Phosphorylation of the Oncofetal Variant of the Human Bile Salt-dependent Lipase
2001; Elsevier BV; Volume: 276; Issue: 15 Linguagem: Inglês
10.1074/jbc.m008658200
ISSN1083-351X
AutoresAlain Vérine, Josette Le Petit‐Thévenin, Laurence Panicot‐Dubois, Annick Valette, Dominique Lombardo,
Tópico(s)Mass Spectrometry Techniques and Applications
ResumoIn 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. 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 (1Lindstrom M.B. Sternby B. Bogrström B. Biochim. Biophys. Acta.. 1988; 959: 178-184Google Scholar, 2Lombardo D. Guy O. Biochim. Biophys. Acta.. 1980; 611: 147-155Google Scholar, 3Howles P.N. Carter C.P. Hui D.Y. J. Biol. Chem... 1996; 271: 7196-7202Google Scholar, 4Shamir R. Johnson W.J. Zolfaghari R.L. Lee H.S. Fisher E.A. Biochemistry.. 1995; 34: 6351-6358Google Scholar). This enzyme is found in the pancreatic secretions of all species examined up to now, from fishes to humans (5Gjellesvik D.R. Lombardo D. Walther B.T. Biochim. Biophys. Acta.. 1992; 1124: 123-134Google Scholar, 6Lombardo D. Guy O. Figarella C. Biochim. Biophys. Acta.. 1978; 527: 142-149Google Scholar). 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 theN-glycosylation at Asn187, which occurs in the endoplasmic reticulum (7Sugo T. Mas E. Abouakil N. Endo T. Escribano M.J. Kobata A. Lombardo D. Eur. J. Biochem... 1993; 216: 799-805Google Scholar). The second post-translational modification is the O-glycosylation of each tandemly repeated sequence present in the C-terminal domain of BSDL (8Reue K. Zambaux J. Wong H. Lee G. Leete T.H. Ronk M. Shively J.E. Sternby B. Borgström B. Ameis D. Schotz M.C. J. Lipid Res... 1991; 32: 267-276Google Scholar). During its intracellular traffic from the endoplasmic reticulum to the trans-Golgi network, BSDL is associated with intracellular membranes (9Bruneau N. Lombardo D. J. Biol. Chem... 1995; 270: 13524-13533Google Scholar, 10Bruneau N. Lechêne de la Porte P. Sbarra V. Lombardo D. Eur. J. Biochem... 1995; 133: 209-218Google Scholar), 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 (11Pasqualini E. Caillol N. Valette A. Lloubès R. Vérine A. Lombardo D. Biochem. J... 2000; 345: 121-128Google Scholar). This third post-translational modification remains poorly documented, but it appeared essential to the secretion of the enzyme (12Pasqualini E. Caillol N. Mas E. Bruneau N. Lexa D. Lombardo D. Biochem. J... 1997; 327: 527-535Google Scholar) 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 (12Pasqualini E. Caillol N. Mas E. Bruneau N. Lexa D. Lombardo D. Biochem. J... 1997; 327: 527-535Google Scholar). Sequence comparison of BSDL (5Gjellesvik D.R. Lombardo D. Walther B.T. Biochim. Biophys. Acta.. 1992; 1124: 123-134Google Scholar, 13Han J.H. Stratowa C. Rutter W.J. Biochemistry.. 1987; 26: 1617-1625Google Scholar, 14Kyger E.M. Wiegand R.C. Lange L.G. Biochem. Biophys. Res. Commun... 1989; 164: 1302-1309Google Scholar, 15Colwell N.S. Aleman-Gomez J.A. Kumar B.V. Biochim. Biophys. Acta.. 1993; 1172: 175-180Google Scholar, 16Sbarra V. Bruneau N. Mas E. Hamosh M. Lombardo D. Hamosh P. Biochim. Biophys. Acta.. 1998; 1393: 80-89Google Scholar) 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 (5Gjellesvik D.R. Lombardo D. Walther B.T. Biochim. Biophys. Acta.. 1992; 1124: 123-134Google Scholar) up to 39 in gorilla (17Madeysski K. Lidberg U. Bjursell G. Nilsson J. Gene ( Amst. ).. 1999; 239: 273-282Google Scholar). Furthermore, the fetoacinar pancreatic protein (FAPP) is a phosphorylated oncofetal variant of the human BSDL (18Escribano M.J. Imperial S. J. Biol. Chem... 1989; 267: 21865-21871Google Scholar) that has only six repeated sequences instead of the 16 normally present in human enzyme (8Reue K. Zambaux J. Wong H. Lee G. Leete T.H. Ronk M. Shively J.E. Sternby B. Borgström B. Ameis D. Schotz M.C. J. Lipid Res... 1991; 32: 267-276Google Scholar). 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. 2A. Verine, J. Le Petit-Thevenin, L. Panicot-Dubois, A. Valette, and D. Lombardo, unpublished observations. 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 (19Pasqualini E. Caillol N. Panicot L. Mas E. Lloubès R. Lombardo D. J. Biol. Chem... 1998; 273: 28208-28218Google Scholar). 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 fromCLONTECH (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 (20Abouakil N. Rogalska E. Bonicel J. Lombardo D. Biochim. Biophys. Acta.. 1988; 961: 299-308Google Scholar) and were purified on protein A-Sepharose. These antibodies also recognized FAPP (21Mas E. Abouakil N. Roudani S. Mirallès F. Guy-Crotte O. Figarella C. Escribano M.J. Lombardo D. Biochem. J... 1993; 289: 609-615Google Scholar). 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. Transcripts of FAPP, obtained by reverse transcriptase-PCR (19Pasqualini E. Caillol N. Panicot L. Mas E. Lloubès R. Lombardo D. J. Biol. Chem... 1998; 273: 28208-28218Google Scholar), were digested by HindIII andEcoRI 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. 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 −80 °C until use. Under these conditions, no loss of esterolytic activity was observed for at least 4 weeks. CHO cells transfected with the cDNA of FAPP (19Pasqualini E. Caillol N. Panicot L. Mas E. Lloubès R. Lombardo D. J. Biol. Chem... 1998; 273: 28208-28218Google Scholar) 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 (M r 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 mmEDTA 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. 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 (6m) 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. Esterase activity was determined on p-nitrophenyl hexanoate as already described (22Vérine A. Bruneau N. Valette A. Le Petit-Thevenin J. Pasqualini E. Lombardo D. Biochem. J... 1999; 342: 179-187Google Scholar) 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 (23Goldberg E. J. Biol. Chem... 1972; 247: 2044-2048Google Scholar), and protein content was determined with the bicinchoninic acid test from Pierce using BSA as a standard. SDS-PAGE was performed in 10% polyacrylamide and 0.1% SDS as described by Laemmli (24Laemmli U.K. Nature.. 1970; 227: 680-685Google Scholar), 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 −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). 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 0.1m Tris/HCl buffer (pH 9.5), 100 mm NaCl, and 1 mm MgCl2. The pSecFAPPw transcript, initially used for the transfection of the CHO cells with the cDNA coding for FAPP (19Pasqualini E. Caillol N. Panicot L. Mas E. Lloubès R. Lombardo D. J. Biol. Chem... 1998; 273: 28208-28218Google Scholar), was subcloned in Escherichia colicells 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. (25Ansaldi M. Lepelletier M. Méjean V. Anal. Biochem... 1996; 234: 110-111Google Scholar). 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 GTCGCC GAG GAG GAC TTC TAC-3′ and 5′-CAGCGG 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 fromCLONTECH. 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 withDpnI 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 competentE. 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-β-d-galactopyranoside/5-bromo-4-chloro-3-indolyl-β-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 withNotI 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. 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. 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 (19Pasqualini E. Caillol N. Panicot L. Mas E. Lloubès R. Lombardo D. J. Biol. Chem... 1998; 273: 28208-28218Google Scholar). 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 (19Pasqualini E. Caillol N. Panicot L. Mas E. Lloubès R. Lombardo D. J. Biol. Chem... 1998; 273: 28208-28218Google Scholar) or BSDL cDNA (8Reue K. Zambaux J. Wong H. Lee G. Leete T.H. Ronk M. Shively J.E. Sternby B. Borgström B. Ameis D. Schotz M.C. J. Lipid Res... 1991; 32: 267-276Google Scholar), 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 (11Pasqualini E. Caillol N. Valette A. Lloubès R. Vérine A. Lombardo D. Biochem. J... 2000; 345: 121-128Google Scholar). This motif is present in the BSDL sequence of all species examined up to now except salmon enzyme (16Sbarra V. Bruneau N. Mas E. Hamosh M. Lombardo D. Hamosh P. Biochim. Biophys. Acta.. 1998; 1393: 80-89Google Scholar) and in that of FAPP (19Pasqualini E. Caillol N. Panicot L. Mas E. Lloubès R. Lombardo D. J. Biol. Chem... 1998; 273: 28208-28218Google Scholar). Amino acid numbering is given according to Reue et al. (8Reue K. Zambaux J. Wong H. Lee G. Leete T.H. Ronk M. Shively J.E. Sternby B. Borgström B. Ameis D. Schotz M.C. J. Lipid Res... 1991; 32: 267-276Google Scholar). 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 (19Pasqualini E. Caillol N. Panicot L. Mas E. Lloubès R. Lombardo D. J. Biol. Chem... 1998; 273: 28208-28218Google Scholar), 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. 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. 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. 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). 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 (10Bruneau N. Lechêne de la Porte P. Sbarra V. Lombardo D. Eur. J. Biochem... 1995; 133: 209-218Google Scholar). 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
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