The mammalian target of rapamycin complex 2 controls folding and stability of Akt and protein kinase C

Abstract

The target of rapamycin (TOR), as part of the rapamycinsensitive TOR complex 1 (TORC1), regulates various aspects of protein synthesis. Whether TOR functions in this process as part of TORC2 remains to be elucidated. Here, we demonstrate that mTOR, SIN1 and rictor, components of mammalian (m)TORC2, are required for phosphorylation of Akt and conventional protein kinase C (PKC) at the turn motif (TM) site. This TORC2 function is growth factor independent and conserved from yeast to mammals. TM site phosphorylation facilitates carboxyl-terminal folding and stabilizes newly synthesized Akt and PKC by interacting with conserved basic residues in the kinase domain. Without TM site phosphorylation, Akt becomes protected by the molecular chaperone Hsp90 from ubiquitination-mediated proteasome degradation. Finally, we demonstrate that mTORC2 independently controls the Akt TM and HM sites in vivo and can directly phosphorylate both sites in vitro. Our studies uncover a novel function of the TOR pathway in regulating protein folding and stability, processes that are most likely linked to the functions of TOR in protein synthesis.

mTORC 2 phosphorylates protein kinase Cζ to regulate its stability and activity

“…F The TM of PKCf is dephosphorylated in cells treated with mTOR kinase inhibitors. 293T cells were treated with Torin1 (100 nM, lanes 1-4) or PP242 (1 lM, lanes [5][6][7][8] for the indicated time.…”

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…More recent evidence has demonstrated that the mammalian Target of Rapamycin (mTOR) pathway regulates TM phosphorylation of some, but not all, PKCs [59][60][61]. mTOR is a serine/threonine kinase that associates with other proteins to form multi-protein complexes.…”

“…One of these complexes, mTORC1, is sensitive to the drug rapamycin while the other complex, mTORC2, is insensitive to acute rapamycin treatment [62]. mTORC2 has been implicated in TM phosphorylation of cPKCs ( , I , II , ) and PKC in murine embryonic fibroblasts (MEFs) [59,60]. In MEFs that lack critical components of the mTORC2 complex, the levels of expression of cPKCs and PKC are vastly reduced and TM phosphorylation is also absent on the residual pool of PKC (A-loop and HM phosphorylation are also absent).…”

“…12 levels of expression of PKC , , , and are unaffected in cells that lack mTORC2-specific components [59,60]. This implies that other signalling pathways or autophosphorylation may regulate these isoforms at this site.…”

“…Interestingly, while the levels of expression of PKC are unaffected following deletion of mTORC2 components in MEFs [59,60], phosphorylation of PKC (and PKC * ) at the HM motif is sensitive to rapamycin treatment in human embryonic kidney cells, which therefore implicates the mTORC1 complex in phosphorylation of these isoforms [73,66]. It is clear therefore that mTORC1, mTORC2 and possibly other pathways impact on the HM of different…”