From the Departments of Anatomy and Cell Biology and
Biology, McGill University, Montreal, Quebec H3A 2B2, Canada,
** Biotechnology Research Institute, National Research Council of
Canada, Montreal, Quebec H4P 2R2, Canada, and § Glaxo
Wellcome Research and Development, Gunnels Wood Road, Stevenage,
Hertfordshire SG1 2NY, United Kingdom
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ABSTRACT |
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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.
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INTRODUCTION |
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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
ER1-associated kinase(s) (1).
This phosphorylation was exclusively on serine residues and by
controlled protease digestion found to be cytosolically oriented (1,
2). 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 (1, 2).
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 1-3 (3-13).
Purification of the ER-associated kinase that phosphorylated calnexin in microsomes led to the identification of CK2 (14). The properties of this kinase were consistent with the conditions that originally revealed calnexin phosphorylation (1, 14). Furthermore, purified CK2 has been found to phosphorylate calnexin on putative CK2 sites found within the cytosolic domain of calnexin (14, 15).
Calnexin is phosphorylated in vivo (16-18). Phosphorylated
calnexin has been shown to associate with the null Hong Kong mutant of
-1-antitrypsin, coinciding with retention of this misfolded glycoprotein within the lumen of the ER (16). 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 (17). 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
(19). 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 (18). Remarkably,
calnexin phosphorylation also increased 3-fold when McCoy cells were
treated with Clostridium difficile cytotoxin B (18), a
protein that glucosylates Rho proteins of the Ras superfamily (20).
Although some progress has been made on the kinases and sites of phosphorylation of calnexin after in vitro phosphorylation of intact microsomes (14, 15) 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.
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EXPERIMENTAL PROCEDURES |
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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 > 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.
Media and Cell Lines-- 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 CO2 and used when they were 80% confluent.
In vivo [32P]O4 Labeling of Cultured Cells and Immunoprecipitation-- Cells were radiolabeled as described previously (16, 17) 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 (3). 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 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). 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 (1). Radioactive bands were visualized by radioautography at room temperature.
Phosphoamino Acid Analysis--
Phosphoamino acid analysis was
performed as described (21). 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 6 N 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.
In Gel Digestion and Mass Spectrometry (DE-MALDI MS and nano-ESI
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. 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 mM
dithiothreitol 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 (22). 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) (22). An aliquot (0.4 µl) was spotted onto a stainless steel target precoated with -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 (23).
Briefly, in gel trypsin-digested calnexin was desalted via a POROS R2
capillary column (22, 23) (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 (24, 25) 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 between
m/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 at
m/z 79 (26, 27). Molecular ions for these peptides were recorded by scanning the first quadrupole between m/z 300 and 1400 with the third quadrupole set to
transmit m/z 79 only (precursors
m/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 of
m/z 49 profiled phosphopeptides by loss of
H3PO4 (molecular mass of 98) from doubly
charged molecular ions (Constant Neutral Loss scans) (28). 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.
Computer Analysis-- Candidate kinases for the cytosolic serine residues of calnexin were predicted based on consensus sequence motifs employing the PROSITE data base of EXPASY2 (29). Predicted peptide m/z values were evaluated employing tools provided by ProteinProspector.3 Calnexin and calmegin sequence alignments were initially generated by BLASTp (30), FASTA (31), and MSA (32) and optimized manually.
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RESULTS |
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Calnexin Is in Vivo Phosphorylated on Serine Residues in HepG2 and
MDCK Cells--
Calnexin was originally identified as a major
substrate of ER-associated kinase(s) by in vitro
phosphorylation of intact microsomes with [-32P]GTP as
phosphate donor (1). 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). 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.
1B, respectively). The phosphorylated residues were not altered with longer (up to 24 h) in vivo radiolabeling
(data not shown). Hence, calnexin was exclusively in vivo
phosphorylated on serine residues in both cell types.
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Analyses of Tryptic Digests of Calnexin by DE-MALDI-ToF MS-- 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. 3A 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+ (2), conditions employed during in gel trypsin digestion.
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Analyses of Tryptic Digests of Calnexin by Nano-ESI MS: Identity and in Vivo Sites of Phosphorylation-- The Q1 positive ion spectrum between m/z 800 and 1150 of HepG2 calnexin tryptic digests displays four ions (P1-P4) tentatively assigned as tryptic fragments of calnexin by comparison with mock in gel trypsin digest (Figs. 4, A and B). By comparison of the observed masses and the cDNA predicted calculated average masses (33), P1 (m/z 817) and P2 (m/z 886.3) correspond to the doubly positively charged states of the tryptic fragments 91ESKLPGDKGLVLMSR105 (calculated average mass, 1631.0 atomic mass units) and 42APVPTGEVYFADSFDR57 (calculated average mass, 1771.9 atomic mass units), respectively, of calnexin. The P3 (m/z 903) and P4 (m/z 910) ions correspond to the triply positively charged states of the tryptic fragments 151TPELNLDQFHDKTPYTIMFGPDK173 (calculated average mass, 2709.1 atomic mass units) and 191TGIYEEKHAKRPDADLKTYFTDK213 (calculated average mass, 2728.0 atomic mass units), respectively, of calnexin. The tentative assignment of the major non-trypsin tryptic peptide ion P2 (m/z 886.3) was confirmed by generation of sequence-specific sequence tags by MS/MS. Prominent peptide fragment ions of 1120.6, 1306.4, 1407.6, and 1504.8 m/z values correspond to y-ion series of singly positively charged ions, y9 (calculated m/z 1120.2), y11 (calculated m/z 1306.4), y12 (calculated m/z 1407.5), y13 (calculated m/z 1504.6) for collision-induced fragmentation of the calnexin tryptic peptide 42APVPTGEVYFADSFDR57 (using ProteinProspector search tools). This identification is unambiguous (search parameters: mass tolerance of 0.5 Da for both parent and fragmented ions using ProteinProspector tools), and the partial sequence ... (Val44)-Pro-Thr-(Gly-Glu)-(Val49) ... (weak assignments shown in parentheses) can be assigned from the MS/MS spectrum, taking into account potential weak signals for y8 (calculated m/z 1021.1), y10 (calculated m/z 1249.3) and y14 (calculated m/z 1603.7) (Fig. 4, inset).
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DISCUSSION |
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A recently uncovered family of resident ER proteins has revealed properties of novel lectin-like molecular chaperones. These recognize N-linked glycoproteins and couple N-linked oligosaccharide modification with productive glycoprotein folding (3-13, 43). The family is composed of calnexin, a type I transmembrane protein of the ER (1, 2); calreticulin, a KDEL-terminated soluble ER-resident protein (44); and calmegin, a testis-specific ER transmembrane protein with sequence conservation at the predicted luminal domain (45).
Major differences among the three proteins are found at their COOH termini. Mammalian calnexins reveal 89 cytosolically oriented residues (1, 2, 33, 42), which were here shown to be phosphorylated at three of the four invariant serine residues. The calmegin deduced protein sequences predict 119 amino acids cytosolically oriented (45, 46) with six conserved (human and mouse) potential serine phosphorylation sites. The calmegin conserved potential serine phosphorylation sites are, as with the observed sites of serine phosphorylation in calnexin, also in the COOH-terminal half of the respective cytosolic domain. Five of the six potential serine phosphorylation sites of calmegin are within motifs similar to those observed for calnexin; three are in CK2 motifs, one is in a PKC motif, and another (mouse sequence only) is in a PDK motif (Fig. 9). Thus, calmegin is predicted to be phosphorylated on equivalent serines to those in calnexin (Fig. 9). The alignment of the cytosolic domains of calnexins and calmegins identifies three major (and three minor) loops (boxed in Fig. 9) that are unique to the calmegins. Furthermore, this alignment reveals that the cytosolic domains of both calnexin and calmegin can be divided into four subdomains: a juxtamembrane basic, lysine-rich subdomain; a central acidic, glutamic acid-rich subdomain; a phosphorylation signaling subdomain; and a putative COOH-terminal ER retrieval subdomain (47) (Fig. 9). Calreticulin is a luminal ER-resident protein that has no cytosolically oriented sequences but rather a COOH-terminal KDEL ER retrieval signal (48).
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For yeast, greater evolutionary divergence has occurred at the cytosolic domain of calnexin as opposed to the intraluminal domain. The 48-amino acid cytosolic domain of the calnexin homologue Cnx1 in the fission yeast, Schizosaccharomyces pombe, is phosphorylated in vivo.5 The S. pombe calnexin gene is essential for viability, but the cytosolic domain is dispensable for that essential feature (49). The Saccharomyces cerevisiae calnexin homologue Cne1p reveals only one potential cytosolic amino acid (Thr482), and this calnexin gene, CNE1, is nonessential for S. cerevisiae viability (50). The riddle of the evolution of a cytosolic domain for S. pombe calnexin coincident with essential function(s) for viability for which the cytosolic domain appears to be dispensable may be ultimately resolved by an analysis of the signaling cascades that phosphorylate calnexin in mammalian species and in S. pombe.5
The cytosolic domains of all known mammalian calnexins display 83% identity with four invariant serines (Fig. 9). As a consequence of this high degree of sequence identity, we set out to identify the sequences constitutively phosphorylated in two cell lines, i.e. human HepG2 and canine MDCK cells. By phosphoamino acid analyses, both calnexins were exclusively in vivo phosphorylated on serine residues.
The first cytosolic serine residue, Ser484 and Ser485 of HepG2 and MDCK calnexins, respectively, is a juxtamembrane serine residue that is invariant among mammalian calnexins (Fig. 9) and corresponds to a potential PKC phosphorylation site. A tryptic peptide containing the transmembrane domain and thus the juxtamembrane serine residue was not detected by MS analyses. Low recovery of this tryptic fragment may be due to poor extractability (51) of such a peptide and/or due to partial proteolysis generating very large hydrophobic poorly extractable fragments probably linked to the protease-resistant Ca2+ luminal core of calnexin (2). The next COOH-terminal serine residue was conserved in HepG2 (Ser490, a potential CK2 phosphorylation site) and MDCK (Ser491, a potential CK2/PDK phosphorylation site) calnexins but not rodent calnexins (Fig. 9). This site was detected only as a nonphosphorylated fragment by MS analysis of the HepG2 tryptic digests (Fig. 3B).
As summarized in Fig. 8, only Ser534, Ser544, and Ser563 (human numbering, invariant in mammalian calnexins; see Fig. 9) were in vivo phosphorylated as detected by two selective nano-ESI MS techniques for detection of phosphorylated peptides. Two of these three invariant serine phosphorylation sites, Ser534 and Ser544, are within well recognized CK2 motifs (34). This coincides with earlier observations that calnexin in microsomes was in vitro phosphorylated by CK2 (14, 15) and that CK2 was purified as an ER membrane-associated kinase (14). The identification of a third site of calnexin phosphorylation (Ser563 in HepG2; Ser564 in MDCK) was not predicted from previous in vitro studies (1, 14, 15). This site, invariant in mammalian calnexins, is within a motif potentially recognized by either PKC (35, 36) or PDK (37, 38).
We have presented evidence for diphosphorylated and triphosphorylated (Figs. 7 and 8) calnexins but no conclusive data for a monophosphorylated form, i.e. with only one of the two CK2 sites or only the S563PR site being phosphorylated, since no singly phosphorylated fragments encompassing the three sites were identified. Nonphosphorylated peptides encompassing the observed two CK2 sites of serine phosphorylation were identified by DE-MALDI-ToF MS analyses: 532QKSDAEEDGGTVSQEEEDRKPK553 of HepG2 calnexin and 535SDAEEDGGTASQEEDDRKPK554 of MDCK calnexin (Figs. 2 and 3, and Table II). The strongest evidence for only one of the two CK2 sites being phosphorylated was observed from MDCK trypsinized calnexin (Fig. 3A and Table II). The peptide ion, m/z 2130.9 (Fig. 3A and Table II) corresponds to the partial tryptic fragment 533pyro-QKSDAEEDGGTASQEEDDR551, containing one phosphate group and cyclization of the NH2-terminal glutamine (calculated average mass, 2130.9) (52). The cyclization of the NH2-terminal glutamine of this tryptic fragment was suggested also by Cala and co-workers (15). For both calnexins, the two phosphorylated CK2 sites (Ser534 and Ser544, HepG2 calnexin) are contained within the same tryptic fragment, and thus by our strategies, these two phosphorylation sites could not be characterized individually. Evidence for a nonphosphorylated state of the most COOH-terminal serine residue was not obtained. This may be a consequence of complete proteolytic digestion of this nonphosphorylated form and subsequent loss of the corresponding tryptic tripeptide, (R)S563PR, during desalting/washing steps prior to MS analyses. However, the S563PR site in the diphosphorylated large partial tryptic fragment (Fig. 7) that contains three potential sites of phosphorylation may correspond to a nonphosphorylated Ser563 site. On this basis, there are six potentially different (three mono- and three diphosphorylated) partially phosphorylated states of calnexin that probably represent a regulatory mechanism for calnexin action.
The phosphorylation results presented in this paper extend the previous
finding by Capps and Zuniga (17) and Le et. al. (16) that a
significant proportion of calnexin was phosphorylated in
vivo. The identification of phosphorylated calnexin in association with a subset of incompletely folded major histocompatibility complex
class I allotypes (17) or of the misfolded null Hong Kong mutant of
-1-antitrypsin (16) is suggestive of a coincident and perhaps
regulatory role with the action of the luminal domain of calnexin in
glycoprotein folding and quality control. Conservation of the three
serine targets of protein kinases as elucidated here predicts that this
conservation and their phosphorylation are under strict control.
Elucidation of the signaling cascades that trigger calnexin
phosphorylation at the PKC/PDK site as well as the CK2 sites may lead
to new insights in the regulation of cargo folding and transport from
the ER. These studies may also lead ultimately to a rationale for the
evolution of three distinct genes in mammals that encode this family of
molecular chaperones.
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ACKNOWLEDGEMENTS |
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We thank Dr. Louise Larose for advice and assistance in phosphoamino acid analysis and Pamela H. Cameron for technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the Medical Research Council of Canada and Glaxo Wellcome (to J. J. M. B. and D. Y. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of the postdoctoral fellowship from Association de Recherche Contre le Cancer.
To whom correspondence should be addressed. Tel.: 514-398-6351;
Fax: 514-398-5047; E-mail: eh14{at}musica.mcgill.ca.
1 The 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.
2 Available on the World Wide Web at http://expasy.hcuge.ch.
3 Available on the World Wide Web at http://prospector.ucsf.edu.
4 M. A. Ward, unpublished observations.
5 H. N. Wong, E. Chevet, A. W. Bell, D. Y. Thomas, and J. J. M. Bergeron, unpublished observations.
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REFERENCES |
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