine cell size. The mean FSC-H +/− S.E. of the G₁-phase FITC+ cell population was determined. The WT bar is from quadruplicate samples from two independent transfections; pRK7 and KD bars are from five samples from three independent transfections. Statistical comparisons are shown with brackets. Equivalent expression of the transfected HA-S6K1s in this experiment is shown by immunoblot (inset). (B) Cells were cotransfected with the indicated plasmids (10 μg) plus CD20 (1 μg), and the mean FSC-H +/− S.E. of the FITC+ cell population from triplicate transfections was determined, as above. *P < 0.05 versus vector control. (C) The mean FSC-H of single transfections with the indicated plasmids (8 μg of pMV7 or pMV7/eIF4E + 2 μg of pACTAG2 or pACT/AA-4EBP1) plus CD20 (1 μg) is shown, as above. (D) Cells were cotransfected with the indicated combinations of plasmids (5 μg + 5 μg) plus CD20 (1 μg), and the mean FSC-H +/− S.E. of triplicate samples is shown, as above. Statistical comparisons are shown with brackets. Although the P-value comparing S6K1 + pMV7 versus S6K1 + eIF4E (P = 0.06) falls just outside our defined value for statistical significance (P < 0.05), we consistently observe in many experiments that S6K1 + eIF4E cotransfection results in a larger cell size than S6K1 alone. (E) Cells were cotransfected with the indicated plasmids (10 μg) plus CD20 (1 μg), and the mean FSC-H +/− S.E. of FITC+ cells from triplicate transfections is shown, as above. Statistical comparisons are shown with brackets. (F) Equivalent amounts of protein (same transfected cells as in E) were resolved on SDS-PAGE and analyzed by anti-HA-4EBP1 immunoblot (upper) or incubated with m⁷GTP-sepharose beads, and the amount of endogenous eIF4E and transfected HA-4EBP1 bound to the beads was determined (lower).

Figure 6

Overexpression of S6K1 or eIF4E individually increases cell size, coexpression of both S6K1 and eIF4E cooperate to increase cell size further, and overexpression of a dominant mutant of 4EBP1 reduces cell size. Cap-dependent translation likely mediates the eIF4E and 4EBP1 effects on cell size. (A) U2OS cells were transiently cotransfected with CD20 (1 μg) plus pRK7 vector control, wild-type (WT), or kinase dead (KD) S6K1 constructs (10 μg), cultured for 72 h in DMEM/FBS, and assayed by flow cytometry to determine cell size. The mean FSC-H +/− S.E. of the G₁-phase FITC+ cell population was determined. The WT bar is from quadruplicate samples from two independent transfections; pRK7 and KD bars are from five samples from three independent transfections. Statistical comparisons are shown with brackets. Equivalent expression of the transfected HA-S6K1s in this experiment is shown by immunoblot (inset). (B) Cells were cotransfected with the indicated plasmids (10 μg) plus CD20 (1 μg), and the mean FSC-H +/− S.E. of the FITC+ cell population from triplicate transfections was determined, as above. *P < 0.05 versus vector control. (C) The mean FSC-H of single transfections with the indicated plasmids (8 μg of pMV7 or pMV7/eIF4E + 2 μg of pACTAG2 or pACT/AA-4EBP1) plus CD20 (1 μg) is shown, as above. (D) Cells were cotransfected with the indicated combinations of plasmids (5 μg + 5 μg) plus CD20 (1 μg), and the mean FSC-H +/− S.E. of triplicate samples is shown, as above. Statistical comparisons are shown with brackets. Although the P-value comparing S6K1 + pMV7 versus S6K1 + eIF4E (P = 0.06) falls just outside our defined value for statistical significance (P < 0.05), we consistently observe in many experiments that S6K1 + eIF4E cotransfection results in a larger cell size than S6K1 alone. (E) Cells were cotransfected with the indicated plasmids (10 μg) plus CD20 (1 μg), and the mean FSC-H +/− S.E. of FITC+ cells from triplicate transfections is shown, as above. Statistical comparisons are shown with brackets. (F) Equivalent amounts of protein (same transfected cells as in E) were resolved on SDS-PAGE and analyzed by anti-HA-4EBP1 immunoblot (upper) or incubated with m⁷GTP-sepharose beads, and the amount of endogenous eIF4E and transfected HA-4EBP1 bound to the beads was determined (lower).

Figure 7

Model depicting the role of mTOR signaling in control of cell size through its downstream targets S6K1 and 4EBP1/eIF4E. Nutrient- and mitogen-dependent signaling likely regulate cell growth and cell size in response to extracellular conditions via the cooperative action of both mTOR- and PI3K-dependent signaling. Although not shown in this model for the sake of simplicity, Akt (PKB) is an important regulator of cell size downstream of PI3K. Although Akt (PKB) has been reported to signal to S6K1, 4EBP1, and mTOR, the precise relationship of Akt to these signaling molecules remains unclear.