p11, a Unique Member of the S100 Family of Calcium-binding Proteins, Interacts with and Inhibits the Activity of the 85-kDa Cytosolic Phospholipase A2 *

Using a two hybrid system screen of a human cDNA library, we have found that p11, a unique member of the S100 family of calcium-binding proteins, interacts with the carboxyl region of the 85-kDa cytosolic phospholipase A2(cPLA2). p11 synthesized in a cell-free system interacts with cPLA2 in vitro. The p11-cPLA2complex is detectable from a human bronchial epithelial cell line (BEAS 2B). Furthermore, p11 inhibits cPLA2 activity in vitro. Selective inhibition of p11 expression in the BEAS 2B cells by antisense RNA results in an increased PLA2activity as well as an increased release of prelabeled arachidonic acid. This study demonstrates a novel mechanism for the regulation of cPLA2 by an S100 protein.

Phospholipase A 2 s (PLA 2 , 1 EC 3.1.1.4) are a group of enzymes that catalyze the hydrolysis of the sn-2-ester bond of phospholipids, resulting in the production of free fatty acid and lysophospholipids. These lipid products may serve as intracellular second messengers or can be further metabolized to potent inflammatory mediators. The release of arachidonic acid (AA) from membranes by PLA 2 and its subsequent conversion into leukotrienes, prostaglandins, and other eicosanoids play a key role in the process leading to inflammation. Several mammalian PLA 2 s, including the 14-kDa secreted form PLA 2 and the 85-kDa cytosolic PLA 2 (cPLA 2 ), have been proposed to mediate AA release and eicosanoid generation in various cell types (1)(2)(3).
The mammalian low molecular mass 14-kDa PLA 2 s mainly include two types, type I and type II. Although the type I PLA 2 is mainly found in pancreatic secretion, it has also been found in other mammalian tissues, and the physiological role of this extra-pancreatic type I PLA 2 remains unclear. The type II PLA 2 has been suggested to be involved in the pathogenesis of inflammatory reactions (1)(2). It is found associated with several cells and tissues or is present extracellularly when released in response to proinflammatory mediators such as interleukin-1, interleukin-6, or tumor necrosis factor. The soluble type II 14-kDa PLA 2 found in inflammatory fluids, tissue exudates, or serum has been suggested to propagate inflammation through membrane digestion and liberation of proinflammatory lipid mediators. The cell-associated type II PLA 2 has been assumed to release AA for lipid mediator generation (1)(2)(3). However, because the 14-kDa PLA 2 lacks selectivity for the sn-2 fatty acids of phospholipids and requires much higher Ca 2ϩ (mM) than normal cellular Ca 2ϩ level (nM-M) for activation, it was not certain whether this type of PLA 2 contributes to the release of AA and production of eicosanoids. Studies using antisense inhibition and selective inhibitors of different forms of PLA 2 s have demonstrated that the type II PLA 2 participates in AA release and prostaglandin generation in the P388D 1 macrophages (4 -6). These findings have established an important role of the 14-kDa PLA 2 for AA metabolism.
In an effort to further investigate the regulation of the 85-kDa cPLA 2 , this study was designed to identify proteins interacting with the NH 2 -terminal or COOH-terminal region of cPLA 2 protein by screening a human cDNA library using the yeast two hybrid system. An 11-kDa protein (p11), the unique member of S100 family of calcium-binding proteins, was found to interact with the COOH-terminal region of cPLA 2 . Further studies demonstrated that p11 associated with cPLA 2 and in-hibited the cPLA 2 activity in vitro. The p11-cPLA 2 complex was precipitated from a human bronchial epithelial cell line (BEAS 2B cell) expressing both p11 and cPLA 2 . Reduction of p11 expression in the BEAS 2B cells by antisense RNA reversed the inhibition of p11 on cPLA 2 , which resulted in an increased PLA 2 activity as well as an increased AA release. This study revealed a previously unrecognized mechanism of cPLA 2 regulation by the S100 protein family.

EXPERIMENTAL PROCEDURES
Construction of Plasmids for Two Hybrid System Screen-Plasmid pGBT9 (CLONTECH, Palo Alto, CA), containing the sequences for the GAL4 DNA-binding domain (amino acids 1-147) as well as sequences for tryptophan (TRP1), was used to express the fusion proteins. Two cPLA 2 cDNA fragments (540 bp corresponding to the NH 2 terminus and 1707 bp corresponding to the COOH terminus) were amplified by polymerase chain reaction using human cPLA 2 cDNA plasmid as the template (8). The obtained cPLA 2 cDNA fragments were cloned into pGBT9 vector in the correct orientation and with the correct reading frame to express the GAL4-cPLA 2 fusion proteins. To obtain the cPLA 2 cDNA fragments, two primer pairs were synthesized according to the cPLA 2 cDNA sequence (8). The primer pair for the 540-bp cPLA 2 cDNA fragment was composed of the following sequences: 5Ј primer-GTC-GACTGATGTCATTTATAGATCCTTACCAGCAC (starting from the translation start site; corresponding to the ϩ1 to ϩ27 of the coding region; containing the underlined SalI site and TG dinucleotides for in frame cloning); 3Ј primer-GTCGACTCCTTCACTATTCTTTGGAC-CCAAGAGTTT (corresponding to the ϩ540 to ϩ511 of the coding region; containing the underlined SalI site). The primer pair for the 1707-bp cPLA 2 cDNA fragment was composed of the following sequences: 5Ј primer-GTCGACTGTTGCATTCTGCACGTGATGTGCCTGTG (corresponding to ϩ541 to ϩ567 of the coding region; containing the underlined SalI site and TG dinucleotides for in frame cloning); 3Ј primer-GTCGACTGCTTTGGGTTTACTTAGAAACTCCTTGTT (corresponding to the ϩ2247 to ϩ2218 of the coding region and containing the underlined SalI site). The cDNA was amplified via polymerase chain reaction utilizing Thermus aquaticus DNA polymerase. The amplified fragments were initially cloned into the pCR TM 3 vector using the TA cloning kit (Invitrogen). The inserts were then released from the pCR TM 3 vector by restriction enzyme digestion with SalI and were cloned into the pGBT9 vector. The identity and orientation of the constructs were confirmed by DNA sequencing. The plasmid containing the 540-bp cPLA 2 cDNA fragment corresponding to the NH 2 -terminal cPLA 2 protein was designated as plasmid pGBT9-cPLA2N. The plasmid containing the 1707-bp cPLA 2 cDNA fragment corresponding to the COOH-terminal cPLA 2 protein was designated as plasmid pGBT9-cPLA2C. These plasmids were used to transform host HF7c cells. The transformants containing the pGBT9-cPLA2N or pGBT9-cPLA2C were selected for tryptophan prototrophy and examined for ␤-galactosidase activity using a filter assay. The ␤-galactosidase activity was undetectable in colonies containing only pGBT9-cPLA2N or pGBT9-cPLA2C. The HF7c cells containing pGBT9-cPLA2N or pGBT9-cPLA2C were then lysed for Western blotting analysis using the rabbit anti-cPLA 2 antibody (Genetics Institute, Boston, MA). The identity of the expressed fusion proteins was confirmed by the observation that both the NH 2terminal cPLA 2 fusion protein (approximately 40 kDa) and COOHterminal cPLA 2 fusion protein (approximately 90 kDa) reacted with the polyclonal anti-cPLA 2 antibody.
Two Hybrid System Screening of Human cDNA Library-A human leukocyte MATCHMAKER cDNA library constructed in pGAD10 plasmid (CLONTECH) was screened to identify proteins interacting with cPLA 2 . The Saccharomyces cerevisiae HF7c reporter strain was used for transformation with the library and plasmid pGBT9-cPLA2N or plasmid pGBT9-cPLA2C by the lithium/acetate method (31). The HF7c yeast cells were sequentially transformed with pGBT9-cPLA2N or pGBT9-cPLA2C followed by transformation with DNA from the MATCHMAKER human leukocyte cDNA library. Double transformants were plated in the SD synthetic medium lacking histidine, tryptophan, and leucine at a density of approximately 10,000 colonies per 150-mm plate (20 plates for each of pGBT9-cPLA2N and pGBT9-cPLA2C). Colonies appeared after incubation at 30°C for 3 days. The colonies were transferred to VWR grade 413 paper filters and assayed for ␤-galactosidase activity using 5-bromo-4-chloro-3-indolyl-␤-D-galactoside (X-Gal) as substrate. Filters were placed in liquid nitrogen for 30 s, thawed at room temperature, and then incubated in buffer containing X-Gal. If transformants expressed ␤-galactosidase activity, the X-Gal was cleaved and colony replicates turned blue. Positive colonies were purified by restreaking on selection medium, and well isolated colonies were reassayed for ␤-galactosidase activity.
Plasmids encoding the candidate interacting GAL4 activation domain/cPLA 2 binding protein were extracted from the positive HF7c clones using a previously described method (32). The extracted plasmid DNA was transformed to an Escherichia coli strain (HB101) carrying a leuB mutation to select the activation domain hybrid plasmid that contains yeast LEU2 gene. The transformed E. coli cells were plated on M9 agar medium containing a 1 ϫ mixture of amino acids (lacking leucine), 50 g/ml ampicillin, 40 g/ml proline, and 1 mM thiamine HCl. Individual colonies were isolated, and the plasmid DNA was extracted using a standard plasmid mini-prep procedure (33).
Sequence Analysis of pGAD10-cDNA Clones-Initial sequence of the cDNA inserts was obtained by dideoxy sequencing using a 17-mer oligonucleotide primer (5Ј-TACCACTACAATGGATG-3Ј), which corresponds to the GAL4 activation domain sequences and reads toward the junction of the GAL4 activation domain and the cloned candidate interacting protein. Specific oligonucleotide primers corresponding to the insert sequence were subsequently generated to obtain the full-length sequence of the inserts.
Expression of p11 Protein in Bacteria-To construct plasmid for p11 protein expression in bacteria, human p11 cDNA was amplified by reverse transcription and polymerase chain reaction using human lung poly(A) ϩ RNA. The primer pair, synthesized according to the human p11 cDNA sequence (34), was composed of the following sequences: 5Ј primer-GGATCCCCATCTCAAATGGAACACGCCATG (corresponding to 115-138 of the cDNA sequence; adjacent to the translation start site; containing the underlined BamHI site) and 3Ј primer-CTCGAGTAT-CAGGGAGGAGCGAACTGCTCA (corresponding to 413-436 of the cDNA sequence; located in 3Ј-untranslated region; containing the underlined XhoI site). The amplified fragments were initially cloned into the pCR TM 3 vector using the TA cloning kit. The inserts were then released from the pCR TM 3 vector by restriction enzyme digestion (BamHI and XhoI) and subsequently cloned into the prokaryotic expression vector pET30a (Novagen), giving rise to p11-pET30a. The identity and orientation of the construct was confirmed by DNA sequencing. The pET30a-p11 vector expressed p11 protein as a fusion protein containing a stretch of 6 consecutive histidine residues (His-tag) at the NH 2 terminus. The His-tag, removable by digestion with protease (e.g. enterokinase), was used for efficient purification of the recombinant p11 proteins under gentle, native conditions for maintaining activity of soluble proteins (35). The plasmid was transformed into E. coli strain BL21 (DE3), a lon mutant strain containing the T7 polymerase under the control lacUV5 promoter. Addition of isopropyl-␤-D-thiogalactopyranoside induces the expression of p11. The expressed p11 was purified using histidine-binding resin (Novagen) according to the manufacturer's instruction. The His-tag-p11 was digested with enterokinase, and the released p11 without His-tag was collected and further purified by gel filtration using Bio-Gel P-30 (Fig. 1). The purified p11 Protein expression was induced in bacteria transformed with His-tag-p11 expression plasmid according to standard methods (see "Experimental Procedures"). The expressed protein was purified with histidine-binding resin. The resin containing His-tag-p11 was digested with enterokinase, and the recombinant p11 without His-tag was released into the supernatant. Aliquots of the supernatants and resins were subjected to SDS-PAGE, and the gel was stained with Coomassie Blue. Prior to enterokinase digestion, the expressed fusion protein exists in the resin (lane 4) but not in the supernatant (lane 2). Two fragments, the His-tag and free p11 without His-tag, were generated by proteolytic digestion with enterokinase (lane 5, resin; lane 3, supernatant). The positions of size markers (lane 1), His-tag-p11 fusion protein, p11 without His-tag, and His-tag fragment were indicated. The free p11 in the supernatant was collected for further gel filtration purification.
was used for subsequent studies of the regulatory effects of p11 on cPLA 2 enzyme activity.
In Vitro PLA 2 Activity Assay Using Purified cPLA 2 and p11-As the in vitro assayed cPLA 2 activity is dependent on the composition of the liposome (36), we used the natural membrane vesicles prepared from the BEAS 2B cells as cPLA 2 substrate. To obtain the natural membrane vesicles, the BEAS 2B cells grown in 175-cm 2 flasks were labeled with [5,6,8,9,11,12,14, H]arachidonic acid (214 Ci/mmol) in LHC-8 media. As incorporation of AA to membrane phospholipid classes may be dependent on the length of labeling period, the cells were labeled twice with [ 3 H]AA (18-h and 10-min labeling periods) to ensure even distribution of radiolabeled AA in various classes of phospholipids (5)(6)37). The cells were first labeled with [ 3 H]AA for 18 h to allow [ 3 H]AA to equilibrate among phospholipids. Under these conditions, phosphatidylethanolamine accounts for the majority of esterified [ 3 H]AA in phospholipids, the remainder being esterified in phosphatidylcholine, phosphatidylinositol, and phosphatidylserine. After the 18-h labeling period, the cells were labeled again with [ 3 H]AA for 10 min. At this short labeling term, the distribution of AA esterified in phospholipids differs from that seen at long incubation times in that phosphatidylcholine, not phosphatidylethanolamine, is the phospholipid that incorporates most of the radiolabeled AA. The dual-labeled cells were harvested and resuspended in 0.5 ml of homogenization buffer (50 mM HEPES, pH 7.4, 1 mM EGTA, 50 g/ml leupeptin, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 M phosphoramidon, 10 g/ml soybean trypsin inhibitor, 100 g/ml aprotinin). Cells were disrupted by repeated freeze/thaw cycles. The lysate was centrifuged at 1000 ϫ g for 5 min to remove nuclei, unbroken cells, and debris. The low speed supernatant was then centrifuged at 100,000 ϫ g for 1 h to produce a crude cytosolic fraction (supernatant) and a membrane fraction (pellet). The crude cytosol was discarded, and the pellet was washed once, resuspended in 50 mM HEPES buffer (pH 7.4, without EGTA and protease inhibitors), and stored in aliquots at Ϫ80°C for future use as cPLA 2 substrate. Membrane phospholipid concentration was estimated after separation of phospholipids by thin layer chromatography on a silica gel G plate (Alltech, Dearfield, IL) developed with chloroform/methanol/ acetic acid/water, 50:30:8:4, using standards of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol (Sigma). The phospholipids were visualized by development with 2,7-dichlorofluorescein (Aldrich).
In vitro cPLA 2 activity was measured using 3 H-labeled BEAS 2B cell membrane (7.5 ϫ 10 3 dpm/6.5 g protein/assay) and purified human cPLA 2 in a final volume of 100 l (50 mM HEPES buffer, pH 7.4). The cPLA 2 was kindly provided by the Genetics Institute and is purified from U937 cells. For accurate control of the Ca 2ϩ concentration, a CaCl 2 /EGTA buffering system was used (38). As the physiological cellular Ca 2ϩ level is normally at micromolar level (0.1 M in normal resting cells and 0.3-1.0 M in activated cells) and the cPLA 2 activity is measurable under micromolar concentrations of Ca 2ϩ , a physiologically relevant Ca 2ϩ concentration (0.5 M free Ca 2ϩ ) was used for all the assays (1 mM EGTA and 0.96 mM CaCl 2 ). The reaction was started by the addition of substrate to the reaction mixture. The assays were then incubated at 37°C for 1 h and terminated by the addition of 300 l of 2:1 chloroform/methanol containing 1% acetic acid and 1 mg/ml free AA. Release of free fatty acid was analyzed using silica gel H thin layer chromatography plates as described previously (23)(24). The effect of p11 was expressed as a percent of the arachidonate liberated by cPLA 2 alone in each experiment (500 -580 dpm).
Binding of Purified p11 and cPLA 2 in Vitro-The 35 S-labeled p11 (carrying the His-tag at the NH 2 -terminal) was synthesized from the pET30a-p11 vector by the coupled in vitro transcription and translation using the Single Tube Protein System 2 (Novagen). For each synthesis reaction, 0.5 g of pET30a-p11 plasmid template was transcribed in 10 l at 30°C for 15 min, followed by the addition of 40 l of translation mixture with [ 35 S]methionine and continued incubation for 60 min. Products from individual synthesis reactions were pooled and stored in aliquots at Ϫ20°C for future use.
To allow formation of the p11-cPLA 2 complex, 5 l of the synthesized [ 35 S]p11 (3.4 ϫ 10 6 dpm) was incubated with 100 ng of purified human cPLA 2 in 1 ml of HBSS (with calcium and magnesium) at 4°C for 30 min. The cPLA 2 was provided by Genetics Institute, Boston, MA, and is purified from U937 cells. 10 l of rabbit anti-human cPLA 2 antibody (Genetics Institute) and 25 l of Protein G Plus/Protein A-agarose (Pierce) were then added, and the mixture was incubated at 4°C on a rotating device for 4 h, followed by centrifugation in a microcentrifuge at 2500 rpm for 5 min at 4°C. The supernatant was aspirated, and the pellet was washed four times with 1.0 ml of PBSTDS (1 ϫ phosphatebuffered saline, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) by repeated centrifugation. Following four washings with PBSTDS, 20 l of protein loading buffer was added to the pellet, and the sample was boiled for 10 min before electrophoresis on 18% polyacrylamide gels (Novex, San Diego, CA) using Tris glycine/ SDS buffer. The gel was then dried under vacuum and exposed to the x-ray film.
Immunoprecipitation of Native p11 Protein from BEAS 2B Cell Lysate Using Anti-human cPLA 2 Antibody-For isolation of the crude cytosolic proteins, the BEAS 2B cells grown on 175-cm 2 culture flasks were digested with 0.1% collagenase for 10 min. The cells were then lysed in 0.5 ml of homogenization buffer containing 1 mM EGTA and protease inhibitors, and the crude cytosolic protein was isolated as described above. The crude cytosolic samples isolated from individual flasks were pooled and stored in aliquots at Ϫ80°C for future use. For immunoprecipitation, the isolated crude cytosolic fraction (100 l, 200 g of protein) was added to an Eppendorf tube containing 1 ml of HBSS (with calcium and magnesium) and 10 l of rabbit anti-human cPLA 2 antibody. The samples were preincubated at 4°C for 30 min. 25 l of Protein G Plus/Protein A-agarose was then added to each sample, and the mixture was incubated at 4°C on rotating devices for 4 h, followed by centrifugation in a microcentrifuge at 2500 rpm for 5 min at 4°C. The supernatant was aspirated, and the pellet was washed four times with 1.0 ml of PBSTDS by repeating centrifugation. Following four washings with PBSTDS, 20 l of protein loading buffer was added to the pellet, and the sample was boiled for 10 min before electrophoresis on 16% polyacrylamide gels (Novex) using Tris glycine/SDS buffer. The separated proteins were electrophoretically transferred onto a nitrocellulose membrane, which was blocked with 5% nonfat milk overnight, and then probed with a 1:1000 dilution of mouse anti-p11 monoclonal antibody. The blots were then probed with a 1:1000 dilution of horseradish peroxidase-labeled goat anti-mouse IgG and detected using the enhanced chemiluminescence Western Blotting Detection System.
Immunoprecipitation of Native cPLA 2 Protein from BEAS 2B Cells Using Anti-p11 Antibodies-Crude cytosolic extracts were prepared as described above using BEAS 2B cells. 100 l (200 g, protein) of the cytosol was immunoprecipitated with 2.5 mg of mouse anti-p11 monoclonal antibody (Transduction Science) and Protein G/Protein A-agarose. Following washing of the Protein G/Protein A-agarose pellet with PBSTDS, 100 l of loading buffer was added to the pellet, and the sample was boiled for 5 min before electrophoresis of 20 l on 6% polyacrylamide gels using Tris glycine/SDS buffer. The separated proteins were electrophoretically transferred onto a nitrocellulose membrane that was blocked with 5% nonfat milk overnight and then probed with a 1:1000 dilution of rabbit anti-cPLA 2 antibody (Genetics Institute). The blots were then probed with a 1:1000 dilution of horseradish peroxidase-labeled goat anti-rabbit IgG and detected using the Pierce Super Signal detection system.
Stable Transfection of Antisense p11 Plasmid in BEAS 2B Cells-To construct the antisense p11 expression vector, full-length human p11 cDNA was released from one of the three isolated plasmids encoding the fusion protein GAL4 activation domain/p11 protein and then cloned into the pCI-neo mammalian expression plasmid in the antisense orientation (pCI-neo-p11AS vector). The pCI-neo vector carries the human cytomegalovirus immediate-early enhancer/promoter region to promote strong, constitutive expression of the cloned p11 inserts in mammalian cells. The vector also contains the SV40 enhancer and early promoter region to control the expression of the neomycin phosphotransferase gene and the SV40 origin of replication to allow efficient replication in cell lines that express SV40 large T antigen (e.g. BEAS 2B cells). The BEAS-2B cells grown in 175-cm 2 flasks were exposed to 250 l of LipofectAMINE Reagent with 20 g of p11AS-pCI-neo plasmid. Control cells were transfected with pCI-neo expression plasmid alone. Cells were exposed to the mixture of LipofectAMINE and plasmid for 2 h. Following removal of the transfection reagent, fresh medium was added and the incubation was continued for 24 h. The cells were then plated in medium containing 800 g/ml geneticin (G418 sulfate). Colonies of resistant cells appeared after approximately 12 days, and the cells were subcultured at 16 days. Subsequent cultures of the selected cells were routinely grown in the presence of selective pressure.
To determine the protein levels of p11, cPLA 2 , and annexin II, crude cytosolic protein was isolated from cells transfected with pCI-neo vector alone or transfected with p11AS-pCI-neo vector. The cells were grown on 175-cm 2 culture flasks. Each flask contained approximately 3 ϫ 10 7 cells when the culture reached confluence. The cells were lysed in the homogenization buffer containing 1 mM EGTA and protease inhibitors, and the crude cytosolic protein was isolated as described above. The obtained cytosolic protein was subjected to SDS-PAGE and Western blotting analysis using a 1:1000 dilution of mouse anti-p11 (Transduc-tion Laboratories), rabbit anti-cPLA 2 , or mouse anti-annexin II (Transduction Laboratories) antibodies. The blots were then probed with a 1:1000 dilution of horseradish peroxidase-labeled goat anti-mouse IgG (for detection of p11 and annexin II) or Protein A (for detection of cPLA 2 ) and finally detected using the enhanced chemiluminescence Western Blotting Detection System.
Immunoprecipitation of Native p11 Protein and Annexin II Heavy Chain from Transfected BEAS 2B Cells Using Anti-human cPLA 2 Antibody-Crude cytosolic extracts were prepared, and immunoprecipitation with anti-cPLA2 antibody was performed as described above using BEAS 2B cells stably transfected with either the antisense p11 plasmid or the pCI neo plasmid (vector). Following washing of the Protein G/Protein A-agarose pellet with PBSTDS, 100 l of loading buffer was added to the pellet, and the sample was boiled for 5 min before electrophoresis of 20 l on 18% polyacrylamide gels using Tris glycine/SDS buffer. The separated proteins were electrophoretically transferred onto a nitrocellulose membrane that was blocked with 5% nonfat milk overnight and then probed with a 1:1000 dilution of either mouse anti-p11 monoclonal antibody or mouse anti-annexin II monoclonal antibody. The blots were then probed with a 1:1000 dilution of of horseradish peroxidase-labeled goat anti-mouse IgG and detected using the Pierce Super Signal detection system. PLA 2 Activity and Arachidonic Acid Release from Cells Transfected with Antisense p11 Plasmid-Cellular PLA 2 activity was assayed as described previously (23)(24). Equal numbers of the control cells transfected with pCI-neo vector alone or cells transfected with the plasmid pCI-neo-p11AS were grown on 175-cm 2 culture flasks coated with type I rat tail collagen. Each flask contained approximately 3 ϫ 10 7 cells when culture reached confluence. The number of cells in each culture dish was monitored by measuring cellular DNA level using bis-benzimidazole (Hoechst 33258) (39).
For [ 3 H]AA release, equal numbers of the control cells transfected with pCI-neo vector alone and the cells transfected with the antisense p11 plasmid pCI-neo-p11AS were grown in 175-cm 2 culture flasks and, when the cells were nearly confluent, labeled for 18 h with 1 Ci/ml [5,6,8,9,11,12,14, H]arachidonic acid (214 Ci/mmol) in LHC-8 media. For studies of basal release the cells were then washed with culture media and fresh media replaced and collected after 4 hours of culture. The samples were extracted by Sep-Pak C 18 cartridges and chromatographed by reverse phase high pressure liquid chromatography (HPLC) as described previously (23)(24). The arachidonic acid fraction of HPLC elution was collected and measured for radioactivity. For studies of [ 3 H]AA release after calcium ionophore stimulation, equal numbers of the control cells transfected with pCI-neo vector alone and the cells transfected with the antisense p11 plasmid pCI-neo-p11AS were grown on 60-mm culture dishes coated with type I rat tail collagen. Each dish contained approximately 4 ϫ 10 6 cells when culture reached confluence. Experiments were performed when the cells were nearly confluent. The cells were labeled for 18 h with 1 Ci/ml [5,6,8,9,11,12,14, H]arachidonic acid (214 Ci/mmol) in LHC-8 media. Following repeated washing with HBSS, 5 ml of HBSS with calcium ionophore A23187 (10 Ϫ6 M) was added to each dish, and the cells were incubated at 37°C for 15 min. The supernatants collected from four dishes were pooled as one sample for further analysis. The samples were extracted by Sep-Pak C 18 cartridges and chromatographed by reverse phase high pressure liquid chromatography (HPLC) as described above. The arachidonic acid fraction of HPLC elution was collected and measured for radioactivity.

RESULTS
Two Hybrid Screen Indicated that p11 Binds to the C-terminal Region of cPLA 2 -In an effort to identify proteins that interact with either the NH 2 -terminal regulatory Ca 2ϩ -binding domain or the COOH-terminal catalytic domain of the 85-kDa cPLA 2 protein, different regions of the cPLA 2 cDNA were cloned into pGBT9 vector to express the fusion proteins containing the common GAL4 DNA-binding domain and the NH 2terminal or COOH-terminal fragment of the cPLA 2 protein.
Either the plasmid pGBT9-cPLA2N (containing the 540-bp cPLA 2 cDNA fragment corresponding to the NH 2 -terminal cPLA 2 protein) or pGBT9-cPLA2C (containing the 1707-bp cPLA 2 cDNA fragment corresponding to the COOH-terminal cPLA 2 protein) was used to transform the host HF7c cells. The HF7c strain containing pGBT9-cPLA2N or pGBT9-cPLA2C was used for subsequent transformation with DNA prepared from a human leukocyte MATCHMAKER cDNA library con-structed in the pGAD10 plasmid. By this approach, four clones were identified to interact with the COOH-terminal region of cPLA 2 protein. However, no clone was found to interact with the NH 2 -terminal region of the cPLA 2 .
To verify the true positives, the isolated four plasmids were retransformed back into the original HF7c yeast host strain in the presence or absence of plasmid pGBT9 containing GAL4 DNA-binding domain alone, plasmid pGBT9-cPLA2N, or plasmid pGBT9-cPLA2C. All four positive clones exhibited positive results in the ␤-galactosidase activity assay only when cotransformed with the plasmid pGBT9-cPLA2C. These plasmids were also cotransformed into another host S. cerevisiae strain SFY526. Similar results in the ␤-galactosidase activity assay were obtained in SFY526. In SFY526, the lacZ reporter gene is under the control of a promoter different from that used to control lacZ in HF7c. While these two promoters share only GAL4-responsive elements in common, the rest of the two promoter sequences differ significantly. Any positive two hybrid interaction observed in both HF7c and SFY526 reporter strains likely requires binding of the GAL4 DNA-binding domain specifically to the GAL4 responsive elements (40). Therefore, we concluded that the isolated four clones represent the true positive.
DNA sequences were obtained for inserts of the four clones identified in the genetic screen. Three of the clones were identical and contained the full-length cDNA sequence of human p11 (34), a member of the S100 protein family.
p11 Inhibits cPLA 2 Enzyme Activity-The findings from the two hybrid screen indicated that p11 binds to cPLA 2 protein. To study the possible regulatory effect of p11 on cPLA 2 enzyme activity, experiments were designed to examine the direct effect of p11 on cPLA 2 enzyme activity in vitro. The natural membrane substrate was incubated with 100 ng of purified human cPLA 2 with various concentrations of recombinant human p11. Cytochrome c was used as a control protein for the in vitro PLA 2 activity assays as described previously (41). As shown in Fig. 2, while cytochrome c exhibited no effect, p11 caused a dose-dependent inhibition of the cPLA 2 activity. These results demonstrate that p11 interacts with the cPLA 2 FIG. 2. Dose-dependent effect of p11 on cPLA 2 activity in vitro. 3 H-Labeled membrane substrate (0.25 nmol of phospholipid) was incubated with 100 ng of purified human cPLA 2 in the presence of increasing concentrations of recombinant human p11 or cytochrome c. The assays were performed in 0.5 M free Ca 2ϩ in a final volume of 100 l. Reactions were initiated by the addition of substrate. The assays were incubated at 37°C for 1 h and terminated by the addition of 300 l of 2:1 chloroform/methanol containing 1% acetic acid and 1 mg/ml free AA. Release of free fatty acid was determined using thin layer chromatography as described under "Experimental Procedures." Data were expressed as percentage of PLA 2 activity from 3 to 6 individual experiments. enzyme and inhibits the cPLA 2 activity. To test whether the inhibitory effect of p11 is related to substrate concentrations, the inhibition of cPLA 2 by p11 was evaluated under various amounts of substrate (2.5-20 M phospholipid). As shown in Fig. 3A, p11 exhibited similar inhibitory effects under different substrate concentrations. These findings suggest that the inhibition of cPLA 2 by p11 is unlikely through an interaction between p11 and the phospholipid substrate.
As p11 is often associated with the 36-kDa annexin II to form annexin II heterotetramer in cells, we also examined whether the inhibitory effect of p11 on cPLA 2 activity may be altered by the presence of annexin II. The effect of annexin II alone or combined annexin II and p11 on cPLA 2 activity was studied under the same in vitro conditions. As shown in Fig. 3B, annexin II alone exhibited some inhibitory effect on cPLA 2 activity. This effect was not unexpected because annexin II might sequester substrate from its access to various PLA 2 s under the in vitro assay conditions. While annexin II alone exhibited certain inhibitory effect on cPLA 2 activity, this effect was less remarkable than the inhibition by p11. The combination of annexin II and p11 appeared to exhibit additive inhibition. These results indicate that annexin II does not significantly alter the inhibitory effect of p11 on cPLA 2 activity. p11 itself is sufficient to bind and inhibit cPLA 2 , and the formation of p11-annexin II complex is not required for the inhibition.
p11 Binds to cPLA 2 in Vitro-To further demonstrate the interaction between p11 and cPLA 2 , we used the in vitro translated human p11 and purified human cPLA 2 to examine their binding ability in vitro. The bacterial expression plasmid (pET30a-p11) containing the His-tag and full-length human p11 cDNA sequences was used as the template to synthesize 35 S-labeled p11 proteins by the coupled in vitro transcription and translation procedures. The synthesized protein included the His-tag-p11 fusion protein as well as other smaller size proteins that were likely to be the translational products from internal translation start sites (Fig. 4). The in vitro translated 35 S-labeled p11 was incubated with purified cPLA 2 protein to allow the formation of p11-cPLA 2 complex. As shown in Fig. 4, p11 protein was not precipitated by anti-cPLA 2 in the absence of cPLA 2 protein, but it was precipitated if preincubated with cPLA 2 . This result demonstrates the formation of p11-cPLA 2 protein complex in vitro.
Identification of p11-cPLA 2 Complex from a Human Bron-chial Epithelial Cell Line (BEAS 2B Cell)-The above results demonstrated that p11 binds to cPLA 2 in vivo (based on two hybrid screen) and in vitro (based on the in vitro binding of synthesized p11 and purified cPLA 2 ). To further investigate the interaction between p11 and cPLA 2 in human cells, additional experiments were designed to detect the existence of p11-cPLA 2 complex in cells expressing both p11 and cPLA 2 . Our previous studies revealed the existence of cPLA 2 in the BEAS 2B human bronchial epithelial cell line (23)(24). Using immunoblotting analysis, the expression of p11 was also found in the BEAS 2B cells (see below, Fig. 6). Therefore, we reasoned that the p11-cPLA 2 complex might be detected from the BEAS 2B  (shown in B, n ϭ 3-6), the 3 H-labeled membrane (0.25 nmol of phospholipid) was incubated with 100 ng of purified cPLA 2 in the presence of 20 ng of recombinant human p11 and/or 20 ng of purified bovine annexin II. The reaction was started by the addition of substrate to the reaction mixture, and the assays were incubated at 37°C for 1 h. Release of free fatty acid was analyzed using thin layer chromatography as described under the "Experimental Procedures." cells under appropriate conditions. As shown in Fig. 5, p11 was precipitated from the BEAS 2B cell lysate by anti-human cPLA 2 antibody. This result demonstrates the existence of p11-cPLA 2 complex in the BEAS 2B cells. Attempts to precipitate the complex with a monoclonal antibody to p11 were not successful (data not shown).
Antisense Inhibition of p11 Increases PLA 2 Activity and Arachidonic Acid Release from the BEAS 2B Cells-We have shown that p11 binds to cPLA 2 and inhibits the cPLA 2 activity assayed in vitro. To study further the effect of p11 on PLA 2 activity and AA release in human cells, we used the antisense RNA approach to selectively inhibit the expression of p11 and then to examine the PLA 2 activity and AA release in the BEAS 2B cells. The expression of p11 protein was successfully reduced by the antisense inhibition (Fig. 6). The antisense inhibition of p11 expression did not alter cellular production of annexin II. In addition, the interaction of p11 and of annexin II heavy chain with cPLA 2 from vector control cells and from cells transfected with the p11 antisense vector was studied. Immunoprecipitation of cytosol preparations from vector control cells and from cells transfected with the antisense vector was performed using the polyclonal anti-cPLA 2 antibody. Western blotting was then performed, and the blots were probed with antibody to p11 or to annexin II heavy chain. While the antisense cells contained reduced p11 but comparable amounts of annexin II heavy chain, immunoprecipitation of cytosol with anti-cPLA 2 antibody demonstrated less p11 and less annexin II bound to cPLA 2 in the antisense cells than in vector control cells (Fig. 7). We then determined the PLA 2 activity and AA release from these permanently transfected cells. The in vitro assayed PLA 2 activity in cells transfected with pCI-neo-p11AS vector (91.7 Ϯ 5.8 pmol/mg protein) was significantly higher than in cells transfected with pCI-neo vector alone (46.8 Ϯ 3.4 pmol/mg protein) (p Ͻ 0.01, n ϭ 3). The release of prelabeled AA from cells transfected with pCI-neo-p11AS was studied under two conditions. First, unstimulated or basal release of AA from both cell lines was studied over a period of 4 h after labeling with [ 3 H]AA for 18 h. Without stimulation, cells permanently transfected with vector alone released 68 Ϯ 8% of the arachidonate released from cells transfected with the p11-AS vector (n ϭ 6, p Ͻ 0.01). Second, because cPLA2 may be acti-vated by increases in intracellular calcium concentrations, AA release after treatment with the calcium ionophore A23187 was studied. After treatment with A23187, the release of prelabeled AA from cells transfected with pCL-neo-p11AS was significantly higher than from cells transfected with pCI-neo vector alone (Fig. 8). These results indicate that p11 inhibits PLA 2 activity and thus decreases AA release in normal BEAS 2B cells. Decreased expression of p11 in the antisense cells reduces the inhibitory effect of cPLA 2 by p11, which results in an increase in PLA 2 activity and AA release. DISCUSSION This study reveals a novel mechanism for the regulation of 85-kDa cPLA 2 . We demonstrate that p11, a unique member of the S100 family of calcium-binding proteins, interacts with the  1, 2, and 4, 300 g of crude cytosolic protein; lanes 5, 6, and 8, 75 g of crude cytosolic protein; lanes 3 and 6, no crude cytosolic protein) were preincubated with 10 l of rabbit anti-human cPLA 2 (lanes 3, 4, 7, and 8) or 10 l of rabbit IgG (lanes 2 and 6) in 1 ml of HBSS at 4°C for 30 min. Lanes 1 and 4 contained only the crude cytosolic protein without rabbit anti-human cPLA 2 or rabbit IgG. 25 l of Protein G Plus/Protein A-agarose beads was then added to each sample, and the mixture was incubated at 4°C on a rotating device for 4 h. The beads were collected and washed four times with 1.0 ml of PBSTDS by repeated centrifugation. Protein loading buffer was then added to the beads, and the samples were boiled for 10 min and subjected to SDS-PAGE. The precipitated p11 protein was then detected by Western blotting analysis using mouse anti-human p11 monoclonal antibody and horseradish peroxidase-conjugated goat anti-mouse IgG as described under "Experimental Procedures." The position of p11 protein was indicated.  1-3), or pCI-neo-p11AS containing the antisense p11 cDNA sequence (lanes 4 -6).

FIG. 7. Western blot of p11 and annexin II.
Crude cytosolic protein from cell lysates of BEAS 2B cells permanently transfected with antisense p11 expression vector or with vector alone was immunoprecipitated with rabbit polyclonal anti-cPLA 2 antibody and Protein G/Protein A-agarose. Following electrophoresis of the washed immunoprecipitates on 18% polyacrylamide gels using Tris glycine/SDS buffer, the separated proteins were electrophoretically transferred onto a nitrocellulose membrane that was blocked and then probed with a 1:1000 dilution of either mouse anti-p11 monoclonal antibody or mouse antiannexin II monoclonal antibody. The blots were then probed with a 1:1000 dilution of horseradish peroxidase-labeled goat anti-mouse IgG and detected using the Pierce Super Signal detection system. carboxyl region of cPLA 2 and inhibits the cPLA 2 activity. The interaction between p11 and cPLA 2 was recognized by screening a human cDNA library using a yeast two hybrid system. Further experiments demonstrated that p11 associated with cPLA 2 in vitro. Using natural membrane vesicles as substrate, p11 was found to inhibit the activity of purified cPLA 2 in vitro. The inhibition of cPLA 2 activity by p11 was independent of substrate concentrations. These results demonstrated that p11 directly interacts with cPLA 2 and inhibits the cPLA 2 enzyme activity. Additional experiments were performed to study the role of p11 in intact human cells. The p11-cPLA 2 complex was identified from the BEAS 2B human bronchial epithelial cell line expressing both cPLA 2 and p11. Selective inhibition of p11 expression in the BEAS 2B cells by antisense RNA caused a reduction of cellular p11 and resulted in an increased PLA 2 activity as well as an increased release of prelabeled AA. Taken together, the above in vitro and in vivo observations demonstrate a novel mechanism for the regulation of cPLA 2 by the S100 protein family.
p11, a Unique Member of the S100 Family of Calcium-binding Proteins, Interacts with the COOH-terminal Region of cPLA 2 -The S100 proteins are a group of low molecular mass (approximately 10 -12 kDa) acidic Ca 2ϩ -binding proteins, so named after the solubility of the first isolated protein in 100% saturated ammonium sulfate (42)(43)(44). The most striking conserved feature of these proteins is the presence of EF-hands (a term representing the two ␣-helices, E and F, and the intervening Ca 2ϩ binding loop), which function as the Ca 2ϩ -binding sites. While other Ca 2ϩ -binding proteins such as the parvalbumin protein family contain three EF-hands and the calmodulin protein family and troponin C protein family contain four EFhands per monomer molecule, the S100 proteins have two EF-hands (NH 2 -terminal and COOH-terminal EF-hands). The evident amino acid sequence homology, conservation of calcium-binding sites, and similarities of gene organization suggest that this group of proteins evolved from a common ancestral EF-hand gene (42)(43)(44). The biological functions of the S100 proteins are less clear. No specific enzymatic property has been ascribed to any of the proteins to date. The binding to calcium induces a conformational change in the S100 proteins, and this may then affect the secondary effector proteins. This mode of protein-protein interaction and modulation of the activity of the secondary effector protein is similar to that seen with calmodulin, another family of calcium-binding proteins containing the EF-hands (42)(43)(44). As the distribution of particular S100 proteins is dependent on specific cell types, the S100 proteins may be involved in transducing the signal of an increase in intracellular calcium in a cell type-specific fashion (42)(43)(44). p11 was first isolated as a potential substrate for the epidermal growth factor-receptor tyrosine kinase (45). Although p11 shares considerable sequence homology with the S100 family, it does not bind calcium because of amino acid deletions and substitutions in the two EF-hand motifs (46). This is an exception to the general Ca 2ϩ -binding properties displayed by other S100 proteins. Instead, p11 interacts with another protein, the 36-kDa annexin II (47)(48). The p11 and annexin II proteins are linked to each other in a tight heterotetrameric complex that is present in many eukaryotic tissues and cells. p11 itself forms a tight dimer that bridges two annexin II molecules thus mediating the formation of a heterotetrameric annexin II 2 p11 2 complex. While annexin II exists as a monomer or binds to p11 forming the heterotetrameric complex, p11 has often been found as part of a complex with annexin II in many p11positive tissues where annexin II is present in equimolar amounts or in excess (47)(48). However, this phenomenon may vary with different cell types. The expression of p11 and annexin II is not always coordinated, and the ratio of p11 to annexin II varies with different cell types (49). In the F9 teratocarcinoma cells, there exists a significant amount of p11 but almost no annexin II both at the RNA and protein levels, with the unbound p11 distributed in the cytoplasm (50). Differentiation of these cells results in a large increase in annexin II and the formation of the annexin II 2 p11 2 heterotetramer (50). The formed heterotetrameric complex becomes associated with plasma membrane and submembranous cytoskeleton through the Ca 2ϩ -dependent interaction of annexin II with the negatively charged phospholipids and components of the cytoskeleton. The interaction of annexin II with actin and membrane phospholipids is dependent upon Ca 2ϩ and p11. p11 binding alters the Ca 2ϩ and/or phospholipid-binding properties of annexin II, with the tetrameric complex having a more than 100-fold increased affinity toward Ca 2ϩ /phospholipid compared with monomeric annexin II (51).
It remains unknown whether p11 is also able to associate with any other protein. Our results demonstrate that the 85-kDa cPLA 2 , a unique calcium-binding enzyme, is a novel protein that interacts with p11. Results from the two hybrid screen demonstrated that p11 associated with cPLA 2 in vivo. While the exact amino acid residues involved in the interaction is unknown, only the COOH-terminal region of the cPLA 2 is found to interact with the p11. The association of p11 and cPLA 2 was also found under in vitro conditions. By using purified human cPLA 2 protein and the in vitro translated human p11 protein, we found that p11 and cPLA 2 interact with each other in vitro. Furthermore, the formation of p11-cPLA 2 complex was also identified from cells expressing both p11 and cPLA 2 by immunoprecipitation. Taken together, these results establish a direct interaction between p11 and the COOH region of cPLA 2 . In addition to annexin II, cPLA 2 represents the only other protein that binds to p11.
p11 Inhibits cPLA 2 Activity and Decreases the Release of Prelabeled AA from BEAS 2B Cells-To examine how the binding of p11 to cPLA 2 may alter the catalytic activity of cPLA 2 , we studied the effects of recombinant p11 on the activity of purified human cPLA 2 under in vitro assay conditions. Two aspects of the assays deserve comment. The first aspect is the use of membrane vesicles containing a mixture of phospholipids as cPLA 2 substrate to mimic the in vivo interaction between cPLA 2 , p11, and membrane. This method was developed by Clark and colleagues (8) who used the U937 cell membrane vesicles as cPLA 2 substrate. In this study, the membrane vesicles labeled with [ 3 H]AA were prepared from the BEAS 2B cells. Another aspect of the assay is the relatively low Ca 2ϩ concentration used in the assay. As the cPLA 2 is activated by micromolar concentrations of Ca 2ϩ and the physiological cellular Ca 2ϩ level is normally at micromolar level as well (0.1 M in normal resting cells and 0.3-1.0 M in activated cells), we used the physiologically relevant Ca 2ϩ concentration (0.5 M free Ca 2ϩ in the Ca 2ϩ -EGTA buffer) for all the assays. Under these assay conditions, we found that p11 inhibited cPLA 2 activity in a dose-dependent fashion (Fig. 2), and this inhibitory effect is not dependent on substrate concentration (Fig. 3A). These results demonstrate that p11 binds to cPLA 2 and inhibits the cPLA 2 enzyme activity in vitro. Although the mechanism for the inhibition of cPLA 2 by p11 is not yet known, it is unlikely that this effect is due to the interaction between p11 and phospholipid substrate because 1) p11 does not bind to phospholipids (46 -47) and 2) the p11-mediated cPLA 2 inhibition is not dependent on substrate concentration. Instead, we demonstrate a direct interaction between p11 and cPLA 2 , and it is likely that the binding of p11 to cPLA 2 results in the inhibition of cPLA 2 activity. However, it remains to be determined how the binding of p11 to cPLA 2 inhibits the cPLA 2 activity. The possibilities may include blockage of the catalytic process of cPLA 2 , conformational change of cPLA 2 , or alteration of calciumdependent association of cPLA 2 with membrane substrate.
To study further the effect of p11 on PLA 2 activity and AA release in human cells, we used the antisense RNA approach to selectively inhibit the expression of p11 and then to examine the PLA 2 activity and AA release in the BEAS 2B cells. The release of prelabeled AA from cells with reduced p11 expression was significantly higher than from the control cells (Fig. 7). The crude cytosolic fraction isolated from the p11-reduced cells also showed an increased PLA 2 activity. These results demonstrate that p11 inhibits PLA 2 activity and decreases AA release in the BEAS 2B cells. Reduced p11 expression reverses the inhibitory effect of p11 on cPLA 2 and thus increases the PLA 2 activity and increases the release of [ 3 H]AA from the [ 3 H]AA-prelabeled cells. These results suggest that p11 decreases the release of AA for eicosanoid production in intact cells by inhibiting cellular cytosolic PLA 2 activity. p11-induced cPLA 2 Inhibition Does Not Depend on Annexin II-It is well known that p11 binds to annexin II, a member of the annexin protein family, which includes at least 13 relatively abundant and structurally related proteins (47)(48)(52)(53)(54)(55)(56)(57). The annexins are widely distributed in eukaryotic cells. The roles of the annexins are not well understood, but they have been suggested to function in a broad range of physiological processes including inhibition of PLA 2 (54). The biochemical hallmark of members of the annexin protein family is their Ca 2ϩ -dependent binding to negatively charged phospholipids, most likely reflecting their association with cellular membranes in vivo. This common property is mediated by the conserved Ca 2ϩ /phospholipid-binding sites that comprise four to eight repeats of 70 -80 amino acid segments with a highly conserved 17-amino acid consensus sequence in each repeat. Typically, the annexins are monomeric proteins with the exception of annexin II which can exist as monomer or heterotetramer. The annexin II heterotetramer is composed of two subunits of 36-kDa annexin II and two subunits of p11 (47)(48). The annexin II consists of two functional domains. The aminoterminal domain contains the first 30 amino acids and includes the binding site for p11 and both the serine and tyrosine phosphorylation sites. The first NH 2 -terminal 14 amino acids of annexin II molecule bind to p11 forming an amphipathic ␣-helix upon complex formation. The serine and tyrosine residues can be phosphorylated by protein kinase C and the tyrosine kinase encoded by the src oncogene, respectively (45). The remaining carboxyl domain of annexin II contains four repeats of the conserved amino acid segments that bind to Ca 2ϩ , phospholipids, and F-actin. Although p11 does not directly bind to calcium and phospholipids, annexin II has all Ca 2ϩ -and lipidbinding properties typical of members of the annexin family.
For the treatment of inflammatory diseases, considerable research efforts have been made to identify selective inhibitors of PLA 2 . The annexins, formerly called lipocortins or calpactins, were found to inhibit cellular PLA 2 activity in various cell types (54,58). In vitro studies using the 14-kDa PLA 2 demonstrated that the PLA 2 activity was inhibited by annexins, and this led to the hypothesis that the inhibition of 14-kDa PLA 2 by annexins was the mechanism of the anti-inflammatory action for glucocorticoids (54, 58 -61). However, subsequent studies failed to demonstrate any direct interaction between the 14-kDa PLA 2 and annexins. Instead, it was found that the in vitro inhibition of 14-kDa PLA 2 by annexins could be overcome by increasing the substrate concentrations (41,(62)(63)(64). The extent of inhibition was more closely related to the inhibitor:substrate rather than the inhibitor:enzyme ratio, and the kinetics of inhibition were not compatible with a simple competitive or noncompetitive pattern. Based on these observations, the inhibitory action of annexins toward 14-kDa PLA 2 was ascribed to substrate sequestration rather than to direct interaction with the 14-kDa cPLA 2 .
We have demonstrated that p11 interacts with the COOHterminal region of cPLA 2 and inhibits the cPLA 2 enzyme activity. As the inhibition of cPLA 2 by p11 is independent of substrate concentration, this effect is likely due to a direct interaction between p11 and cPLA 2 . Because p11 is often associated with the 36-kDa annexin II to form annexin II heterotetramer in cells, we also examined whether the inhibitory effect of p11 on cPLA 2 activity may be altered by the presence of annexin II. The effect of annexin II alone or combined annexin II and p11 on cPLA 2 activity was studied under the in vitro conditions (Fig. 3B). We found that annexin II alone exhibited some inhibitory effect on cPLA 2 activity. This effect was as expected because annexin II might sequester substrate from its access to various PLA 2 s under the in vitro assay conditions. While annexin II alone exhibited certain inhibitory effect on cPLA 2 activity, this effect was less remarkable than the inhibition by p11. The combination of annexin II and p11 appeared to exhibit additive inhibition. These results indicated that annexin II does not significantly alter the inhibitory effect of p11 on cPLA 2 activity. p11 itself is sufficient to bind and inhibit cPLA 2 .