From INSERM Unité 260, Faculté de Médecine, 27 Blvd. Jean Moulin, 13385 Marseille Cedex 5, France
Received for publication, September 21, 2000, and in revised form, January 5, 2001
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ABSTRACT |
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In this paper, we report, for the first time, the
localization of the phosphorylation site of the fetoacinar pancreatic
protein (FAPP), which is an oncofetal variant of the pancreatic bile
salt-dependent lipase. Using Chinese hamster ovary
(CHO) cells transfected with the cDNA encoding FAPP, we
radiolabeled the enzyme with 32P, and then the
protein was purified by affinity chromatography on cholate-immobilized
Sepharose column and submitted to a CNBr hydrolysis. Analysis of
peptides by high pressure liquid chromatography, associated with the
radioactivity profile, revealed that the phosphorylation site is
located at threonine 340. Site-specific mutagenesis experiments, in
which the threonine was replaced by an alanine residue, were used to
invalidate the phosphorylation of FAPP and to study the influence of
the modification on the activity and secretion of the enzyme. These
studies showed that CHO cells, transfected with the mutated cDNA of
FAPP, kept all of their ability to synthesize the protein, but the loss
of the phosphorylation motif prevented the release of the protein in
the extracellular compartment. However, the mutated enzyme, which was
sequestrated in the transfected CHO cells, remains active on bile
salt-dependent lipase substrates.
The bile salt-dependent lipase
(BSDL1; EC 3.1.1.13) is an
enzyme implicated in the duodenal hydrolysis of cholesteryl esters (1-4). This enzyme is found in the pancreatic secretions of all species examined up to now, from fishes to humans (5, 6). BSDL is
synthesized within the endoplasmic reticulum of acinar cells and
follows the secretory pathway of these cells before its release into
the pancreatic juice. To be secreted, the enzyme experiences co- and
post-translational modifications. The first one is the
N-glycosylation at Asn187, which occurs in the
endoplasmic reticulum (7). The second post-translational modification
is the O-glycosylation of each tandemly repeated sequence
present in the C-terminal domain of BSDL (8). During its intracellular
traffic from the endoplasmic reticulum to the trans-Golgi
network, BSDL is associated with intracellular membranes (9, 10), from
which the enzyme is dissociated upon the phosphorylation of a
hydroxylated amino acid residue of the protein, such as threonine or
serine, by the action of a protein kinase casein kinase II (11).
This third post-translational modification remains poorly documented,
but it appeared essential to the secretion of the enzyme (12) and
should occur once the sequential glycosylation of the protein was
achieved. We have further determined that the stoichiometry of the
phosphorylation is about 1.2 ± 0.5 mol of phosphorus/mol of
secreted BSDL (12). Sequence comparison of BSDL (5, 13-16) indicated
that this protein differs from species to species at the level of the
C-terminal domain that encompasses a variable amount of tandemly
repeated identical sequences. The number of these repeated sequences
varies from none in salmon (5) up to 39 in gorilla (17). Furthermore, the fetoacinar pancreatic protein (FAPP) is a phosphorylated oncofetal variant of the human BSDL (18) that has only six repeated sequences instead of the 16 normally present in human enzyme (8). As a
consequence of the variability of the C-terminal domain of the protein,
the phosphorylation site should be located within the N-terminal domain
of BSDL. Sequence analysis of BSDL, using the ExPASy Prosite program of
the Swiss Institute of Bioinformatics, suggests that as many as eight
putative CK II phosphorylation sites are present on BSDL sequence, all
located within the N-terminal domain of the
protein.2
The aim of these studies was to investigate the role of the
phosphorylation step in the BSDL behavior and most particularly (i) to
determine the nature and the location of the amino acid involved in
phosphorylation process, (ii) to analyze the influence of the
phosphorylation step vis-à-vis of the secretion. For this purpose, the phosphorylation site was invalidated by site-directed mutagenesis. Due to the low amount of identical motifs coding for
repeated sequences of the protein, FAPP cDNA sequence was used
instead of that of BSDL. The former cDNA would be easier to
manipulate for site-directed mutagenesis experiments. Consequently, we
used a vector including the cDNA encoding FAPP that leads to a
secreted protein upon transfection in CHO cells (19).
Materials--
Unless otherwise stated, all A grade chemicals
were purchased from Sigma. Culture medium Ham F-12 was from Life
Technologies, Inc. Taq polymerase was purchased from
CLONTECH (Palo Alto, CA) and was a part of the
GC-rich PCR kit. [32P]Orthophosphoric acid was from
PerkinElmer Life Sciences. Polyclonal antibodies
(pAbL64) against BSDL purified from human pancreatic juice
were raised in our laboratory in rabbit (20) and were purified on
protein A-Sepharose. These antibodies also recognized FAPP
(21).
Cell Cultures--
Transfected CHO cells were routinely cultured
in Ham's F-12 medium, supplemented with 10% (by volume) fetal calf
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells, in
100-mm diameter culture dishes, were maintained under 5%
CO2 atmosphere at 37 °C.
Transfection--
Transcripts of FAPP, obtained by reverse
transcriptase-PCR (19), were digested by HindIII and
EcoRI restriction enzymes and ligated into pSecTag
(Invitrogen), an expression vector that carries the V-J2-C region of
the mouse IgK chains driving expressed proteins toward secretion. This
material, referred to as pSecFAPPw ("w" represents "wild"), was
then transfected into CHO-K1 cell line using LipofectAMINE according to
the manufacturer's procedure (Life Technologies). Transfected cells
were first stabilized in Ham's F-12 medium supplemented with zeocin
(500 µg/ml). The different clones were then isolated by end dilution
procedure and maintained under zeocin selection for at least 6 weeks.
Control cells were transfected, according to the same protocol, with
the empty pSecTag vector, and corresponding positive clones
(CHO-control) were selected as indicated previously.
Cell Protein Preparation--
Transfected cells were grown to
about 80% confluence, and then they were washed twice with incomplete
PBS buffer (10 mM sodium phosphate buffer at pH 7.4 with
0.15 M NaCl and without Mg2+ and
Ca2+ ions) and scraped with a rubber policeman. Cells were
resuspended in this buffer and pelleted by low speed centrifugation.
The supernatant was removed, and the sedimented cell pellet was
homogenized in the complete PBS buffer (0.5 ml for cells obtained from
a 100-mm diameter dish culture) by sonication (15 s, 4 watts, 4 °C).
Homogenates were quickly cleared by centrifugation at 14,000 × g for 30 min at 4 °C, and the supernatants were
immediately used for enzymatic assays or frozen and stored at
Metabolic 32P Labeling of FAPP and Purification on
Cholate-immobilized Sepharose Affinity Column--
CHO cells
transfected with the cDNA of FAPP (19) were grown until 80%
confluence in Ham's F-12 medium. Cells were washed twice at room
temperature with incomplete PBS and cultured for 3 h in
phosphorus-free Ham's F-12 medium. After this preincubation was used
to deplete CHO cells of phosphorus, the medium was removed and replaced
by 3 ml of the phosphorus-free fresh Ham's F-12 medium in which was
added 0.33 mCi/ml of sodium [32P]orthophosphate, and
cells were incubated overnight at 37 °C. At the end of the
incubation time, the cell culture medium, containing free radioactivity
and radiolabeled secreted proteins, was withdrawn. Free radioactivity
and salts were removed by ultrafiltration, using Amicon devices
(Mr cut-off 10,000), and dialyzed against water.
Then the concentrated cell culture medium, containing radiolabeled proteins, was lyophilized.
The 32P-radiolabeled FAPP was then isolated from
concentrated cell culture medium by affinity chromatography on a
cholate-immobilized Sepharose column equilibrated in a 25 mM Tris-HCl buffer, pH 9.0, containing 2 mM
EDTA and 1 mM benzamidine. For this purpose, lyophilized material was solubilized in the equilibrating buffer and mixed with the
affinity gel. After incubation overnight at 4 °C on a rotating
shaker, the gel was poured into a Bio-Rad Econopack column and
extensively washed until the absorbance (measured at 280 nm), and the
radioactivity reached the background level. Absorbed 32P
material was then eluted by competition using the equilibrating buffer,
supplemented with sodium cholate (2% w/v). Eluted material was then
extensively dialyzed against cold ammonium hydrogenocarbonate solution
(5 mM, pH 8.0) and finally concentrated by lyophilization. Unlabeled FAPP was also isolated according to the same protocol.
Cyanogen Bromide Digestion and Amino Acid Sequence
Analysis--
The purified radiolabeled material was mixed with 500 µg of pure nonradiolabeled enzyme, used as a vector, and dissolved
into 0.2 ml of 70% formic acid. The reaction was started by adding CNBr (12 mg), and the mixture was incubated, under slow agitation, for
6 h at room temperature and for an additional 12 h at
4 °C. The reaction was stopped by adding 1 ml of distilled water and 0.2 ml of ethanol. The medium was then evaporated to dryness under vacuum.
Protein fragments, dissolved in 0.17 ml of guanidinium HCl (6 M) in 0.1% trifluoroacetic acid, were separated by HPLC
using a C8 reversed-phase column and eluted with a gradient of
acetonitrile from 0 up to 70% in 0.1% trifluoroacetic acid. Peptide
collection was monitored by recording the absorbance at 214 nm (Applied
Biosystems absorbance detector model 785A). The radioactivity of
each fraction was measured by liquid scintillation. The fraction,
containing the radioactive peptide, was once again chromatographed
under identical conditions using the same column and lyophilized, and its amino acid sequence was determined.
Enzyme Activity and Protein Determinations--
Esterase
activity was determined on p-nitrophenyl hexanoate as
already described (22) and defined as the difference between the
activity levels in assays performed without (control) and with added
bile salts (sodium taurocholate, 4 mM).
The lactate dehydrogenase activity was determined as described by
Goldberg (23), and protein content was determined with the
bicinchoninic acid test from Pierce using BSA as a standard.
SDS-PAGE and Immunoblotting--
SDS-PAGE was performed in 10%
polyacrylamide and 0.1% SDS as described by Laemmli (24), using a
Bio-Rad Mini Protean II apparatus. After electrophoretic migration,
proteins were electrotransferred onto nitrocellulose membranes at 4 mA/cm2 for 18 h. The efficiency of the electrotransfer
was checked by staining the nitrocellulose membrane with 2% Ponceau S solution.
Nitrocellulose membranes were air-dried, and transferred proteins were
detected by autoradiography for 24 h at
For immunodetection assays, membranes were blocked for 1 h in
Tris/HCl buffer (5 mM, pH 8.0) containing 150 mM NaCl and 3% bovine serum albumin. The immunodetection
was carried out for 1 h using pAbL64 (1 µg/ml). After
incubation for 1 h in blocking buffer containing 0.05% Tween 20, membranes were rinsed and incubated for another 1 h in a solution
containing alkaline phosphatase-conjugated goat anti-rabbit IgG. After
several washings with PBS supplemented with 0.05% Tween 20, membranes
were developed for 10 min with a mixture of nitro blue tetrazolium and
5-bromo-4-chloro-3-indoyl phosphate (0.5 mM each) in 0.1 M Tris/HCl buffer (pH 9.5), 100 mM NaCl, and 1 mM MgCl2.
Site-directed Mutagenesis--
The pSecFAPPw transcript,
initially used for the transfection of the CHO cells with the cDNA
coding for FAPP (19), was subcloned in Escherichia coli
cells and mutated to prevent the expression of the threonine residue
bearing the phosphorus motif.
For this purpose, a pair of primers, designed to cover the sequence
encoding this threonine, was used according to the method described by
Ansaldi et al. (25). Their sequences were modified to
replace the threonine codon (ACG) with the alanine codon (GCC). The two
primers had the following sequences: 5'-AAC AAG GGC AAC AAG AAA GTC
GCC GAG GAG GAC TTC TAC-3' and 5'-CAG
CGG CTC CTC CTG AAG ATG TTC GAC CAG TCA CTC AAG
3'. Underlined letters indicate the modified bases. The
oligonucleotide primers, each complementary to opposite strands of the
pSecFAPPw vector, were extended using the GC-rich PCR kit from
CLONTECH. The amount of the initial substrate was
maintained below 100 ng. The amplification was performed on a
PerkinElmer Life Sciences 2400 GeneAmp PCR system using a 15-reaction cycle program as follows: denaturation (94 °C, 0.5 min), annealing (52 °C, 0.5 min), and extension (68 °C, 4 min). The reaction was terminated by an incubation at 68 °C for 8 min. A treatment with DpnI endonuclease was used to eliminate the methylated
parental DNA template. After digestion by DpnI, only the
newly synthesized DNA containing the desired mutation remained.
About 5 µl of the digested material was used to transform competent
E. coli cells (Top10 F' strain), which were spread and
incubated at 37 °C overnight on agarose plates containing the
appropriate antibiotic (ampicillin) and the
isopropyl-1-thio- Detection of Glycosylated Motifs on the Mutated FAPP--
Cells
were grown to confluence and washed twice with PBS buffer, and BSDL,
present in clarified lysate (450 µl), was purified on a
cholate-immobilized Sepharose column previously equilibrated at pH 9.0 in 25 mM Tris-HCl, 2 mM EDTA, and 1 mM benzamidine buffer (TBE buffer). After incubation
overnight at 4 °C, Sepharose beads (50 µl by assay) were
sedimented by centrifugation and washed with 1 ml of TBE buffer
supplemented with 0.5% Triton X-100 and 6-fold with TBE buffer alone.
Sepharose beads were sedimented and then added to 30 µl of Laemmli's
sample buffer and boiled for 3 min. After a rapid centrifugation, the
supernatant was submitted to SDS-PAGE. Proteins were transferred onto
nitrocellulose membranes. Each assay was performed in duplicate on
the same nitrocellulose membrane, which was divided into two
parts. Proteins present on the first half of the membrane were revealed
by Western blot using pAbL64 as primary antibody. The
second half was treated for 6 h at 37 °C with 0.1 unit/ml of
neuraminidase (Sigma) in acetate buffer (100 mM, pH 5.2)
and probed with biotin-conjugated peanut agglutinin Arachis
hypogaea (PNA) lectin (10 µl/ml) and antibodies to biotin
linked to alkaline phosphatase and developed as Western blottings.
Localization of the Phosphorylated Site of FAPP
Proteins of CHO cells transfected with the pSecFAPPw vector were
metabolically labeled with [32P]orthophosphoric acid.
After an overnight incubation, the cell-free medium containing about
1.1 ± 0.4 mg (n = 32) of radioactive protein was
recovered. The radiolabeled secreted [32P]FAPP was
purified by affinity chromatography. After exhaustive washings, only
2% of the total loaded radioactive material was eluted with
competitive sodium cholate (~40 µg of 32P-labeled
FAPP). The eluted material was then analyzed, and after SDS-PAGE
followed by electrotransfer on nitrocellulose membrane, only one band
can be detected after Ponceau S development or autoradiography. This
protein is associated with a molecular mass of 78 kDa, which correlates
with that of FAPP (19). After concentration by lyophilization, the
affinity-purified [32P]FAPP was subjected to CNBr
hydrolysis. When the digested peptides were separated by reverse phase
HPLC, the elution profile revealed many peaks (Fig.
1). Major peaks, detected by UV
absorption at 214 nm, were eluted near the end of the gradient under
weak polarity conditions. The radioactivity of each fraction was
quantified, and the radioactivity profile was superimposed to the
peptide elution pattern. It appeared that the radioactivity was mainly recovered under a single peak, associated with the fraction number 47 (Fig. 1, arrow). Several analyses were performed, under the same chromatographic conditions, and all fractions, containing the
radioactive peptide, were pooled and once again chromatographed on the
same HPLC column. This allows to collect enough radioactive material
for further sequencing. One major sequence of 16 amino acids,
PAINKGNKKVXEEDFY, was determined. Comparison of this sequence with the
amino acid sequence deduced from the FAPP cDNA (19) or BSDL
cDNA (8), revealed a high degree of homology with the sequence
PAINKGNKKVTEEDFY, located between Pro330 and
Tyr345 in the N-terminal domain of FAPP and BSDL. The
missing amino acid residue, X, in the sequence of the
radioactive peptide can be identified as Thr340, which
therefore could represent the phosphorylation site. Furthermore, the
sequence around this Thr residue is representative of a consensus motif
that can be phosphorylated by a protein kinase casein kinase II (11).
This motif is present in the BSDL sequence of all species examined up
to now except salmon enzyme (16) and in that of FAPP (19). Amino acid
numbering is given according to Reue et al. (8).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C until use. Under these conditions, no loss of esterolytic
activity was observed for at least 4 weeks.
70 °C (BioMax MR,
Eastman Kodak Co.) and/or by immunodetection using polyclonal antibodies pAbL64 specific to human pancreatic
BSDL/FAPP. Autoradiograms were analyzed by densitometric scanning and
quantified using the NIH Image program (National Institutes of Health,
Bethesda, MD).
-D-galactopyranoside/5-bromo-4-chloro-3-indolyl-
-D- galactopyranoside mixture to perform the blue/white selection. The
white and light blue colonies were picked up and cultured in
Luria-Bertani medium supplemented with 50 µg/ml ampicillin. Cells
were pelleted, and plasmid cDNA was isolated using a miniprep kit
method (QIAprep Spin Miniprep kit; Qiagen). The presence of the desired
PCR product within the plasmid was checked by digestion with
NotI restriction enzyme. Positive plasmids were totally
sequenced to detect any undesired mutation within the FAPP cDNA.
The plasmid, bearing the desired mutation, was then transfected into
CHO-KI cells and selected, as above described, to give the
CHO-FAPPT340A clone.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Separation on HPLC of peptides produced by
CNBr hydrolysis of 32P-labeled BSDL.
FAPP expressed by CHO-FAPPw cells was metabolically
32P-labeled and purified on a cholate-immobilized Sepharose
column. The purified material was treated with CNBr and chromatographed
on an HPLC C8 reversed-phase column. The radioactive peptide, indicated
by the arrow, was isolated and rechromatographed on the same
column, and its amino acid sequence was determined.
Site-directed Mutagenesis
The definitive proof that Thr340 is the
phosphorylation site of FAPP was obtained by site-directed mutagenesis.
In this experiment, a pair of primers was designed to create a mutation
at the threonine 340 codon. The pair of primers was used in a PCR
experiment to amplify the pSecFAPPw vector and to replace the threonine
340 residue by alanine. At the end of the PCR experiment, the
methylated parental vector was digested by DpnI. The
remaining material was used to transform the Top 10 F' E. coli strain to obtain enough material for sequencing and
expression in CHO cells. The sequence of the vector does not differ
from that given by the manufacturer. The sequence of the reverse
transcriptase-PCR fragment inserted into the vector matches that of
FAPP (19), except at the level of the codon encoding Thr340
(not shown). This mutated vector will be referred to as pSecFAPPT340A. CHO cells were then stably transfected with the pSecFAPPw,
pSecFAPPT340A, and empty pSecTag vector. After selection in zeocin, the
transfected cells were cultured until confluence, and the expression of
FAPP was determined by Western blotting performed on cell lysates. As
many as 12 pSecFAPPw and pSecFAPPT340A clones were selected in the
presence of zeocin, and all expressed FAPP, which migrates as a 78-kDa
protein. However, cells transfected with the empty pSecTag did not
express the protein (Fig. 2,
upper panel). A clone representative of each
transfection experiment was selected to give CHO-FAPPw, CHO-FAPPT340A
and CHO-control cell clones, respectively. As also shown on Fig. 2
(lower panel), FAPP expressed either by CHO-FAPPw
or CHO-FAPPT340A is quantitatively retained on, and consequently can be
isolated by, a cholate-immobilized Sepharose column. Further, this
result suggests that mutated, as wild, FAPP is still capable of
interacting with bile salt.
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Threonine 340 Is the Phosphorylation Site of BSDL--
By
using the chemical method, we have previously shown that 1.2 ± 0.5 mol of phosphorus was present per mol of BSDL. If this result is
very interesting, it may appear also somewhat ambiguous and may
indicate the presence of one or perhaps two phosphoryl group(s) on
FAPP. The addition of sodium [32P]orthophosphate in
culture medium has been used to confirm that threonine 340 is the
unique phosphorylatable site on the N-terminal domain of FAPP.
CHO-FAPPw, CHO-FAPPT340A, and CHO-control cells were radiolabeled in
[32P]orthophosphate-supplemented medium. At the end of
the incubation, cell-free medium was withdrawn, dialyzed, and
concentrated, whereas cells were harvested and lysed. Same amounts of
proteins, contained in the cell-free medium and in the cleared cell
lysate, were loaded on the cholate-immobilized Sepharose column. After
washing of the affinity gel, retained FAPP was eluted from beads by
boiling in Laemmli sample buffer and separated on SDS-PAGE,
electrotransferred on nitrocellulose membranes, and analyzed by
autoradiography. As indicated in Fig. 3
(left panel), 32P-phosphorylated FAPP
can be detected as a 78-kDa protein in the cell-free medium of
CHO-FAPPw clone, whereas radioactive FAPP was undetectable in the
cell-free medium of CHO-control and CHO-FAPPT340A clones. The scanning
and the quantitation, by the NIH program, of the area around 78 kDa
showed that the dark intensity, associated with FAPPT340A, represented
less than 0.1% of that of FAPP. This result suggested that FAPPT340A
may not be phosphorylated and, possibly, not secreted. Therefore, we
attempted to determine the phosphorylation state of FAPP and FAPPT340A
in cell lysates. Analyses of FAPP present in cell lysates (Fig. 3,
right panel) indicate that phosphorylated FAPP
can be purified from CHO-FAPPw cell lysate but was not detected in
lysates of CHO-control cells. The radioactivity of the material
isolated from CHO-FAPPT340A cell lysate located around 78 kDa was
lowered by more than 80-90% compared with the corresponding band
detected in the CHO-FAPPw cell lysate. Consequently, FAPP expressed by
CHO-FAPPT340A (arrow) appeared very poorly phosphorylated or
even unphosphorylated.
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Although the N-terminal domain of FAPP displays eight putative sites for phosphorylation by casein kinase II protein kinase, threonine 340 appeared as the unique phosphorylatable site of FAPP and, under our experimental conditions, one may rule out the phosphorylation of any other putative sites.
The Phosphorylation of Threonine 340 Is Essential for the Secretion
of FAPP--
As shown above, phosphorylated FAPP cannot be detected in
cell-free medium of CHO-FAPPT340A cells. This result indicated that FAPP secretion could be dependent upon phosphorylation. Therefore, the
influence of the phosphorylation of the threonine 340 on the secretion
of FAPP was analyzed by immunodetection of the protein in cell-free
medium of CHO-FAPPw and CHO-FAPPT340A cells. For this purpose,
nitrocellulose membranes, used in Fig. 3 to determine the
phosphorylation state of FAPP and FAPPT340A, were submitted to an
immunodetection with pAbL64. As shown on Fig.
4, left panel, no
band was immunodetected in the cell-free medium of CHO control transfected with the empty pSecTag vector. However, a protein, migrating at 78 kDa and reactive with pAbL64, was isolated
by affinity chromatography on the cholate-immobilized Sepharose column from the cell-free medium of the CHO-FAPPw clone, which ascertained that this clone had the capacity to synthesize and secrete FAPP. On the
other hand, mutated FAPP, expressed by the CHO-FAPPT340A clone, cannot
be immunodetected in the culture medium of this clone. This suggests
that mutated FAPP was absent of the cell-free medium of CHO-FAPPT340A
cells and, consequently, cannot be isolated by affinity chromatography
from this medium. A Western blotting performed directly on the
cell-free medium of CHO-FAPP and CHO-FAPPT340A cells indicated that the
protein can be detected in the culture medium of the former cells and
was absent in that of the latter clone (see Fig. 2).
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Cell homogenates showed a pattern somewhat different (Fig. 4, right panel); as expected, no band corresponding to FAPP was immunodetected in CHO-control cells. However, following affinity chromatography, FAPP can be immunodetected as a doublet of protein migrating around 78 kDa in CHO-FAPPw and CHO-FAPPT340A clones. This doublet probably corresponds to different states of maturation of the glycosylation of this protein (10). Another band was immunodetected in the lower molecular mass range and might correspond to a degradation product. When the amount of FAPP expressed by each clone was quantitated by densitometric scanning, it appeared that the amount of FAPP expressed by the CHO-FAPPT340A clone was decreased by some 40% when compared with that expressed by the CHO-FAPPw clone. All of these results indicate that FAPPT340A, which cannot be phosphorylated, might not be secreted by CHO-FAPPT340A cells.
FAPP Expressed by CHO-FAPPT340A Remains Active-- The levels of esterase activity were determined in the intra- and extracellular media obtained from CHO-FAPPw and CHO-FAPPT340A cells (Table I). In cell culture medium of CHO-FAPPw, once corrected for the esterase activity present in culture medium of CHO control cells, the esterase activity was about 10-fold the level recorded in the cell-free medium of CHO-FAPPT340A cells. The amount of BSDL activity detected in cell-free medium of CHO-FAPPT340A cells represents some 3.5 ± 1.3% of the total intracellular and extracellular activities. This value is close to that of lactate dehydrogenase activity present in the cell-free medium of CHO-FAPPT340A cells that accounts for 4.4 ± 2.1% of the total lactate dehydrogenase activity. Consequently, BSDL activity detected in cell culture medium of the CHO-FAPPT340A clone might be due to some cell lysis. Furthermore, the amount of BSDL activity recorded in cell culture medium of CHO-FAPPw clone is close to that already found (19). Thus, it is clear that the invalidation of the phosphorylation site of FAPP inhibited the release of T340A mutated protein in the cell-free medium.
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In the cleared cell homogenate of CHO cells either transfected with pSecFAPPw or with pSecFAPPT340A vectors, a significant level of bile salt-dependent lipase activity can be detected (Table I). However, a lower activity, compared with that of CHO-FAPPw clone, was detected in the homogenate of the CHO-FAPPT340A clone. This lower activity could be due to the expression of a partially inactive enzyme, consecutive to the mutation of the phosphorylation site. However, the decrease in activity of some 40% observed, after correction with the control clone activity, paralleled the decrease in expression level of the mutated protein by the CHO-FAPPT340A clone (see Fig. 4). From this observation, it can be concluded that FAPP and mutated FAPP displayed the same specific activity. Consequently, the activity of FAPP did not depend upon phosphorylation of the threonine 340 residue. Furthermore, the mutation T340A did not alter the activity of the enzyme and its modulation by bile salts.
FAPPT340A Is O-Glycosylated--
We have shown that, during its
maturation, BSDL was successively N- and
O-glycosylated. We have further hypothesized that O-glycosylation of the protein masks PEST sequences (Pro-,
Glu-, Ser-, and Thr-rich sequences) that are a signal for rapid
degradation (26), and, in fine, this glycosylation,
regulates the secretion of the enzyme (27). The protein that is not
accurately glycosylated should be degraded. Nevertheless, in this
maturation process, the timing of phosphorylation step with regard to
the glycosylation remained uncertain. To clarify this point, we
examined the glycosylation state of mutated FAPP. For this purpose,
the mutated protein produced by the CHO-FAPPT340A clone was isolated
by a cholate-immobilized Sepharose column. The retained material was
then submitted to SDS-PAGE, transferred onto nitrocellulose membrane,
and probed with pAbL64 and with biotin-labeled PNA lectin.
As shown in Fig. 5, pAbL64 and
PNA lectin detected material migrating with the same molecular mass
suggesting that, albeit not phosphorylated, mutated FAPP is
O-glycosylated.
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This result may be interpreted in two ways; first, phosphorylation
occurs after the O-glycosylation of the protein, and second, O-glycosylation may proceed independently of the phosphorylation.
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DISCUSSION |
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Recent studies (9, 10) on bile salt-dependent lipase
have demonstrated that, during its secretion route in the pancreatic cell, the enzyme is associated with intracellular membranes. This association allowed a correct and complete glycosylation of the enzyme
or participated in the inhibition of the enzyme activity that could be
harmful to the cell. Thereafter, it has been shown that the enzyme is
phosphorylated by a protein kinase cascade. For instance, it is known
that this protein kinase system was insensitive to protein kinase A, C,
and G inhibitors. The phosphorylation process is favored by okadaic
acid, which is known to inhibit protein phosphatases 1 and 2A.
Nevertheless, the phosphorylation of BSDL was inhibited by the
5,6-dichloro-1--D-ribofuranosylbenzimidazole, a specific
inhibitor of protein casein kinase II. This impairment of BSDL
phosphorylation is associated with a significant decrease of enzyme
secretion (11). We have further shown that the phosphorylation process
appeared as an essential step for the dissociation of the enzyme from
intracellular membranes (12). The importance of this step is
ascertained by the fact that there is only 1.2 ± 0.5 mol of
phosphorus/mol of enzyme. The nature of the amino acid bearing the
phosphorylation motif remained unknown and might be a threonine or
serine residue (11). On BSDL, and on its oncofetal variant FAPP, eight
putative casein kinase II phosphorylation sites were located at the
N-terminal domain of the protein. Despite this important number of
potential sites, it appeared that only one site could be phosphorylated
and sufficient for the secretion of the enzyme (12). In the present
study, we show that the threonine residue, at position 340, is the
phosphorylation site of BSDL. The replacement of this threonine residue
by an amino acid of similar size did not prevent the expression of
enzyme, albeit that it cannot be phosphorylated. It appeared
that invalidation of the phosphorylation site at Thr340
preserved the esterase activity of BSDL but abolished the secretion of
the enzyme. All of these data confirm our previous studies that have
proposed that the release of BSDL from intracellular membranes was
consecutive to its phosphorylation (11), an event that is essential to
the enzyme secretion. A direct link between phosphorylation and enzyme
secretion is established in this study. It appeared now that, before
its secretion, the enzyme must be successively N- and
O-glycosylated and phosphorylated. The question that now
arises is how these different processes are organized in time. This
point may be resolved by the study of O-glycosylation of the
mutated FAPP. Our results, showing the presence of
O-glycosylated motifs on mutated FAPP, and the demonstration
that the phosphorylation occurs in a genistein-sensitive compartment,
which could be the trans-Golgi network (11), and that this
modification also releases the enzyme from intracellular membranes (11,
12) strongly suggest that the phosphorylation step takes place after
glycosylation processes. Consequently, phosphorylation could be the
ultimate step driving the enzyme toward the secretion pathway. One
possibility is that only secretion-competent BSDL molecules are
phosphorylated and targeted toward secretion, whereas others could be degraded.
It has been shown that the inhibition of the phosphorylation of BSDL by
genistein induces an accumulation of enzyme in a cell compartment where
it was colocated with the 58-kDa Golgi protein (11). Confocal
microscopy suggests that the cellular retention compartment may be the
endoplasmic reticulum (ER) of human pancreatic tumoral cells (30).
However, FAPP expressed by these latter cells carries out the glycanic
oncofetal J28 epitope. The building of this epitope requires
fucosyltransferase activities located in the trans-Golgi
compartment (31). Furthermore, FAPP is phosphorylated.2 In
this study, we suggested that the phosphorylation of FAPP (and probably
that of BSDL) occurred after O-glycosylation, probably in
the trans-Golgi network, where the protein should accumulate upon phosphorylation deficiency. This apparent discrepancy may be
explained by a retrograde transport pathway of the nonphosphorylated BSDL from the trans-Golgi network back to the ER. We have,
recently, shown that BSDL secretion is depending upon Rab6 cycling
(28). The fact that Rab6 retrieves secretory cargo vesicles from the trans-Golgi back to the ER (29) supports the possible
retrograde transport of nonphosphorylated BSDL to ER. This route may be
used to transport nonphosphorylated BSDL molecules back to the ER, where the enzyme could be degraded by the proteasome machinery (32).
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ACKNOWLEDGEMENTS |
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We thank M. Bain for expert assistance and fruitful discussions and Dr. E. Pasqualini for the release of the pSecFAPP construct.
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FOOTNOTES |
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* This study was supported in part by Association pour la Recherche sur la Cancer (ARC, Villejuif, France) Grant 9912 (to A. V.).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.
To whom correspondence should be addressed. Tel.: 33 491 324 400;
Fax: 33 491 830 187; E-mail: alain.verine@medecine.univ-mrs.fr.
Published, JBC Papers in Press, January 8, 2001, DOI 10.1074/jbc.M008658200
2 A. Verine, J. Le Petit-Thevenin, L. Panicot-Dubois, A. Valette, and D. Lombardo, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: BSDL, bile salt-dependent lipase; FAPP, fetoacinar pancreatic protein; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; ER, endoplasmic reticulum peanut agglutinin Arachis hypogaea.
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