title: Conserved in vivo phosphorylation of calnexin at casein kinase II sites as well as a protein kinase C/proline-directed kinase site.
abstract: Calnexin is a lectin-like chaperone of the endoplasmic reticulum (ER) that couples temporally and spatially N-linked oligosaccharide modifications with the productive folding of newly synthesized glycoproteins. Calnexin was originally identified as a major type I integral membrane protein substrate of kinase(s) associated with the ER. Casein kinase II (CK2) was subsequently identified as an ER-associated kinase responsible for the in vitro phosphorylation of calnexin in microsomes (Ou, W-J., Thomas, D. Y., Bell, A. W., and Bergeron, J. J. M. (1992) J. Biol. Chem. 267, 23789-23796). We now report on the in vivo sites of calnexin phosphorylation. After 32PO4 labeling of HepG2 and Madin-Darby canine kidney cells, immunoprecipitated calnexin was phosphorylated exclusively on serine residues. Using nonradiolabeled cells, we subjected calnexin immunoprecipitates to in gel tryptic digestion followed by nanoelectrospray mass spectrometry employing selective scans specific for detection of phosphorylated fragments. Mass analyses identified three phosphorylated sites in calnexin from either HepG2 or Madin-Darby canine kidney cells. The three sites were localized to the more carboxyl-terminal half of the cytosolic domain: S534DAE (CK2 motif), S544QEE (CK2 motif), and S563PR. We conclude that CK2 is a kinase that phosphorylates calnexin in vivo as well as in microsomes in vitro. Another yet to be identified kinase (protein kinase C and/or proline-directed kinase) is directed toward the most COOH-terminal serine residue. Elucidation of the signaling cascade responsible for calnexin phosphorylation at these sites in vivo may define a novel regulatory function for calnexin in cargo folding and transport to the ER exit sites.
authors.affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 2B2, Canada.
authors.forename: H N
authors.initials: HN
authors.lastname: Wong
authors.forename: M A
authors.initials: MA
authors.lastname: Ward
authors.forename: A W
authors.initials: AW
authors.lastname: Bell
authors.forename: E
authors.initials: E
authors.lastname: Chevet
authors.forename: S
authors.initials: S
authors.lastname: Bains
authors.forename: W P
authors.initials: WP
authors.lastname: Blackstock
authors.forename: R
authors.initials: R
authors.lastname: Solari
authors.forename: D Y
authors.initials: DY
authors.lastname: Thomas
authors.forename: J J
authors.initials: JJ
authors.lastname: Bergeron
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D047390:Casein Kinase II; D002460:Cell Line; D004285:Dogs; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D038461:Proline-Directed Protein Kinases; D011493:Protein Kinase C / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D002135:Calcium-Binding Proteins; D037281:Calnexin; D012694:Serine; D038461:Proline-Directed Protein Kinases; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D014357:Trypsin