title: Protein kinase C inhibits autophagy and phosphorylates LC3.
abstract: During autophagy, the microtubule-associated protein light chain 3 (LC3), a specific autophagic marker in mammalian cells, is processed from the cytosolic form (LC3-I) to the membrane-bound form (LC3-II). In HEK293 cells stably expressing FLAG-tagged LC3, activation of protein kinase C inhibited the autophagic processing of LC3-I to LC3-II induced by amino acid starvation or rapamycin. PKC inhibitors dramatically induced LC3 processing and autophagosome formation. Unlike autophagy induced by starvation or rapamycin, PKC inhibitor-induced autophagy was not blocked by the PI-3 kinase inhibitor wortmannin. Using orthophosphate metabolic labeling, we found that LC3 was phosphorylated in response to the PKC activator PMA or the protein phosphatase inhibitor calyculin A. Furthermore, bacterially expressed LC3 was directly phosphorylated by purified PKC in vitro. The sites of phosphorylation were mapped to T6 and T29 by nanoLC-coupled tandem mass spectrometry. Mutations of these residues significantly reduced LC3 phosphorylation by purified PKC in vitro. However, in HEK293 cells stably expressing LC3 with these sites mutated either singly or doubly to Ala, Asp or Glu, autophagy was not significantly affected, suggesting that PKC regulates autophagy through a mechanism independent of LC3 phosphorylation.
authors.affiliation: Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY 14214, United States.
authors.forename: Houbo
authors.initials: H
authors.lastname: Jiang
authors.forename: Dongmei
authors.initials: D
authors.lastname: Cheng
authors.forename: Wenhua
authors.initials: W
authors.lastname: Liu
authors.forename: Junmin
authors.initials: J
authors.lastname: Peng
authors.forename: Jian
authors.initials: J
authors.lastname: Feng
mesh_terms: D001343:Autophagy; D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008387:Marine Toxins; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D010080:Oxazoles / Q000494:pharmacology; D010588:Phagosomes / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; C477012:MAP1LC3A protein, human; D008387:Marine Toxins; D008869:Microtubule-Associated Proteins; D010080:Oxazoles; C016658:phorbolol myristate acetate; C059041:calyculin A; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate
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