title: Protein kinase Cζ exhibits constitutive phosphorylation and phosphatidylinositol-3,4,5-triphosphate-independent regulation.
abstract: Atypical protein kinase C (aPKC) isoenzymes are key modulators of insulin signalling, and their dysfunction correlates with insulin-resistant states in both mice and humans. Despite the engaged interest in the importance of aPKCs to type 2 diabetes, much less is known about the molecular mechanisms that govern their cellular functions than for the conventional and novel PKC isoenzymes and the functionally-related protein kinase B (Akt) family of kinases. Here we show that aPKC is constitutively phosphorylated and, using a genetically-encoded reporter for PKC activity, basally active in cells. Specifically, we show that phosphorylation at two key regulatory sites, the activation loop and turn motif, of the aPKC PKCζ in multiple cultured cell types is constitutive and independently regulated by separate kinases: ribosome-associated mammalian target of rapamycin complex 2 (mTORC2) mediates co-translational phosphorylation of the turn motif, followed by phosphorylation at the activation loop by phosphoinositide-dependent kinase-1 (PDK1). Live cell imaging reveals that global aPKC activity is constitutive and insulin unresponsive, in marked contrast to the insulin-dependent activation of Akt monitored by an Akt-specific reporter. Nor does forced recruitment to phosphoinositides by fusing the pleckstrin homology (PH) domain of Akt to the kinase domain of PKCζ alter either the phosphorylation or activity of PKCζ. Thus, insulin stimulation does not activate PKCζ through the canonical phosphatidylinositol-3,4,5-triphosphate-mediated pathway that activates Akt, contrasting with previous literature on PKCζ activation. These studies support a model wherein an alternative mechanism regulates PKCζ-mediated insulin signalling that does not utilize conventional activation via agonist-evoked phosphorylation at the activation loop. Rather, we propose that scaffolding near substrates drives the function of PKCζ.
authors.affiliation: Department of Pharmacology, University of California San Diego, La Jolla, CA 92093, U.S.A. Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, CA 92093, U.S.A.
authors.forename: Irene S
authors.initials: IS
authors.lastname: Tobias
authors.affiliation: Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, U.S.A.
authors.forename: Manuel
authors.initials: M
authors.lastname: Kaulich
authors.affiliation: Department of Biochemistry and Molecular Biology, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ 08854, U.S.A.
authors.forename: Peter K
authors.initials: PK
authors.lastname: Kim
authors.affiliation: Department of Pharmacology, University of California San Diego, La Jolla, CA 92093, U.S.A.
authors.forename: Nitya
authors.initials: N
authors.lastname: Simon
authors.affiliation: Department of Biochemistry and Molecular Biology, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ 08854, U.S.A.
authors.forename: Estela
authors.initials: E
authors.lastname: Jacinto
authors.affiliation: Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, U.S.A.
authors.forename: Steven F
authors.initials: SF
authors.lastname: Dowdy
authors.affiliation: Department of Pediatrics, University of California San Diego, La Jolla, CA 92093, U.S.A.
authors.forename: Charles C
authors.initials: CC
authors.lastname: King
authors.affiliation: Department of Pharmacology, University of California San Diego, La Jolla, CA 92093, U.S.A. anewton@ucsd.edu.
authors.forename: Alexandra C
authors.initials: AC
authors.lastname: Newton
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D002478:Cells, Cultured; D007328:Insulin / Q000494:pharmacology; D000076225:Mechanistic Target of Rapamycin Complex 2; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D000099221:Protein Kinase C zeta
chemical_list: D064413:3-Phosphoinositide-Dependent Protein Kinases; D007328:Insulin; D000076225:Mechanistic Target of Rapamycin Complex 2; D046912:Multiprotein Complexes; D018129:Phosphatidylinositol Phosphates; D011493:Protein Kinase C; D058570:TOR Serine-Threonine Kinases; D000099221:Protein Kinase C zeta; C060974:phosphatidylinositol 3,4,5-triphosphate
keywords: atypical protein kinase C; insulin; mTOR complex; phosphatidylinositol signalling; phosphatidylserine; phosphorylation