title: Isozyme-specific interaction of protein kinase Cδ with mitochondria dissected using live cell fluorescence imaging.
abstract: PKCδ signaling to mitochondria has been implicated in both mitochondrial apoptosis and metabolism. However, the mechanism by which PKCδ interacts with mitochondria is not well understood. Using FRET-based imaging, we show that PKCδ interacts with mitochondria by a novel and isozyme-specific mechanism distinct from its canonical recruitment to other membranes such as the plasma membrane or Golgi. Specifically, we show that PKCδ interacts with mitochondria following stimulation with phorbol esters or, in L6 myocytes, with insulin via a mechanism that requires two steps. In the first step, PKCδ translocates acutely to mitochondria by a mechanism that requires its C1A and C1B domains and a Leu-Asn sequence in its turn motif. In the second step, PKCδ is retained at mitochondria by a mechanism that depends on its C2 domain, a unique Glu residue in its activation loop, intrinsic catalytic activity, and the mitochondrial membrane potential. In contrast, of these determinants, only the C1B domain is required for the phorbol ester-stimulated translocation of PKCδ to other membranes. PKCδ also basally localizes to mitochondria and increases mitochondrial respiration via many of the same determinants that promote its agonist-evoked interaction. PKCδ localized to mitochondria has robust activity, as revealed by a FRET reporter of PKCδ-specific activity (δCKAR). These data support a model in which multiple determinants unique to PKCδ drive a specific interaction with mitochondria that promotes mitochondrial respiration.
authors.affiliation: Department of Pharmacology, University of California San Diego, La Jolla, California 92093, USA.
authors.forename: Alyssa X
authors.initials: AX
authors.lastname: Wu-Zhang
authors.forename: Anne N
authors.initials: AN
authors.lastname: Murphy
authors.forename: Mackenzie
authors.initials: M
authors.lastname: Bachman
authors.forename: Alexandra C
authors.initials: AC
authors.lastname: Newton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism*; D057054:Molecular Imaging / Q000379:methods*; D008969:Molecular Sequence Data; D009154:Mutation; D032448:Myoblasts, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010101:Oxygen Consumption / Q000187:drug effects; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011485:Protein Binding; D051745:Protein Kinase C-delta / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D017386:Sequence Homology, Amino Acid
chemical_list: C465664:2-(1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl)-3-(1H-indol-3-yl)maleimide; D007211:Indoles; D007527:Isoenzymes; D008164:Luminescent Proteins; D008301:Maleimides; D047428:Protein Kinase Inhibitors; D015240:Phorbol 12,13-Dibutyrate; D051745:Protein Kinase C-delta