Prolyl 4-hydroxylase alpha-2 directly activates mTOR kinase

Abstract

Mammalian target of rapamycin (mTOR) kinase functions as a central regulator of cell growth and metabolism, and its complexes mTORC1 and mTORC2 phosphorylate distinct substrates1-3. Dysregulation of mTOR signaling is commonly implicated in human diseases, including cancer4,5. Despite three decades of active research in mTOR, much remains to be determined1. In fact, how mTOR kinase is directly activated and recognize distinct substrates are still unknown. Here, we demonstrate that prolyl 4-hydroxylase alpha-2 (P4HA2) can hydroxylate mTOR protein and thereby activating mTOR signal. By performing co-immunoprecipitation, GST pull-down assays combined with mass spectrometry-based proteomic analysis in 293T cells, we found that P4HA2 binds directly to mTOR and hydroxylates one highly conserved proline 2341 (P2341) within a kinase domain of mTOR. Moreover, we discover that the hydroxylation of P2341 strengthens mTOR stability and allows mTOR to accurately recognize its substrates such as S6K and AKT. Our study reveals an undiscovered hydroxylation-regulatory mechanism by which P4HA2 directly activates mTOR kinase, providing insights for therapeutically targeting mTOR kinase-driven cancers.