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mTORC2 Facilitates the Maturation of Protein Kinase C by a Phosphorylation‐Independent Mechanism
Author(s) -
Baffi Timothy R.,
Gould Chistine M.,
Wang Zhiyong,
Gutkind Silvio,
Newton Alexandra C.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.lb570
Subject(s) - mtorc2 , protein kinase c , microbiology and biotechnology , phosphorylation , chemistry , biochemistry , biology , mtorc1 , protein kinase b
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 yield 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 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 of PKC; however, expression of Sin1 in Sin1−/− MEFs rescues the defect, allowing PKC to become phosphorylated and fold into the mature, closed conformation. Furthermore, we demonstrate that PKC is constitutively active in the absence of mTORC2. 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 of PKC that each confer mTORC2‐dependence. Our data support a model in which mTORC2 serves as a molecular chaperone to permit PKC to be processed by phosphorylation, modifications that allow the enzyme to adopt an autoinhibited conformation. Support or Funding Information This work was supported by NIH GM43154, UCSD Graduate Training Program in Cellular and Molecular Pharmacology NIH T32 GM007752, and the PhRMA Foundation Pre‐Doctoral Fellowship in Pharmacology/Toxicology.