title: Calcineurin interacts with PERK and dephosphorylates calnexin to relieve ER stress in mammals and frogs.
abstract: BACKGROUND
  The accumulation of misfolded proteins within the endoplasmic reticulum (ER) triggers a cellular process known as the Unfolded Protein Response (UPR). One of the earliest responses is the attenuation of protein translation. Little is known about the role that Ca2+ mobilization plays in the early UPR. Work from our group has shown that cytosolic phosphorylation of calnexin (CLNX) controls Ca2+ uptake into the ER via the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) 2b.
  
  
  RESULTS
  Here, we demonstrate that calcineurin (CN), a Ca2+ dependent phosphatase, associates with the (PKR)-like ER kinase (PERK), and promotes PERK auto-phosphorylation. This association, in turn, increases the phosphorylation level of eukaryotic initiation factor-2 alpha (eIF2-alpha) and attenuates protein translation. Data supporting these conclusions were obtained from co-immunoprecipitations, pull-down assays, in-vitro kinase assays, siRNA treatments and [35S]-methionine incorporation measurements. The interaction of CN with PERK was facilitated at elevated cytosolic Ca2+ concentrations and involved the cytosolic domain of PERK. CN levels were rapidly increased by ER stressors, which could be blocked by siRNA treatments for CN-Aalpha in cultured astrocytes. Downregulation of CN blocked subsequent ER-stress-induced increases in phosphorylated elF2-alpha. CN knockdown in Xenopus oocytes predisposed them to induction of apoptosis. We also found that CLNX was dephosphorylated by CN when Ca2+ increased. These data were obtained from [gamma32P]-CLNX immunoprecipitations and Ca2+ imaging measurements. CLNX was dephosphorylated when Xenopus oocytes were treated with ER stressors. Dephosphorylation was pharmacologically blocked by treatment with CN inhibitors. Finally, evidence is presented that PERK phosphorylates CN-A at low resting levels of Ca2+. We further show that phosphorylated CN-A exhibits decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is re-established.
  
  
  CONCLUSIONS
  Our data suggest two new complementary roles for CN in the regulation of the early UPR. First, CN binding to PERK enhances inhibition of protein translation to allow the cell time to recover. The induction of the early UPR, as indicated by increased P-elF2alpha, is critically dependent on a translational increase in CN-Aalpha. Second, CN dephosphorylates CLNX and likely removes inhibition of SERCA2b activity, which would aid the rapid restoration of ER Ca2+ homeostasis.
authors.affiliation: Instituto de Investigación Médica Mercedes y Martín Ferreyra (INIMEC CONICET), Córdoba, Argentina.
authors.forename: Mariana
authors.initials: M
authors.lastname: Bollo
authors.forename: R Madelaine
authors.initials: RM
authors.lastname: Paredes
authors.forename: Deborah
authors.initials: D
authors.lastname: Holstein
authors.forename: Nadezhda
authors.initials: N
authors.lastname: Zheleznova
authors.forename: Patricia
authors.initials: P
authors.lastname: Camacho
authors.forename: James D
authors.initials: JD
authors.lastname: Lechleiter
mesh_terms: D000818:Animals; D001001:Anura; D017209:Apoptosis / Q000187:drug effects; D001253:Astrocytes / Q000378:metabolism; D019703:Calcineurin / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D037281:Calnexin / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006706:Homeostasis / Q000187:drug effects; D006801:Humans; D051379:Mice; D009865:Oocytes / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D051381:Rats; D013312:Stress, Physiological / Q000187:drug effects; D013997:Time Factors; D014415:Tunicamycin / Q000494:pharmacology; D014981:Xenopus; D019892:eIF-2 Kinase / Q000378:metabolism*
chemical_list: D019703:Calcineurin; D002118:Calcium; D037281:Calnexin; D015852:Eukaryotic Initiation Factor-2; D014415:Tunicamycin; D019892:eIF-2 Kinase