MTORC2 Regulates the Maturation of Protein Kinase C by Folding and Phosphorylation

Abstract

Protein Kinase C (PKC) is primed by a series of ordered phosphorylations and conformational transitions to yield an autoinhibited enzyme that is positioned to respond to second messengers. For most PKC isozymes, this maturation to produce a signaling‐competent enzyme depends on the mammalian Target of Rapamycin Complex 2 (mTORC2); however, the molecular mechanisms by which mTORC2 regulates PKC are unknown. Here we use a variety of Förster resonance energy transfer (FRET)‐based live cell imaging and pulse‐chase biochemical assays to examine the role of mTORC2 in the the folding, phosphorylation, and activity of overexpressed PKCbII. We show that in cells lacking Sin1, a critical component of mTORC2, PKC exists in an unprimed, unphosphorylated, and open conformation, with fully‐exposed membrane‐targeting modules. In contrast, overexpression of PKCbII in cells with functional mTORC2 results in accumulation of a fully‐phosphorylated and autoinhibited enzyme. Introduction of Glu at the two phosphorylated residues on the C‐terminal tail is insufficient to bypass the requirement for mTORC2 in the folding or rate‐limiting processing step of PKC. We also show that PDK‐1 is involved in the mTORC2‐regulated rate‐limiting step of PKC processing, as overexpression of PDK‐1 in the absence of mTORC2 results in phosphorylation of the hydrophobic motif, but not the turn motif. Finally, analysis of chimeric proteins of an mTORC2‐dependent (PKCbII) and mTORC2‐independent (PKCd) enzyme, coupled with peptide array experiments, identify two critical regions in the kinase domain and C‐terminal tail, respectively, that each confer mTORC2‐dependence. Taken together, our data support a model in which mTORC2 serves two independent functions in the processing of PKC: 1) as the direct kinase of the turn motif, and 2) in the regulation of the rate‐limiting conformational change that allows the enzyme to autophosphorylate at the hydrophobic motif and adopt an autoinhibited conformation.Support or Funding InformationThis work was funded by NIH GM43154 to AN, NIH Pharmacological Sciences Training Grant NIH T32GM007752 to TB (AN), and PhRMA Foundation Pre Doctoral Fellowship in Pharmacology/Toxicology to TB (AN).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.