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

Mee-Sup Yoon,Yu Jiang,Yuting Sun,Jie Chen,Edwin Arauz

doi:10.1074/jbc.m111.262816

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.

Highlights

S6K1, S6 kinase 1; TSC, tuberous sclerosis complex; PLD, phospholipase D; PA, phosphatidic acid; PC, phosphatidylcholine; FKBP12-rapamycin binding domain (FRB), FKBP12-rapamycin binding
PA interacts with the FKBP12-rapamycin binding domain (FRB) of mammalian target of rapamycin (mTOR) with remarkable specificity, and this interaction is disrupted by FKBP12-rapamycin [14]
PA Stimulates mammalian target of rapamycin complex 1 (mTORC1) Kinase Activity—To evaluate a potential effect of PA on the kinase activity of mTOR in cells, we examined the phosphorylation of mTOR on Ser-2481, an autophosphorylation site that has recently been reported to monitor mTORC-specific catalytic activities [27]

Summary

Introduction

S6K1, S6 kinase 1; TSC, tuberous sclerosis complex; PLD, phospholipase D; PA, phosphatidic acid; PC, phosphatidylcholine; FRB, FKBP12-rapamycin binding. In Vitro mTOR Kinase Assays—mTORC1 and mTORC2 were immunoprecipitated using anti-raptor and anti-rictor antibodies, respectively, followed by incubation with protein G agarose beads. PA Disrupts FKBP38-mTOR Interaction—To probe into the mechanism by which PA activates mTORC1 kinase, we considered the role of FKBP38 as an endogenous inhibitor of mTORC1 [13].

Results

Conclusion