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In mammalian cells, the mammalian target of rapamycin (mTOR) forms an enzyme complex with raptor (together with other proteins) named mTOR complex 1 (mTORC1), of which a major target is the p70 ribosomal protein S6 kinase (p70S6K). A second enzyme complex, mTOR complex 2 (mTORC2), contains mTOR and rictor and regulates the Akt kinase. Both mTORC1 and mTORC2 are regulated by phosphorylation, complex formation and localization. So far, the role of p70S6K-mediated mTOR S2448 phosphorylation has not been investigated in detail. Here, we report that endogenous mTOR phosphorylated at S2448 binds to both, raptor and rictor. Experiments with chemical inhibitors of the mTOR kinase and of the phosphatidylinositol-3-kinase revealed that downregulation of mTOR S2448 phosphorylation correlates with decreased mTORC1 activity but can occur decoupled of effects on mTORC2 activity. In addition, we found that the correlation of the mTOR S2448 phosphorylation status with mTORC1 activity is not a consequence of effects on the assembly of mTOR protein and raptor. Our data allow new insights into the role of mTOR phosphorylation for the regulation of its kinase activity.

The cyclin-dependent kinase inhibitor p27Kip1 (p27) is a major gatekeeper of the mammalian cell cycle progression known to be regulated by both, its subcellular localization and its degradation. To allow entrance into S phase and thereby mammalian cell cycle progression p27 must be degraded by a skp2-containing E3 ubiquitin ligase whose task is to target p27 for degradation by the proteasome. The tumor suppressor gene product tuberin directly binds to p27 and protects it from degradation via skp2. Whereas, p27 and tuberin are known to be localized to both, the cytoplasm and the nucleus, the localization of skp2 remained elusive. Here we demonstrate that skp2 is a cytoplasmic and nuclear protein. In addition we found an inverse correlation of the endogenous protein levels of skp2 with p27 and tuberin in different transformed cells and under different growth conditions. These data allow new important insights into this molecular network of cell cycle control.

Mutations in the genes TSC1 or TSC2 cause the autosomal dominantly inherited tumor suppressor syndrome tuberous sclerosis, which is characterized by the development of tumors, named hamartomas, in different organs. The TSC gene products, hamartin and tuberin, form a complex, of which tuberin is assumed to be the functional component. Both, hamartin and tuberin have been implicated in the control of the cell cycle by activating the cyclin-dependent kinase inhibitor p27 and in cell size regulation by inhibiting the mammalian target of rapamycin (mTOR) a regulator of the p70 ribosomal protein S6 kinase (p70S6K) and its target the ribosomal protein S6. The tuberin/hamartin complex was shown to protect p27 from protein degradation. Within the mTOR signaling pathway tuberin harbors GTPase activating (GAP) potential toward Rheb, which is a potent regulator of mTOR. In this study, we have analyzed the protein levels of tuberin, p27, cyclin D1, mTOR and phospho mTOR Ser2448 (activated mTOR), S6 and phospho S6 Ser240/244 (activated S6) and as controls alpha-tubulin and topoisomerase IIbeta, in ten different cells, including primary normal cells, immortalized and transformed cell lines.

The discovery of amniotic fluid stem cells initiated a new and very promising field in stem cell research. In the last four years amniotic fluid stem cells have been shown to express markers specific to pluripotent stem cells, such as Oct-4. Due to their high proliferation potential, amniotic fluid stem cell lineages can be established. Meanwhile, they have been shown to harbor the potential to differentiate into cells of all three embryonic germ layers. It will be a major aim for the future to define the potential of this new source of stem cells for therapies related to specific diseases.