title: Protein kinase Cδ-mediated phosphorylation of Connexin43 gap junction channels causes movement within gap junctions followed by vesicle internalization and protein degradation.
abstract: Phosphorylation of gap junction proteins, connexins, plays a role in global signaling events involving kinases. Connexin43 (Cx43), a ubiquitous and important connexin, has several phosphorylation sites for specific kinases. We appended an imaging reporter tag for the activity of the δ isoform of protein kinase C (PKCδ) to the carboxyl terminus of Cx43. The FRET signal of this reporter is inversely related to the phosphorylation of serine 368 of Cx43. By activating PKC with the phorbol ester phorbol 12,13-dibutyrate (PDBu) or a natural stimulant, UTP, time lapse live cell imaging movies indicated phosphorylated Ser-368 Cx43 separated into discrete domains within gap junctions and was internalized in small vesicles, after which it was degraded by lysosomes and proteasomes. Mutation of Ser-368 to an Ala eliminated the response to PDBu and changes in phosphorylation of the reporter. A phosphatase inhibitor, calyculin A, does not change this pattern, indicating PKC phosphorylation causes degradation of Cx43 without dephosphorylation, which is in accordance with current hypotheses that cells control their intercellular communication by a fast and constant turnover of connexins, using phosphorylation as part of this mechanism.
authors: From the National Center for Microscopy and Imaging Research.
authors: Angela C
authors: AC
authors: Cone
authors: Gabriel
authors: G
authors: Cavin
authors: Cinzia
authors: C
authors: Ambrosi
authors: Hiroyuki
authors: H
authors: Hakozaki
authors: Alyssa X
authors: AX
authors: Wu-Zhang
authors: Maya T
authors: MT
authors: Kunkel
authors: Alexandra C
authors: AC
authors: Newton
authors: Gina E
authors: GE
authors: Sosinsky
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D018031:Connexin 43 / Q000737:chemistry / Q000378:metabolism*; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009068:Movement / Q000187:drug effects; D010703:Phorbol Esters / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation / Q000187:drug effects; D051745:Protein Kinase C-delta / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D012694:Serine / Q000378:metabolism
chemical_list: D018031:Connexin 43; D010703:Phorbol Esters; D047428:Protein Kinase Inhibitors; D012694:Serine; D051745:Protein Kinase C-delta; D010744:Phosphoric Monoester Hydrolases
keywords: Cell Junctions; Connexin; Gap Junctions; Hemichannels; Intercellular Communication; Membrane Protein Degradation; Membrane Protein Trafficking; Protein Kinase C (PKC); Protein Phosphorylation