Oncogene (2001) 20, 5219 ± 5224 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc
An extracellular ligand increases the speci®c activity of the receptor-like protein tyrosine phosphatase DEP-1 Maria SoÈrby1, Jill SandstroÈm1 and Arne OÈstman*,1 1
Ludwig Institute for Cancer Research, Box 595, S-751 24 Uppsala, Sweden
Cellular growth, dierentiation and migration is regulated by protein tyrosine phosphorylation. Receptor-like protein tyrosine phosphatases are thus likely to be key regulators of vital cellular processes. The regulation of these enzymes is in general poorly understood. Ligands have been identi®ed only for a small subset of the receptor-like protein tyrosine phosphatases and in no case has upregulation of the speci®c activity by extracellular ligands been demonstrated. Prompted by earlier ®ndings of ligands for receptor-like protein tyrosine phosphatases in extracellular matrix we investigated if MatrigelTM, a preparation of extracellular matrix proteins, contained modulators of the speci®c activity of the receptor-like protein tyrosine phosphatase DEP-1. MatrigelTM stimulation of cells increased the speci®c activity of immunoprecipitated DEP-1. Also, incubation of immunoprecipitated DEP-1 with MatrigelTM led to an increase in DEP-1 activity, which was blocked by soluble DEP-1 extracellular domain. Finally, immunoprecipitated DECD-DEP-1, a mutant form of DEP-1 lacking most of the extracellular domain, failed to respond to MatrigelTM stimulation. These experiments identify MatrigelTM as a source of DEP-1 agonist(s) and provide the ®rst evidence for upregulation of the speci®c activity of receptor-like protein tyrosine phosphatases by extracellular ligands. Oncogene (2001) 20, 5219 ± 5224. Keywords: DEP-1; MatrigelTM; protein tyrosine phosphatase
Tyrosine phosphorylation of proteins controls cellular growth, dierentiation and migration, and is regulated by the concerted action of kinases and phosphatases. The net tyrosine phosphorylation is thus determined by the expression levels, speci®c activities and localization of tyrosine kinases and protein tyrosine phosphatases (PTPs). Consequently, the regulation of PTPs constitutes a central biological control mechanism. Receptor-like protein tyrosine phosphatases (rPTPs) are composed of an extracellular domain, a single transmembrane segment and one or two intracellular *Correspondence: A OÈstman; E-mail:
[email protected] Received 11 April 2001; revised 12 April 2001; accepted 30 April 2001
catalytic domains (Brady-Kalnay and Tonks, 1995; Van Vactor, 1998). These structural features suggest that rPTPs are regulated by extracellular ligands. For most rPTPs the ligands remain uncharacterized. Known ligands for rPTPs include the extracellular domains of PTPm and PTPk, that act as homophilic ligands (Brady-Kalnay et al., 1993; Gebbink et al., 1993; Sap et al., 1994), and contactin which interacts with PTPb/z and PTPa (Peles et al., 1995; Zeng et al., 1999), as well as the extracellular matrix proteins tenascin and the laminin-nidogen complex which binds PTPb/z and LAR, respectively (Barnea et al., 1994, O'Grady et al., 1998). The recent observation of pleiotrophin-mediated inactivation of PTPb/z provided the ®rst evidence for regulation of rPTPs by extracellular ligands (Meng et al., 2000). However, upregulation of speci®c activity by extracellular ligands has not yet been demonstrated for any rPTP. The rPTP DEP-1, also designated PTPZ and CD148, is composed of eight ®bronectin type III repeats in its extracellular domain, and a single intracellular catalytic domain (Honda et al., 1994; OÈstman et al., 1994). Immunohistochemical stainings have revealed a broad tissue distribution of DEP-1; prominent expression was found in thymus, and also on many epithelial cell types with glandular or endocrine dierentiation (Autschbach et al., 1999). DEP-1 expression is enhanced in high cell density cultures of ®broblasts and in contactinhibited endothelial cells (Borges et al., 1996; OÈstman et al., 1994), and heterologous expression of DEP-1 in bovine aortic endothelial cells leads to inhibition of cfos and Cyclin A promotor activity (Suzuki et al., 2000). DEP-1 expression has also been shown to be upregulated after dierentiation of breast cancer cells (Keane et al., 1996) and thyroid cells (Martelli et al., 1998; Zhang et al., 1997). Re-expression of DEP-1 in transformed thyroid cells suppress the transformed phenotype and leads to increased levels of cyclindependent kinase inhibitor p27 (Trapasso et al., 2000). Recent studies have also indicated a role of DEP-1 in lymphocyte signal transduction (de la Fuente-GarcõÂ a et al., 1998; Tangye et al., 1998a,b). The inhibitory eects of DEP-1 on T-cell receptor signaling are associated with reduced tyrosine phosphorylation of the adaptor protein LAT and of phospholipase Cg1 (Baker et al., 2001). The physiological substrates of DEP-1 remain unknown. However, after transfection into plateletderived growth factor (PDGF) b-receptor expressing
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cells, DEP-1 mediates site-selective dephosphorylation of a subset of PDGF b-receptor autophosphorylation sites (Kovalenko et al., 2000). Prompted by earlier ®ndings of rPTP ligands in extracellular matrix we investigated if MatrigelTM, a preparation of extracellular matrix proteins, contained modulators of the speci®c activity of DEP-1. X23 cells, i.e. porcine aortic endothelial (PAE) cells with tetracycline regulated expression of human DEP-1 with a carboxyterminal VSV-G epitope (Kovalenko et al., 2000), were initially used to investigate the eects of MatrigelTM on DEP-1 activity. DEP-1 expressing or non-expressing X23 cells were taken to suspension and equal number of cells were incubated with either a 1 : 1 mix of MatrigelTM and culture medium or culture
a
medium alone. After stimulation, fractions of lysates were subjected to anti-VSV-G immunoprecipitations and PTP activity in immunoprecipitates was determined. As shown in Figure 1a stimulation with MatrigelTM increased DEP-1 activity more than twofold. No increase in the unspeci®cally recovered PTP activity from non-induced cells was observed. Analysis of immunoprecipitates by anti-DEP-1 immunoblotting demonstrated that equal amounts of DEP-1 was recovered from unstimulated cells and cells stimulated with MatrigelTM (Figure 1b), suggesting that the change in DEP-1 activity after MatrigelTM stimulation re¯ects an increase in speci®c activity of DEP-1. To con®rm that MatrigelTM also increased the activity of endogenously expressed DEP-1, a similar
b
Figure 1 Stimulation of DEP-1 transfected PAE cells with MatrigelTM enhances DEP-1 activity. X23 cells cultured under DEP-1 inducing (+) or non-inducing (7) conditions were treated with versene and pelleted by centrifugation. Cells were resuspended and incubated with a 1 : 1 mix of MatrigelTM and culture medium (+ stimulation) or culture medium only (7 stimulation). (a) PTP activity of anti-VSV-G immunoprecipitates was analysed. PTP activity recovered in immunoprecipitates from unstimulated DEP-1 expressing cells was set to 100% and corresponds to dephosphorylation of 4% of added substrate. Error bars indicate s.e.m.. The experiment was performed four times. (b) Anti-VSV-G immunoprecipitates from lysates of stimulated or unstimulated cells cultured under DEP-1 inducing or non-inducing conditions were subjected to immunoblotting with anti-DEP-1 antibody. The migratory position of marker proteins are indicated. Materials and methods: Culture of X23 cells, i.e. PAE cells with a VSV-G tagged form of human DEP-1 expressed under the control of a tTA regulated promotor, and induction of DEP-1 expression were performed as described previously (Kovalenko et al., 2000). Prior to stimulation cells were brought to suspension by 10 min incubation in 378C with versene (137 mM NaCl, 2.7 mM KCl, 1.42 mM KH2PO4 8.42 mM Na2HPO4 and 1.0 mM EDTA). After pelleting by centrifugation (1000 g, 4 min) cells were resuspended in a small volume of DMEM or a 1 : 1 mix of DMEM and MatrigelTM (Becton-Dickinson) and incubated for 1 h at 48C. The samples were diluted ten times with DMEM and an aliquot of the sample was taken for cell counting and staining of cells with trypan blue before pelleting of cells by centrifugation (1000 g, 4 min). The cells were lysed in lysis buer containing 0.5% Triton X-100, 0.5% Na-deoxycholate, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM EDTA, 30 mM Na2P2O7, 1% Trasylol, 1 mM phenyl methyl sulfonyl ¯uoride (PMSF) and 2 mM dithiothreitol (DTT). Lysates were clari®ed by centrifugation (16 000 g, 15 min). DEP-1 was immunoprecipitated from X23 cells using anti-VSV-G mouse monoclonal antibody (Sigma) and rabbit anti-mouse IgG antibody (Dako). Immunocomplexes were collected on Protein-A-Sepharose beads and washed three times with lysis buer and once in PTP assay buer (20 mM Imidazole, 0.1 mg/ml bovine serum albumin and 10 mM DTT). For the PTP assay Src optimal peptide (Songyang Z et al., 1995) was radioactivelly labeled at its tyrosine residue using g-32P-ATP and recombinant human c-Src (Upstate Biotechnology Incorporated). Assays were performed with an initial phosphotyrosine concentration of 3 mM in a volume of 60 ml, reactions were terminated after 6 min at 308C by addition of 290 ml charcoal mixture (Streuli et al., 1989). After centrifugation (16 000 g, 2 min), free phosphate in the supernatant was measured by scintillation counting. Assays were performed with duplicates of samples. For immunoblotting, immunocomplexes were ®rst resolved by SDS-polyacrylamide gel electrophoresis (SDS ± PAGE) and after transfer to Hybond Super nitrocellulose membrane analysed with 143.41 DEP-1 monoclonal antibodies recognizing the extracellular domain of DEP-1 (de la Fuenta-Garcia et al., 1998). Detection was done with ECL reagent (Amersham Pharmacia Biotech) Oncogene
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experiment was performed on HaCaT cells expressing DEP-1 as determined by PTP assays of anti-DEP-1 immunoprecipitates (Figure 2) and by immunoblotting (data not shown). Stimulation of HaCaT cells with MatrigelTM also increased DEP-1 activity (Figure 2). PTP assays of control (mIgG) immunoprecipitations were also performed and showed no change in activity after stimulation. The changes in DEP-1 activity was not caused by Matrigel induced changes in DEP-1 expression levels (data not shown). From these experiments we conclude that MatrigelTM contains a component(s) with the ability to directly or indirectly modulate the speci®c activity of DEP-1. To investigate whether the enhancement of DEP-1 activity after MatrigelTM stimulation is due to direct interaction of MatrigelTM component(s) with DEP-1, stimulation of immunoprecipitated DEP-1 was performed. Protein-A-Sepharose immobilized anti-VSV-G immunoprecipitates from DEP-1 expressing or nonexpressing X23 cells were divided and incubated with either a 1 : 1 mix of MatrigelTM and culture medium or culture medium alone. Stimulation of immunoprecipitated DEP-1 with MatrigelTM led to an enhancement of the PTP activity, in contrast no increase was observed in the unspeci®cally recovered PTP activity from noninduced cells (Figure 3a). Analysis of anti-VSV-G immunoprecipitates by immunoblotting with anti-DEP1 and anti-VSV-G antibodies, recognizing the extra-
Figure 2 Stimulation of HaCaT cells with MatrigelTM enhances DEP-1 activity. HaCaT cells were stimulated with a 1 : 1 mix of MatrigelTM and culture medium (+stimulation) or culture medium only (7stimulation). Cell lysates were subjected to immunoprecipitations with a mouse IgG fraction (ctrl.) or antiDEP-1 monoclonal antibody (anti-DEP-1) and rabbit anti-mouse IgG. PTP activity recovered in DEP-1 immunoprecipitates from unstimulated HaCaT cells was set to 100% and corresponds to dephosphorylation of 8% of added substrate. Error bars indicate s.e.m. The experiment was performed ®ve times. Materials and methods: HaCaT cells, human keratinocytes, were cultured in DMEM supplemented with 10% fetal calf serum, 2 mM glutamine, 1000 U/1 penicillin and 1 mg/l streptomycin. Stimulation with Matrigel, preparation of cell lysates and PTP assays was performed as in Figure 1. DEP-1 was immunoprecipitated with the mouse monoclonal antibody 143.41
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cellular domain and the carboxy-terminal VSV-G epitope of DEP-1, respectively, showed that MatrigelTM stimulation did neither aect the recovery of DEP-1 nor induce proteolytic cleavage of DEP-1 (Figure 3b). Since no detectable PTP activity was recovered in immunoprecipitates of a catalytically inactive form of DEP-1 (data not shown), we can exclude the possibility that the eect of Matrigel occurs through modulation of a PTP associating with DEP-1. The experiments presented in Figure 3 thus strongly suggest that the agonistic eect of MatrigelTM on DEP-1 speci®c activity occurs through direct interactions between a MatrigelTM component(s) and DEP-1. Two experiments designed to investigate if the MatrigelTM DEP-1 agonist is interacting with the extracellular domain of DEP-1 were performed. In the ®rst experiment the eect of preincubation of MatrigelTM with a recombinant GST-fusion form of the DEP-1 extracellular domain was investigated. As a control for unspeci®c eects caused by the GSTdomain, a recombinant GST-fusion form of the PDGF b-receptor was used. Preincubation of MatrigelTM with DEP-1 extracellular domain consistently reduced the MatrigelTM induced increase in DEP-1 speci®c activity (Figure 4a). Although some inhibitory eects of the PDGFR-b-ECD-GST protein was observed, this eect was in all experiments lesser than the inhibitory eect of the DEP-1 extracellular domain. Analysis by immunoblotting of DEP-1 levels after the various stimulation con®rmed that equal amounts of DEP-1 was analysed in PTP assays (Figure 4b). Preincubation of the control medium with the recombinant GSTfusion form of the DEP-1 extracellular domain did not aect DEP-1 speci®c activity (data not shown). In the second experiment DECD-DEP-1, an aminoterminally truncated form of DEP-1 lacking the major part of the extracellular domain of DEP-1, was used to evaluate the role of the extracellular domain of DEP-1 for MatrigelTM stimulation of speci®c activity. Comparision of transiently expressed and immunoprecipitated DEP-1 and DECD-DEP-1 did not indicate any dierences in speci®c activity between DEP-1 and DECD-DEP-1 (data not shown). DEP-1 and DECDDEP-1 were transiently expressed in COS cells, recovered by immunoprecipitations and subjected to stimulation with either a 1 : 1 mix of MatrigelTM and culture medium or culture medium only (Figure 4c). As in previous experiments wild-type DEP-1 was activated by MatrigelTM stimulation. In contrast, MatrigelTM stimulation did not increase the activity of DECDDEP-1. Analysis by immunoblotting of immunoprecipitated DEP-1 and DECD-DEP-1 con®rmed that equal amounts of proteins were used for control stimulation and for MatrigelTM stimulation (Figure 4d). Together the experiments presented in Figure 4 provide strong support for the notion that the MatrigelTM component(s) responsible for alterations of DEP-1 speci®c activity exerts its eects through interaction with the extracellular domain of DEP-1. Molecular mechanisms proposed to regulate the speci®c activity of rPTPs include serine/threonine
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a
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Figure 3 Stimulation of immunoprecipitated DEP-1 with MatrigelTM increases the speci®c activity of DEP-1. (a) Anti-VSV-G immunoprecipitates from lysates of X23 cells cultured under DEP-1 inducing (+) or non-inducing (7) conditions were stimulated with a 1 : 1 mix of MatrigelTM and culture medium (+stimulation) or culture medium only (7stimulation) and PTP activity of immunoprecipitates was measured. PTP activity recovered in control stimulated immunoprecipitates from DEP-1 expressing cells was set to 100% and corresponds to dephosphorylation of 14% of added substrate. Error bars indicate s.e.m. The experiment was performed 18 times. (b) Stimulated and unstimulated anti-VSV-G immunoprecipitates from lysates of cells cultured under DEP-1 inducing or non-inducing conditions were divided in fractions corresponding to 33 and 66% of the total immunoprecipitates and subjected to immunoblotting with anti-DEP-1 antibody or anti-VSV-G antibody. The migratory position of marker proteins are indicated. Materials and methods: Culture of X23 cells and anti-VSV-G immunoprecipitations were performed as described in Figure 1. After washing, immunoprecipitates were incubated 1 h at 48C with DMEM or mixes of DMEM and MatrigelTM. After stimulation, immunoprecipitates were washed before PTP assays or immunoblotting were performed as described in Figure 1
phosphorylation (Wang et al., 1999), tyrosine phosphorylation (Stover et al., 1991; Stover and Walsh, 1994), as well as dimerization (Bilwes et al., 1996; Jiang et al., 1999; Majeti et al., 1998). Whether the DEP-1 agonist(s) in MatrigelTM acts by any of these mechanisms remain presently unknown and merits further investigations. Although our study does not address the biological consequences of MatrigelTM mediated stimulation of DEP-1 activity, the identi®cation of MatrigelTM as a source of an agonistic DEP-1 ligand should aid in Oncogene
future studies aiming at identifying cellular processes regulated by DEP-1. The expression of DEP-1 in many epithelial cells with glandular dierentiation (Autschbach et al., 1999), which are juxtaposed to extracellular-rich basement membranes suggests that regulated expression of a DEP-1 agonist in extracellular matrix constitutes a biological control mechanism. MatrigelTM is a preparation of extracellular matrix proteins secreted by Englebreth ± Holm ± Swarm mouse sarcoma. Among the known components of matrigel
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c d
Figure 4 The enhancement of DEP-1 activity by MatrigelTM stimulation is due to interactions of MatrigelTM component(s) with the extracellular domain of DEP-1. (a) Anti-VSV-G immunoprecipitates from X23 cells were incubated for 1 h at 48C with either a 1 : 1 mix of MatrigelTM and culture medium (+ stimulation) or culture medium only (7 stimulation) that had or had not been preincubated with 10 mg/ml of recombinant DEP-ECD-GST or PDGFR-b-ECD-GST. PTP activity of immunoprecipitates was subsequently measured. PTP activity recovered in control stimulated immunoprecipitates from DEP-1 expressing cells was set to 100% and corresponds to dephosphorylation of 13% of added substrate. Error bars indicate s.e.m. The experiment was performed three times. (b) Stimulated and unstimulated anti-VSV-G immunoprecipitates from lysates of cells cultured under DEP-1 inducing or non-inducing conditions were subjected to immunoblotting with anti-DEP-1 antibody. The migratory position of marker proteins are indicated. (c) COS cells transiently expressing HA-tagged forms of wild-type DEP-1 or DEP-1 lacking most of the extracellular domain (DECD-DEP-1) were lysed and subjected to anti-HA immunoprecipitations. PTP activity of immunoprecipitates was determined after stimulation with either a 1 : 1 mix of MatrigelTM and culture medium (+ stimulation) or culture medium only (7 stimulation). PTP activity of immunoprecipitates was subsequently measured. PTP activity recovered in control stimulated immunoprecipitates from DEP-1 or DECD-DEP-1 expressing cells was set to 100% and corresponds to dephosphorylation of 8 and 11% respectively, of added substrate. Error bars indicate s.e.m. The experiment was performed three times. (d) Stimulated and unstimulated anti-HA immunoprecipitates from lysates of cells expressing DEP-1 or D-ECD-DEP-1 were subjected to immunoblotting with anti-HA antibody. The migratory position of marker proteins are indicated. Materials and methods: Analysis of DEP-1 immunoprecipitated from X23 cells were performed as described in Figure 3. DEP-ECD-GST is a recombinant protein, puri®ed from the conditioned media of transfected CHO cells, composed of the extracellular domain of DEP-1 and a carboxy-terminal GST-domain. The expression and puri®cation of DEP-ECD-GST will be described in detail elsewhere. The recombinant GST-fusion form of the PDGF b-receptor extracellular domain have been described earlier (LeppaÈnen et al., 2000). The cDNA encoding human carboxy-terminally HA-tagged DEP-1 was generated by insertion, through site-directed mutagenesis, of an MscI restriction site spanning the stop codon of DEP-1. The MscI site was used for ligation with a fragment encoding three consecutive HA-epitopes followed by a stop codon. The cDNA encoding HA-tagged DEP-1 was subsequently inserted as an Eco RI/Eco RI insert into the pSV7d expression vector (Truett et al., 1985). The DECD-DEP-1 pcDNA3 plasmid encodes a protein composed of the IgG heavy chain signal sequence followed by amino acid residues 895 ± 1336 of DEP-1 and carboxy-terminal HAepitopes. The construct was generated after introduction, by site-directed mutagenesis, of a BamHI restriction site spanning codons 893 and 894 of HA-tagged DEP-1. COS cells were transfected using standard calcium phosphate procedure. Transiently expressed HA-tagged DEP-1 and DECD-DEP-1 was recovered by immunoprecipitation with a polyclonal anti-HA rabbit antiserum, a kind gift from Lars RoÈnnstrand, Ludwig Institute for Cancer Research (Uppsala, Sweden). Immunoblotting of HA-tagged DEP-1 and DECD-DEP-1 was performed with a monoclonal HA-antibody (12CA5, Boehringer ± Mannheim). Immunoprecipitations, MatrigelTM stimulation, and PTP assays of transiently expressed DEP-1 and DECD-DEP-1 were performed as described in Figure 3 Oncogene
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are laminin, collagen IV and entactin. In preliminary studies we have not obtained any evidence that laminin or collagen IV exerts DEP-1 agonistic activity (data not shown). The puri®cation and identi®cation of the DEP-1 agonist(s) in MatrigelTM represent an obvious goal for future studies. In conclusion, our experiments demonstrate that MatrigelTM contains a DEP-1 agonist that exerts its eect through interactions with the DEP-1 extracellular domain. Our experiments provide the ®rst evidence of upregulation of the speci®c activity of a rPTP by an extracellular ligand. We expect that our ®ndings, together with the recent demonstration of pleiotrophin
as an extracellular PTPb/z antagonist (Meng et al., 2000), will stimulate additional studies addressing the possibility that other rPTPs also are regulated by extracellular agonists or antagonists.
Acknowledgments We thank Antoni Gaya for kindly providing us with the 143.41 monoclonal antibody and Olli LeppaÈnen for the recombinant PDGF b-receptor GST fusion protein. We would also like to thank Carl-Henrik Heldin, Lars RoÈnnstrand and members of the Growth Regulation group for critical reading of the manuscript.
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