In mammalian cells, the mTOR pathway has been shown to be a pivotal regulator of both cell cycle progression and cell size. Accordingly, studies have been performed before the discovery that mTOR is also part of a second kinase complex named mTORC2. In addition, in many of the underlying experiments mTOR activity has been modulated via rapamycin (see e.g. 7
). Although originally believed to exclusively block mTORC1, rapamycin has been shown to also affect mTORC2 activity at least under specific experimental conditions (10
). Accordingly, with regard to mTOR's role in cell size and cell cycle control in mammalian cells, two major question remained elusive: (i) Is the regulation of mTORC2 essentially involved in mTORC1-mediated cell cycle and/or cell size control? (ii) Is mTORC2 itself capable of regulating mammalian cell cycle and/or cell size?
To answer these questions we felt it to be essential to use an optimal biological cell system. In the past, many data on mTOR regulation were obtained from ectopic overexpression experiments in transformed cells or using in vitro
kinase assays. Varying results on mTOR regulation have been reported in many different immortalized or transformed cell lines, what is mainly because of the fact that a wide variety of upstream regulators of mTOR are deregulated in many different types of diseases and tumors (3
). All experiments presented in this study have been performed using non-transformed, non-immortalized primary human diploid IMR-90 fibroblasts harboring a finite lifetime and a normal karyotype (11
). In addition, with the exception of ectopic expression of myristoylated Akt no overexpression experiment is included, but we exclusively modulated endogenous activities.
It was already reported earlier that mTORC1's effects on cell cycle and cell size are separable (7
). We have confirmed and expanded this knowledge: (i) The approach of two-dimensional contour blot analyses of FSC versus DNA content allowed the demonstration that blocking endogenous mTORC1 via rapamycin reduces the size of cells in all cell cycle phases. (ii) Identical results were obtained in cells, in which endogenous mTORC1 was diminished via raptor-specific siRNAs. (iii) Rapamycin time-course experiments revealed that the effects of downregulated mTORC1 on cell size occur far before the cell cycle deregulation. (iv) Re-stimulation experiments with and without rapamycin showed that blocking mTORC1 activity delays S-phase induction with smaller G1 cells starting to replicate.
Four independent experimental data support our conclusion that mTORC1-mediated cell cycle regulation does not need to involve effects on mTORC2 activity. (i) Rapamycin treatment of logarithmically growing cells triggers accumulation of G0/G1 cells accompanied by a downregulation of the amount of S-phase cells without effects on endogenous mTORC2 activity. (ii) A rapamycin time-course experiment revealed that after 24 h blocking mTORC1 activity cyclin A levels, cyclin D1 levels and % S-phase cells decreased, but no effects could be detected on mTORC2 activity. (iii) Serum re-stimulation approaches revealed that a rapamycin-mediated block of mTORC1 significantly delays S-phase entry without any effects on mTORC2 activity. (iv) Downregulation of endogenous mTORC1 activity via raptor-specific siRNAs in logarithmically growing cells also triggered accumulation of G0/G1 without negatively affecting mTORC2. That mTORC2 deregulation is also not playing an essential role for mTORC1-triggered cell size control was demonstrated (i) by showing that rapamycin triggers size reduction in all cell cycle phases of cycling cells without affecting mTORC2; (ii) by obtaining the same results upon blocking of mTORC1 via raptor-specific siRNAs; (iii) by detecting deregulated cell size control in a rapamycin time-course experiment without effects on endogenous mTORC2 activity; (iv) by demonstrating that rapamycin-treated cells enter the replicative phase at smaller size but do not exhibit any modulation of endogenous mTORC2 activity. In summary, these findings allow the conclusion that in mammalian cells mTORC1 can regulate cell cycle progression and cell size control independent of any effects on endogenous mTORC2 activity.
Next, we found that downregulating endogenous mTORC2 activity via rictor-specific siRNAs causes an accumulation of G0/G1 cells accompanied by a decrease of S-phase cells. Our here obtained data using primary non-transformed, non-immortalized human fibroblasts are in perfect agreement with a recent study reporting the same cell cycle phenotype upon knockdown of rictor in the MCF7 breast cancer and PC3 prostate cancer cell lines (28
), suggesting that this potential of mTORC2 might be independent of the transformation status of the cell. Still, that mTORC2 itself is involved in tumor development is suggested by the recent finding that many gliomas overexpress rictor accompanied with elevated mTORC2 activities (29
Drosophila mutants removing the critical TORC2 components, rictor and sin1, showed minor growth impairment (30
). The aspect whether mTORC2 is involved in mammalian cell size regulation remained elusive so far. We report here that siRNA-induced knockdown of endogenous mTORC2 activity triggers strong effects on cell size regulation of non-transformed primary human cells. Several independent experiments revealed that rictor-siRNA-mediated size reduction to be comparable to the effects of a raptor-specific knockdown. Two-dimensional contour blot analyses of FSC versus DNA content demonstrated that mTORC2-mediated cell size regulation is not a consequence of its effects on cell cycle progression described above. We found that knockdown of rictor to trigger a size reduction in all cell cycle phases very comparable to the effects of raptor-specific siRNAs. Accordingly, we conclude mTORC2 to be a potent cell size regulator and to be able to control cell size of cells in all phases of the mammalian cell cycle.
mTORC2 is a major regulator of Akt kinase activity. One Akt substrate that has been reported to be a pivotal regulator of mammalian cell cycle and cell size is the tuberous sclerosis gene product TSC2. Akt is known to regulate growth by directly phosphorylating TSC2 (1
). To investigate whether TSC2 is essentially involved in the mTORC2-mediated cell cycle and/or cell size regulation, we analysed the effects of TSC2-specific siRNA treatment on the rictor-siRNA-induced deregulations described above. Several independent experiments revealed that the rictor-siRNA-triggered accumulation of G0/G1 cells heavily depends on the endogenous levels of TSC2. The same was found to be true for the effects of rictor knockdown on the size of cells in all different cell cycle phases.
In the past, different observations were reported regarding the question whether mTORC2 affects the mTORC1-induced phosphorylation of p70S6K. Very likely, owing to the usage of different approaches to modulate mTORC2 and of different transformed immortalized cells, modulating mTORC2 was described to be either without any effects on p70S6K phosphorylation, or to trigger its up- or downregulation (31
). However, whenever we downregulated endogenous mTORC2 activity via rictor-specific siRNAs in non-transformed, non-immortalized primary human cells, we observed a very pronounced downregulation of endogenous mTORC1-mediated p70S6K phosphorylation on T389. This is one observation that provides evidence for the mTORC1/p70S6K cascade to be involved mTORC2-induced cell size and cell cycle regulation. As discussed above, we proved TSC2 to be of essential relevance for these mTORC2-dependent regulations and TSC2 is very well known to exert its cell size effects via regulation of mTORC1/p70S6K. Knockdown of TSC2 reverted the cell cycle and cell size effects of rictor-specific siRNAs accompanied with a reactivation of endogenous T389 p70S6K phosphorylation. In addition, this finding shows that the effects of downregulated mTORC2 activity on mTORC1 are indeed mediated via the TSC2-involving cascade. Knockdown of PRAS40 could not reactivate p70S6K phosphorylation and also did not have any influence on the mTORC2-triggered cell size and cell cycle effects.
All these findings provide strong evidence that mTORC2 affects cell size regulation via modulating the potential of TSC2 to regulate mTORC1. This potential of TSC2 is known to be mediated via Rheb (1
). We found that TSC2's potential to counteract mTORC2-mediated cell size control indeed depends on functional Rheb. Taken together with our here reported observation that downregulation of mTORC2 via rictor siRNAs cannot control mammalian cell size in cells harboring constitutive Akt kinase activity, these findings allow the conclusion that mTORC2 is a potent regulator of mammalian cell size via a mechanism involving the Akt/TSC2/Rheb cascade.