From the Departments of Chemistry and Biochemistry,
University of Washington, Seattle, Washington 98195, the
§ Department of Pharmacology, University of Oxford,
Mansfield Road, Oxford OX1 3QT, United Kingdom, the
Laboratory
of Thrombosis, Centro de Biofisica y Bioquimica, IVIC, Caracas,
Venezuela, and ¶ Lilly Research Laboratories,
Indianapolis, Indiana 46285
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ABSTRACT |
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The present study identifies the phosphorylation
sites of the 85-kDa cytosolic phospholipase A2
(cPLA2) in human platelets and HeLa cells. Tryptic digests
of 32P-phosphorylated and -immunoprecipitated
cPLA2 were analyzed by microbore high performance liquid
chromatography and two-dimensional phosphopeptide mapping against
synthetic phosphopeptide standards. Thrombin stimulated significant
phosphorylation of platelet cPLA2 at two sites, Ser-505 and
Ser-727. Exclusive phosphorylation on these two sites was also seen in
collagen-stimulated platelets and HeLa cells stimulated with
interferon- or arsenite; no tyrosine phosphorylation was detected.
The inhibitor of the 38-kDa stress-activated protein kinase
(p38mapk), SB 203580, reduced phosphorylation of both Ser-505
and Ser-727 by 50 and 60%, respectively, in thrombin-stimulated
platelets. An additional p38mapk inhibitor SB 202190 also
partially (60%) inhibited the phosphorylation of cPLA2 in
arsenite-stimulated HeLa cells. These studies extend the previous work
on the identification of multiple phosphorylation sites on
cPLA2 expressed in a baculovirus/insect cell system to cPLA2 in mammalian cells stimulated with physiological
agonists. They also underscore the necessity of high resolution
phosphopeptide mapping combined with microbore high performance liquid
chromatography for quantification of phosphorylation levels, which has
lead to the conclusion that Ser-505 and Ser-727 are common
phosphorylation sites on cPLA2 in different
mammalian cells stimulated with multiple agonists.
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INTRODUCTION |
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Cytosolic phospholipase A2 (cPLA2)1 catalyzes the cleavage of arachidonic acid from the sn-2 position of phospholipids (1, 2). The 85-kDa enzyme is present in many mammalian cells (3), and strong evidence is accumulating for the role of cPLA2 in the generation of tissue mediators that are metabolites of arachidonic acid, such as prostaglandins, leukotrienes, and thromboxanes. In contrast to the small molecular weight phospholipase A2s that are secreted and are active on the outside of cells, cPLA2 is regulated by intracellular signals that are propagated from surface receptors. One important regulatory mechanism appears to be a rise in the intracellular Ca2+ concentration which causes translocation of cPLA2 from the cytosol to internal membranes (4-7) where it binds through a Ca2+-dependent lipid-binding domain (8). A second, well established mechanism of the regulation of cPLA2 activity is by phosphorylation on Ser-505 through a mitogen-activated protein kinase (MAPK) (9) which modestly increases the intrinsic activity of the lipase measured in vitro (3, 10, 11). Phosphorylation of cPLA2 together with release of arachidonic acid has been observed in a variety of cells (11-16).
A thorough characterization of the phosphorylation sites of human cPLA2 heterologously expressed in Spodoptera frugiperda (Sf9) cells by high performance liquid chromatography (HPLC), mass spectrometry, and protein sequencing has revealed four sites of phosphorylation: Ser-437, Ser-454, Ser-505, and Ser-727 (17). The kinases phosphorylating the three novel serine residues in Sf9 cells are not known. Interestingly, the protein phosphatase inhibitor okadaic acid causes a marked increase in arachidonic acid release from these Sf9 cells and a selective increase in the phosphorylation of cPLA2 at Ser-727 (17). Moreover, phosphorylation of Ser-727 decreased the electrophoretic mobility of cPLA2 on an SDS-polyacrylamide gel, a characteristic that has been frequently used in numerous previous studies of cPLA2 phosphorylation in mammalian cells as a "selective" method to investigate Ser-505 phosphorylation. Thus, detailed phosphopeptide mapping and phosphorylation quantification studies are needed to obtain reliable information about the pattern of cPLA2 phosphorylation in agonist-stimulated cells.
We have previously studied the regulation of cPLA2 in human platelets stimulated by collagen and thrombin, two key physiological platelet agonists. Studies using the p42/p44mapk kinase inhibitor PD 98059 (18, 19) clearly showed that these kinases do not mediate the phosphorylation of cPLA2 stimulated by thrombin or collagen, and that they are not involved in the process of arachidonic acid release from platelets (20). In addition, protein kinase C does not seem to directly regulate platelet cPLA2 (21). In contrast, inhibition of the 38-kDa stress-activated protein kinase (p38mapk) by the anti-inflammatory drug 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole (SB 203580) (22, 23) reduced both thrombin-stimulated incorporation of 32P into cPLA2 and thrombin-stimulated increase in cPLA2 activity (measured in platelet lysates) by 50% (24). Under these conditions of partial inhibition by SB 203580 in the presence of thrombin, the cPLA2 SDS-polyacrylamide gel electrophoresis (PAGE) gel shift, which is the commonly used assay of cPLA2 phosphorylation, was completely blocked by treatment with the kinase inhibitor (24). In contrast, both 32P incorporation into cPLA2 and in vitro activity were nearly completely inhibited by SB 203580 when platelets were stimulated with collagen (25). Similar results were also obtained by the use of agonists that only partially stimulate platelet responses (26, 27). These results and the observation of multiple phosphorylation sites on cPLA2 expressed in Sf9 cells raise the possibility that platelet cPLA2 is phosphorylated on one or more residues other than Ser-505, and they also leave doubts about the usefulness of the indirect gel shift assay for assessing the level and site(s) of cPLA2 phosphorylation. Thus, high resolution phosphopeptide mapping studies are needed, and these are described in the present study.
Recent studies suggest that cPLA2 is phosphorylated on
tyrosine in HeLa cells stimulated with interferon- (28).
cPLA2 could be co-immunoprecipitated with the protein
tyrosine kinase Jak1, and transfection studies suggest that Jak1 is
necessary for cPLA2 activation in this system. In light of
studies in other cell types showing phosphorylation of
cPLA2 only on serine, the possibility of cPLA2
tyrosine phosphorylation was investigated in the present study using
phosphopeptide mapping.
Analysis of cPLA2 phosphorylation described here was carried out in part with microbore HPLC (mbHPLC) for the accurate quantification of the levels of site-specific cPLA2 phosphorylation when only small amounts of protein obtained from non-transfected mammalian cells are available. It is shown that mbHPLC gives reliable quantification data; two-dimensional phosphopeptide mapping on cellulose plates does not because of sample losses.
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MATERIALS AND METHODS |
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Reagents and Antibodies--
Polyclonal anti-cPLA2
antiserum was obtained as described previously (11, 17). Antibody
against MAPK-activated protein kinase 2 (MAPKAP-K2) was purchased from
Upstate Biotechnology Inc. (TCS Biologicals Ltd., Bucks, UK), and
substrate peptide was synthesized by Alta Bioscience Ltd. (Birmingham,
UK). SB 203580 was kindly provided by Dr. J. C. Lee (SmithKline
Beecham, King of Prussia, PA), and SB 202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole) was
purchased from Calbiochem. 32Pi (dissolved in
H2O, 8,500-9,120 Ci/mmol) and [-32P]ATP
(3,000 Ci/mmol) were obtained from NEN Life Science Products Inc.
Sequencing grade, modified trypsin came from Promega and activated
recombinant p42mapk from Calbiochem. mbHPLC standard peptides
S437-PP (cPLA2 amino acids 428-445, Ser-437
phosphorylated), S454-PP (446-467, Ser-454 phosphorylated), and
S727-PP (721-736, Ser-727 phosphorylated) were synthesized by Multiple
Peptide Systems (San Diego, CA). Peptides were purified by reverse
phase HPLC, and their structures were confirmed by electrospray mass
spectrometry. Prostacyclin was kindly donated by Wellcome Laboratories
(Beckenham, Kent, UK). Bovine thrombin, staurosporine, genistein,
okadaic acid, aprotinin, leupeptin, and pepstatin A were purchased from
Sigma, and microcystin-LR came from Calbiochem. Collagen was obtained from Nycomed Arzneimittel (Munich, Germany). Grace's supplemented medium was obtained from Life Technologies, Inc. Phosphate-free Grace's medium with glutamine was obtained from Speciality Media Inc.
(catalog GR-005-B) and supplemented with yeastolate (catalog IM-123-D)
and lactalbumin hydrolysate (catalog IM-113-D) from the same
company.
Phosphorylation Studies on cPLA2 Derived from Sf9 Cells-- cPLA2 expressed in Sf9 cells was radiolabeled with 32Pi essentially as described (17) except that the following amounts of cells and 32Pi were used. To each of two T-75 flasks was added 10 ml of Sf9 cells (0.9 × 106/ml) in Grace's supplemented medium containing 10% fetal bovine serum and penicillin G/streptomycin/fungizone (100-fold dilution of catalog number 15240-062 from Life Technologies, Inc.) at 27 °C for 1 h. Adherent cells were infected with the recombinant baculovirus expressing human cPLA2 (29) at a multiplicity of infection of 18. Twenty-four hours after infection, cells in each flask were rinsed twice in Grace's supplemented phosphate-free medium containing 10% fetal bovine serum and penicillin G/streptomycin/fungizone and were labeled with 10 mCi of 32Pi at 27 °C. After 48 h, cells were rinsed twice in phosphate-free medium, and then stimulated with 1 µM okadaic acid for 3 h. After two rinses, cells from each flask were lysed on ice in 1 ml of lysis buffer (50 mM HEPES, pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 100 µM leupeptin, 200 µM Na3VO4, 10 mM Na4P2O7, 3 µM p-nitrophenyl phosphate) and insoluble material was removed by microcentrifugation. cPLA2 was immunoprecipitated using polyclonal anti-cPLA2 antiserum (R11783 antiserum, 1:7.5 dilution) as described (17). The combined supernatants from each immunoprecipitate were loaded into a 55-mm wide preparative well (or two 28-mm wide wells) of a 1 mm-thick 10% SDS-PAGE mini-gel (Bio-Rad Mini-PROTEAN II). Gels were run at 130 V until the bromphenol blue dye front just reached the bottom, and the bottom 2 cm of the gels were discarded for safety reasons. Gels were dried and phosphorylation was visualized by autoradiography.
Preparation of 32Pi Radiolabeled Platelets-- Blood (100 ml) was drawn on the day of experiment from healthy volunteers by venepuncture using acidic citrate dextrose solution as anticoagulant (21). Platelet-rich plasma was separated from red blood cells by centrifugation at 200 × g for 20 min, and platelets were isolated by centrifugation at 1,000 × g for 10 min in the presence of 0.1 µg/ml prostacyclin at 30 °C. The platelet pellet was gently resuspended in 1 ml of phosphate-free platelet buffer (25 mM HEPES, pH 7.3, 145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose), and indomethacin (10 µM) was added to block cyclooxygenase. Platelets were radiolabeled with 25 mCi of 32Pi at 30 °C for 1.5-2 h. The platelet suspension was washed with platelet buffer containing acidic citrate dextrose solution and prostacyclin and centrifuged at 1,000 × g for 10 min. The pellet was resuspended in platelet buffer at an approximate concentration of 1 × 109 platelets/ml. Indomethacin (10 µM) was added, and platelets were left at 30 °C for 30 min prior to experimentation. EGTA (1 mM) was added just before stimulation.
Stimulation of Platelets and Immunoprecipitation of cPLA2-- The platelet suspension was distributed into 500-µl aliquots and prewarmed to 37 °C. When required, platelets were incubated in the presence of Me2SO (1%) or SB 203580 (20 µM) for 15 min. Platelets were gently shaken on a moving platform during stimulation with thrombin or stirred in an aggregometer at 1,200 rpm during stimulation with collagen. After addition of 500 µl of ice-cold lysis buffer (final concentrations: 1% Triton X-100, 0.5% SDS, 0.75% deoxycholate, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 100 µg/ml aprotinin, 10 µM pepstatin A, 1 µM microcystin-LR, 5 µM staurosporine, 5 µM genistein, 10 mM Na4P2O7, 50 mM sodium fluoride, 200 µM Na3VO4), insoluble material was removed by microcentrifugation, and lysates were pre-cleared using 20 µl of protein A-Sepharose CL-4B (1:2 in H2O, v/v). cPLA2 was immunoprecipitated at 4 °C overnight using 2 µl of polyclonal anti-cPLA2 antiserum (11) and 20 µl of protein A-Sepharose CL-4B slurry. Immunoprecipitates were washed as described previously (21), extracted with Laemmli sample buffer, and proteins were resolved by 10% SDS-PAGE. For the preparation of cPLA2 for mbHPLC analysis, the extracts of 8 immunoprecipitates, derived from 8 aliquots of platelet lysate, were pooled in a 55-mm wide preparative well. To control for the degree of phosphorylation in each experiment, single immunoprecipitates from stimulated and non-stimulated platelets were put onto a separate gel.
Phosphorylation of cPLA2 by p42mapk in
Vitro--
Wild-type human cPLA2 was expressed in
Sf9 cells and purified (29). His-tagged S505A cPLA2
mutant was expressed in Sf9 cells and purified using standard
Ni(II) immobilized-ion affinity chromatography procedures (30). Both
proteins were dialyzed against 25 mM HEPES, pH 7.4. Ten
micrograms of dialyzed cPLA2 was incubated with 4 units of
activated p42mapk (1 unit = 1 pmol of phosphorylation of
myelin basic protein/min) at 30 °C in kinase buffer (25 mM HEPES, pH 7.4, 1 mM dithiothreitol, 10 mM MgCl2, 1 mM EGTA, 100 µM ATP, 100 µM
Na3VO4, 200 µCi/ml [-32P]ATP) in a total volume of 25 µl. The kinase
reaction was stopped after 30 min by the addition of 4 × Laemmli
sample buffer. Samples were boiled, subjected to 10% SDS-PAGE, and
gels were dried.
Preparation of Tryptic Digests of cPLA2-- The exact position of cPLA2 on dried gels was located by autoradiography. 32P of cPLA2 in dried SDS-PAGE gels was quantified either with a PhosphorImager or by densitometry analysis of autoradiographs obtained with different exposure times. cPLA2 was eluted from the gel, precipiated with trichloroacetic acid, washed with acetone, and digested with trypsin as described (17). Yields were constantly monitored by Cerenkov counting. Fifty-two to 60% of the counts/min of platelet cPLA2 (67-75% of cPLA2 from in vitro kinase experiments) was eluted from the gel slices (two elutions), and >95% of the eluted cPLA2 was recovered after precipitation with trichloroacetic acid and washing with acetone.
mbHPLC-- Each 200-µl frozen tryptic digest was thawed and analyzed by mbHPLC. To the digest was added a mixture of synthetic peptide standards (S437-PP, S454-PP, and S727-PPox), and the entire mixture was injected onto the mbHPLC system. Sufficient amounts of synthetic phosphopeptides were added to give an approximate full-scale detector response using the mbHPLC system described below (predetermined by analyzing the standards alone). mbHPLC was carried out with a Titanium Dynamax SD-200 HPLC system (Rainin) with PEEK tubing. The eluant from the pumps was connected to a low dead volume PEEK splitter (VICI Instruments MY1CPK). One outlet was connected to the inlet of a titanium Rheodyne injector (1-ml PEEK sample loop), and the other outlet to a standard analytical HPLC column (218TP52, Vydac Inc., virtually any analytical column can be used since its purpose is to provide back pressure to the splitter). The outlet of the injector was connected to the mbHPLC column (Vydac 901-3151-00, 0.5 × 150 mm, Michrom BioResources, Inc.). The outlet of the mbHPLC column was connected to the UV detector (Dionex detector equipped with a 2-µl PEEK flow cell). The outlet of the flow cell was connected to a 12-inch length of narrow-bore PEEK tubing (0.005 inch inner diameter) to provide sufficient back pressure to prevent outgassing in the flow cell. The flow rate through the mbHPLC column was adjusted to give 30 µl/min (total system flow rate was about 0.2 ml/min). The column was developed with 100% solvent A (0.06% CF3COOH in Milli-Q H2O (Millipore)) for 15 min, to 30% solvent B (0.06% CF3COOH in CH3CN (HPLC grade, Baker)) over 60 min, to 60% solvent B over 30 min, to 100% solvent B over 10 min, and then for 45 min at 100% solvent B. Eluting peptides were detected by monitoring the absorbance at 210 nm with the detector range set at 0.1-0.2 absorbance units full scale. Fractions were collected every 15 min during the first hour and then every 2 min into silicanized polypropylene tubes (VWR number 20172-935). Radioactivity was monitored by Cerenkov counting. Appropriate radioactive fractions were pooled for oxidation and/or two-dimensional phosphopeptide mapping.
The yield of eluted phosphopeptides from the mbHPLC column was determined by summing the counts/min from all mbHPLC fractions from 0 to 160 min and dividing this number by the counts/min injected onto the column. This latter number was obtained by Cerenkov counting of the tryptic digest tube before and after its contents were injected onto the mbHPLC. Typically, >90% of the counts/min in the digest was injected onto mbHPLC. The yield of eluted phosphopeptides in platelet experiments where less than 3,000 cpm were applied onto the column was 82-98%.Phosphopeptide Maps-- Pooled mbHPLC fractions or tryptic digests were dried in a Speed-Vac. Phosphopeptides were dissolved in 10 µl of glacial CH3COOH and spotted onto 20 × 20-cm cellulose thin-layer chromatography (TLC) plates (Kodak number 13255), which were developed by electrophoresis (1,400 V for 20 min) and ascending chromatography as described (17). 32P-Phosphorylated peptides were located with a PhosphorImager or by autoradiography.
Oxidation of Peptides with Performic Acid-- mbHPLC fractions were dried in a Speed-Vac at low temperature. Oxidation with performic acid was as described (31). Peptides were dissolved in 10 µl of glacial CH3COOH for two-dimensional maps or in 50 µl of H2O for mbHPLC, and solubility was checked by Cerenkov counting.
Measurement of MAPKAP-K2 Activity--
MAPKAP-K2 was
immunoprecipitated from platelet lysates (5 × 108
platelets/lysate) for 2.5 h at 4 °C using 1 µl of antiserum
and 20 µl of protein A-Sepharose CL-4B slurry. Immunoprecipitates were collected by microcentrifugation and washed three times in 20 mM Tris, pH 7.6, containing 137 mM NaCl and
0.1% Tween 20. For the in vitro kinase assay,
immunoprecipitates were incubated with 40 µl of 50 mM
Tris, pH 7.0, 100 µM EGTA, 10 mM
MgCl2, 100 µM ATP, 50 µCi/ml
[-32P]ATP, and 30 µM substrate peptide
KKLNRTLSVA (32) at 37 °C. After 10 min, samples were briefly
microcentrifuged. The kinase reaction was stopped by pipetting 30 µl
of supernatant onto P-81 phosphocellulose paper (Whatman) and washing
these in 75 mM phosphoric acid. Radioactivity of the dried
filter papers was determined by liquid scintillation counting.
Studies with HeLa Cells--
HeLa S3 cells were obtained from
ATCC and cultured in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.; 11965-092) containing 10% fetal bovine serum in a
humidified atmosphere of 5% CO2 at 37 °C. For
radiolabeling, cells were grown in 25-cm2 flasks (T25) to
70% confluence. Cells were washed three times with
phosphate-free Dulbecco's modified Eagle's medium (Life
Technologies, Inc.; 11971-025) containing 0.5% bovine serum
albumin and no fetal bovine serum and then labeled in 1 ml of the same
medium containing 1 or 5 mCi of carrier-free
32Pi. After a 4-h labeling period, cells were
either left untreated or stimulated for 20 min with 1,000 units/ml
interferon- (Accurate Chemical & Scientific Inc.) or for 30 min with
0.5 mM sodium arsenite. Stimulations were stopped by
placing the flasks on ice, and cells were washed twice with ice-cold
medium. Cells were lysed with 0.5 ml of cold lysis buffer, and the
cleared supernatant was treated with 1 µl of R11683
anti-cPLA2 antiserum to immunoprecipitate cPLA2
as described (17). Immunoprecipitates were submitted to SDS-PAGE, and
gel bands were processed for trypsin as described above for platelet
studies. Half of each digest was submitted to mbHPLC, one-third to
two-dimensional phosphopeptide mapping, both as described above for
platelets, and one-sixth to phosphoamino acid analysis as described
(17). In these mbHPLC studies a conventional analytical flow cell was
used in the detector (Rainin Dynamax with a 7200-075 flow cell). The
delay between UV detection and emergence of counts/min from the tubing
outlet was measured by co-injecting bovine serum albumin and
32Pi and monitoring UV in real time and
counts/min in 2-min fractions.
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RESULTS |
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Analysis of the Tryptic Digest of cPLA2 Produced in Sf9 Cells-- The previous characterization of phosphorylation sites of cPLA2 expressed in Sf9 cells has been performed on a C18 reverse phase HPLC column with samples that contained approximately 100,000 cpm (17). To be able to analyze the 32P-phosphorylation of cPLA2 derived from native cells, where the amount of cPLA2 comprises only a fraction of the cPLA2 expressed in Sf9 cells, we set up a C18 mbHPLC column. As internal standards, the three phosphopeptides S437-PP, S454-PP, and S727-PP corresponding to possible phosphorylated tryptic digest fragments of cPLA2 (17) were synthesized. The peptide bearing the MAPK phosphorylation site Ser-505 (residues 489-533) was too long to be readily chemically synthesized, and thus it was generated by trypsin digestion of cPLA2 that had been phosphorylated in vitro with p42mapk (see below). Typical retention times of the standard peptides from the mbHPLC column were 70 min for S437-PP, 84 min for S727-PP, and 84 and 85 min for S454-PP, which was resolved as a doublet (due to the presence of an impurity). Since S727-PP was previously found to be oxidized on a cysteine residue during the process of its isolation (17), it was oxidized (S727-PPox) with performic acid which resulted in a retention time of 79-80 min and thus well separated from S454-PP. The retention times of these standard peptides were highly reproducible between runs (variation < 1 min), and they were co-injected with trypsin digests in every run. The standard peptides eluted from the mbHPLC column in the same order as seen using a Vydac 218TP52 C18 column (S437-PP 63 min, S727-PPox 73 min, S454-PP 77 min; not shown) but the retention times were delayed by 7 min. We therefore expected the Ser-505 containing peptide to elute well after 105 min, the retention time on the 218TP52 column (17).
To evaluate the mbHPLC method and to generate authentic cPLA2 tryptic peptides, studies were first carried out with cPLA2 from Sf9 cells, and the results compared with the earlier study (17). Tryptic digests of 32P-phosphorylated cPLA2 from okadaic acid-stimulated Sf9 cells were prepared and subjected to mbHPLC together with a mixture of the three standard peptides. Major radioactive fractions eluted with retention times of 72, 90, and 134 min (peaks), and the standard peptides were detected at 70.7 (S437-PP), 80.4 (S727-PPox), and 85.1 min (S454-PP) (Fig. 1A). The first radioactive peak is the Ser-437-phosphorylated peptide. The third radioactive peak observed after 130-140 min co-eluted with a large peptide peak detected by UV absorbance (132.2 min) on the HPLC chart (Fig. 1A). This is similar to the co-elution of the Ser-505-containing tryptic fragment around 105 min with a major peptide peak from the 218TP52 column (17). This 130-140-min peak co-eluted with the radiolabeled tryptic phosphopeptide prepared by in vitro phosphorylation of cPLA2 with p42mapk (see below).
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Two Major Phosphorylation Sites of cPLA2 in Thrombin-stimulated Human Platelets-- Having shown that the mbHPLC system is reliable for the analysis of cPLA2-derived phosphopeptides, we turned to the identification of the phosphorylation sites of cPLA2 from human platelets. After 32Pi labeling of platelets and stimulation with thrombin, cPLA2 was purified by immunoprecipitation and SDS-PAGE, digested with trypsin, and subjected to mbHPLC. Two radioactive fractions eluted at 82-88 min and at 130-138 min (Fig. 2). The column was eluted for up to 180 min (0.06% CF3COOH in CH3CN from 115 to 180 min), and there were no further phosphopeptides detected. The yield of counts/min in all mbHPLC fractions was typically 82-98% of those applied, and approximately 6% (6-11% in other runs) of the counts/min eluted in the void volume. Thus it can be concluded that the two phosphopeptides detected represent the major sites of phosphorylation on cPLA2 from thrombin-stimulated platelets. The ratio of radioactivity of the two peaks was approximately 1:1, as determined by summing up the counts/min of the 4-5 radioactive fractions constituting each peak. This ratio did not change when platelets were stimulated with thrombin for 2 min instead of 5 min, or in the presence of 1 mM external Ca2+ instead of EGTA. Also, doubling the amount of trypsin in the digest did not alter this pattern (not shown), which suggests that the phosphopeptides are not partial tryptic fragments.
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mbHPLC Analysis of cPLA2 Phosphorylated by p42mapk in Vitro-- To further confirm that the late eluting radioactive peak from the mbHPLC column in the platelet experiments is due to phosphorylation at Ser-505, we performed in vitro assays with activated p42mapk using wild-type human cPLA2 and the S505A cPLA2 mutant as substrates. Following SDS-PAGE and autoradiography, prominent 32P-phosphorylation was seen in cPLA2/p42mapk samples, but a small amount of cPLA2 phosphorylation was also detected in kinase reactions that lacked exogenous p42mapk (not shown). The tryptic digests of cPLA2 eluted from the gel were then analyzed on mbHPLC (Fig. 5). The major radioactive fraction of the cPLA2/p42mapk sample (retention time 130-140 min) co-eluted with a prominent OD210 peptide peak (133.2 min) and represented 85% of the total counts/min eluting from the mbHPLC column. Its retention time was the same as that of the Ser-505-containing phosphopeptide seen in the platelet and Sf9 cell experiments. On two-dimensional maps, the phosphopeptides recovered from this mbHPLC peak moved in positions of spots 4 and 4a (not shown). In the absence of p42mapk there was a trace of radioactivity detectable with a similar retention time (132-138 min). However, this peak was not seen in experiments where the mutated S505A cPLA2 was used as substrate for p42mapk (Fig. 5). This suggests that the minor peak is caused by a MAPK contamination in the purified cPLA2 used in these in vitro experiments.
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Effect of the p38mapk Inhibitor SB 203580 on the Phosphorylation of cPLA2 in Thrombin-activated Platelets-- Our previous studies on the phosphorylation of cPLA2 in human platelets treated with thrombin showed that the p38mapk inhibitor SB 203580 inhibits cPLA2 phosphorylation by 50% (24). To verify the effect of SB 203580 on p38mapk activity in platelets, the activity of MAPKAP-K2, an in vivo substrate of p38mapk, was determined. SB 203580 inhibited activation of MAPKAP-K2 by thrombin in a concentration dependent manner with an IC50 of 0.2 µM, and inhibition was complete with 10 µM inhibitor (not shown). SB 203580 did not interfere with vital platelet responses such as activation of protein kinase C, 5-hydroxytryptamine release, and tyrosine phosphorylation of platelet proteins.2
When platelets were incubated with SB 203580 (20 µM) before stimulation with thrombin (1 unit/ml, 2 min), 32P-phosphorylation of cPLA2 was reduced by approximately 50% (Fig. 6A) which is consistent with our previous results (24). The separation of the tryptic digest of cPLA2 on mbHPLC resulted in three radioactive fractions with retention times of 82, 86, and 134 min (peaks) in both the thrombin and the SB 203580/thrombin sample (Fig. 6B). The 82-min peak co-eluted with the S727-PPox standard, the 86-min peak with unoxidized S727-PP (which co-elutes with S454-PP, see above), and the retention time of the 134-min peak corresponded to the authentic Ser-505 phosphopeptide (Fig. 5). Based on these results, TLC analysis, and the previous studies (17), it is clear that the S727-PP undergoes variable oxidation in different preparations. This behavior further confirms that the platelet-derived phosphopeptide is S727-PP.
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Phosphorylation of cPLA2 in Collagen-activated Platelets and Effect of the 38mapk Inhibitor SB 203580-- Collagen (100 µg/ml) stimulated 32P-phosphorylation of cPLA2 by 2.6-fold over basal levels (Fig. 7A). Radioactivity was incorporated into 2 tryptic fragments, as resolved by mbHPLC (Fig. 7B), and the retention times of these fractions (peaks: 86 and 134 min) were virtually identical to those of the two phosphopeptides obtained from thrombin-activated platelet cPLA2 (Fig. 6B). The phosphopeptide recovered from the first peak was oxidized by performic acid and subjected to mbHPLC, which resulted in a shift of the peak to a shorter retention time (80 min), now co-eluting with the S727-PPox standard (retention time 80.1 min; Fig. 7B).
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Phosphorylation of cPLA2 in HeLa S3 Cells--
In
light of the cPLA2 phosphorylation results with Sf9
cells and platelets, we were intrigued by the recent report that
cPLA2 in interferon--stimulated HeLa S3 cells is
phosphorylated on tyrosine (28). Thus, we carried out mbHPLC studies of
cPLA2 derived from HeLa cells. HeLa S3 cells were
radiolabeled with 32Pi in the presence and
absence of 1,000 units/ml interferon-
. HeLa cells were also
stimulated with sodium arsenite, which is a known activator of
stress-activated protein kinases such as p38mapk. As shown in
Fig. 8A, two radioactive
tryptic peptides were detected with both stimulants. The earlier peak
eluted with the S727-PP standard, and the later peak eluted in the
region expected for the Ser-505 phosphopeptide. These assignments were
confirmed by submitting each mbHPLC peak to two-dimensional
phosphopeptide mapping (not shown). Furthermore, two-dimensional
phosphopeptide mapping of the entire tryptic digest (without mbHPLC)
revealed only these two phosphopeptides (not shown). Treatment of HeLa cells with interferon-
resulted in an increase in
cPLA2 phosphorylation. Cerenkov counting of the
SDS-PAGE bands from immunoprecipitates gave 5,000 cpm in the control
sample, and 6,400 cpm in the stimulated sample. cPLA2 from
arsenite-treated cells contained 11,300 cpm. Interferon-
treatment
led to a 2-fold increase in phosphorylation at Ser-727 and a 1.6-fold
increase in phosphorylation at Ser-505, and the corresponding increases
with arsenite treatment were 6.5-fold for Ser-727 and 1.9-fold for
Ser-505 (Fig. 8A). To explore the possibility that a
putative phosphotyrosine peptide was lost in the mbHPLC analysis or in
the transfer of the tryptic digest to the two-dimensional TLC plate,
the entire tryptic digest was submitted to phosphopeptide amino acid
analysis (performed in the same tube as the trypsin digestion). As
shown in Fig. 8B, only phosphoserine was detected, and the
non-radiolabeled standards (phosphoserine, -threonine, and -tyrosine)
were clearly visible as well defined spots by ninhydrin detection. When
HeLa cells were labeled with 5 mCi of 32Pi,
phosphorylation of cPLA2 in control cells (28,500 cpm) was increased to 46,700 cpm in the presence of arsenite, and co-treatment with SB 202190 (20 µM) and arsenite resulted in
incorporation of 32,000 cpm of phosphate into cPLA2. Thus,
as seen with platelets, SB 202190 causes partial inhibition of
cPLA2 phosphorylation.
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DISCUSSION |
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All of the phosphorylation sites of cPLA2 expressed in a baculovirus/Sf9 cell system have been previously determined by a combination of HPLC, electrospray mass spectrometry, and Edman sequencing (17). As pointed out in this previous study, two-dimensional phosphopeptide mapping analysis is problematic for accurately quantifying the amount of each phosphopeptide, and this point should be considered in any analysis of protein phosphorylation. This is because the multiple lyophilizations needed to remove buffer salt prior to analysis causes a significant fraction of the radiolabeled phosphopeptides to irreversibly adhere to the walls of the tube, and thus the counts/min of each phosphopeptide spot cannot be used for quantification. In this previous study, quantification was carried out by conventional HPLC analysis of trypsin digests since the yield of counts/min from the reverse phase column was >80%. In these studies, tens of micrograms of cPLA2 were available (typically 100,000 cpm), and thus no extraordinary measures were needed to handle the phosphopeptides. This approach had to be modified for the present study by use of mbHPLC to analyze relatively tiny amounts of phosphopeptides derived from immunoprecipitates of platelet and HeLa cell cPLA2. The small diameter of the mbHPLC column (0.5 mm) enabled us to elute samples containing 100-3,000 cpm with a yield of over 90%. Thus, mbHPLC is a valuable tool for phosphopeptide mapping and quantification of phosphorylation of proteins present at natural abundance in mammalian cells.
From mbHPLC and two-dimensional phosphopeptide maps we conclude that in
thrombin- and collagen-stimulated platelets, cPLA2 is
phosphorylated on Ser-505 and Ser-727. Tryptic phosphopeptide fragments
generated from the Sf9 baculovirus expression system (17) were
compared with those from platelets and were an important component for
identification of the phosphorylation sites of platelet cPLA2. Our firm demonstration that cPLA2 is not
phosphorylated on tyrosine in interferon-- and arsenite-stimulated
HeLa S3 cells despite the fact that both agents increase
cPLA2 phosphorylation is at odds with the published
conclusion that cPLA2 is tyrosine phosphorylated in these
cells (28). In the previous study, phosphotyrosine formation was
assessed only by immunoblotting with a tyrosine phosphate-specific
antibody, whereas in the present studies it was assessed by direct
protein chemical approaches including phosphoamino acid analysis. It is
inconceivable that a putative tyrosine phosphate-containing cPLA2 peptide was lost due to surface absorption during
sample handing because the entire tryptic digest was hydrolyzed with HCl in the same tube as the trypsin digest itself. By labeling HeLa S3
cells with 5 mCi of 32Pi, we were able to
obtain immunoprecipitated cPLA2 that contained tens of
thousands of counts/min, and thus even if 1% of these counts/min
represented tyrosine phosphate this amino acid would have been detected
by phosphoamino acid analysis. Although we cannot say with absolute
certainty that cPLA2 is not phosphorylated on tyrosine (our
HeLa S3 cells are probably not genetically identical to the ones used
by Flati et al. (28), and handling of the cells could be
somewhat different in the two laboratories), our direct structural
results cast significant doubt that cPLA2 is tyrosine phosphorylated in interferon-
-treated HeLa S3 cells. Phosphorylation of cPLA2 on tyrosine has been claimed to occur in
transforming growth factor-
-stimulated HEL-30 keratinocytes, but
again this is based only on an anti-phosphotyrosine immunoblot
(33).
In the present study we have found that SB 203580 inhibited
phosphorylation of cPLA2 on both Ser-505 and Ser-727 by 60 and 50%, respectively, in thrombin-stimulated platelets. This partial inhibition occurs under conditions in which MAPKAP-K2 activation is
completely inhibited by SB 203580. These results suggest either that a
SB 203580-sensitive p38mapk is only partially responsible for
cPLA2 phosphorylation, or that the Km
for cPLA2 as a substrate for p38mapk is relatively
low compared with other substrates. We cannot address the last
possibility at the present time because of the limited solubility of SB
203580 and SB 202190, which precludes testing at concentrations >20
µM. The recently described stress-activated protein
kinases SAPK3 and SAPK4, neither of which is inhibited by SB 203580 and
SB 202190 (34), or an unidentified kinase may be responsible for a
portion of the cPLA2 phosphorylation that occurs in
thrombin-stimulated platelets. The recently described isoform of
p38mapk, p38, which is inhibited by SB 203580 and SB 202190 (35), is also a candidate kinase for cPLA2
phosphorylation.
The reduction of cPLA2 phosphorylation on Ser-727 by SB 203580 was surprising since this site does not lie within a MAPK consensus sequence. Indeed, p42mapk did not phosphorylate cPLA2 at Ser-727 in vitro (Fig. 5). Moreover, S505A cPLA2 was a poor substrate for the partially-purified platelet p38mapk preparation that phosphorylated wild type cPLA2 (26), which suggests that Ser-727 is not directly phosphorylated by p38mapk. The following possibilities may account for the inhibition of Ser-727 phosphorylation by SB 203580: 1) Ser-505 and Ser-727 are phosphorylated by different kinases and both are inhibited by SB 203580; 2) the Ser-727 kinase is downstream of the SB 203580-sensitive Ser-505 kinase in the activation pathway; 3) phosphorylation of Ser-505 by a SB 203580-sensitive kinase (p38mapk or an isoform) leads to a conformational change in cPLA2 that exposes Ser-727 to the same kinase (this would explain why platelet-derived p38mapk did not phosphorylate S505A cPLA2 in vitro (27)) or to a distinct kinase. Clearly, additional work is needed to firmly identify the kinase(s) responsible for Ser-505 and Ser-727 phosphorylation.
We have previously shown that the kinase inhibitor staurosporine completely blocks phosphorylation of cPLA2 in thrombin-activated platelets without inhibiting the release of arachidonic acid (21). However, staurosporine increases the thrombin-induced rise of intracellular Ca2+ which might counteract the effect of inhibition of cPLA2 phosphorylation on arachidonic acid formation. In light of the fact that specific inhibitors which fully block cPLA2 phosphorylation at Ser-505 and Ser-727 in platelets and HeLa cells are not available, the role of cPLA2 phosphorylation in promoting agonist-induced arachidonic acid release cannot yet be conclusively resolved.
Our results underscore the importance of direct chemical approaches for analyzing the sites of cPLA2 phosphorylation. Furthermore, mbHPLC provides the level of phosphorylation to be accurately quantified, and this is critical for the study of the effect of kinase inhibitors on cPLA2 phosphorylation. The widely used SDS-PAGE gel shift analysis may not be reliable for determination of the sites or levels of cPLA2 phosphorylation.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. G. S. de Carvalho for contributions to preliminary studies, F. Ghomashchi for help with mbHPLC analyses, Dr. S. Pasquet for help with in vitro phosphorylation assays, and B. Haubold for comments on the manuscript. We are grateful to Dr. J. Lee for SB 203580 and Dr. C. C. Leslie for cPLA2 antiserum and advice on Sf9 cell labeling.
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FOOTNOTES |
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* This work was supported by the National Institutes of Health Grant HL50040 (to M. H. G.) and the British Heart Foundation (to A. B.-H. and S. P. W.).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.
** Royal Society Research Fellow.
To whom correspondence should be addressed: Depts. of Chemistry
and Biochemistry, Box 351700, University of Washington, Seattle, WA 98195. Fax: 206-685-8665; E-mail: gelb{at}chem.washington.edu (or A. G. Börsch-Haubold, Dept. of Pharmacology,
Mansfield Road, Oxford OX1 3QT, UK. Fax: 44-1865-271853; E-mail:
angelika.borsch{at}pharmacology.oxford.ac.uk).
1 The abbreviations used are: cPLA2, cytosolic phospholipase A2; MAPK, mitogen-activated protein kinase; Sf9 cell, Spodoptera frugiperda cell; HPLC, high performance liquid chromatography; p38mapk, 38-kDa stress-activated protein kinase; SB 203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole; PAGE, polyacrylamide gel electrophoresis; mbHPLC, microbore HPLC; MAPKAP-K2, MAPK-activated protein kinase 2; SB 202190, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl) imidazole; S437-PP, serine 437-containing phosphopeptide; S454-PP, serine 454-containing phosphopeptide; S727-PP, serine 727-containing phosphopeptide; S505A cPLA2, cPLA2 with serine 505 mutated to alanine; Me2SO, dimethyl sulfoxide.
2 A. G. Börsch-Haubold and S. P. Watson, unpublished data.
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REFERENCES |
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