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.
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February 2016
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Wild type Neuroligin3 (red) localizes to the cell surface in PC12 Tet-on cells, in contrast to proteins within the endoplasmic reticulum (calnexin, green). See pp. 423–434 for further details. Image kindly provided by Ulbrich et al. - PDF Icon PDF LinkFront Matter
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Research Article|
February 09 2016
Protein kinase Cζ exhibits constitutive phosphorylation and phosphatidylinositol-3,4,5-triphosphate-independent regulation
Irene S. Tobias;
Irene S. Tobias
*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.
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Manuel Kaulich;
Manuel Kaulich
‡Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, U.S.A.
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Peter K. Kim;
Peter K. Kim
§Department of Biochemistry and Molecular Biology, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ 08854, U.S.A.
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Nitya Simon;
Nitya Simon
*Department of Pharmacology, University of California San Diego, La Jolla, CA 92093, U.S.A.
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Estela Jacinto;
Estela Jacinto
§Department of Biochemistry and Molecular Biology, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ 08854, U.S.A.
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Steven F. Dowdy;
Steven F. Dowdy
‡Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, U.S.A.
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Charles C. King;
Charles C. King
║Department of Pediatrics, University of California San Diego, La Jolla, CA 92093, U.S.A.
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Alexandra C. Newton
Alexandra C. Newton
1
*Department of Pharmacology, University of California San Diego, La Jolla, CA 92093, U.S.A.
1To whom correspondence should be addressed (email anewton@ucsd.edu).
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Publisher: Portland Press Ltd
Received:
October 08 2014
Revision Received:
November 23 2015
Accepted:
December 03 2015
Accepted Manuscript online:
December 03 2015
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2016 Authors; published by Portland Press Limited
2016
Biochem J (2016) 473 (4): 509–523.
Article history
Received:
October 08 2014
Revision Received:
November 23 2015
Accepted:
December 03 2015
Accepted Manuscript online:
December 03 2015
Citation
Irene S. Tobias, Manuel Kaulich, Peter K. Kim, Nitya Simon, Estela Jacinto, Steven F. Dowdy, Charles C. King, Alexandra C. Newton; Protein kinase Cζ exhibits constitutive phosphorylation and phosphatidylinositol-3,4,5-triphosphate-independent regulation. Biochem J 15 February 2016; 473 (4): 509–523. doi: https://doi.org/10.1042/BJ20151013
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