Tsc and Rheb are two important players of the PI3K/AKT/TSC/mTOR signaling pathway that has been shown to regulate cell cycle progression in addition to other cellular processes including proliferation, tumorigenesis, angiogenesis, differentiation, and anti-apoptosis. Tsc1 and Tsc2 form a complex that functions as a GTPase activating protein (GAP) for Rheb. Mutations in the Tsc1 or Tsc2 genes have been implicated in tuberous sclerosis, a genetic disorder marked by the appearance of benign tumors called hamartomas in multiple organs. Previously we used Tsc2-/- MEFs as a model to investigate the function of the TSC/Rheb/mTOR pathway. We have shown that Tsc2-/- MEFs escape cell cycle arrest in G0/G1 at high confluency and in serum-starved conditions (Gau et al., AACR meeting, 2004). However, the mechanism of how TSC/Rheb/mTOR induces cell cycle progression is not known. Our analysis of in vitro kinase assays revealed that CDK2 kinase activity is significantly increased in Tsc2-null MEFs. We have also examined expression levels of various cyclins, CDKs, and CDK inhibitors. No significant differences in the level of expression of CDK2, p27, and p21 were detected between Tsc2+/+ and Tsc2-/- MEFs. However, we did observe a difference in the cellular localization of p27 between the two cells. Biochemical fractionation experiments showed that p27 is localized in both cytoplasmic and nuclear fractions in Tsc2+/+ MEFs. In contrast, p27 was localized only in the cytoplasmic fraction in Tsc2-/- MEFs. There is no change in the localization of p21. This lack of nuclear localization of p27 may explain the increased activity of CDK2 in Tsc2-/- MEFs. It is interesting to point out that cytoplasmic translocation of p27 has been detected in a number of cancer cells. In addition, cytoplasmic translocation of p27 has been detected in breast cancer and HEK293T cells upon Akt activation. Furthermore, TSC2 was found to bind p27 to protect it from proteasomal degradation. Rheb is clearly implicated in cell cycle progression, as overexpression of Rheb in HEK293 cells promoted an S phase progression. Since the Tsc1/Tsc2 complex negatively regulates Rheb, a question arises regarding the role of Rheb in cell cycle progression. Further analysis of how Rheb regulates G1/S cell cycle associated proteins will be discussed. These results provide insight into how Tsc and Rheb are involved in the regulation of cell cycle progression. This also implicates the possibility of taking advantage of drugs such as farnesyltransferase inhibitors that target Rheb to influence cell cycle progression.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]