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PTPRH activity is regulated by reversible dimerization
BBRC Biochemical and Biophysical Research Communications 331 (2005) 497–502 www.elsevier.com/locate/ybbrc
Sap-1/PTPRH activity is regulated by reversible dimerization Se´bastien Wa¨lchli 1, Xavier Espanel 2, Rob Hooft van Huijsduijnen * Serono Pharmaceutical Research Institute, 14, chemin des Aulx, Plan-les-Ouates/Geneva, Switzerland Received 3 March 2005 Available online 7 April 2005
Abstract Sap-1/PTPRH, a receptor protein tyrosine phosphatase (RPTP), is a ubiquitously expressed enzyme that is upregulated in human gastrointestinal cancers. Using both chemical cross-linkers and co-immunoprecipitation we show that overexpressed full-length Sap1 is present as a stable homodimer. Unlike a number of adhesion RPTPs which have tandem catalytic domains that are involved in dimerization, Sap-1 has a single catalytic domain, and we show that this domain is not required for Sap-1 dimerization, which is mediated instead by the large extracellular and transmembrane domains. Exposing cells that express the receptor to a reducing environment reversibly disrupts the Sap-1 dimer, suggesting that cysteine bonds play a role in dimer formation/stabilization. The switch between Sap-1 dimers and monomers is accompanied by an increase in catalytic activity as judged by its capacity to dephosphorylate and activate c-src, which we identify as a novel substrate for this phosphatase. 2005 Elsevier Inc. All rights reserved. Keywords: Protein tyrosine phosphatase; Sap-1; Dimerization; Signaling cascade
Many protein tyrosine phosphatases (PTPs) are type I transmembrane proteins (‘‘receptor-PTPs’’ or RPTPs). Most RPTPs have two domains with homology to phosphatase catalytic domains, where only the plasma membrane-proximal (D1) domain has significant catalytic activity. In a mechanism that is the exact opposite of how many receptor kinases are activated, some RPTPs are known to be active as monomers but inactive in the dimerized state. Although evidence for dimerization has been obtained for a number of PTPs, it is by no means clear if all receptor PTPs undergo dimerization or how this relates to activity [1]. PTP-DEP-1 has been shown to undergo activation upon incubation with Matrigel, but the exact nature of the ligand or whether this activation is associated with dimerization is known [2]. So far, only a single bona fide soluble ligand, midkine, has been reported for a receptor PTP (PTP-f) [3].
Many other receptor PTPs have adhesion-like extracellular domains and are likely involved in homo- or heterotypic intercellular binding [4–8]. While receptor PTPs with two catalytic domains have been well studied, very little is known about multimerization of receptor PTPs that have a single domain, such as PTP-Sap-1 (stomach cancer-associated phosphatase/ PTPRH; [9]. This PTP was initially discovered in colorectal cancers [10,11]. More recent studies conclude that the protein is downregulated in advanced hepatic carcinomas [12] and that it induces apoptosis [13] with Lck as a potential substrate [14]. In the present study, we investigate if Sap-1 may form higher-order complexes, and whether these complexes are associated with altered catalytic activity.
Materials and methods *
Corresponding author. Fax: +41 22 7946965. E-mail address: [email protected] (R.H. van Huijsduijnen). 1 Present address: Institute for Cancer Research, The Norwegian Radium Hospital, Montebello 0310 Oslo, Norway. 2 Present address: Sanofi-Synthelabo, 31676 LABEGE, France. 0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.196
Antibodies, peptides, and chemical reagents. Western blot detection of HA-tags was performed with mouse monoclonal anti-HA.11 (BAbCO) or rabbit polyclonal anti-HA (Santa Cruz). Immunoprecipitation was carried out with anti-HA.11 (BAbCO). Anti-Myc:
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mouse monoclonal anti-Myc9E10 (Santa Cruz). Anti-SRC327 was a gift from Dr. J. Brugge; rabbit polyclonal anti-SRCPAN, rabbit polyclonal anti-SRCPY530 were from Biosource and rabbit polyclonal anti-SRC was from Santa Cruz. Mouse monoclonal anti-His was from Dianova. DSS and BS3 were from Pierce, dithiothreitol, DTT, iodoacetamide, and IODA were from Sigma. Cell culture, transfection. COS7 cells were grown under 5% CO2 in DulbeccoÕs modified EagleÕs medium, DMEM, with 10% FCS, penicillin at 105 U/L, streptomycin at 105 U/L, and L-glutamine. Transfections were performed with Fugene-6 reagent (Roche) following the manufacturerÕs instructions. BS3, DSS, and DTT treatments. Twenty-four after transfection in COS7 cells, the medium was changed and a solution of BS3 in PBS (2.5 mg/ml) or PBS alone was added and left for 1 h at 4 C. The crosslinking reaction was stopped by the addition of PBS containing 0.15 M Tris, pH 7.5, for 15 min at 4 C followed by lysis. Treatment with DSS was almost the same except that the product was dissolved in DMSO at a concentration of 25 mM (10·) and diluted in PBS; control cells were incubated with 0.1· DMSO. Cells treated with DTT were serum-starved for 8 h after transfection (with PTP-Sap-1 constructs or vector), and incubated for 30 min in serum-free medium with various amounts of DTT. Before lysis in a buffer containing 150 mM IODA, cells were washed with ice-cold PBS containing 50 mM IODA in order to block free DTT. Samples were run under reducing or non-reducing conditions, with or without b-mercaptoethanol in the sample buffer, respectively. PTP-Sap-1 constructs. PTP-Sap-1 FL construct was as described [15]. The complete cDNA with the poly(A) tail was subcloned into several vectors using XbaI and HindIII restriction enzymes. Most of the experiments presented herein were done with the pcDNA4a vector (Invitrogen). The pcDNA4-Sap-1 DECD HA clone is a fusion of the signal peptide sequence of PTP-Sap-1, followed by a HA-tag and the PTP sequence from the N-terminal part of the transmembrane region. It was prepared as follows: the full cytoplasmic domain of PTP-Sap-1 was amplified by polymerase chain reaction (PCR) with Herculase Polymerase (Stratagene) using an antisense primer from the vector and a sense primer containing the HA-tag fused to the sequence encoding the five extracellular amino acids on the N-terminal side of the transmembrane (5 0 -TAC CCA TAC GAC GTC CCA GAC TAC GCT CAC ACC GAG AGT GCA GGG GT-3 0 ). On the other side, the signal peptide was amplified with a reverse primer fused to the HAtag in the C-terminal part of the sequence (5 0 -AGC GTA GTC TGG GAC GTC GTA TGG GTA GGG GGC AGG CGC CCT GGC CCC T-3 0 ) and the forward primer was from the vector. The two PCR products were mixed and amplified again with external primers. Two XbaI sites (one generated and the other one from the original vector) surrounding the amplicon were used to clone the intracellular part of PTP-Sap-1 fused to the signal peptide and the HA-tag. The full-length construct fused in C-terminal frame with an HA-tag was prepared with an antisense primer containing the sequence of an XhoI site, an HAtag, and the end of the PTP-Sap-1 sequence with the STOP codon (5 0 TAC TCG AGT TAA GCG TAG TCT GGG ACG TCG TAT GGG TAG ACC TCC AAC TTG TGG GCC T-3 0 ). The full-length Sap-1HA fusion construct was amplified using a plasmid specific forward primer in a long run PCR using Herculase in the presence of 5% DMSO and Hotstart conditions. The D-intra construct was obtained by amplifying the extracellular domain sequence with a primer complementary to the intracellular proximal transmembrane region coding sequence (about 30 bp downstream) in order to keep the targeting sequence (5 0 -ATG AAT TCA GCG GCC CAT CTG GCT GCC TCT TTC TCA GGA AGA AAA TCA-3 0 ) and adding a, EcoRI site. When the amplicon was fused to pCDNA4b, it was in-frame with the two tags (His and c-Myc) and a STOP codon. PTP-Sap-1 att HA (membrane associated) consisted of a fusion of the PTP-Sap-1 cytoplasmic region with an HA-tag in its C-term part (amplification of the pcDNA4-PTP-Sap-1FLHA construct). A primer containing the Lck myristoylation site (underlined) and a start codon surrounded by a
Kozak sequence ATA AGC TTA CCa tgg gct gtg gct gca gct cac acc cgg aag atg act ggA AGA GGA GGA ATA AGA AGA AG was used with a vector primer to amplify the cytoplasmic fragment of PTP-Sap1. The amplicon was cloned into pcDNA4 using HindIII restriction site of the primer for the 5 0 end ligation. All constructs were checked by DNA sequencing. c-src constructs. c-src cDNA was a gift from Dr. K. Maundrell. It was subcloned into pcDNA4 (Invitrogen). The c-src Y530F mutant was made using by site-directed mutagenesis with the following primers (5 0 -AGT TCC AGC CCG GGG AGA ACC TC-3 0 and 5 0 GAG GTT CTC CCC GGG CTG GAA CT-3 0 ). Immunoprecipitation. Every immunoprecipitation (IP) was performed with the same buffer and optimal dilution of the antibodies. Cells were incubated in RIPA lysis buffer (PBS 1·, 1% IGEPAL, 0.5% Na–deoxycholate, 0.1% SDS supplemented with a complete protease inhibitor cocktail tablet (Roche), and sodium orthovanadate 1 mM) or NP-40 buffer for anti-myc (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 25 mM b-glycerophosphate, 1 mM sodium pyrophosphate, 1% NP-40, and supplemented with a complete protease inhibitor cocktail tablet (Roche)), both buffers being used ice-cold. Cells were mechanically broken by one cycle of freeze–thawing and passed through a G21 syringe. Lysates were clarified by centrifugation (14,000 rpm) for 10 min at 4 C and precleared with normal antibody (Santa Cruz) and 20 ll of precoated protein A/G–Sepharose beads (Santa Cruz). The supernatant from centrifugation was recovered and incubated with the specific antibody (that had been precoated for 2 h on protA/G beads) overnight at 4 C. The beads were finally washed once with lysis buffer and twice with PBS–0.1% Triton X-100 (the volume of the washes corresponds to two volumes of the lysate). Western blot. Proteins were separated on SDS–polyacrylamide gel (PAGE) using the NuPAGE-gel system from Novex (InVitroGen), following the manufacturerÔs protocols. Proteins were transferred to PVDF membrane and detected by Western blot. Kinase assay. After the last wash of immunoprecipitation, the kinase was dissolved in phosphatase buffer (20 mM Tris, pH 7.5, 150 mM NaCl, and 1 mM EDTA). 1/10 of this solution was run on a SDS–PAGE to perform a control Western blot, while the rest was used for a kinase assay. The immunoprecipitate was directly incubated with MBP, sodium orthovanadate 1 mM, and 35 lCi [c-32P]ATP. In addition, the buffer was brought to a final concentration of 5 mM MgCl2, 2.5 mM MnCl2, and 2.5 mM DTT. The reaction was stopped after 30 min at 30 C by the addition of protein-sample buffer and run on a SDS–PAGE. Bands were visualized by autoradiography using Kodak X-OMAT films.
Results Sap-1-multimers can be revealed by chemical cross-linkers The chemical cross-linkers BS3 and DSS were used to detect protein–protein interactions. BS3 cannot pass cell membranes and can only cross-link extracellular protein. By contrast, DSS is cell-permeable and it can cross-link both extra- and intracellular proteins. Upon incubation with DSS, receptor-PTP-a has been shown to shift its migration profile when run on a SDS–polyacrylamide gel (SDS–PAGE; [16]). We have studied Sap-1 for evidence of dimerization. Fig. 1A shows schematically the structure of wildtype Sap-1 (left) and a number of deletion constructs that were used in this study. Sap-1 has a single intracellular catalytic domain (red) and eight extracellular fibro-
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Fig. 1. Chemical cross-linking of Sap-1. (A) PTP-Sap-1 constructs used in this work. FL: full-length construct; FL HA: idem, with a C-terminal HAtag; DECD: lacking extracellular domain, with N-terminal HA-tag; and att HA: intracellular domain with C-terminal HA-tag, lacking transmembrane domain and attached to the plasma membrane by a membrane attachment domain. D-intra: lacking all intracellular sequences, carrying an internal myc/His6 tag. (B) COS7 cells were transfected with expression vectors for FL HA, or a DECD and treated with cross-linking agent BS3, a non-cell-permeable compound. (C) Cross-linking with DSS, a chemical cross-linker that is cell-permeable. In this assay, Sap-1 att-HA is also tested for dimerization. Cross-linked products are indicated by asterisks.
nectin-III-like domains (blue). The various constructs carry intra- or extracellular HA (green) or Myc/His6 (yellow) epitope tags. When (full-length) PTP-Sap-1 overexpressing COS-7 cells were incubated with either BS3 or DSS, a band shift was observed upon SDS–PAGE analysis (Fig. 1B, lanes 3 and 4). The cross-linked material migrated at more than 210 kDa, which corresponds to dimers of PTP-Sap-1 (120 kDa). The smearing may result from additional cross-linking to receptor ligands or to other membrane proteins. Similar results were obtained with the untagged full-length construct (data not shown). When cells overexpressed DECD (which lacks all of the extracellular fibronectin III-like domains), no additional products were observed (Fig. 1B, lanes 1 and 2). When transfected cells were treated with the cell-permeable cross-linker DSS, a very similar result was obtained (Fig. 1C, left panel). We also tested a construct (att-HA) that lacked the transmembrane region, in addition to all extracellular sequences. This construct was directed to the cell membrane through the Lck myristoylation site (Fig. 1C, right panel). Neither the intracellular form of PTP-Sap-1 nor the membrane-targeted construct produced multimers. We also verified, using immunohistochemistry, that the membrane-targeted constructs were predominantly localized at the cell membrane (Supplementary Fig. 1). In the case of the att-HA construct, we found that many cells showed a rounded phenotype (Supplementary Fig. 1, right top panel inset). This may result from an increased cellular phosphatase activity of this construct, followed by activation of src (see below). We conclude that at least a fraction of Sap-1 forms dimers, and that the extracellular domain is essential
for protein–protein interaction as seen after chemical cross-linking. This situation contrasts with PTP-a, where dimerization is mediated by the N-terminal D1 (active) catalytic domains and transmembrane regions [16]. While the PTP-a catalytic domains form spontaneous dimers [17], we have observed, in large-scale purification of the Sap-1 cytoplasmic domain, that this protein forms no dimers, as judged by size exclusion chromatography (data not shown). Sap-1 dimerizes through its extracellular domain In order to test whether Sap-1 chemical cross-linking was due to homophilic interaction between two Sap-1 molecules (homodimerization) or by binding to other proteins only, we have performed immunoprecipitation (IP) of the Sap-1 complex from cells that co-expressed differently tagged Sap-1. We have used the full-length HA-tagged construct and His6-tagged Sap-1 extracellular domain construct (D-intra; see Fig. 1A). First, the HA-tagged Sap-1 constructs were immunoprecipitated with anti-HA; then, the presence of the His-tagged D-intra was probed with an anti-His6 antibody. As shown in Fig. 2 (left panel), the FL-HA construct efficiently coimmunoprecipitated with the D-intra construct, but not at all with the construct that only expressed the intracellular domains plus transmembrane region (DECD; lane 1), even though this construct was efficiently expressed, as shown in the control gel for the same IPs (Fig. 2, right panel). We conclude that Sap-1 forms homodimers and confirm that this dimerization requires two extracellular domains. This interaction is quite stable, since this immunoprecipitation experiment was performed without the use of chemical cross-linkers.
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Fig. 2. Sap-1 forms homodimers. Left panel: cells were transfected to co-express full-length, His6-tagged Sap-1 extracellular domains (D-intra) plus various HA-tagged Sap-1 constructs. HA-tagged protein was immunoprecipitated, and the presence of His-tagged Sap-1 was detected with an antibody. Right panel: the same samples as shown in the left panel were re-tested, as a control, on Western blot for the expression of HA-tagged constructs. The identity of several bands is indicated on the right.
One or more unpaired cysteines is essential for dimerization The Sap-1 extracellular domain contains an unpaired cysteine (after we corrected 13 amino acids in the originally published sequence [15], and as the corrected sequence now appears in GenBank as D15049). Since extracellular cysteines are normally engaged in disulfide bridges, we assessed whether one of the 13 cysteines in the Sap-1 extracellular domain could be involved in
dimerization between Sap-1 partners. In order to analyze the role of free Sap-1 cysteines, we overexpressed the tagged, full-length Sap-1 protein and lysed the cells in the presence of iodoacetamide (IODA), which irreversibly binds to free-sulfhydryl (-SH) groups, followed by SDS–PAGE electrophoresis in the presence or absence of reducing agent. As shown in Fig. 3, full-length Sap-1 migrated partially as a dimer in non-reducing gel (left panel). The construct that lacked the extracellular domain (DECD) did not show dimerization nor was dimerization observed upon migration in standard gel (Fig. 3A, right panel). We also exposed Sap-1 expressing cells to DTT, followed by lysis in IODA and non-reducing SDS–PAGE. As shown in Fig. 3B, treatment of cells with increasing amounts of DTT reduces the extent of Sap-1 dimerization (lanes 1–4; Sap-1 dimer is marked with an asterisk). Disruption of dimerization by DTT is reversible; when cells that had been incubated in 50 mM DTT were washed in medium that lacked DTT, the Sap-1 dimer re-formed (Fig. 3B, lane 6). We conclude that one (or perhaps more) cysteines in the Sap-1 extracellular domain are essential for the formation of stable Sap-1 dimers, probably by forming a disulfide bridge between two Sap-1 proteins. Dimerization results in a reduction of Sap-1 enzymatic activity In order to probe the catalytic activity of various Sap1 constructs we co-transfected cells with c-src, which is a
Fig. 3. Involvement of unpaired cysteine(s) in Sap-1 multimerization. (A) COS7 cells transfected with Sap-1 constructs (FL or DECD) or vector were lysed in buffer containing iodoacetamide (IODA). The protein extracts were SDS–PAGE electrophoresed under reducing or non-reducing conditions. As a positive control, a BS3 cross-linking experiment was performed in parallel. (B) Left panel: COS7 cells that overexpress HA-tagged Sap-1 were incubated for 30 min with the indicated concentration of dithiothreitol (DTT) and lysates were analyzed as described in (A). Right panel: after incubation for 30 min, DTT was washed out and cells were incubated for a further 15 min in DTT-free medium. Cross-linked products are indicated by asterisks.
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substrate of Sap-1 (S.W., unpublished). Src was subsequently immunoprecipitated from lysates and tested for kinase activity by autophosphorylation in the presence of [c-32P]ATP. As shown in Fig. 4, Sap-1 co-transfection resulted in increased src kinase activity (quantification shown in Fig. 4B). The increase in src activity was larger for the Sap-1 construct that lacked the extracellular domain (DECD), even though controls indicated that it was expressed at somewhat lower levels than the Sap-1 full-length construct (Fig. 4A, right panel). Since we know that DECD does not form dimers, this suggests that monomeric Sap-1 is significantly more active than the dimeric form. In order to test more directly that Sap-1 activity is associated with its dimerization state, we took advantage of the possibility to disrupt cysteine bridges by treatment with DTT. COS7 cells were co-transfected for expression of c-src and full-length PTP-Sap-1, and were incubated with varying amounts of the reducing agent. Here again we observed a reproducible increase in c-src activation, supporting the notion that the dimerization of Sap-1 inhibits its catalytic activity (data not shown).
Fig. 4. Sap-1 that lacks the extracellular domain has increased phosphatase activity. (A) COS7 cells transiently co-expressed c-src and either DECD or full-length Sap-1. c-Src was immunoprecipitated and tested for autophosphorylation activity using [c-32P]ATP. The same lysates were also tested for overall src expression (center panel) and expression of the Sap-1 constructs (right panel). (B) Quantification of data shown in (A), left panel. The units on the vertical axis are in pixel count (thousands).
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Discussion In the work described here, we show that the Sap-1 extracellular domain induces protein dimerization and reduces the enzymeÕs catalytic activity. While this dimerization state can be reversibly controlled by changing the redox state of the extracellular environment, we do not know whether physiological regulators exist. We also show that Sap-1 efficiently activates c-src (Fig. 4) and that the expression of a Sap-1 construct that lacks the ECD results in a rounded phenotype of the cells (Supplementary Fig. 1), which is associated with dephosphorylation of the c-src autoinhibitory C-terminal tyrosine phosphate (S.W., unpublished). These data provide a rationale for Sap-1Õs role in gastrointestinal cancer and suggest that blocking Sap-1, either by inhibitors directed against its catalytic domain, or by agents that lock or stabilize dimerization, may be useful in the treatment of gastrointestinal cancers. Enhanced Sap-1 dimerization and enzymatic inactivation could be accomplished or stabilized with bivalent antibodies or multimeric, fibronectin-binding compounds. In the case of CD-45, it has been shown that antibodies against this PTPÕs extracellular domain do indeed have the potential to inhibit CD45 catalytic activity [18,19]. Although dimerization of receptor PTPs has now been established for a number of cases, Sap-1 is unique in that it is the first PTP with a single catalytic domain for which this is shown and, more importantly, this dimerization appears to be driven by its extracellular domains rather than by the catalytic domains. We have also investigated the full-length PTP-b, a PTP related (by sequence) to Sap-1, for its ability to form dimers under the same conditions (chemical cross-linking or DTT treatment). We were unable to see PTP-b dimerization when tested in parallel with Sap-1 (data not shown). DEP1, another relative of Sap-1, does not have an odd number of cysteines in its predicted extracellular domain, suggesting that the regulation of Sap-1 is unique. A survey among 13,049 mammalian extracellular protein domains showed that the majority of these (7321) lack cysteines altogether while among those that do have cysteines, an odd number (4145) is over 2.5-fold more frequent than an even number (1592; M. Ibberson, unpublished observations). However, our finding that two halves of the ECD can each homodimerize argues against a single cysteine being involved. One can speculate about the involvement of a ligand that helps catalyze the covalent cross-linking of receptor subunits, as has been demonstrated for the CSF-1 [20] and PDGF [21] receptors. Our chemical cross-linking experiments do not allow us to say what percentage of Sap-1 molecules is naturally dimerized, because the irreversible cross-linking event may shift and ‘‘freeze’’ the natural equilibrium between Sap-1 di- and monomeric states. It is also unclear to what extent this equilibrium depends on absolute
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Sap-1 expression levels or whether natural modulators to control Sap-1 activity exist. Nevertheless, the striking difference in catalytic activity between full-length and intracellular domain-only Sap-1 constructs (Fig. 4) is suggestive of a physiological ‘‘switch.’’ Interestingly, it has been reported that a significant fraction of the growth hormone receptor (GHR) pool is exported to the plasma membrane in an already dimerized form [22]; a similar mechanism may apply to Sap-1. If Sap-1 is also being recycled, as many receptors are [23], then this might be one process whereby the cell can modulate Sap-1 dimerization and (re)activation.
Acknowledgments We thank J.S. Brugge for the src antiserum, K. Maundrell for src cDNA, and M. Ibberson for the bioinformatics analysis of receptor extracellular domains. We are grateful to Dr. Matozaki for the Sap-1 cDNA and the rabbit anti-serum. We thank Prof. Kirsten Sandvig (The Norwegian Radium Hospital, Oslo, Norway) for support. Part of S.W.Õs work was as a recipient of a postdoctoral fellowship from the Medical Faculty of the University of Oslo.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.bbrc.2005.03.196.
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Sap-1/PTPRH activity is regulated by reversible dimerization Se´bastien Wa¨lchli 1, Xavier Espanel 2, Rob Hooft van Huijsduijnen * Serono Pharmaceutical Research Institute, 14, chemin des Aulx, Plan-les-Ouates/Geneva, Switzerland Received 3 March 2005 Available online 7 April 2005
Abstract Sap-1/PTPRH, a receptor protein tyrosine phosphatase (RPTP), is a ubiquitously expressed enzyme that is upregulated in human gastrointestinal cancers. Using both chemical cross-linkers and co-immunoprecipitation we show that overexpressed full-length Sap1 is present as a stable homodimer. Unlike a number of adhesion RPTPs which have tandem catalytic domains that are involved in dimerization, Sap-1 has a single catalytic domain, and we show that this domain is not required for Sap-1 dimerization, which is mediated instead by the large extracellular and transmembrane domains. Exposing cells that express the receptor to a reducing environment reversibly disrupts the Sap-1 dimer, suggesting that cysteine bonds play a role in dimer formation/stabilization. The switch between Sap-1 dimers and monomers is accompanied by an increase in catalytic activity as judged by its capacity to dephosphorylate and activate c-src, which we identify as a novel substrate for this phosphatase. 2005 Elsevier Inc. All rights reserved. Keywords: Protein tyrosine phosphatase; Sap-1; Dimerization; Signaling cascade
Many protein tyrosine phosphatases (PTPs) are type I transmembrane proteins (‘‘receptor-PTPs’’ or RPTPs). Most RPTPs have two domains with homology to phosphatase catalytic domains, where only the plasma membrane-proximal (D1) domain has significant catalytic activity. In a mechanism that is the exact opposite of how many receptor kinases are activated, some RPTPs are known to be active as monomers but inactive in the dimerized state. Although evidence for dimerization has been obtained for a number of PTPs, it is by no means clear if all receptor PTPs undergo dimerization or how this relates to activity [1]. PTP-DEP-1 has been shown to undergo activation upon incubation with Matrigel, but the exact nature of the ligand or whether this activation is associated with dimerization is known [2]. So far, only a single bona fide soluble ligand, midkine, has been reported for a receptor PTP (PTP-f) [3].
Many other receptor PTPs have adhesion-like extracellular domains and are likely involved in homo- or heterotypic intercellular binding [4–8]. While receptor PTPs with two catalytic domains have been well studied, very little is known about multimerization of receptor PTPs that have a single domain, such as PTP-Sap-1 (stomach cancer-associated phosphatase/ PTPRH; [9]. This PTP was initially discovered in colorectal cancers [10,11]. More recent studies conclude that the protein is downregulated in advanced hepatic carcinomas [12] and that it induces apoptosis [13] with Lck as a potential substrate [14]. In the present study, we investigate if Sap-1 may form higher-order complexes, and whether these complexes are associated with altered catalytic activity.
Materials and methods *
Corresponding author. Fax: +41 22 7946965. E-mail address: [email protected] (R.H. van Huijsduijnen). 1 Present address: Institute for Cancer Research, The Norwegian Radium Hospital, Montebello 0310 Oslo, Norway. 2 Present address: Sanofi-Synthelabo, 31676 LABEGE, France. 0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.196
Antibodies, peptides, and chemical reagents. Western blot detection of HA-tags was performed with mouse monoclonal anti-HA.11 (BAbCO) or rabbit polyclonal anti-HA (Santa Cruz). Immunoprecipitation was carried out with anti-HA.11 (BAbCO). Anti-Myc:
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mouse monoclonal anti-Myc9E10 (Santa Cruz). Anti-SRC327 was a gift from Dr. J. Brugge; rabbit polyclonal anti-SRCPAN, rabbit polyclonal anti-SRCPY530 were from Biosource and rabbit polyclonal anti-SRC was from Santa Cruz. Mouse monoclonal anti-His was from Dianova. DSS and BS3 were from Pierce, dithiothreitol, DTT, iodoacetamide, and IODA were from Sigma. Cell culture, transfection. COS7 cells were grown under 5% CO2 in DulbeccoÕs modified EagleÕs medium, DMEM, with 10% FCS, penicillin at 105 U/L, streptomycin at 105 U/L, and L-glutamine. Transfections were performed with Fugene-6 reagent (Roche) following the manufacturerÕs instructions. BS3, DSS, and DTT treatments. Twenty-four after transfection in COS7 cells, the medium was changed and a solution of BS3 in PBS (2.5 mg/ml) or PBS alone was added and left for 1 h at 4 C. The crosslinking reaction was stopped by the addition of PBS containing 0.15 M Tris, pH 7.5, for 15 min at 4 C followed by lysis. Treatment with DSS was almost the same except that the product was dissolved in DMSO at a concentration of 25 mM (10·) and diluted in PBS; control cells were incubated with 0.1· DMSO. Cells treated with DTT were serum-starved for 8 h after transfection (with PTP-Sap-1 constructs or vector), and incubated for 30 min in serum-free medium with various amounts of DTT. Before lysis in a buffer containing 150 mM IODA, cells were washed with ice-cold PBS containing 50 mM IODA in order to block free DTT. Samples were run under reducing or non-reducing conditions, with or without b-mercaptoethanol in the sample buffer, respectively. PTP-Sap-1 constructs. PTP-Sap-1 FL construct was as described [15]. The complete cDNA with the poly(A) tail was subcloned into several vectors using XbaI and HindIII restriction enzymes. Most of the experiments presented herein were done with the pcDNA4a vector (Invitrogen). The pcDNA4-Sap-1 DECD HA clone is a fusion of the signal peptide sequence of PTP-Sap-1, followed by a HA-tag and the PTP sequence from the N-terminal part of the transmembrane region. It was prepared as follows: the full cytoplasmic domain of PTP-Sap-1 was amplified by polymerase chain reaction (PCR) with Herculase Polymerase (Stratagene) using an antisense primer from the vector and a sense primer containing the HA-tag fused to the sequence encoding the five extracellular amino acids on the N-terminal side of the transmembrane (5 0 -TAC CCA TAC GAC GTC CCA GAC TAC GCT CAC ACC GAG AGT GCA GGG GT-3 0 ). On the other side, the signal peptide was amplified with a reverse primer fused to the HAtag in the C-terminal part of the sequence (5 0 -AGC GTA GTC TGG GAC GTC GTA TGG GTA GGG GGC AGG CGC CCT GGC CCC T-3 0 ) and the forward primer was from the vector. The two PCR products were mixed and amplified again with external primers. Two XbaI sites (one generated and the other one from the original vector) surrounding the amplicon were used to clone the intracellular part of PTP-Sap-1 fused to the signal peptide and the HA-tag. The full-length construct fused in C-terminal frame with an HA-tag was prepared with an antisense primer containing the sequence of an XhoI site, an HAtag, and the end of the PTP-Sap-1 sequence with the STOP codon (5 0 TAC TCG AGT TAA GCG TAG TCT GGG ACG TCG TAT GGG TAG ACC TCC AAC TTG TGG GCC T-3 0 ). The full-length Sap-1HA fusion construct was amplified using a plasmid specific forward primer in a long run PCR using Herculase in the presence of 5% DMSO and Hotstart conditions. The D-intra construct was obtained by amplifying the extracellular domain sequence with a primer complementary to the intracellular proximal transmembrane region coding sequence (about 30 bp downstream) in order to keep the targeting sequence (5 0 -ATG AAT TCA GCG GCC CAT CTG GCT GCC TCT TTC TCA GGA AGA AAA TCA-3 0 ) and adding a, EcoRI site. When the amplicon was fused to pCDNA4b, it was in-frame with the two tags (His and c-Myc) and a STOP codon. PTP-Sap-1 att HA (membrane associated) consisted of a fusion of the PTP-Sap-1 cytoplasmic region with an HA-tag in its C-term part (amplification of the pcDNA4-PTP-Sap-1FLHA construct). A primer containing the Lck myristoylation site (underlined) and a start codon surrounded by a
Kozak sequence ATA AGC TTA CCa tgg gct gtg gct gca gct cac acc cgg aag atg act ggA AGA GGA GGA ATA AGA AGA AG was used with a vector primer to amplify the cytoplasmic fragment of PTP-Sap1. The amplicon was cloned into pcDNA4 using HindIII restriction site of the primer for the 5 0 end ligation. All constructs were checked by DNA sequencing. c-src constructs. c-src cDNA was a gift from Dr. K. Maundrell. It was subcloned into pcDNA4 (Invitrogen). The c-src Y530F mutant was made using by site-directed mutagenesis with the following primers (5 0 -AGT TCC AGC CCG GGG AGA ACC TC-3 0 and 5 0 GAG GTT CTC CCC GGG CTG GAA CT-3 0 ). Immunoprecipitation. Every immunoprecipitation (IP) was performed with the same buffer and optimal dilution of the antibodies. Cells were incubated in RIPA lysis buffer (PBS 1·, 1% IGEPAL, 0.5% Na–deoxycholate, 0.1% SDS supplemented with a complete protease inhibitor cocktail tablet (Roche), and sodium orthovanadate 1 mM) or NP-40 buffer for anti-myc (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 25 mM b-glycerophosphate, 1 mM sodium pyrophosphate, 1% NP-40, and supplemented with a complete protease inhibitor cocktail tablet (Roche)), both buffers being used ice-cold. Cells were mechanically broken by one cycle of freeze–thawing and passed through a G21 syringe. Lysates were clarified by centrifugation (14,000 rpm) for 10 min at 4 C and precleared with normal antibody (Santa Cruz) and 20 ll of precoated protein A/G–Sepharose beads (Santa Cruz). The supernatant from centrifugation was recovered and incubated with the specific antibody (that had been precoated for 2 h on protA/G beads) overnight at 4 C. The beads were finally washed once with lysis buffer and twice with PBS–0.1% Triton X-100 (the volume of the washes corresponds to two volumes of the lysate). Western blot. Proteins were separated on SDS–polyacrylamide gel (PAGE) using the NuPAGE-gel system from Novex (InVitroGen), following the manufacturerÔs protocols. Proteins were transferred to PVDF membrane and detected by Western blot. Kinase assay. After the last wash of immunoprecipitation, the kinase was dissolved in phosphatase buffer (20 mM Tris, pH 7.5, 150 mM NaCl, and 1 mM EDTA). 1/10 of this solution was run on a SDS–PAGE to perform a control Western blot, while the rest was used for a kinase assay. The immunoprecipitate was directly incubated with MBP, sodium orthovanadate 1 mM, and 35 lCi [c-32P]ATP. In addition, the buffer was brought to a final concentration of 5 mM MgCl2, 2.5 mM MnCl2, and 2.5 mM DTT. The reaction was stopped after 30 min at 30 C by the addition of protein-sample buffer and run on a SDS–PAGE. Bands were visualized by autoradiography using Kodak X-OMAT films.
Results Sap-1-multimers can be revealed by chemical cross-linkers The chemical cross-linkers BS3 and DSS were used to detect protein–protein interactions. BS3 cannot pass cell membranes and can only cross-link extracellular protein. By contrast, DSS is cell-permeable and it can cross-link both extra- and intracellular proteins. Upon incubation with DSS, receptor-PTP-a has been shown to shift its migration profile when run on a SDS–polyacrylamide gel (SDS–PAGE; [16]). We have studied Sap-1 for evidence of dimerization. Fig. 1A shows schematically the structure of wildtype Sap-1 (left) and a number of deletion constructs that were used in this study. Sap-1 has a single intracellular catalytic domain (red) and eight extracellular fibro-
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Fig. 1. Chemical cross-linking of Sap-1. (A) PTP-Sap-1 constructs used in this work. FL: full-length construct; FL HA: idem, with a C-terminal HAtag; DECD: lacking extracellular domain, with N-terminal HA-tag; and att HA: intracellular domain with C-terminal HA-tag, lacking transmembrane domain and attached to the plasma membrane by a membrane attachment domain. D-intra: lacking all intracellular sequences, carrying an internal myc/His6 tag. (B) COS7 cells were transfected with expression vectors for FL HA, or a DECD and treated with cross-linking agent BS3, a non-cell-permeable compound. (C) Cross-linking with DSS, a chemical cross-linker that is cell-permeable. In this assay, Sap-1 att-HA is also tested for dimerization. Cross-linked products are indicated by asterisks.
nectin-III-like domains (blue). The various constructs carry intra- or extracellular HA (green) or Myc/His6 (yellow) epitope tags. When (full-length) PTP-Sap-1 overexpressing COS-7 cells were incubated with either BS3 or DSS, a band shift was observed upon SDS–PAGE analysis (Fig. 1B, lanes 3 and 4). The cross-linked material migrated at more than 210 kDa, which corresponds to dimers of PTP-Sap-1 (120 kDa). The smearing may result from additional cross-linking to receptor ligands or to other membrane proteins. Similar results were obtained with the untagged full-length construct (data not shown). When cells overexpressed DECD (which lacks all of the extracellular fibronectin III-like domains), no additional products were observed (Fig. 1B, lanes 1 and 2). When transfected cells were treated with the cell-permeable cross-linker DSS, a very similar result was obtained (Fig. 1C, left panel). We also tested a construct (att-HA) that lacked the transmembrane region, in addition to all extracellular sequences. This construct was directed to the cell membrane through the Lck myristoylation site (Fig. 1C, right panel). Neither the intracellular form of PTP-Sap-1 nor the membrane-targeted construct produced multimers. We also verified, using immunohistochemistry, that the membrane-targeted constructs were predominantly localized at the cell membrane (Supplementary Fig. 1). In the case of the att-HA construct, we found that many cells showed a rounded phenotype (Supplementary Fig. 1, right top panel inset). This may result from an increased cellular phosphatase activity of this construct, followed by activation of src (see below). We conclude that at least a fraction of Sap-1 forms dimers, and that the extracellular domain is essential
for protein–protein interaction as seen after chemical cross-linking. This situation contrasts with PTP-a, where dimerization is mediated by the N-terminal D1 (active) catalytic domains and transmembrane regions [16]. While the PTP-a catalytic domains form spontaneous dimers [17], we have observed, in large-scale purification of the Sap-1 cytoplasmic domain, that this protein forms no dimers, as judged by size exclusion chromatography (data not shown). Sap-1 dimerizes through its extracellular domain In order to test whether Sap-1 chemical cross-linking was due to homophilic interaction between two Sap-1 molecules (homodimerization) or by binding to other proteins only, we have performed immunoprecipitation (IP) of the Sap-1 complex from cells that co-expressed differently tagged Sap-1. We have used the full-length HA-tagged construct and His6-tagged Sap-1 extracellular domain construct (D-intra; see Fig. 1A). First, the HA-tagged Sap-1 constructs were immunoprecipitated with anti-HA; then, the presence of the His-tagged D-intra was probed with an anti-His6 antibody. As shown in Fig. 2 (left panel), the FL-HA construct efficiently coimmunoprecipitated with the D-intra construct, but not at all with the construct that only expressed the intracellular domains plus transmembrane region (DECD; lane 1), even though this construct was efficiently expressed, as shown in the control gel for the same IPs (Fig. 2, right panel). We conclude that Sap-1 forms homodimers and confirm that this dimerization requires two extracellular domains. This interaction is quite stable, since this immunoprecipitation experiment was performed without the use of chemical cross-linkers.
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Fig. 2. Sap-1 forms homodimers. Left panel: cells were transfected to co-express full-length, His6-tagged Sap-1 extracellular domains (D-intra) plus various HA-tagged Sap-1 constructs. HA-tagged protein was immunoprecipitated, and the presence of His-tagged Sap-1 was detected with an antibody. Right panel: the same samples as shown in the left panel were re-tested, as a control, on Western blot for the expression of HA-tagged constructs. The identity of several bands is indicated on the right.
One or more unpaired cysteines is essential for dimerization The Sap-1 extracellular domain contains an unpaired cysteine (after we corrected 13 amino acids in the originally published sequence [15], and as the corrected sequence now appears in GenBank as D15049). Since extracellular cysteines are normally engaged in disulfide bridges, we assessed whether one of the 13 cysteines in the Sap-1 extracellular domain could be involved in
dimerization between Sap-1 partners. In order to analyze the role of free Sap-1 cysteines, we overexpressed the tagged, full-length Sap-1 protein and lysed the cells in the presence of iodoacetamide (IODA), which irreversibly binds to free-sulfhydryl (-SH) groups, followed by SDS–PAGE electrophoresis in the presence or absence of reducing agent. As shown in Fig. 3, full-length Sap-1 migrated partially as a dimer in non-reducing gel (left panel). The construct that lacked the extracellular domain (DECD) did not show dimerization nor was dimerization observed upon migration in standard gel (Fig. 3A, right panel). We also exposed Sap-1 expressing cells to DTT, followed by lysis in IODA and non-reducing SDS–PAGE. As shown in Fig. 3B, treatment of cells with increasing amounts of DTT reduces the extent of Sap-1 dimerization (lanes 1–4; Sap-1 dimer is marked with an asterisk). Disruption of dimerization by DTT is reversible; when cells that had been incubated in 50 mM DTT were washed in medium that lacked DTT, the Sap-1 dimer re-formed (Fig. 3B, lane 6). We conclude that one (or perhaps more) cysteines in the Sap-1 extracellular domain are essential for the formation of stable Sap-1 dimers, probably by forming a disulfide bridge between two Sap-1 proteins. Dimerization results in a reduction of Sap-1 enzymatic activity In order to probe the catalytic activity of various Sap1 constructs we co-transfected cells with c-src, which is a
Fig. 3. Involvement of unpaired cysteine(s) in Sap-1 multimerization. (A) COS7 cells transfected with Sap-1 constructs (FL or DECD) or vector were lysed in buffer containing iodoacetamide (IODA). The protein extracts were SDS–PAGE electrophoresed under reducing or non-reducing conditions. As a positive control, a BS3 cross-linking experiment was performed in parallel. (B) Left panel: COS7 cells that overexpress HA-tagged Sap-1 were incubated for 30 min with the indicated concentration of dithiothreitol (DTT) and lysates were analyzed as described in (A). Right panel: after incubation for 30 min, DTT was washed out and cells were incubated for a further 15 min in DTT-free medium. Cross-linked products are indicated by asterisks.
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substrate of Sap-1 (S.W., unpublished). Src was subsequently immunoprecipitated from lysates and tested for kinase activity by autophosphorylation in the presence of [c-32P]ATP. As shown in Fig. 4, Sap-1 co-transfection resulted in increased src kinase activity (quantification shown in Fig. 4B). The increase in src activity was larger for the Sap-1 construct that lacked the extracellular domain (DECD), even though controls indicated that it was expressed at somewhat lower levels than the Sap-1 full-length construct (Fig. 4A, right panel). Since we know that DECD does not form dimers, this suggests that monomeric Sap-1 is significantly more active than the dimeric form. In order to test more directly that Sap-1 activity is associated with its dimerization state, we took advantage of the possibility to disrupt cysteine bridges by treatment with DTT. COS7 cells were co-transfected for expression of c-src and full-length PTP-Sap-1, and were incubated with varying amounts of the reducing agent. Here again we observed a reproducible increase in c-src activation, supporting the notion that the dimerization of Sap-1 inhibits its catalytic activity (data not shown).
Fig. 4. Sap-1 that lacks the extracellular domain has increased phosphatase activity. (A) COS7 cells transiently co-expressed c-src and either DECD or full-length Sap-1. c-Src was immunoprecipitated and tested for autophosphorylation activity using [c-32P]ATP. The same lysates were also tested for overall src expression (center panel) and expression of the Sap-1 constructs (right panel). (B) Quantification of data shown in (A), left panel. The units on the vertical axis are in pixel count (thousands).
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Discussion In the work described here, we show that the Sap-1 extracellular domain induces protein dimerization and reduces the enzymeÕs catalytic activity. While this dimerization state can be reversibly controlled by changing the redox state of the extracellular environment, we do not know whether physiological regulators exist. We also show that Sap-1 efficiently activates c-src (Fig. 4) and that the expression of a Sap-1 construct that lacks the ECD results in a rounded phenotype of the cells (Supplementary Fig. 1), which is associated with dephosphorylation of the c-src autoinhibitory C-terminal tyrosine phosphate (S.W., unpublished). These data provide a rationale for Sap-1Õs role in gastrointestinal cancer and suggest that blocking Sap-1, either by inhibitors directed against its catalytic domain, or by agents that lock or stabilize dimerization, may be useful in the treatment of gastrointestinal cancers. Enhanced Sap-1 dimerization and enzymatic inactivation could be accomplished or stabilized with bivalent antibodies or multimeric, fibronectin-binding compounds. In the case of CD-45, it has been shown that antibodies against this PTPÕs extracellular domain do indeed have the potential to inhibit CD45 catalytic activity [18,19]. Although dimerization of receptor PTPs has now been established for a number of cases, Sap-1 is unique in that it is the first PTP with a single catalytic domain for which this is shown and, more importantly, this dimerization appears to be driven by its extracellular domains rather than by the catalytic domains. We have also investigated the full-length PTP-b, a PTP related (by sequence) to Sap-1, for its ability to form dimers under the same conditions (chemical cross-linking or DTT treatment). We were unable to see PTP-b dimerization when tested in parallel with Sap-1 (data not shown). DEP1, another relative of Sap-1, does not have an odd number of cysteines in its predicted extracellular domain, suggesting that the regulation of Sap-1 is unique. A survey among 13,049 mammalian extracellular protein domains showed that the majority of these (7321) lack cysteines altogether while among those that do have cysteines, an odd number (4145) is over 2.5-fold more frequent than an even number (1592; M. Ibberson, unpublished observations). However, our finding that two halves of the ECD can each homodimerize argues against a single cysteine being involved. One can speculate about the involvement of a ligand that helps catalyze the covalent cross-linking of receptor subunits, as has been demonstrated for the CSF-1 [20] and PDGF [21] receptors. Our chemical cross-linking experiments do not allow us to say what percentage of Sap-1 molecules is naturally dimerized, because the irreversible cross-linking event may shift and ‘‘freeze’’ the natural equilibrium between Sap-1 di- and monomeric states. It is also unclear to what extent this equilibrium depends on absolute
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Sap-1 expression levels or whether natural modulators to control Sap-1 activity exist. Nevertheless, the striking difference in catalytic activity between full-length and intracellular domain-only Sap-1 constructs (Fig. 4) is suggestive of a physiological ‘‘switch.’’ Interestingly, it has been reported that a significant fraction of the growth hormone receptor (GHR) pool is exported to the plasma membrane in an already dimerized form [22]; a similar mechanism may apply to Sap-1. If Sap-1 is also being recycled, as many receptors are [23], then this might be one process whereby the cell can modulate Sap-1 dimerization and (re)activation.
Acknowledgments We thank J.S. Brugge for the src antiserum, K. Maundrell for src cDNA, and M. Ibberson for the bioinformatics analysis of receptor extracellular domains. We are grateful to Dr. Matozaki for the Sap-1 cDNA and the rabbit anti-serum. We thank Prof. Kirsten Sandvig (The Norwegian Radium Hospital, Oslo, Norway) for support. Part of S.W.Õs work was as a recipient of a postdoctoral fellowship from the Medical Faculty of the University of Oslo.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.bbrc.2005.03.196.
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