Research ArticleKinases

mTORC2 controls the activity of PKC and Akt by phosphorylating a conserved TOR interaction motif

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Science Signaling  13 Apr 2021:
Vol. 14, Issue 678, eabe4509
DOI: 10.1126/scisignal.abe4509

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mTORC2 marks kinases for maturity

The activity of members of the AGC family of kinases, which includes Akt and PKC, requires multiple phosphorylation events at different sites. As part of the mTORC2 complex, the kinase mTOR has been proposed to catalyze the phosphorylation of one of these sites termed the hydrophobic motif. Baffi et al. identified a distinct and evolutionarily conserved motif in Akt and PKC termed the TOR interaction motif (TIM) whose phosphorylation by mTORC2 was required for catalytic competence. Newly synthesized PKC dimerized through the TIM, and phosphorylation of the TIM in PKC by mTORC2 abolished this dimerization, ultimately leading to autophosphorylation of the hydrophobic motif. These results resolve long-standing conflicts about the role of mTORC2 in AGC kinase maturation and highlight a potential negative effect of mTOR kinase inhibitors that are now in clinical development.

Abstract

The complex mTORC2 is accepted to be the kinase that controls the phosphorylation of the hydrophobic motif, a key regulatory switch for AGC kinases, although whether mTOR directly phosphorylates this motif remains controversial. Here, we identified an mTOR-mediated phosphorylation site that we termed the TOR interaction motif (TIM; F-x3-F-pT), which controls the phosphorylation of the hydrophobic motif of PKC and Akt and the activity of these kinases. The TIM is invariant in mTORC2-dependent AGC kinases, is evolutionarily conserved, and coevolved with mTORC2 components. Mutation of this motif in Akt1 and PKCβII abolished cellular kinase activity by impairing activation loop and hydrophobic motif phosphorylation. mTORC2 directly phosphorylated the PKC TIM in vitro, and this phosphorylation event was detected in mouse brain. Overexpression of PDK1 in mTORC2-deficient cells rescued hydrophobic motif phosphorylation of PKC and Akt by a mechanism dependent on their intrinsic catalytic activity, revealing that mTORC2 facilitates the PDK1 phosphorylation step, which, in turn, enables autophosphorylation. Structural analysis revealed that PKC homodimerization is driven by a TIM-containing helix, and biophysical proximity assays showed that newly synthesized, unphosphorylated PKC dimerizes in cells. Furthermore, disruption of the dimer interface by stapled peptides promoted hydrophobic motif phosphorylation. Our data support a model in which mTORC2 relieves nascent PKC dimerization through TIM phosphorylation, recruiting PDK1 to phosphorylate the activation loop and triggering intramolecular hydrophobic motif autophosphorylation. Identification of TIM phosphorylation and its role in the regulation of PKC provides the basis for AGC kinase regulation by mTORC2.

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