Conserved in VivoPhosphorylation of Calnexin at Casein Kinase II Sites as Well as a Protein Kinase C/Proline-directed Kinase Site
1998; Elsevier BV; Volume: 273; Issue: 27 Linguagem: Inglês
10.1074/jbc.273.27.17227
ISSN1083-351X
AutoresHetty N. Wong, Malcolm Ward, Alexander W. Bell, Éric Chevet, Satty Bains, Walter Blackstock, Roberto Solari, David Y. Thomas, John Bergeron,
Tópico(s)Heat shock proteins research
ResumoCalnexin is a lectin-like chaperone of the endoplasmic reticulum (ER) that couples temporally and spatiallyN-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 thein 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 sitesin vivo may define a novel regulatory function for calnexin in cargo folding and transport to the ER exit sites. Calnexin is a lectin-like chaperone of the endoplasmic reticulum (ER) that couples temporally and spatiallyN-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 thein 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 sitesin vivo may define a novel regulatory function for calnexin in cargo folding and transport to the ER exit sites. Calnexin was originally identified and purified as a constituent of a complex of four co-isolated integral membrane proteins, two of which were phosphorylated in microsomes by ER 1The abbreviations used are: ER, endoplasmic reticulum; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; FBS, fetal bovine serum; DE-MALDI, delayed extraction matrix-assisted laser desorption ionization; ToF, time of flight; MS, mass spectrometry; nano-ESI, nanoelectrospray ionization; CNL, constant neutral loss; TLC, thin layer cellulose; MS/MS, tandem mass spectrometry; CK2, casein kinase II; MDCK, Madin-Darby canine kidney; PKC, protein kinase C; PDK, proline-directed kinase; PVDF, polyvinylidene difluoride. -associated kinase(s) (1Wada I. Rindress D. Cameron P.H. Ou W-J. Doherty, II J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Abstract Full Text PDF PubMed Google Scholar). This phosphorylation was exclusively on serine residues and by controlled protease digestion found to be cytosolically oriented (1Wada I. Rindress D. Cameron P.H. Ou W-J. Doherty, II J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Abstract Full Text PDF PubMed Google Scholar,2Ou W-J. Bergeron J.J.M. Li Y. Kang C.Y. Thomas D.Y. J. Biol. Chem. 1995; 270: 18051-18059Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). As a consequence of the cDNA cloning of this phosphoprotein, it was predicted and subsequently confirmed to be a type I integral membrane protein with extensive sequence similarity in its luminal domain to the ER luminal resident protein, calreticulin (1Wada I. Rindress D. Cameron P.H. Ou W-J. Doherty, II J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Abstract Full Text PDF PubMed Google Scholar, 2Ou W-J. Bergeron J.J.M. Li Y. Kang C.Y. Thomas D.Y. J. Biol. Chem. 1995; 270: 18051-18059Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Calnexin and then calreticulin were found to be novel molecular chaperones of the ER. These chaperones act as lectins to couple oligosaccharide modifications to newly synthesized N-linked glycoproteins with productive glycoprotein folding. The lectin specificity of these chaperones has been identified as the recognition of high mannose oligosaccharides terminating in monoglucosyl residues linked α1–3 (3Ou W-J. Cameron P.H. Thomas D.Y. Bergeron J.J.M. Nature. 1993; 364: 771-776Crossref PubMed Scopus (488) Google Scholar, 4Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (721) Google Scholar, 5Bergeron J.J.M. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (465) Google Scholar, 6Hebert D.N. Foellmer B. Helenius A. 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Michalak M. Bergeron J.J.M. Thomas D.Y. J. Biol. Chem. 1998; 273: 6009-6012Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). Purification of the ER-associated kinase that phosphorylated calnexin in microsomes led to the identification of CK2 (14Ou W-J. Thomas D.Y. Bell A.W. Bergeron J.J.M. J. Biol. Chem. 1992; 267: 23789-23796Abstract Full Text PDF PubMed Google Scholar). The properties of this kinase were consistent with the conditions that originally revealed calnexin phosphorylation (1Wada I. Rindress D. Cameron P.H. Ou W-J. Doherty, II J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Abstract Full Text PDF PubMed Google Scholar, 14Ou W-J. Thomas D.Y. Bell A.W. Bergeron J.J.M. J. Biol. Chem. 1992; 267: 23789-23796Abstract Full Text PDF PubMed Google Scholar). Furthermore, purified CK2 has been found to phosphorylate calnexin on putative CK2 sites found within the cytosolic domain of calnexin (14Ou W-J. Thomas D.Y. Bell A.W. Bergeron J.J.M. J. Biol. Chem. 1992; 267: 23789-23796Abstract Full Text PDF PubMed Google Scholar, 15Cala S.E. Ulbright C. Kelley J.S. Jones L.R. J. Biol. Chem. 1993; 268: 2969-2975Abstract Full Text PDF PubMed Google Scholar). Calnexin is phosphorylated in vivo (16Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar, 17Capps G.G. Zuniga M.C. J. Biol. Chem. 1994; 269: 11634-11639Abstract Full Text PDF PubMed Google Scholar, 18Schue V. Green G.A. Girardot R. Monteil H. Biochem. Biophys. Res. Commun. 1994; 203: 22-28Crossref PubMed Scopus (8) Google Scholar). Phosphorylated calnexin has been shown to associate with the null Hong Kong mutant of α-1-antitrypsin, coinciding with retention of this misfolded glycoprotein within the lumen of the ER (16Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar). Phosphorylated calnexin was also found in association with newly synthesized major histocompatibility complex class I allotypes, which egressed from the ER at slow rates. Those allotypes transported to the Golgi apparatus at more rapid rates were associated preferentially with nonphosphorylated calnexin (17Capps G.G. Zuniga M.C. J. Biol. Chem. 1994; 269: 11634-11639Abstract Full Text PDF PubMed Google Scholar). Prolonged association of newly synthesized major histocompatibility complex class I heavy chains with calnexin was found in a B lymphoblastoid cell line transfected with HLA-B701 after incubation with the phosphatase inhibitor cantharidin or okadaic acid (19Tector M. Zhang Q. Salter R.D. J. Biol. Chem. 1994; 269: 25816-25822Abstract Full Text PDF PubMed Google Scholar). Furthermore, when human synovial epithelial (McCoy) cells were treated with okadaic acid, the major cellular protein whose phosphorylation was shown to increase (based on two-dimensional gels followed by protein microsequencing) was calnexin (18Schue V. Green G.A. Girardot R. Monteil H. Biochem. Biophys. Res. Commun. 1994; 203: 22-28Crossref PubMed Scopus (8) Google Scholar). Remarkably, calnexin phosphorylation also increased 3-fold when McCoy cells were treated with Clostridium difficile cytotoxin B (18Schue V. Green G.A. Girardot R. Monteil H. Biochem. Biophys. Res. Commun. 1994; 203: 22-28Crossref PubMed Scopus (8) Google Scholar), a protein that glucosylates Rho proteins of the Ras superfamily (20Just I. Selzer J. Wilm M. von Eichel-Streiber C. Mann M. Aktories K. Nature. 1995; 375: 500-503Crossref PubMed Scopus (885) Google Scholar). Although some progress has been made on the kinases and sites of phosphorylation of calnexin after in vitro phosphorylation of intact microsomes (14Ou W-J. Thomas D.Y. Bell A.W. Bergeron J.J.M. J. Biol. Chem. 1992; 267: 23789-23796Abstract Full Text PDF PubMed Google Scholar, 15Cala S.E. Ulbright C. Kelley J.S. Jones L.R. J. Biol. Chem. 1993; 268: 2969-2975Abstract Full Text PDF PubMed Google Scholar) little is known of the sites of calnexin phosphorylation in vivo. Here we report on their identification in two mammalian cell lines, HepG2 cells (human) and Madin-Darby canine kidney MDCK cells. Both cell lines revealed phosphorylation of the cytosolic domain of calnexin exclusively on serine residues within CK2 motifs as well as a protein kinase C (PKC) and/or proline-directed kinase (PDK) motif. Rabbit antibodies raised against a synthetic peptide, corresponding to amino acid residues 487–505 of canine calnexin, described previously were used (3Ou W-J. Cameron P.H. Thomas D.Y. Bergeron J.J.M. Nature. 1993; 364: 771-776Crossref PubMed Scopus (488) Google Scholar).32P-Orthophosphoric acid (specific activity > 8000 Ci/mmol) was purchased from NEN Life Science Products. Protein A-Sepharose beads were from Amersham Pharmacia Biotech. Pyridine, phenylmethylsulfonyl fluoride, aprotinin, leupeptin, and HEPES were from Sigma. Sequencing grade bovine trypsin was from Boehringer Mannheim. TLC microcrystalline plates (0.1-mm thickness) were from EM Science (Gibbstown, NJ). Kodak XAR-5 OMAT film was purchased from Picker International Canada Inc. (Montreal, Quebec). Reagents for SDS-PAGE and protein determination were from Bio-Rad. All other reagents were from Sigma, Anachemia Canada Inc. (Lachine, Quebec), or Boehringer Mannheim. Dulbecco's modified Eagle's medium, phosphate-deficient Dulbecco's modified Eagle's medium, dialyzed FBS, penicillin, and streptomycin were purchased from Life Technologies, Inc. FBS was obtained from HyClone Laboratories, Inc. (Logan, UT). Both human hepatoma (HepG2) cells and MDCK cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) FBS, 500 units/ml penicillin, and 500 μg/ml of streptomycin. Cells were maintained in a 37 °C incubator with 5% atmospheric CO2and used when they were 80% confluent. Cells were radiolabeled as described previously (16Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar, 17Capps G.G. Zuniga M.C. J. Biol. Chem. 1994; 269: 11634-11639Abstract Full Text PDF PubMed Google Scholar) with the following modifications. Briefly, cells were washed in phosphate-free Dulbecco's modified Eagle's medium supplemented with 1% dialyzed FBS followed by incubation in the same medium for 1 h at 37 °C. Cells were then labeled by the addition of 2 mCi/ml [32P]orthophosphate for 3 h. At the end of labeling, the cells were lysed as described previously (3Ou W-J. Cameron P.H. Thomas D.Y. Bergeron J.J.M. Nature. 1993; 364: 771-776Crossref PubMed Scopus (488) Google Scholar). Briefly, cells were washed twice with ice-cold phosphate-buffered saline (20 mm NaPO4, pH 7.5, 150 mm NaCl) and once with ice-cold HEPES-buffered saline (50 mm HEPES, pH 7.6, 200 mm NaCl) before harvesting. Cells were then lysed in 2% CHAPS/HEPES-buffered saline lysis buffer (2% (w/v) CHAPS, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml each leupeptin and aprotinin, 10 mmNaF, 10 mm NaPPi, 0.4 mmNaVO4, 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 (3Ou W-J. Cameron P.H. Thomas D.Y. Bergeron J.J.M. Nature. 1993; 364: 771-776Crossref PubMed Scopus (488) Google Scholar). Immunoprecipitated calnexin was isolated by SDS-PAGE. The gel was dried for 2 h at 80 °C under vacuum, or the proteins were transferred onto PVDF membrane (1Wada I. Rindress D. Cameron P.H. Ou W-J. Doherty, II J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Abstract Full Text PDF PubMed Google Scholar). Radioactive bands were visualized by radioautography at room temperature. Phosphoamino acid analysis was performed as described (21Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar). Briefly, in vivo phosphorylated calnexin was resolved by SDS-PAGE and electroblotted onto a PVDF membrane. 32P-Labeled calnexin was detected by radioautography, and the corresponding PVDF bands were excised. The membrane containing phosphorylated calnexin was washed extensively with distilled water and subjected to acid hydrolysis, immersed in 6n HCl at 110 °C for 60 min. The hydrolysate was transferred to a microcentrifuge tube, lyophilized, and dissolved in pH 1.9 buffer (88% formic acid, glacial acetic acid, H2O; 2.5:7.8:89.7 (v/v/v)). Phosphoamino acid analysis was by two-dimensional electrophoresis on TLC plates in the presence of phosphoamino acid standards; phosphoserine, phosphothreonine, and phosphotyrosine. First dimension electrophoresis was carried out in pH 1.9 buffer for 20 min at 1.3 kV employing a Hunter thin layer electrophoresis system (C.B.S. Scientific, Del Mar, CA). Second dimension electrophoresis was carried out in pH 3.5 buffer (pyridine, glacial acetic acid, H2O; 0.5:5:94.5 (v/v/v)) at 1.5 kV for 20 min. The standards were visualized by spraying a 0.25% (w/v) ninhydrin acetone solution followed by incubation at 65 °C for 10 min. The radiolabeled amino acids were detected by radioautography with an enhancing screen at −70 °C. Recovery from each step was monitored by Cerenkov counting. 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. Coomassie stain was removed by extraction (twice) with 50% (v/v) acetonitrile/H2O, followed by two cycles each of extracting with acetonitrile and swelling with 100 mm NH4HCO3. Calnexin was in gel reduced and alkylated with 15 mmdithiothreitol and 1.3 mm iodoacetamide and then in gel digested with 13 μg/ml bovine trypsin in the presence of 5 mm CaCl2 as described (22Wilm M. Shevchenko A. Houthaeve T. Breit S. Schweigerer L. Fotsis T. Mann M. Nature. 1996; 379: 466-469Crossref PubMed Scopus (1507) Google Scholar). Tryptic peptides were first extracted with acetonitrile, followed by two cycles each of swelling with 100 mm NH4HCO3 and extracting with acetonitrile and then two cycles each of swelling with 5% formic acid and extracting with acetonitrile. The efficiency of the extraction of calnexin tryptic phosphorylated fragments from gel pieces was evaluated with radiolabeled calnexin and Cerenkov counting. Greater than 70% of the radioactivity was extracted and recovered (data not shown). For DE-MALDI-ToF MS, dried peptide extracts were redissolved in 5% formic acid containing 5% methanol (10 μl) (22Wilm M. Shevchenko A. Houthaeve T. Breit S. Schweigerer L. Fotsis T. Mann M. Nature. 1996; 379: 466-469Crossref PubMed Scopus (1507) Google Scholar). An aliquot (0.4 μl) was spotted onto a stainless steel target precoated with α-cyano-4-hydroxycinnamic acid. The target was allowed to air dry before being washed with an aqueous solution containing 1% trifluoroacetic acid. Excess wash solution was blown off the target using compressed air. The target was loaded into the mass spectrometer for analysis by DE-MALDI-ToF mass spectrometry using a Bruker Reflex instrument fitted with a 337-nm nitrogen laser. All spectra were acquired with the instrument in reflector mode. Acquisition parameters were set as follows: sampling frequency, 500 MHz; gain, 50 mV; source voltage, 24,000 V; reflector voltage, 24850 V. DE-MALDI-ToF mass spectra were acquired for each digest and compared with mock in gel trypsin digest. Nano-ESI experiments were performed as described (23Betts J.C. Blackstock W.P. Ward M.A. Anderton B.H. J. Biol. Chem. 1997; 272: 12922-12927Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Briefly, in gel trypsin-digested calnexin was desalted via a POROS R2 capillary column (22Wilm M. Shevchenko A. Houthaeve T. Breit S. Schweigerer L. Fotsis T. Mann M. Nature. 1996; 379: 466-469Crossref PubMed Scopus (1507) Google Scholar, 23Betts J.C. Blackstock W.P. Ward M.A. Anderton B.H. J. Biol. Chem. 1997; 272: 12922-12927Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) (PerSeptive Biosystems, Farmingham, MA); dried in a vacuum centrifuge; and resuspended in 10 μl of spraying solution (50% (v/v) methanol, 5% (v/v) ammonia in water for negative ion mode or 50% (v/v) methanol, 1% (v/v) formic acid in water for positive ion mode). 1 μl was inserted into a homemade capillary needle. Spraying capillary needles were made from borosilicate glass capillaries (Clark Electromedical Instruments, Pangbourne, Reading, UK) employing a micropipette puller (Sutter Instrument Co., Novato, CA), and gold was sputtered employing a vapor desorption instrument. A PE-Sciex API III triple quadrupole mass spectrometer (Perkin-Elmer) fitted with a nano-ESI source (24Wilm M. Mann M. Anal. Chem. 1996; 68: 1-8Crossref PubMed Scopus (1719) Google Scholar, 25Wilm M.S. Mann M. Int. J. Mass Spectrom. Ion Processes. 1994; 136: 167-180Crossref Scopus (878) Google Scholar) was used to acquire all electrospray mass spectra. The ion spray voltage was set to 600–650 V with an orifice potential of 60–70 V. Instrument polarity was set appropriate to the detection of positive or negative ions. Q1 scans were used to mass analyze ions relating to the intact peptides in the mixture. These were recorded by scanning the first quadrupole betweenm/z 400 and 2000 using a 1.0-ms dwell time and a 0.1-Da step size. Peptides containing phosphate produce a characteristic PO3− ion fragment atm/z 79 (26Wilm M. Neubauer G. Mann M. Anal. Chem. 1996; 68: 527-533Crossref PubMed Scopus (280) Google Scholar, 27Carr S.A. Huddleston M.J. Annan R.S. Anal. Biochem. 1996; 239: 180-192Crossref PubMed Scopus (330) Google Scholar). Molecular ions for these peptides were recorded by scanning the first quadrupole betweenm/z 300 and 1400 with the third quadrupole set to transmit m/z 79 only (precursorsm/z 79 scans). Argon gas was used in the collision cell (quadrupole 2) at a collision gas thickness of 250 units. Scanning the first and third quadrupoles with an offset ofm/z 49 profiled phosphopeptides by loss of H3PO4 (molecular mass of 98) from doubly charged molecular ions (Constant Neutral Loss scans) (28Covey T. Shushan B. Bonner R. Schroder W. Hucho F. Jornall H. Hoog J.O. Gustavsson A.M. Methods in Protein Sequence Analysis. Birkhaeuser Verlag, Basel, Switzerland1991: 249-256Crossref Google Scholar). For MS/MS detection, Q1 was set to transmit a mass window of 2 Da for product ion scans. Product ion spectra were accumulated with a 0.2-Da mass step size. Dwell time was 1.0 ms, and collision energy was optimized to obtain the MS/MS spectra. Spectra interpretation was performed using BioMultiView (Sciex) software. Candidate kinases for the cytosolic serine residues of calnexin were predicted based on consensus sequence motifs employing the PROSITE data base of EXPASY 2Available on the World Wide Web athttp://expasy.hcuge.ch. (29Appel R.D. Bairoch A. Hochstrasser D.F. Trends. Biochem. Sci. 1994; 19: 258-260Abstract Full Text PDF PubMed Scopus (512) Google Scholar). Predicted peptide m/z values were evaluated employing tools provided by ProteinProspector. 3Available on the World Wide Web athttp://prospector.ucsf.edu. Calnexin and calmegin sequence alignments were initially generated by BLASTp (30Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar), FASTA (31Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Crossref PubMed Scopus (9393) Google Scholar), and MSA (32Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar) and optimized manually. Calnexin was originally identified as a major substrate of ER-associated kinase(s) by in vitrophosphorylation of intact microsomes with [γ-32P]GTP as phosphate donor (1Wada I. Rindress D. Cameron P.H. Ou W-J. Doherty, II J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Abstract Full Text PDF PubMed Google Scholar). In order to determine the in vivo sites of phosphorylation for calnexin, both HepG2 and MDCK cells werein 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. 1 A, lanes 1 and 2). The bands corresponding to phosphorylated calnexin were excised from the PVDF membranes and subjected to phosphoamino acid analyses. Radioautograms of the two-dimensional TLC plates for both human and canine calnexins revealed only 32P-labeled serine that comigrated with the nonradiolabeled phosphoserine standard as detected by ninhydrin staining (left and right parts of Fig. 1 B, respectively). The phosphorylated residues were not altered with longer (up to 24 h) in vivo radiolabeling (data not shown). Hence, calnexin was exclusively in vivophosphorylated on serine residues in both cell types. In isolated ER microsomes, in vitro phosphorylation by ER-associated kinase(s) is exclusively on the cytosolic domain of calnexin (1Wada I. Rindress D. Cameron P.H. Ou W-J. Doherty, II J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Abstract Full Text PDF PubMed Google Scholar). The cytosolic COOH-terminal domains of canine (MDCK) and human (HepG2) calnexins (1Wada I. Rindress D. Cameron P.H. Ou W-J. Doherty, II J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Abstract Full Text PDF PubMed Google Scholar, 33David V. Hochstenbach F. Rajagopalan S. Brenner M.B. J. Biol. Chem. 1993; 268: 9585-9592Abstract Full Text PDF PubMed Google Scholar) share 94% identity (Fig. 2). The cytosolic domain of canine calnexin contains six serines, and the cytosolic domain of human calnexin contains five equivalent serines and a threonine. The five conserved cytosolic serine residues are in primary sequence motifs that are predicted to be recognized by CK2 (34Pinna L.A. Biochim. Biophys. Acta. 1990; 1054: 267-284Crossref PubMed Scopus (806) Google Scholar), PKC (35Kishimoto A. Nishiyama K. Nakanishi H. Uratsuji Y. Nomura H. Takeyama Y. Nishizuka Y. J. Biol. Chem. 1985; 260: 12492-12499Abstract Full Text PDF PubMed Google Scholar, 36Woodgett J.R. Gould K.L. Hunter T. Eur. J. Biochem. 1986; 161: 177-184Crossref PubMed Scopus (400) Google Scholar), PDK (37Nigg E.A. BioEssays. 1995; 17: 471-480Crossref PubMed Scopus (797) Google Scholar, 38Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2286) Google Scholar), or protein kinase A (39Feramisco J.R. Glass D.B. Krebs E.G. J. Biol. Chem. 1980; 255: 4240-4245Abstract Full Text PDF PubMed Google Scholar, 40Glass D.B. el-Maghrabi M.R. Pilkis S.J. J. Biol. Chem. 1986; 261: 2987-2993Abstract Full Text PDF PubMed Google Scholar) (Table I). To identify the in vivo sites of phosphorylation of calnexin we proceeded to mass spectrometry.Table IPotential serine phosphorylation site(s) of the cytosolic domains of calnexinsCanine calnexinaCK2, predicted casein kinase II site; PKC, predicted protein kinase C site; PDK, predicted proline-directed kinase site; PKA, predicted protein kinase A site.Ser485Ser490Ser491Ser535Ser545Ser564CK2−−+++−PKC+−−−−+PDK−−(+)−−+PKA−(+)−−−−Human calnexinaCK2, predicted casein kinase II site; PKC, predicted protein kinase C site; PDK, predicted proline-directed kinase site; PKA, predicted protein kinase A site.Ser484Ser490Ser534Ser544Ser563CK2−+++−PKC+−−−+PDK−−−−+Parenthesis indicate motif residues that are not conserved between human and canine calnexins.a CK2, predicted casein kinase II site; PKC, predicted protein kinase C site; PDK, predicted proline-directed kinase site; PKA, predicted protein kinase A site. Open table in a new tab Parenthesis indicate motif residues that are not conserved between human and canine calnexins. Calnexin was immunoprecipitated from nonradiolabeled cell lysates from both cultured MDCK and HepG2 cells. After separation by SDS-PAGE and visualization by Coomassie staining, calnexin was in gel digested with trypsin as described under “Experimental Procedures.” DE-MALDI-ToF mass spectra for the tryptic peptides of calnexin from MDCK and HepG2 were collected (Fig. 3,A and B). Peptide masses not observed in the mock in gel digest (data not shown), were employed to confirm the identity of MDCK and HepG2 calnexins. Total coverage for calnexin from MDCK cells was 165 of 573 (28.8%) amino acid residues, and coverage from HepG2 cells was 171 of 572 (29.9%) amino acid residues. With respect to the cytosolic domain of calnexin, however, the degree of coverage was 58.4 and 33.7% from MDCK and HepG2 cells, respectively. This coverage by DE-MALDI-ToF included phosphopeptides observed as peak 1 and peak 7 in Fig. 3 A for MDCK calnexin and as summarized in Table II. Coverage of the cytosolic domain was increased by nano-ESI MS and was greater than 90% by combination of both mass spectrometric techniques (see below). Poor coverage of the luminal domain was consistent with the generation of a protease-resistant core in the presence of Ca2+ (2Ou W-J. Bergeron J.J.M. Li Y. Kang C.Y. Thomas D.Y. J. Biol. Chem. 1995; 270: 18051-18059Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), conditions employed during in gel trypsin digestion.Table IISummary of DE-MALDI-ToF mass spectral data (Fig. 3, A and B)PeakObserved massCalculatedResiduesModificationaPO4, phosphate group; Met-ox, methionine sulfoxide; pyro-Q, pyroglutamic acid.Mi massAverage massMDCK (Fig. 3 A) 11508.761508.671509.5555–5661 PO4 21584.851584.761585.8175–186 31602.861602.791603.8427–440 41707.751707.831708.9383–396 51811.761811.871812.969–84 61835.901835.921837.1382–396 72130.902129.782130.9533–551Pyro-Q, 1 PO4 82163.982163.932165.2535–554 92240.142240.142241.6364–381 102254.862255.012256.4256–273 112259.102259.012260.3498–516 122479.042479.102480.8341–361HepG2 (Fig. 3 B) 1943.42943.42944.1488–495 21616.841616.811617.8426–439 31735.871735.831736.9382–395 41770.871
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