Inhibition of Cell Growth and Spreading by Stomach Cancer-associated Protein-tyrosine Phosphatase-1 (SAP-1) through Dephosphorylation of p130cas*

Tetsuya NoguchiDagger, Masahiro Tsuda, Hitoshi Takeda, Toshiyuki Takada, Kenjiro Inagaki, Takuji Yamao, Kaoru Fukunaga, Takashi Matozaki§, and Masato Kasuga

From the Second Department of Internal Medicine, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan and § Biosignal Research Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-Machi, Maebashi 371-8512, Japan

Received for publication, August 9, 2000, and in revised form, January 11, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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SAP-1 (stomach cancer-associated protein-tyrosine phosphatase-1) is a transmembrane-type protein-tyrosine phosphatase that is abundant in the brain and certain cancer cell lines. With the use of a "substrate-trapping" approach, p130cas, a major focal adhesion-associated phosphotyrosyl protein, has now been identified as a likely physiological substrate of SAP-1. Expression of recombinant SAP-1 induced the dephosphorylation of p130cas as well as that of two other components of the integrin-signaling pathway (focal adhesion kinase and p62dok) in intact cells. In contrast, expression of a substrate-trapping mutant of SAP-1 induced the hyperphosphorylation of these proteins, indicating a dominant negative effect of this mutant. Overexpression of SAP-1 induced disruption of the actin-based cytoskeleton as well as inhibited various cellular responses promoted by integrin-mediated cell adhesion, including cell spreading on fibronectin, growth factor-induced activation of extracellular signal-regulated kinase 2, and colony formation. Finally, the enzymatic activity of SAP-1, measured with an immunocomplex phosphatase assay, was substantially increased by cell-cell adhesion. These results suggest that SAP-1, by mediating the dephosphorylation of focal adhesion-associated substrates, negatively regulates integrin-promoted signaling processes and, thus, may contribute to contact inhibition of cell growth and motility.

    INTRODUCTION
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Dynamic changes in the extent of tyrosine phosphorylation of cellular proteins are fundamental to signal transduction pathways that regulate cell growth and differentiation, the cell cycle, tissue morphogenesis, and cytoskeletal organization. Tyrosine phosphorylation is tightly controlled by coordination of the activities of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs)1 (1). Although much progress has been made in our understanding of the structure, function, and regulation of protein-tyrosine kinases, PTPs are less well characterized.

Similar to protein-tyrosine kinases, PTPs can be divided into two structurally distinct subgroups: cytoplasmic PTPs and transmembrane-type (receptor-like) PTPs (RPTPs) (2). Despite their marked diversity in overall structure, all PTPs possess a core sequence (I/V)HCXAGXXR(S/T)G that contains conserved cysteine and arginine residues critical for enzymatic activity (3). The cellular functions of PTPs are thought to be determined by their substrate specificity, subcellular localization, and interaction with other signaling molecules.

Although initial observations of the effects of overexpression of PTPs suggested that these enzymes might simply counteract the signaling events elicited by protein-tyrosine kinases, more recent genetic and biochemical evidence indicates that PTPs play more complex roles in a wide range of cellular activities (4, 5). For example, SHP-1 and SHP-2, two Src homology 2 domain-containing cytoplasmic PTPs play negative and positive regulatory roles, respectively, in protein-tyrosine kinase signaling (6). Members of the band 4.1 family of cytoplasmic PTPs, including PTPH1 and PTPMEG, are thought to regulate cytoskeletal reorganization (7, 8). In addition, the RPTP PTPalpha affects cell-substratum adhesion and cellular transformation by regulating the activity of the protein-tyrosine kinase Src (9). Finally, members of the LAR family of RPTPs, including PTPsigma (10) and PTPdelta (11) as well as Drosophila DLAR and the related DPTP69D (12, 13), are essential for axon guidance or neuronal differentiation in the developing nervous system.

We have previously cloned a human RPTP termed SAP-1 (stomach cancer-associated PTP-1) (14). This enzyme contains a single catalytic domain in the cytoplasmic region, a single transmembrane domain, and eight fibronectin type III-like domains in the extracellular region. SAP-1 belongs to the class 2 subfamily of RPTPs (15), which includes HPTPbeta (15), DEP-1 (16), PTP-U2 (17), and Drosophila DPTP10D (18, 19). The observations that the fibronectin type III-like domain is present in many neural cell adhesion molecules and that SAP-1 is abundant in the brain suggest that this PTP plays a role in neural cell-cell adhesion signaling (14). SAP-1 is also abundant in a subset of pancreatic and colorectal cancer cell lines and tissues but not in their normal counterparts (14, 20). Furthermore, the SAP-1 gene is located on the long arm of human chromosome 19, at position q13.4, which is close to the locus of the gene for carcinoembryonic antigen at 19q13.2 (14). Expression of SAP-1 may thus also be associated with cancer development. However, the biological roles of this PTP remain unknown.

The identification of physiological substrates of PTPs provides important insight into the functions of these enzymes. However, substrate identification for this class of enzymes has proved problematic because of the low affinity and transient nature of the interaction of PTPs with their substrates. A recently developed substrate-trapping strategy has overcome this limitation and greatly facilitated isolation of specific substrates of PTPs (21-24). With the use of this strategy, we have now identified p130cas, a major phosphotyrosyl protein that is localized to focal adhesions (FAs), as a potential substrate of SAP-1. We also provide evidence that SAP-1 negatively regulates various integrin-promoted cellular responses and that the enzymatic activity of this PTP is up-regulated by cell-cell adhesion.

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Expression Vectors-- The cDNAs encoding a catalytically inactive mutant of SAP-1 (SAP-1C/S), in which Cys1022 is replaced by Ser, and a substrate-trapping mutant of SAP-1 (SAP-1D/A), in which Asp988 is replaced by Ala, were generated by site-directed mutagenesis with human SAP-1 cDNA as a template and a transformer site-directed mutagenesis kit (CLONTECH). The full-length wild-type and mutant SAP-1 cDNAs were then inserted separately into the HindIII site of the pRc/CMV expression vector (Invitrogen). The pSRalpha vector encoding hemagglutinin (HA)-tagged p130cas was kindly provided by H. Hirai (University of Tokyo), the pBabe vector encoding Myc-tagged paxillin-alpha was provided by H. Sabe (Osaka Bioscience Institute), the pRc/CMV vector encoding HA-tagged focal adhesion kinase (FAK) was provided by S. K. Hanks (Vanderbilt University, Nashville, TN), and the pcDNA3 vector encoding HA-tagged extracellular signal-regulated kinase 2 (ERK2) was provided by J. S. Gutkind (National Institutes of Health, Bethesda, MD). The pRc/CMV vector encoding HA-tagged mouse p62dok was generated as described (25).

Cells, Antibodies, and Transfection-- Chinese hamster ovary (CHO) cell lines stably expressing wild-type SAP-1 (SAP-1WT) or SAP-1C/S were generated as described previously (26), and the expression level of each SAP-1 protein was determined by immunoblot analysis with polyclonal antibodies to SAP-1 as described below. CHO-K1 cells and the established cell lines were maintained in Ham's F-12 medium supplemented with 10% fetal bovine serum. 293 human embryonic kidney cells, Panc-1 cells, WiDr cells, Swiss 3T3 cells, and NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Transfection of CHO-K1 cells or 293 cells (~2 × 105 cells/60-mm dish) was performed with the use of a CellPhect Transfection Kit (Amersham Pharmacia Biotech).

Rabbit polyclonal antibodies to SAP-1 (14) and the mouse monoclonal antibody (mAb) 3G5 to SAP-1 were generated in response to a glutathione S-transferase (GST) fusion protein containing the cytoplasmic region of SAP-1 in our laboratory. Rabbit polyclonal antibodies to p62dok were described previously (25). The mAb 12CA5 to the HA epitope tag and the mAb 9E10 to the Myc tag were purified from the culture supernatants of mouse hybridoma cells. Mouse mAbs to p130cas, to paxillin, and to beta -catenin were obtained from Transduction Laboratories; horseradish peroxidase-conjugated mouse mAb PY20 to phosphotyrosine and rabbit polyclonal antibodies to p130cas or to FAK were from Santa Cruz Biotechnology; rabbit polyclonal antibodies that react specifically with tyrosine-phosphorylated ERK or with total ERK protein were from New England BioLabs; a mouse mAb to vinculin was from Sigma; fluorescein isothiocyanate-conjugated sheep polyclonal antibodies to mouse immunoglobulin and Texas red-conjugated donkey polyclonal antibodies to rabbit immunoglobulin were from Amersham Pharmacia Biotech.

Immunoprecipitation and Immunoblot Analysis-- Cells were thawed on ice in 1 ml of ice-cold lysis buffer (20 mM Tris-HCl (pH 7.6), 140 mM NaCl, 1 mM EDTA, 1% (v/v) Nonidet P-40) containing 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride, aprotinin (10 µg/ml), and 1 mM sodium vanadate. The cell lysates were centrifuged at 10,000 × g for 15 min at 4 °C, and the resulting supernatants were subjected to immunoprecipitation and immunoblot analysis. For immunoprecipitation, the supernatants were incubated for 3 h at 4 °C with antibody-coupled protein G-Sepharose beads (20 µl of beads) (Amersham Pharmacia Biotech). The beads were washed three times with 1 ml of WG buffer (50 mM Hepes-NaOH (pH 7.6), 150 mM NaCl, 0.1% (v/v) Triton X-100) and then suspended in Laemmli sample buffer. Immunoblot analysis with various antibodies was performed with the use of the ECL detection system (Amersham Pharmacia Biotech).

Expression and Purification of Recombinant PTPs-- The cytoplasmic regions of SAP-1WT, SAP-1C/S, and SAP-1D/A were produced as GST fusion proteins. The polymerase chain reaction was performed with wild-type or mutant SAP-1 cDNA as template and with 5'-TAGGATCCCCAGGGGACATCCCAGCTGAAG (nucleotides 2436-2457 of SAP-1 cDNA) and 5'-TCGAATTCGGGCTGCCGACCCAGCCCCCTCG (nucleotides 3400-3422) as sense and antisense primers, respectively. The amplification products were digested with BamHI and EcoRI and inserted in-frame into the BamHI and EcoRI sites of pGEX-2T (Amersham Pharmacia Biotech). The encoded GST fusion proteins were then expressed in Escherichia coli and purified with the use of glutathione-Sepharose beads (Amersham Pharmacia Biotech). The recombinant full-length SHP-2 was prepared as described previously (26).

Substrate Trapping-- Panc-1 cells in one 100-mm dish were treated with 100 µM pervanadate for 30 min and then lysed in 1 ml of buffer A (20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100) containing 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 10 µM leupeptin, aprotinin (10 µg/ml), and 5 mM iodoacetic acid. After incubation of the lysate for 30 min at 4 °C with gentle agitation, dithiothreitol was added to a final concentration of 10 mM to inactivate any unreacted iodoacetic acid. The lysate was then centrifuged at 100,000 × g for 30 min at 4 °C, and the resulting supernatant was incubated for 2 h at 4 °C with glutathione-Sepharose beads conjugated with 10 µg of recombinant SAP-1 protein. The beads were washed three times with 1 ml of buffer A, and bound material was subjected to immunoblot analysis. In some experiments, 1 mM orthovanadate was included during the incubation of beads with lysate.

In-blot Dephosphorylation Assay-- Lysates from pervanadate-treated Panc-1 cells were subjected to immunoprecipitation; the resultant precipitates containing tyrosine-phosphorylated proteins were fractionated by SDS-polyacrylamide gel electrophoresis, and the separated proteins were transferred to a nitrocellulose filter. The dephosphorylation reaction was performed by incubating the filter in the absence or presence of recombinant SAP-1 or SHP-2 for 20 min at 30 °C in a solution containing 50 mM Hepes-NaOH (pH 7.1), 150 mM NaCl, 10 mM dithiothreitol, and 2 mM EDTA. The filter was then washed for 5 min at room temperature with phosphate-buffered saline (PBS) containing 500 mM NaCl, 0.5% (w/v) SDS, and 0.1% (v/v) Triton X-100. The extent of tyrosine phosphorylation of each protein was determined by immunoblot analysis with mAb PY20 to phosphotyrosine.

Cell-spreading Assay-- Cells were detached from the culture dish by treatment with 0.025% trypsin, collected by centrifugation, and washed once with serum-free Ham's F-12 medium. The cells were replated in serum-free Ham's F-12 medium at a density of 1 × 105 cells/ml on 60-mm culture dishes coated with fibronectin (10 µg/ml) (Sigma) or poly-L-lysine (10 µg/ml) (Sigma). The cells were then incubated for 2 h at 37 °C in a humidified incubator containing 5% CO2, after which they were examined under a light microscope equipped with phase-contrast optics (model IX 70; Olympus), and random fields were photographed at a total magnification of 200×.

Generation of and Infection with Recombinant Adenoviruses-- The cDNAs encoding SAP-1WT or SAP-1C/S were cloned separately into the SwaI site of pAxCAwt (27), which contains the CAG promoter, and the resulting constructs were introduced together with DNA-terminal protein complex by transfection into 293 cells. The resulting recombinant adenoviruses were screened by immunoblot analysis and cloned by limiting dilution. Adenoviral vectors were propagated by a standard procedure, purified by two rounds of CsCl density gradient centrifugation, and then titrated with a limiting dilution assay in 293 cells. Swiss 3T3 cells seeded on glass coverslips in a 6-well plate were infected with viruses at 37 °C for 1 h, with brief agitation every 20 min, and were then exposed to normal growth medium.

Immunofluorescence Staining-- Cells were washed with PBS, fixed with 3.7% formaldehyde for 20 min, permeabilized with 0.5% (v/v) Triton X-100 in PBS for 5 min, and incubated for 2 h with TBS-T (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.05% (v/v) Tween 20) containing 5% (w/v) nonfat dry milk, 10% fetal bovine serum, and 1% (w/v) bovine serum albumin. The cells were then incubated for 1 h at room temperature with mAb 3G5 to SAP-1 (1 µg/ml) or with polyclonal antibodies to SAP-1 (1 µg/ml), together with either rhodamine-labeled phalloidin (0.1 µg/ml) (Sigma) or a mAb to vinculin (20 µg/ml). Cells were then washed twice with PBS and incubated with fluorescein isothiocyanate- or Texas red-conjugated secondary antibodies for 30 min. After washing three times with PBS, the cells were examined under a laser-scanning confocal microscope (model MRC-1024; Bio-Rad), and built-up images were constructed.

Assay of ERK2 activation-- Transfected 293 cells (60-mm dishes) were deprived of serum for 12 h and then incubated for 5 min with 10 µM lysophosphatidic acid (LPA) (Sigma) or epidermal growth factor (EGF) (100 ng/ml) (Calbiochem) or for 10 min with 1 µM 12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma). Cells were then lysed in 400 µl of a solution containing 50 mM Hepes-NaOH (pH 7.8), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 0.1% (v/v) Triton X-100, 20 mM beta -glycerophosphate, 100 mM NaF, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, aprotinin (10 µg/ml), and 1 mM sodium vanadate. Recombinant ERK2 was immunoprecipitated with mAb 12CA5 to the HA tag and then subjected to immunoblot analysis with antibodies specific for activated ERK or for total ERK protein. The extent of ERK2 phosphorylation was quantified by scanning densitometry with the NIH Image program.

Colony Formation Assay-- CHO-K1 cells or NIH 3T3 cells (~2 × 105 cells/60-mm dish) were transiently transfected with 3 µg of pRc/CMV containing (or not) SAP-1WT or SAP-1C/S cDNA. After 48 h, transfected cells were diluted 1:20 with normal growth medium supplemented with G418 (400 µg/ml) and cultured in 100-mm dishes. The medium was changed every 3 days, and, after 14 days, cells that had formed colonies were fixed with 3.7% formaldehyde and stained with crystal violet. The efficiency of colony formation was quantified by scanning densitometry with the NIH Image program.

Determination of PTP Activity-- The enzymatic activity of recombinant SAP-1 was assayed with p-nitrophenylphosphate (pNPP) as a substrate as described previously (14). Cellular SAP-1 activity immunopurified with mAb 3G5 was assayed with pNPP as described previously (28).

Reproducibility-- All data presented are representative of at least three independent experiments.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Identification of p130cas as a Potential Substrate of SAP-1-- To identify physiological substrates of SAP-1, we first generated two types of SAP-1 mutant: a catalytically inactive mutant, in which the invariant Cys1022 residue is replaced by Ser, and a substrate-trapping mutant, in which the invariant Asp988 residue is replaced by Ala. The latter mutant was expected to be a more powerful tool than was the former because it should not only be catalytically inactive but also retain the ability to bind SAP-1 substrates (22). We then prepared GST fusion proteins that contain the catalytic domains of wild-type SAP-1 (GST-SAP-1WT), catalytically inactive SAP-1 (GST-SAP-1C/S), or the substrate-trapping mutant of SAP-1 (GST-SAP-1D/A). Each recombinant protein was expressed in and purified from bacteria as a 60-kDa polypeptide, as revealed by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining (Fig. 1A).


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Fig. 1.   Association of p130cas and paxillin with a substrate-trapping mutant of SAP-1. A, expression and purification of recombinant SAP-1 proteins. GST fusion proteins containing the cytoplasmic region of either wild-type SAP-1 (GST-SAP-1WT), a catalytically inactive mutant of SAP-1 (GST-SAP-1C/S), or a substrate-trapping mutant of SAP-1 (GST-SAP-1D/A) were expressed in bacteria, purified with the use of glutathione-Sepharose beads, and subjected to SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining. The position of each recombinant protein is indicated by the arrow labeled SAP-1. B, lysates prepared from pervanadate-treated Panc-1 cells were incubated for 2 h at 4 °C with glutathione-Sepharose beads conjugated with each recombinant SAP-1 protein or with GST alone. The bead-bound proteins were then subjected to immunoblot analysis with horseradish peroxidase-conjugated mAb PY20 to phosphotyrosine (alpha PY). The positions of bound phosphotyrosyl proteins are indicated. C, effect of vanadate on the association between mutant SAP-1 and phosphotyrosyl proteins. Cell lysates prepared as in B were incubated with GST-SAP-1C/S or GST-SAP-1D/A in the absence (-) or presence (+) of 1 mM orthovanadate, after which the bead-bound proteins were subjected to immunoblot analysis with mAb PY20. The positions of bound phosphotyrosyl proteins are indicated. The immunoreactive material apparent immediately above p55 (B and C) and p75-85 (C) in the GST-SAP-1D/A lanes corresponds to cross-reactive polypeptides of unknown origin. D, recognition of p125-135 and p75-85 by specific antibodies to p130cas and to paxillin, respectively. The precipitates prepared as described in B were subjected to immunoblot analysis with polyclonal antibodies to p130cas (alpha Cas) or with a mAb to paxillin (alpha paxillin), as indicated.

Whereas GST-SAP-1WT exhibited substantial catalytic activity with the artificial substrate pNPP, GST-SAP-1C/S and GST-SAP-1D/A possessed virtually no activity (Fig. 2A). Each of the three recombinant proteins bound to glutathione-Sepharose beads was then incubated with lysates prepared from pervanadate-treated Panc-1 cells, which express SAP-1 (14). Immunoblot analysis with the mAb PY20 to phosphotyrosine revealed that GST-SAP-1D/A, but not GST-SAP-1WT or GST alone, precipitated several phosphotyrosyl proteins with molecular sizes of 190, 125-135, 75-85, and 55 kDa (hereafter referred to as p190, p125-135, p75-85, and p55, respectively) (Fig. 1B). GST-SAP-1C/S precipitated a similar group of proteins, although to a much lesser extent than did GST-SAP-1D/A (Fig. 1, B and C). The amounts of p190, p125-135, p75-85, and p55 bound to GST-SAP1D/A were markedly reduced by incubation of cell lysates with the recombinant protein in the presence of vanadate, a potent inhibitor of PTPs; in contrast, the amounts of these proteins bound to GST-SAP-1C/S were substantially increased by incubation with vanadate (Fig. 1C). Vanadate ions interact directly with the thiolate anion of the cysteine residue in the catalytic core sequence of PTPs and thereby block enzyme-substrate association (29, 30). These results thus suggest that all of the detected phosphotyrosyl proteins might bind, either directly or indirectly, to the active site of SAP-1.


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Fig. 2.   Dephosphorylation of p130cas by SAP-1 in vitro. A, catalytic activities of recombinant wild-type and the mutant SAP-1 proteins, a GST fusion protein containing full-length SHP-2 (GST-SHP-2), and GST with pNPP as the substrate. Data are expressed as absorbance units at 410 nm and are the means of triplicates from a representative experiment. B, PTP activities toward p130cas, paxillin, and beta -catenin. Lysates of pervanadate-treated Panc-1 cells were subjected to immunoprecipitation with antibodies to p130cas (top panel), to paxillin (middle panel), or to beta -catenin (bottom panel). The resultant precipitates were fractionated by SDS-polyacrylamide gel electrophoresis, after which the separated proteins were transferred to a nitrocellulose membrane and incubated with GST-SAP-1WT (50 ng/ml), GST-SHP-2 (5 µg/ml), or GST (5 µg/ml) for 20 min at 30 °C. The extent of tyrosine phosphorylation of each protein was determined by immunoblot analysis with mAb PY20 to phosphotyrosine (alpha PY).

To identify p125-135, we subjected the proteins precipitated by GST-SAP-1D/A to immunoblot analysis with antibodies specific for either FAK, Ras GTPase-activating protein, or p130cas, all of which are in the size range of 125-135 kDa and are phosphorylated on tyrosine residues. Of these antibodies, the polyclonal antibodies to p130cas specifically recognized a 125- to 135-kDa protein that bound to GST-SAP-1D/A (Fig. 1D). A mAb to p130cas that recognizes a distinct epitope of the protein also reacted with p125-135 (data not shown). These results suggest that p125-135 might be p130cas. We also tested the ability of antibodies to paxillin, cortactin, or SHP-2 to recognize p75-85. A mAb to paxillin specifically reacted with a 75-85-kDa protein that bound to GST-SAP-1D/A (Fig. 1D), suggesting that p75-85 might be paxillin. However, we cannot exclude the possibility that the p125-135 and p75-85 bands contain additional unidentified proteins. The identities of p190 and p55 remain unknown.

We next examined whether SAP-1 dephosphorylates p130cas and paxillin in vitro. The catalytic activity of GST-SAP-1WT with pNPP as substrate was markedly greater than that of the recombinant SHP-2 used as a control (Fig. 2A). Tyrosine-phosphorylated forms of p130cas and paxillin immunopurified from pervanadate-treated Panc-1 cells were also incubated with equal amounts of activity of GST-SAP-1WT and GST-SHP-2, after which their phosphorylation status was determined. GST-SAP-1WT effectively dephosphorylated p130cas and, to a lesser extent, paxillin; in contrast, GST-SHP-2 or GST alone had virtually no effect on the extent of phosphorylation of these proteins (Fig. 2B). The human RPTP PTPkappa dephosphorylates beta -catenin (31); however, SAP-1 did not exhibit detectable catalytic activity with beta -catenin as substrate (Fig. 2B). Together, these results indicate that SAP-1 selectively dephosphorylates p130cas and, to a lesser extent, paxillin in vitro.

We also examined whether SAP-1 dephosphorylates p130cas and paxillin in intact cells. We subjected 293 cells to transient transfection with expression vectors encoding SAP-1 and either HA-tagged p130cas (HA-Cas) or Myc epitope-tagged paxillin-alpha and then evaluated the extent of tyrosine phosphorylation of each tagged recombinant protein. Wild-type SAP-1 (SAP-1WT) expressed in these cells possessed substantial catalytic activity, whereas the catalytically inactive mutant (SAP-1C/S) or the substrate-trapping mutant (SAP-1D/A) did not, as revealed by an immunocomplex phosphatase assay using mAb 3G5 to SAP-1 (data not shown). Coexpression of SAP-1WT, but not that of SAP-1C/S, markedly reduced the extent of tyrosine phosphorylation of HA-Cas (Fig. 3A), suggesting that SAP-1 dephosphorylates p130cas in intact cells. In contrast, coexpression of SAP-1D/A markedly increased the extent of tyrosine phosphorylation of HA-Cas. The presence of equal amounts of recombinant SAP-1 proteins in the different transfected cells was confirmed by immunoblot analysis with antibodies to SAP-1 (Fig. 3F). Given that substrate-trapping mutants of PTPs often induce hyperphosphorylation of the corresponding substrates (21-24), these results indicate that p130cas serves as a direct substrate of SAP-1 in vivo. Coexpression of SAP-1WT also reduced the extent of tyrosine phosphorylation of paxillin-alpha ; however, the substrate-trapping mutant did not increase the extent of tyrosine phosphorylation of this protein (Fig. 3B). Thus, paxillin might not be a physiological substrate of SAP-1.


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Fig. 3.   Effects of overexpression of SAP-1 on tyrosine phosphorylation of p130cas (A), paxillin (B), FAK (C), p62dok (D), and ERK2 (E). 293 cells were transiently transfected with 1 µg of pSRalpha encoding HA-tagged p130cas (A), pBabe encoding Myc epitope-tagged paxillin-alpha (B), pRc/CMV encoding HA-tagged FAK (C) or HA-tagged p62dok (D), or pcDNA3 encoding HA-tagged ERK2 (E) together with 4 µg either of pRc/CMV encoding SAP-1WT (WT), SAP-1C/S (C/S), or SAP-1D/A (D/A) or of the empty vector (Vector). Forty-eight hours after transfection, whole cell lysates were prepared and subjected to immunoprecipitation (IP) with mAb 12CA5 to the HA tag (alpha HA) or with mAb 9E10 to the Myc tag (alpha Myc), as indicated. The resulting precipitates were then subjected to immunoblot analysis either with horseradish peroxidase-conjugated mAb PY20 to phosphotyrosine (alpha PY) (A-D, upper panels) or with polyclonal antibodies specific for tyrosine-phosphorylated ERK (alpha pMAPK) (E, upper panel). Duplicate samples were subjected to immunoblot analysis with antibodies to p130cas (alpha Cas) (A, lower panel), to paxillin (alpha paxillin) (B, lower panel), to FAK (alpha FAK) (C, lower panel), to p62dok (alpha Dok) (D, lower panel), or to total ERK protein (alpha MAPK) (E, lower panel) to determine the amounts of each protein in the immunoprecipitates. F, total cell lysates were also subjected to immunoblot analysis with polyclonal antibodies to SAP-1 (alpha SAP-1) to determine the amount of each SAP-1 protein expressed. The positions of p130cas (Cas), paxillin, FAK, p62dok (Dok), ERK2, and the phosphorylated forms of each of these proteins (Cas-P, Paxillin-P, FAK-P, Dok-P, and ERK2-P) as well as of SAP-1 are indicated.

Effects of Overexpression of Wild-type or Mutant SAP-1 on Tyrosine Phosphorylation of FA-associated Proteins-- p130cas undergoes tyrosine phosphorylation during integrin-mediated cell adhesion and mediates the assembly of FAs (32, 33). If SAP-1-mediated dephosphorylation of p130cas induces the disassembly of FAs, then it might also affect the tyrosine phosphorylation status of other FA-associated proteins. To test this hypothesis, we examined the effects of SAP-1 overexpression on the tyrosine phosphorylation of two other components of the integrin-signaling pathway, FAK and p62dok (25, 34). Coexpression of SAP-1WT, but not that of SAP-1C/S, substantially reduced the extent of tyrosine phosphorylation of HA-tagged forms of FAK (Fig. 3C) and p62dok (Fig. 3D). These recombinant proteins also underwent hyperphosphorylation in cells expressing SAP-1D/A, indicating that this latter mutant exerts a dominant negative effect on tyrosine dephosphorylation of FAK and p62dok. Thus, SAP-1 also mediates the dephosphorylation of FAK and p62dok in vivo; however, these effects may be indirect given that neither FAK nor p62dok were detected as potential substrates of SAP-1 in the substrate-trapping assay. We also examined the tyrosine phosphorylation of ERK, which localizes predominantly to the cytosol and is not associated with FAs. Immunoblot analysis with antibodies specific for tyrosine-phosphorylated (activated) ERK revealed that, although SAP-1WT reduced the extent of tyrosine phosphorylation of HA-tagged ERK2, the latter did not undergo hyperphosphorylation when coexpressed with SAP-1D/A, again demonstrating the specificity of the dominant negative effect of this mutant (Fig. 3E).

SAP-1-induced Disruption of Actin Stress Fibers and FAs-- We next examined the effect of SAP-1 on the actin-based cytoskeleton. Since WiDr cells or Panc-1 cells, which normally express SAP-1, were not suitable for examination of cytoskeletal organization, we could not determine subcellular localization of endogenous SAP-1 by immunofluorescence staining. We therefore subjected Swiss 3T3 cells to infection with adenoviruses encoding SAP-1WT or SAP-1C/S and then examined the cells by confocal microscopy. Infection with a control virus encoding beta -galactosidase had no marked effect on cytoskeletal architecture, with the infected cells showing a staining pattern for filamentous actin similar to that of noninfected cells (data not shown). Immunostaining with mAb 3G5 to SAP-1 revealed that SAP-1WT was distributed diffusely throughout the cell periphery (Fig. 4, A and G). Expression of SAP-1WT resulted in marked disruption of actin stress fibers, with disorganized, thin actin filaments apparent in the subcortical region (Fig. 4C). Furthermore, the number of vinculin-positive patches was markedly decreased in these cells, reflecting disruption of FAs (Fig. 4I). In contrast, SAP-1C/S was predominantly apparent at the leading edge of cells (Fig. 4, B and H), where it was colocalized with filamentous actin (Fig. 4F). The numbers of actin stress fibers and vinculin-positive FAs in the cells expressing SAP-1C/S were similar to those in cells infected with the control virus (Fig. 4, D and J; data not shown). These results together with those of the coexpression experiments indicate that SAP-1 associates with FAs and that it disrupts both actin stress fibers and FAs by mediating the dephosphorylation of FA-associated proteins.


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Fig. 4.   Effects of overexpression of SAP-1 on cytoskeletal architecture. Swiss 3T3 cells seeded on coverslips were infected with adenoviruses encoding SAP-1WT (Ax-SAP-1WT) (A, C, E, G, I, and K) or SAP-1C/S (Ax-SAP-1C/S) (B, D, F, H, J, and L) at a multiplicity of infection of 200 plaque-forming units per cell. Twelve hours after infection, cells were fixed and stained with mAb 3G5 to SAP-1 (A-F) or with polyclonal antibodies to SAP-1 (G-L) together with either rhodamine-labeled phalloidin (A-F) or a mAb to vinculin (G-L). For detection of SAP-1, cells were further incubated with appropriate secondary antibodies conjugated with either fluorescein isothiocyanate (A-F) or Texas red (G-L). For detection of vinculin, cells were also incubated with secondary antibodies conjugated with fluorescein isothiocyanate (G-L). Cells were examined with a confocal microscope. Arrowheads in B and H indicate SAP-1C/S present at the leading edge of a cell. Original magnification, 630×.

Effect of SAP-1 on Cell Spreading on Fibronectin-- To examine the effect of SAP-1 on cell spreading, we established CHO cell lines that stably express SAP-1WT (CHO-SAP-1WT) or SAP-1C/S (CHO-SAP-1C/S) (Fig. 5A). The number and size of colonies formed by cells expressing SAP-1WT were substantially smaller than were those of colonies formed by cells expressing SAP-1C/S or those of colonies formed by cells transfected with the empty vector (see Fig. 7). Furthermore, the amount of SAP-1 in each CHO-SAP-1WT cell line was consistently lower than that in the CHO-SAP-1C/S cell lines (Fig. 5A; data not shown). Exponential growth rates were similar for all cell lines established (data not shown); however, a marked morphological difference was observed. Thus, under normal culture conditions, CHO-SAP-1WT cells were less polarized than were CHO-SAP-1C/S cells or CHO-K1 cells (data not shown). CHO-K1 and CHO-SAP-1C/S cells exhibited an elongated, spindle-like morphology when cultured on fibronectin, a specific integrin ligand; in contrast, CHO-SAP-1WT cells exhibited a flattened, less polarized morphology (Fig. 5B). None of the cells spread substantially when cultured on the nonspecific substrate poly-L-lysine (Fig. 5B). These results suggest that SAP-1 affects integrin-mediated cell spreading in a manner dependent on its catalytic activity.


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Fig. 5.   Effect of ectopic expression of SAP-1 on cell spreading on fibronectin. A, whole cell lysates prepared from CHO-K1 cells or from CHO-K1 cells stably expressing SAP-1WT (CHO-SAP-1WT) cells (two independent lines assayed) or SAP-1C/S (CHO-SAP-1C/S) cells were subjected to immunoblot analysis with polyclonal antibodies to SAP-1 (alpha SAP-1). As a positive control, lysates from WiDr cells were similarly analyzed. B, CHO-K1, CHO-SAP-1WT, or CHO-SAP-1C/S cells were detached from their culture dishes and replated in serum-free Ham's F-12 medium on dishes coated with fibronectin or with poly-L-lysine. After incubation for 2 h at 37 °C, the cells were photographed with a light microscope equipped with phase-contrast optics. Original magnification, 200×.

Inhibition by SAP-1 of Growth Factor-induced ERK Activation-- Cell adhesion to extracellular matrix (ECM) proteins promotes, in an FAK-dependent manner, the activation of ERK induced by various growth factors (35-38). We therefore examined whether SAP-1 affects growth factor-induced ERK activation. 293 cells were transiently cotransfected with vectors encoding HA-ERK2 and SAP-1 and were then exposed to LPA, EGF, or TPA. All of these growth stimulants induced substantial activation of HA-ERK2 in cells cotransfected with an empty vector (Fig. 6). The activity of HA-ERK2 in unstimulated cells was reduced by coexpression of SAP-1WT, suggesting that SAP-1 may inactivate an upstream element required for basal ERK2 activity. Coexpression of SAP-1WT also prevented activation of HA-ERK2 by LPA (Fig. 6A), substantially inhibited HA-ERK2 activation by EGF (Fig. 6B), and inhibited to a lesser extent HA-ERK2 activation by TPA (Fig. 6C). In contrast, coexpression of SAP-1C/S did not markedly affect the activity of HA-ERK2 under basal or stimulated conditions. Whereas LPA and EGF bound to specific membrane receptors and activated ERKs in a protein-tyrosine kinase-dependent manner, TPA activated these enzymes predominantly by a protein-tyrosine kinase-independent pathway that includes protein kinase C and Raf. Together, these results suggest that SAP-1 negatively regulates growth factor-induced ERK activation and that the extent of this effect depends on the signaling pathways triggered by each growth factor.


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Fig. 6.   Effects of overexpression of SAP-1 on growth factor-induced ERK activation. 293 cells were transiently cotransfected with 1 µg of pcDNA3 encoding HA-ERK2 and 4 µg either of pRc/CMV encoding SAP-1WT (WT) or SAP-1C/S (C/S) or of the empty vector (Vector). Forty-eight hours after transfection, cells were deprived of serum for 12 h and then incubated in the absence (-) or presence (+) of 10 µM LPA (A) or EGF (100 ng/ml) (B) for 5 min or of 1 µM TPA for 10 min (C). Whole cell lysates were prepared and subjected to immunoprecipitation with mAb 12CA5 to the HA tag, and the resulting precipitates were subjected to immunoblot analysis with antibodies specific for tyrosine-phosphorylated ERK (top left panels). Duplicate samples were subjected to immunoblot analysis with antibodies to total ERK (left middle panels) to ensure that equal amounts of ERK2 were present in each precipitate. The cell lysates were also subjected to immunoblot analysis with antibodies to SAP-1 to determine the amount of each recombinant SAP-1 protein (left bottom panels). The phosphorylation of HA-ERK2 was quantified by scanning densitometry with the NIH Image program and expressed as a percentage of the value for cells transfected with the empty vector and exposed to each stimulant (right panels).

Inhibition by SAP-1 of Colony Formation-- To gain further insight into the biological consequences of SAP-1-mediated protein dephosphorylation, we next examined the effect of this PTP on colony formation. CHO-K1 or NIH 3T3 cells were transiently transfected with vectors encoding SAP-1WT or SAP-1C/S, and the efficiency of colony formation was evaluated. Expression of SAP-1WT reduced the number of G418-resistant colonies formed by CHO-K1 or NIH 3T3 cells by 65 and 54%, respectively, compared with the value for cells transfected with the empty vector (Fig. 7). Expression of SAP-1C/S also reduced the extent of colony formation by CHO-K1 and NIH 3T3 cells by 30 and 38%, respectively. These results are consistent with the notion that SAP-1 inhibits cell proliferation by reducing ERK activity, but they also suggest that this effect may not depend entirely on the catalytic activity of SAP-1.


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Fig. 7.   Effects of overexpression of SAP-1 on colony formation. CHO-K1 or NIH 3T3 cells were transiently transfected with 3 µg either of pRc/CMV encoding SAP-1WT or SAP-1C/S or of the empty vector (Vector). Forty-eight hours after transfection, cells were exposed to normal growth medium containing G418 (400 µg/ml) for 14 days. Cells were then fixed and stained with crystal violet (A). The efficiency of colony formation by each cell line was quantified by scanning densitometry with the NIH Image program and is expressed as a percentage of the value for cells transfected with the empty vector (B).

Activation of SAP-1 by Cell-Cell Adhesion-- Tyrosine phosphorylation of p130cas is regulated by cell adhesion to the ECM (34). Our observation that SAP-1 catalyzes the dephosphorylation of p130cas suggests that the activity of this PTP might also be regulated by cell-substratum adhesion. The presence of multiple fibronectin type III-like domains in the extracellular region of SAP-1 (14) also suggests that its activity might be regulated by cell-cell adhesion. To investigate these possibilities, we measured cellular SAP-1 activity under adherent or suspension conditions with the in vitro assay. The mAb 3G5 immunoprecipitated PTP activity from an adherent monolayer of either CHO-SAP-1WT or WiDr cells (Fig. 8A). SAP-1 activity in each cell line decreased markedly as a function of time in suspension, reaching a minimal value of ~50% of that for adherent cells after ~15 min (Fig. 8A; data not shown). This effect was not attributable to cell damage because the experimental treatment did not substantially affect the viability of either cell line, as revealed by trypan blue exclusion, for up to 60 min (data not shown).


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Fig. 8.   Up-regulation of SAP-1 activity by cell-cell contact. A, confluent monolayers of CHO-SAP-1WT or WiDr cells were maintained adherent (Ad), or the cells were detached from the culture dish and maintained in suspension for 0-60 min in normal growth medium at 37 °C. Cell lysates were then prepared, normalized for protein concentration, and subjected to immunoprecipitation with mAb 3G5 to SAP-1. The immunopurified SAP-1 was assayed for PTP activity with pNPP as substrate. B, CHO-SAP-1WT or WiDr cells were cultured until the indicated extent of confluence was achieved, after which immunopurified SAP-1 was assayed for PTP activity as in A. The apparent PTP activity obtained with precipitates prepared with an unrelated mouse mAb was subtracted from the activity observed with precipitates prepared with mAb 3G5. Data are means of triplicates from a representative experiment.

Because detachment from the culture dish disrupts not only cell-substratum adhesion but also cell-cell adhesion, we could not conclude from this experiment which type of adhesion regulates SAP-1 activity. We therefore measured the activity of SAP-1 immunopurified from cells cultured at various densities; at the high density, all cells achieved cell-cell adhesion, whereas at the low density, virtually none did. For both cell lines studied, SAP-1 activity in the high density culture was substantially greater than that in the low density culture (Fig. 8B). Thus, cell-cell adhesion may be responsible for up-regulation of SAP-1 activity, although we cannot exclude the possibility that cell-substratum adhesion also has a regulatory role.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have now shown that p130cas, a major FA-associated phosphotyrosyl protein, appears to be a substrate of SAP-1. A substrate-trapping mutant of SAP-1, but not wild-type SAP-1, selectively precipitated p130cas from pervanadate-treated Panc-1 cells, which express endogenous SAP-1 (14). The association of this mutant with p130cas was disrupted by vanadate, indicating that p130cas interacts with the active site of SAP-1. Wild-type SAP-1 effectively and selectively dephosphorylated p130cas both in vitro and in vivo, whereas the substrate-trapping mutant of SAP-1 induced hyperphosphorylation of p130cas in intact cells. These results indicate that p130cas is a physiological substrate of SAP-1. We also showed that 190- and 55-kDa phosphotyrosyl proteins bound to the substrate-trapping mutant of SAP-1, suggesting that these unidentified proteins also might be substrates of this PTP. In addition, pervanadate-treated cells are not thought to contain a complete spectrum of all possible phosphotyrosyl proteins. Thus, additional targets of SAP-1 may remain to be detected.

We showed that overexpression of SAP-1 disrupts both actin stress fibers and FAs. Evidence suggests that p130cas plays an important role in the maintenance of these structures. Thus, the association of p130cas with Src facilitates phosphorylation of tyrosine residues in the substrate domain of p130cas, resulting in the binding of Src homology 2 domain-containing molecules to this protein (39). The Src homology 2 domain-containing adapter protein Crk binds to tyrosine-phosphorylated p130cas and thereby induces the rearrangement of the actin-based cytoskeleton (40). Furthermore, p130cas associates with the actin-binding protein tensin (41), and fibroblasts derived from p130cas knockout mice exhibit impaired formation of actin stress fibers (42). Finally, YopH, a Yersinia PTP capable of dephosphorylating p130cas, disrupts actin stress fibers and FAs (43, 44). Together with these observations, our results indicate that SAP-1 catalyzes the tyrosine dephosphorylation of p130cas and thereby induces the dissociation of this protein from its effector molecules, resulting in the disruption of actin stress fibers and FAs.

Integrin-mediated cell adhesion to the ECM regulates many important cellular responses including activation of protein kinase cascades, cytoskeletal reorganization, proliferation, and survival (45, 46). Our results have now shown that SAP-1 affects cell spreading on fibronectin, a process mediated by integrin engagement. Given that p130cas contributes to cell spreading on the ECM (42), SAP-1-mediated dephosphorylation of p130cas might result in impaired cell spreading on fibronectin. Consistent with this notion, PTP-PEST, which also dephosphorylates p130cas, has been shown to regulate FA disassembly and cell spreading (47, 48).

We have also shown that SAP-1 inhibits growth factor-induced ERK activation, a process supported by cell adhesion to the ECM (35-38). This inhibitory effect of SAP-1 was most pronounced with LPA, less marked with EGF, and marginal with TPA. Given that activation of ERKs by LPA appears to depend on FAs to a greater extent than does that by EGF or by TPA (49), these results indicate that SAP-1 specifically blocks ERK activation mediated through FAs. We further showed that ectopic expression of SAP-1 inhibits colony formation, indicative of a negative effect of this PTP on cell proliferation. In addition to promoting cell growth through ERK activation, integrins initiate various signals required for cell proliferation. Thus, in cooperation with soluble growth factors, they promote cell survival through activation of phosphatidylinositol 3-kinase and Akt, and they activate c-Jun NH2-terminal kinases, which regulate cell cycle progression (46). SAP-1 therefore likely inhibits cell proliferation by inactivating both growth-promoting and survival-promoting signals supported by integrin-mediated cell adhesion.

Our data indicate that cell-cell adhesion up-regulates the enzymatic activity of SAP-1. Cell-cell contact increases the expression or the catalytic activity of various RPTPs, including DEP-1, PTPbeta , and PTPµ (16, 50, 51). Although such effects have been attributed to density-dependent growth arrest or down-regulation of growth factor signaling (52, 53), a lack of knowledge of the specific substrates of each PTP has hindered elucidation of the underlying mechanisms. The activity of Src and the extent of tyrosine phosphorylation of its substrates, including p130cas, paxillin, and FAK, increase in response to cell-cell contact, although again the mechanism and the functional significance of these effects have remained unclear (54, 55). Given that we have now shown that SAP-1 mediates the dephosphorylation of these Src substrates in vivo, this PTP may function to counteract mitogenic signaling initiated by Src. SAP-1 activation induced by cell-cell adhesion may thus contribute to the mechanism by which contact-inhibited cells enter growth arrest.

Expression of the genes for the RPTPs DEP-1 and PTP-U2, which are structurally similar to SAP-1, increases during differentiation of breast cancer cells and monoblastoid leukemia cells (56, 57). Ectopic expression of DEP-1 or PTP-U2 in these cells resulted in growth inhibition or enhanced apoptosis subsequent to terminal differentiation (56, 57). The abundance of SAP-1 mRNA also increases during cancer cell differentiation (56). Together with the inhibitory effect of SAP-1 on cell proliferation, these observations suggest a model in which SAP-1, by mediating the tyrosine dephosphorylation of FA-associated substrates, activates a signal that leads to growth arrest and differentiation of cancer cells. According to this model, cancer progression would be facilitated if SAP-1 expression is lost.

In conclusion, our data indicate that p130cas is a substrate of SAP-1. Overexpression of SAP-1 disrupted the actin-based cytoskeleton. It also inhibited a wide range of cellular responses promoted by integrin-mediated cell adhesion, including cell spreading on fibronectin, growth factor-induced ERK activation, and colony formation. Furthermore, activation of SAP-1 in response to cell-cell adhesion indicates a role for this PTP in contact inhibition of cell growth and motility.

    ACKNOWLEDGEMENTS

We thank H. Hirai, H. Sabe, S. K. Hanks, and J. S. Gutkind for providing vectors.

    FOOTNOTES

* This study was supported by a grant-in-aid for cancer research and a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan and by a grant-in-aid for research for the Future Program from the Japan Society for the Promotion of Science.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.

Dagger To whom correspondence should be addressed. Tel.: 81-78-382-5861; Fax: 81-78-382-2080; E-mail: noguchi@med.kobe-u.ac.jp.

Published, JBC Papers in Press, February 14, 2001, DOI 10.1074/jbc.M007208200

    ABBREVIATIONS

The abbreviations used are: PTP, protein-tyrosine phosphatase; RPTP, receptor-like PTP; SAP-1, stomach cancer-associated protein-tyrosine phosphatase-1; FA, focal adhesion; FAK, FA kinase; CHO, Chinese hamster ovary; mAb, monoclonal antibody; GST, glutathione S-transferase; HA, hemagglutinin epitope; ERK, extracellular signal-regulated kinase; PBS, phosphate-buffered saline; LPA, lysophosphatidic acid; EGF, epidermal growth factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; pNPP, p-nitrophenylphosphate; ECM, extracellular matrix; CMV, cytomegalovirus.

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