The mammalian target of rapamycin complex 1 (mTORC1) on lysosomes and late endosomes (Ly/LEs) integrates intra- and extracellular nutrient signals and regulates metabolic pathways such as protein synthesis, lysosome biogenesis and autophagy. Many factors stimulate mTORC1 activity, including the production of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] by class I phosphatidylinositol 3-kinases (PI3Ks) at the plasma membrane and phosphatidylinositol 3-phosphate [PI(3)P] by class III phosphatidylinositol 3-kinase at endosomes. In contrast, within the course of this thesis phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], synthesized by class II PI3K b was identified as a negative regulator of mTORC1. PI3KC2b is shown to repress mTORC1 activity locally on Ly/LEs, whereas loss of PI3KC2b hyperactivates mTORC1. Growth factor deprivation induces the association of PI3KC2b with the Raptor subunit of mTORC1 and local PI(3,4)P2 synthesis triggers repression of mTORC1 activity through association of Raptor with inhibitory 14-3-3 proteins. We used SILAC (stable isotope labeling with amino acids in cell culture) based quantitative mass spectrometry to identify growth factor dependent interaction partners and post- translational modifications of PI3KC2b. We could show that 14-3-3 protein binding to phosphorylated threonine 279 in the presence of growth factors competes with the recruitment of PI3KC2b to mTORC1 and to Ly/LEs. Furthermore, protein kinase N 2 (PKN2) was found to be the major kinase that phosphorylates PI3KC2b at T279, thereby triggering its complex formation and cytosolic sequestration with inhibitory 14-3-3 proteins to activate mTORC1. Conversely, loss of PKN2 or mutational inactivation of its target phosphorylation site in PI3KC2b repress nutrient signaling via mTORC1. Furthermore, mammalian target of Rapamycin Complex 2 (mTORC2) was shown to activate PKN2, while mTORC2 inhibition mimicked the effects of PKN2-loss upon PI3KC2b. Together, these results uncover a novel mechanism that couples mTORC2-dependent activation of PKN2 to the regulation of mTORC1-mediated nutrient signaling via PI3KC2b- mediated PI(3,4)P2 synthesis.
