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.
Highlights
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
In order to determine the in vivo sites of phosphorylation for calnexin, both HepG2 and MDCK cells were in vivo labeled with [32P]orthophosphate followed by immunoprecipitation with anti-calnexin antibodies
SDS-PAGE-resolved immunoprecipitates were electroblotted onto PVDF membranes and similar levels of phosphorylated calnexin from both cell types were revealed by radioautography (Fig. 1A, lanes 1 and 2)
Summary
Materials—Rabbit antibodies raised against a synthetic peptide, corresponding to amino acid residues 487–505 of canine calnexin, described previously were used [3]. 32P-Orthophosphoric acid (specific activity Ͼ 8000 Ci/mmol) was purchased from NEN Life Science Products. Cells were lysed in 2% CHAPS/HEPES-buffered saline lysis buffer (2% (w/v) CHAPS, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml each leupeptin and aprotinin, 10 mM NaF, 10 mM NaPPi, 0.4 mM NaVO4, and 5 mM NaMoO4) for 30 min on ice. The same procedures were used to immunoprecipitate calnexin from both 32P-labeled and cold phosphate-labeled cellular extracts as described previously [3]. In Gel Digestion and Mass Spectrometry (DE-MALDI MS and nanoESI MS)—Calnexin immunoprecipitates from nonradiolabeled HepG2 and MDCK cell lysates were resolved by SDS-PAGE, visualized by Coomassie Blue staining (stain was 0.2% (w/v) Coomassie Brilliant Blue R250 in 50% (v/v) methanol in water containing 2% (v/v) acetic acid; destain was 50% (v/v) methanol in water containing 2% (v/v) acetic acid); and the corresponding gel slice was excised. Parenthesis indicate motif residues that are not conserved between human and canine calnexins
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