Protein kinase C inhibits autophagy and phosphorylates LC3 |
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Authors: | Houbo Jiang Wenhua Liu Jian Feng |
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Institution: | a Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY 14214, United States b Department of Human Genetics, Center for Neurodegenerative Diseases, Emory University, Atlanta, GA 30322, United States c Emory Proteomics Service Center, Emory University, Atlanta, GA 30322, United States |
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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. |
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Keywords: | LC3 microtubule-associated protein light chain 3 PKC protein kinase C Bis I bisindolylmaleimide I IDB thymeleatoxin Ingenol 3 20-dibenzoate PMA phorbo-12-myristate-13-acetate CA calyculin A mTOR mammalian target of rapamycin Atg autophagy-related gene |
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