Although mTOR has been known to control G1
-phase progression, the roles of specific mTOR-dependent pathways in cell cycle control have remained poorly defined. We demonstrate that restoration of mTOR function by using rapamycin-resistant mutant rescues rapamycin-inhibited G1
-phase progression, as in lower eukaryotes (18
). This result is consistent with the reported requirement for mTOR in G1
-phase progression; microinjection of the isolated FKBP12-rapamycin-binding (FRB) domain of mTOR inhibited serum-stimulated S-phase entry, presumably by functioning as a dominant-negative (57
). Our data furthermore define roles for the mTOR-regulated S6K1 and 4E-BP1/eIF4E pathways in the control of G1
-phase progression by mTOR: activation of either the S6K1 or 4E-BP1/eIF4E pathways partially rescues the inhibitory effect of rapamycin on G0
-to-S-phase cell cycle progression and modestly accelerates cell cycle progression in the absence of drug. mTOR-dependent phosphorylation of 4E-BP1 is required for mTOR's ability to drive the cell cycle, as is the ability of the mTOR/raptor complex to associate with 4E-BP1 via the TOS motif. Both the S6K1 and the 4E-BP1/eIF4E pathways independently mediate mTOR-dependent cell cycle control in parallel, as simultaneous downregulation of these pathways (using RNAi against S6K1 and overexpression of the phosphorylation site mutant AA-4E-BP1) additively inhibits G1
-phase progression compared to downregulation of the pathways individually.
Our finding that RR-S6K1 partially rescues rapamycin-inhibited G1
-phase progression is consistent with previous reports in which RR-S6K1 could partially restore rapamycin-suppressed E2F transcriptional responses in Kit225 cells, a human T-cell line (3
), and could partially rescue rapamycin-inhibited proliferation of vascular endothelial cells, as assayed by measuring [3
H]thymidine incorporation (58
). Importantly, we demonstrate that overexpression of S6K1 accelerates serum-stimulated G1
-phase progression and increases the proliferation rate in low serum, suggesting that the activity of S6K1 is limiting for G1
-phase progression. Since the reduction of S6K1 expression by using RNAi inhibited but did not completely block G1
-phase progression, our data suggest that, although S6K1 participates in cell cycle control, it is not absolutely required for serum-stimulated G1
-phase progression. This result differs from a previous report in which microinjection of anti-S6K1 antibodies blocked serum-stimulated S-phase entry, as well as total protein synthesis and c-Fos induction (26
). Although it is possible that the more modest requirement for S6K1 that we demonstrate here results from our inability to completely inhibit S6K1 signaling with RNAi or results from cell type-specific differences, our data demonstrate complete RNAi-mediated reduction of overexpressed S6K1 and phosphorylation of endogenous ribosomal protein S6. Consistent with our data, deletion of S6K1 in embryonic stem cells reduces, but does not block, the capacity of the cells to proliferate in culture, and rapamycin treatment further reduces proliferation rate of these cells (23
). Furthermore, rapamycin completely inhibits S6K1 and S6K2 activity, yet only modestly reduces total protein synthesis, does not block serum-stimulated c-Fos induction, and only delays G1
-phase progression. Thus, the formation of intracellular S6K1-containing immune complexes formed by antibody microinjection may disrupt cellular functions differently than the mere absence of S6K1 by RNAi. Collectively, the data suggest that, although it is not absolutely required, S6K1 has a positive influence on cell cycle progression and proliferation and that other mTOR-dependent signaling pathways likely contribute to this as well.
That reduction of S6K1 expression does not induce cell cycle arrest is consistent with our finding that the 4E-BP1/eIF4E pathway operates in parallel to S6K1 downstream of mTOR to control cell cycle progression. We report negative and positive roles for 4E-BP1 and eIF4E, respectively, in control of mTOR-regulated G1
-phase progression in mammalian cells. These data are consistent with transformation of rodent fibroblasts by overexpression of eIF4E (27
), which is blocked by cooverexpression of 4E-BP1 (41
), and consistent with the increased cell division time caused by antisense RNA-mediated reduction in eIF4E expression (10
). Our finding that overexpression of eIF4E accelerates G1
-phase progression appears at first to conflict with a report that eIF4E overexpression activates a negative-feedback loop, resulting in the dephosphorylation of S6K1 and 4E-BP1 (24
). It is important to note that eIF4E accelerates the G1
phase in our cell system when it is expressed at low levels from the weak retroviral long terminal repeat promoter; when eIF4E is expressed to much higher levels with a strong CMV promoter, however, G1
-phase progression is inhibited. Therefore, expression level likely determines whether eIF4E activates a negative feedback loop to restrict aberrant cell cycle progression and proliferation. Since simultaneous downregulation of both the S6K1 and 4E-BP1/eIF4E pathways inhibits G1
-phase progression to an extent approaching that of rapamycin, it is likely that these represent the major pathways mediating mTOR-dependent cell cycle control.
We also consistently noted that the combination of KD mTOR plus rapamycin more strongly inhibits 4E-BP1 phosphorylation, G1
-phase progression, and cell size (13
) compared to WT-mTOR plus rapamycin. The trivial explanation for these phenomena is that although rapamycin blocks all mTOR signals, it does so incompletely. A more intriguing possibility is that mTOR also possesses rapamycin-insensitive kinase-dependent functions that, although obviously not inhibited by rapamycin, could be blocked with kinase-inactive and dominant-negative mTOR. In this case, inhibition of the rapamycin-sensitive functions of mTOR by treatment with rapamycin would unmask these rapamycin-insensitive functions of the mTOR kinase. Indeed, the observation that rapamycin completely inhibits S6K1 and endogenous ribosomal prtotein S6 phosphorylation suggests that the blockade of at least some mTOR signals by rapamycin is complete. Furthermore, TOR2 in budding yeast mediates both rapamycin-sensitive and rapamycin-insensitive signals, and one of the proteins (AVO1) found in the rapamycin-insensitive TOR2 complex (TORC2) has a mammalian orthologue of unknown function, mAVO1/hSIN1 (28
). Therefore, although the possibility that mTOR may signal in a rapamycin-insensitive manner needs to be investigated much more carefully, there is precedent for the idea.
We reported previously that mTOR controls cell size, which is mediated, at least in part, by the S6K1 and 4E-BP1/eIF4E signaling pathways (13
). Since both cell size and cell cycle progression are controlled by mTOR and by the same mTOR-dependent signaling pathways, this nutrient- and mitogen-responsive signaling molecule is centrally positioned to couple cell growth with cell division (Fig. ). These data, together with the known dependence of cell cycle progression on a sufficient level of cell growth (22
; for a review, see reference 36
), suggest a model in which mTOR primarily drives cell growth (i.e., macromolecular biosynthesis), and as a secondary consequence promotes cell cycle progression (Fig. , model 1). Although we favor this model, our data do not exclude the possibility that mTOR controls cell cycle progression via a cell growth-independent mechanism (Fig. model 2); for example, mTOR-dependent signaling could directly regulate components of the cell cycle machinery via a fast-acting phosphorylation cascade. Indeed, the phosphatidylinositol 3-kinase/Akt pathway, which coordinates growth factor signaling with mTOR signals, has been reported to have direct effects on the cell cycle machinery (11
FIG. 8. Models. (A) mTOR functions as a central coordinator of mTOR-dependent cell growth/cell size and cell cycle progression. The mTOR kinase regulates at least two downstream signaling pathways, the S6K1 and 4E-BP1/eIF4E pathways, that promote (i) cell growth (more ...)
Our data also show that the increased cell size produced by S6K1 or eIF4E overexpression (13
) results from augmented cell growth and not from delayed cell cycle progression. Conversely, the decreased cell size observed upon 4E-BP1 overexpression results from decreased cell growth and not from accelerated cell cycle progression. We make this point because cell size phenotypes can result from changes in either cell growth rate or cell cycle progression rate (e.g., when the cell cycle is blocked, cells grow to increased cell size; when the cell cycle accelerates in the face of an unchanged rate of cell growth, cells become smaller). Therefore, whenever a cell size phenotype is observed, it is important to determine whether it results from altered cell growth or altered rate of cell cycle progression.
If mTOR-regulated cell growth influences the rate of cell cycle progression, it seems reasonable to speculate that, since the S6K1 and 4E-BP1/eIF4E pathways control translation, increased expression of critical cell cycle regulatory proteins could represent a mechanism by which cell cycle progession is coupled to cell growth. Indeed, the synthesis of the G1
-cyclin CLN3 in budding yeast is controlled by TOR and Cdc33 (an eIF4E orthologue) at the level of translation initiation, effectively coupling the synthesis of a cell cycle regulator to protein biosynthetic rate (2
). A short upstream open reading frame in the 5′ leader of the CLN3 transcript functions as a translational control element to repress CLN3 expression when protein synthesis and cell growth rate are low (38
). Since eIF4E has also been reported to regulate nucleocytoplasmic transport of mRNA transcripts (reviewed in reference 51
), it is also possible that eIF4E drives cell growth and cell cycle progression by increasing the nucleocytoplasmic export rate of mRNAs encoding specific cell cycle regulators instead of or in addition to controlling their translation. Although the identity of the critical eIF4E-controlled cell cycle regulator(s) is not known at this time, eIF4E has been shown to increase the translation of ODC and c-Myc and to increase the expression of cyclin D1 through increased nucleocytoplasmic transport (42
; reviewed in reference 48
The mechanism by which S6K1 drives cell growth and the cell cycle is also unclear. Of course, S6K1-dependent phosphorylation of ribosomal protein S6 may drive ribosome biogenesis and therefore increase protein biosynthetic capacity, resulting in augmented cell growth and promotion of cell cycle progression. Although S6K1 clearly controls cell growth and cell cycle progression, the critical downstream targets responsible for mediating this response are not clearly defined. In two different experiments with two different cell types, we noted that the degree of ribosomal protein S6 phosphorylation was not concordant with the cell cycle phenotype produced by S6K1 overexpression. For example, the S6K1 mutants E389
E and E389
ΔCT accelerated G1
-phase progression, whereas WT-S6K1 did not, and yet overexpression of all three S6K1 constructs increased the phosphorylation of ribosomal protein S6 over vector control. In addition, S6K1 overexpression increased proliferation rate in low serum compared to parental or control cells, and yet in low serum ribosomal protein S6 was phosphorylated to similar levels in all three cell lines. The observation that S6K1-regulated G1
-phase progression and ribosomal protein S6 phosphorylation can be dissociated suggests the intriguing possibility that S6K1 likely has other targets besides the ribosomal protein S6. Consistent with this idea, we have recently identified a novel S6K1-interacting protein and in vivo substrate, SKAR, which bears homology to the ALY/REF family of proteins that couple transcription, splicing, and RNA export (C. Richardson et al., unpublished data). SKAR also controls cell growth or cell size and also regulates the rate of cell cycle progression (Richardson et al., unpublished), suggesting that S6K1 may control cell growth and cell cycle progression through an RNA processing event (Fig. ). Another in vivo target of S6K1 is the cap-binding protein CBP80, which functions in an early step of RNA splicing (60
). Therefore, it is possible that S6K1-regulated cell cycle progression may be due in part to regulation of RNA processing (Fig. ). Alternatively, S6K1 could regulate the cell cycle by a cell growth-independent mechanism via phosphorylation of unidentified targets that would directly regulate the cell cycle machinery (Fig. , model 2).
In the present study, we identify the S6K1 and 4E-BP1/eIF4E pathways as the major mTOR-dependent downstream signaling pathways that mediate mTOR-regulated G1
-phase progression (Fig. ). Since these pathways also mediate mTOR-dependent control of cell growth or cell size (13
), mTOR is uniquely positioned to function as a central coordinator of cell growth and cell division. In the future, we hope to better understand the relationship between mTOR-regulated cell growth and cell division and to move further downstream to determine how mTOR couples these fundamental biological processes.