Phosphatidic Acid Activates Mammalian Target of Rapamycin Complex 1 (mTORC1) Kinase by Displacing FK506 Binding Protein 38 (FKBP38) and Exerting an Allosteric Effect

Abstract

Phosphatidic acid (PA) is a critical mediator of mitogenic activation of mammalian target of rapamycin complex 1 (mTORC1) signaling, a master regulator of mammalian cell growth and proliferation. The mechanism by which PA activates mTORC1 signaling has remained unknown. Here, we report that PA selectively stimulates mTORC1 but not mTORC2 kinase activity in cells and in vitro. Furthermore, we show that PA competes with the mTORC1 inhibitor, FK506 binding protein 38 (FKBP38), for mTOR binding at a site encompassing the rapamycin-FKBP12 binding domain. This leads to PA antagonizing FKBP38 inhibition of mTORC1 kinase activity in vitro and rescuing mTORC1 signaling from FKBP38 in cells. Phospholipase D 1, a PA-generating enzyme that is an established upstream regulator of mTORC1, is found to negatively affect mTOR-FKBP38 interaction, confirming the role of endogenous PA in this regulation. Interestingly, removal of FKBP38 alone is insufficient to activate mTORC1 kinase and signaling, which require PA even when the FKBP38 level is drastically reduced by RNAi. In conclusion, we propose a dual mechanism for PA activation of mTORC1: PA displaces FKBP38 from mTOR and allosterically stimulates the catalytic activity of mTORC1.

Hepatic Gluconeogenesis Is Enhanced by Phosphatidic Acid Which Remains Uninhibited by Insulin in Lipodystrophic Agpat2−/− Mice

“…Recent experiments show that PA could also bind to the FK506-binding protein (FKBP12)-rapamycin-binding domain (11) and, in fact, some studies show that PA enhances mTOR activity (10). However, when primary mouse hepatocytes were incubated with specific PA species (C16:0/18:2, C16:0/18:1 and C18:1/20:4 PA), mTOR and its molecular targets remained unchanged.…”

A Novel Semisynthetic Inhibitor of the FRB Domain of Mammalian Target of Rapamycin Blocks Proliferation and Triggers Apoptosis in Chemoresistant Prostate Cancer Cells

“…The activity of mTOR is modulated by a number of small ligands including phosphatidic acid and rapamycin, both of which bind to the FRB domain of mTOR pointing to a special role of this domain in controlling the catalytic activity of mTOR (Veverka et al, 2008;Yoon et al, 2011;Zoncu et al, 2011). In silico molecular docking did not reveal any significant binding affinity of C-KbBA to S6K1 or PP2A.…”

Stoichiometry and assembly of mTOR complexes revealed by single-molecule pulldown

“…These assays followed previously published procedures (32) and are described in detail in SI Materials and Methods.…”