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

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.

Akt Isoforms: A Family Affair in Breast Cancer

“…HM phosphorylation enhances phosphorylation at the A-loop and increases Akt activity whereas TM phosphorylation induces dephosphorylation of A-loop phosphorylation, thus decreasing Akt activity [39]. A recent study identified additional mTORC2-regulated phosphorylation sites termed TOR interacting motif (TIM) at the C-terminal tail [40]. These sites are conserved among AGC kinases (Akt1-T443, Akt-T444, Akt3-T440) and regulate A-loop phosphorylation by PDK1.…”

“…These sites are conserved among AGC kinases (Akt1-T443, Akt-T444, Akt3-T440) and regulate A-loop phosphorylation by PDK1. Although there are controversies whether mTORC2 directly phosphorylates HM site or not, it can regulate Akt activity by phosphorylating TIM [40].…”

Conventional protein kinase C in the brain: repurposing cancer drugs for neurodegenerative treatment?

“…Following their biosynthesis, conventional PKC isozymes are processed by a series of constitutive phosphorylations that are necessary for the enzymes to adopt an autoinhibited and stable conformation [ 7 ]. These priming phosphorylations are mediated by mTORC2 at a recently identified TOR Interaction Motif and adjacent turn motif on the C-tail, promoting phosphorylation by the phosphoinositide-dependent kinase 1 (PDK1) at the activation loop, in turn triggering an intramolecular autophosphorylation at another key regulatory site in the C-tail, the hydrophobic motif [ 11 ]. In the autoinhibited state, PKC is relatively resistant to dephosphorylation and subsequent degradation, and it has a half-time on the order of days [ 12 ].…”

The Tails of Protein Kinase A

“…Although each AGC kinase is regulated in unique ways by phosphorylation, the assembled Ct-Tail and the ways that it contributes to stabilizing the N-lobe of the kinase core when it is in its active conformation is conserved. The ways in which mTORC contributes to the assembly of the PKC Ct-Tail and the newly discovered TIM motif in the Ct-Tail is just the newest chapter to a long story (Baffi et al, 2021).…”