PTP1B Modulates the Association of β-Catenin with N-cadherin through Binding to an Adjacent and Partially Overlapping Target Site
2002; Elsevier BV; Volume: 277; Issue: 51 Linguagem: Inglês
10.1074/jbc.m206454200
ISSN1083-351X
AutoresGang Xu, Carlos O. Arregui, Jack Lilien, Janne Balsamo,
Tópico(s)Galectins and Cancer Biology
ResumoThe nonreceptor tyrosine phosphatase PTP1B associates with the cytoplasmic domain of N-cadherin and may regulate cadherin function through dephosphorylation of β-catenin. We have now identified the domain on N-cadherin to which PTP1B binds and characterized the effect of perturbing this domain on cadherin function. Deletion constructs lacking amino acids 872–891 fail to bind PTP1B. This domain partially overlaps with the β-catenin binding domain. To further define the relationship of these two sites, we used peptides to compete in vitro binding. A peptide representing the most NH2-terminal 8 amino acids of the PTP1B binding site, the region of overlap with the β-catenin target, effectively competes for binding of β-catenin but is much less effective in competing PTP1B, whereas two peptides representing the remaining 12 amino acids have no effect on β-catenin binding but effectively compete for PTP1B binding. Introduction into embryonic chick retina cells of a cell-permeable peptide mimicking the 8 most COOH-terminal amino acids in the PTP1B target domain, the region most distant from the β-catenin target site, prevents binding of PTP1B, increases the pool of free, tyrosine-phosphorylated β-catenin, and results in loss of N-cadherin function. N-cadherin lacking this same region of the PTP1B target site does not associate with PTP1B or β-catenin and is not efficiently expressed at the cell surface of transfected L cells. Thus, interaction of PTP1B with N-cadherin is essential for its association with β-catenin, stable expression at the cell surface, and consequently, cadherin function. The nonreceptor tyrosine phosphatase PTP1B associates with the cytoplasmic domain of N-cadherin and may regulate cadherin function through dephosphorylation of β-catenin. We have now identified the domain on N-cadherin to which PTP1B binds and characterized the effect of perturbing this domain on cadherin function. Deletion constructs lacking amino acids 872–891 fail to bind PTP1B. This domain partially overlaps with the β-catenin binding domain. To further define the relationship of these two sites, we used peptides to compete in vitro binding. A peptide representing the most NH2-terminal 8 amino acids of the PTP1B binding site, the region of overlap with the β-catenin target, effectively competes for binding of β-catenin but is much less effective in competing PTP1B, whereas two peptides representing the remaining 12 amino acids have no effect on β-catenin binding but effectively compete for PTP1B binding. Introduction into embryonic chick retina cells of a cell-permeable peptide mimicking the 8 most COOH-terminal amino acids in the PTP1B target domain, the region most distant from the β-catenin target site, prevents binding of PTP1B, increases the pool of free, tyrosine-phosphorylated β-catenin, and results in loss of N-cadherin function. N-cadherin lacking this same region of the PTP1B target site does not associate with PTP1B or β-catenin and is not efficiently expressed at the cell surface of transfected L cells. Thus, interaction of PTP1B with N-cadherin is essential for its association with β-catenin, stable expression at the cell surface, and consequently, cadherin function. β-Catenin occupies a central position in cell physiology and development as both a transcriptional co-activator regulated by Wnt signaling (reviewed in Refs. 1Peifer M. Polakis P. Science. 2000; 287: 1606-1609Crossref PubMed Scopus (1139) Google Scholar and 2Seidensticker M.J. Behrens J. Biochim. Biophys. Acta. 2000; 1495: 168-182Crossref PubMed Scopus (235) Google Scholar) and a bridge between the cytoplasmic domain of cadherin cell-cell adhesion molecules and the actin-containing cytoskeleton (reviewed in Ref. 3Wheelock M.J. Knudsen K.A. Johnson K.R. Curr. Top. Membr. 1996; 43: 169-185Crossref Scopus (43) Google Scholar), a connection that is crucial to function (reviewed in Refs. 4Yap A.S. Brieher W.M. Pruschy M. Gumbiner B.M. Curr. Biol. 1997; 7: 308-315Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar and 5Adams C.L. Nelson W.J. Curr. Opin. Cell Biol. 1998; 10: 572-577Crossref PubMed Scopus (237) Google Scholar). It is noteworthy that distinct gene products may carry out these two key roles inCaenorhabditis elegans (6Korswagen H.C. Herman M.A. Clevers H.C. Nature. 2000; 406: 527-532Crossref PubMed Scopus (177) Google Scholar). However, inDrosophila and vertebrates, the same gene products appear to assume both roles (7Peifer M. Trends Cell Biol. 1995; 5: 224-229Abstract Full Text PDF PubMed Scopus (196) Google Scholar). It is not surprising, then, that in the absence of Wnt stimulus the pool of free β-catenin is subject to rapid degradation (8Hart M.J. de los Santos R. Albert I.N. Rubinfeld B. Polakis P. Curr. Biol. 1998; 8: 573-581Abstract Full Text Full Text PDF PubMed Google Scholar, 9Henderson B.R. Nat. Cell Biol. 2000; 2: 653-660Crossref PubMed Scopus (412) Google Scholar), possibly preventing spurious interference between these two key functions. Whereas the details of how these two roles of β-catenin are regulated to integrate function and maintain the integrity of the two pathways are not completely clear, tyrosine phosphorylation of β-catenin may be one key regulatory determinant. Phosphorylation of tyrosine residues on β-catenin has repeatedly been correlated with loss of cadherin adhesive function (reviewed in Refs.10Lilien J. Balsamo J. Arregui C. Xu G. Dev. Dyn. 2002; 224: 18-29Crossref PubMed Scopus (135) Google Scholar and 11Daniel J.M. Reynolds A.B. Bioessays. 1997; 19: 883-891Crossref PubMed Scopus (285) Google Scholar). This, in turn, is correlated with instability of the β-catenin-cadherin bond (12Roura S. Miravet S. Piedra J. de Herreros A.G. Duñach M. J. Biol. Chem. 1999; 274: 36734-36740Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar), uncoupling of cadherin from the actin-containing cytoskeleton (13Balsamo J. Arregui C. Leung T.-C. Lilien J. J. Cell Biol. 1998; 143: 523-532Crossref PubMed Scopus (143) Google Scholar, 14Balsamo J. Leung T.-C. Ernst H. Zanin M.K.B. Hoffman S. Lilien J. J. Cell Biol. 1996; 134: 801-813Crossref PubMed Scopus (194) Google Scholar), and an increase in the free cytosolic pool of tyrosine-phosphorylated β-catenin (15Kinch M.S. Clark G.J. Der C.J. Burridge K. J. Cell Biol. 1995; 130: 461-471Crossref PubMed Scopus (280) Google Scholar, 16Müller T. Choidas A. Reichmann E. Ulrich A. J. Biol. Chem. 1999; 274: 10173-10183Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Tyrosine phosphorylation of β-catenin also increases the interaction of β-catenin with basal transcription factor and increases transcriptional activity of the β-catenin-Tcf complex (12Roura S. Miravet S. Piedra J. de Herreros A.G. Duñach M. J. Biol. Chem. 1999; 274: 36734-36740Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar,17Piedra J. Martinez D. Castaño J. Miravet S. Duñach M. de Herreros A.G. J. Biol. Chem. 2001; 276: 20436-20443Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). The same tyrosine residue, 654, is critical for both instability of the β-catenin-cadherin bond and for enhanced binding to basal transcription factor (12Roura S. Miravet S. Piedra J. de Herreros A.G. Duñach M. J. Biol. Chem. 1999; 274: 36734-36740Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 17Piedra J. Martinez D. Castaño J. Miravet S. Duñach M. de Herreros A.G. J. Biol. Chem. 2001; 276: 20436-20443Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), suggesting that the two functions of β-catenin may be coordinated through the creation of a pool of free β-catenin following tyrosine phosphorylation and dissociation of the β-catenin-cadherin link and a concomitant increase in the potential of this pool to participate in transcription. Our laboratory has focused on the role of the nonreceptor tyrosine phosphatase PTP1B in regulating the phosphorylation of tyrosine residues on β-catenin in N-cadherin-expressing cells. Introduction into L-cells constitutively expressing N-cadherin of a catalytically inactive, dominant-negative construct of PTP1B results in hyperphosphorylation of tyrosine residues on β-catenin, an increase in the free cytosolic pool of tyrosine-phosphorylated β-catenin, and dissociation of the cadherin-actin connection concomitant with loss of cadherin function (13Balsamo J. Arregui C. Leung T.-C. Lilien J. J. Cell Biol. 1998; 143: 523-532Crossref PubMed Scopus (143) Google Scholar, 14Balsamo J. Leung T.-C. Ernst H. Zanin M.K.B. Hoffman S. Lilien J. J. Cell Biol. 1996; 134: 801-813Crossref PubMed Scopus (194) Google Scholar). This dominant negative PTP1B construct also inhibits neurite extension on N-cadherin substrates (18Pathre P. Arregui C. Wampler T. Kue I. Leung T.-C. Lilien J. Balsamo J. J. Neurosci Res. 2001; 63: 143-150Crossref PubMed Scopus (43) Google Scholar). Consistent with these observations, PTP1B is present at adherens junctions and localizes to growth cones (14Balsamo J. Leung T.-C. Ernst H. Zanin M.K.B. Hoffman S. Lilien J. J. Cell Biol. 1996; 134: 801-813Crossref PubMed Scopus (194) Google Scholar, 18Pathre P. Arregui C. Wampler T. Kue I. Leung T.-C. Lilien J. Balsamo J. J. Neurosci Res. 2001; 63: 143-150Crossref PubMed Scopus (43) Google Scholar). Furthermore, PTP1B binds directly to the cytoplasmic domain of N-cadherin (13Balsamo J. Arregui C. Leung T.-C. Lilien J. J. Cell Biol. 1998; 143: 523-532Crossref PubMed Scopus (143) Google Scholar, 14Balsamo J. Leung T.-C. Ernst H. Zanin M.K.B. Hoffman S. Lilien J. J. Cell Biol. 1996; 134: 801-813Crossref PubMed Scopus (194) Google Scholar). Because of the key position PTP1B occupies, it is not surprising that its binding to N-cadherin is also regulated. Indeed, we have previously shown that PTP1B must be tyrosine-phosphorylated on tyrosine 152 in order to bind to N-cadherin (13Balsamo J. Arregui C. Leung T.-C. Lilien J. J. Cell Biol. 1998; 143: 523-532Crossref PubMed Scopus (143) Google Scholar, 14Balsamo J. Leung T.-C. Ernst H. Zanin M.K.B. Hoffman S. Lilien J. J. Cell Biol. 1996; 134: 801-813Crossref PubMed Scopus (194) Google Scholar, 19Rhee J. Lilien J. Balsamo J. J. Biol. Chem. 2001; 276: 6640-6644Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In this paper, we define the target domain for PTP1B on N-cadherin and show that it is adjacent to, and partially overlaps with, the binding site for β-catenin. Introduction into primary embryonic chick neural retina cells of a cell-permeable, "Trojan" peptide that mimics the most distant, nonoverlapping portion of the PTP1B binding site in N-cadherin results in loss of cadherin function and an increase in the free pool of tyrosine-phosphorylated β-catenin. Furthermore, L-cells transfected with N-cadherin lacking the entire site or the portion that does not overlap with the β-catenin binding site show loss of N-cadherin expression at the cell surface. We suggest that the proximity of β-catenin and PTP1B binding sites allows for continuous and rapid removal of phosphate from tyrosine residues, stabilizing and maintaining the integrity of the cadherin-actin cytoskeletal linkage and cadherin function. The anti-N-cadherin antibody NCD-2 (20Hatta K. Takeichi M. Nature. 1986; 320: 447-449Crossref PubMed Scopus (519) Google Scholar) was purified from hybridoma culture medium in our laboratory as described previously (21Balsamo J. Thiboldeaux R. Swaminathan N. Lilien J. J. Cell Biol. 1991; 113: 429-436Crossref PubMed Scopus (35) Google Scholar). Anti-phosphotyrosine (PY20) monoclonal antibody was from Transduction Laboratories (Lexington, KY), anti-FLAG was from Stratagene (La Jolla, CA), and anti-phosphoserine was from Zymed Laboratories Inc. (San Francisco, CA). Anti-PTP1B antibodies were obtained from Transduction Laboratories,Calbiochem, or Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-β-catenin antibodies were a mouse monoclonal IgG from Transduction Laboratories and a rabbit polyclonal antibody directed against a synthetic 15-amino acid sequence (22Balsamo J. Ernst H. Zanin M.K. Hoffman S. Lilien J. J. Cell Biol. 1995; 129: 1391-1401Crossref PubMed Scopus (46) Google Scholar). HRP 1The abbreviations used are: HRP, horseradish peroxidase; GST, glutathione S-transferase; BSA, bovine serum albumin; PVDF, polyvinylidene difluoride; NHS, N-hydroxysuccinimide; GFP, green fluorescent protein-conjugated secondary antibodies were purchased from Organon Teknika Co. (Durham, NC), and antibodies conjugated to magnetic beads, used in immunoprecipitations, were from Polysciences Inc. (Warrington, PA). cDNAs encoding the full cytoplasmic region of N-cadherin (residues 752–912) and truncated constructs were generated by PCR using specific oligonucleotide primers flanked byEcoRI and NcoI restriction sites and chick N-cadherin cDNA as a template. The PCR products were subcloned in PGEX-KG (Amersham Biosciences). To create N-cadherin containing the extracellular, transmembrane and cytoplasmic domain carrying deletions of amino acids 872–891, 878–891, or 884–891, we used a two-step PCR procedure. Primers corresponding to the 5′-end of the N-cadherin cDNA with an added NotI site and the 3′-end with an added SalI site were used with primers flanking the deletion site in two separate PCRs. Products from these two reactions were mixed and used as template for another PCR containing only the 5′ and 3′ primers. This reaction generated N-cadherin cDNA with flankingNotI and SalI and the desired deletions. These constructs were subcloned in pCMV-FLAG-4A (Stratagene, La Jolla, CA) for mammalian expression to generate pCMV-Δ872–891 pCMV-Δ878–891 and pCMVΔ884–891. All cDNA clones were confirmed by sequencing. To prepare N-cadherin fused to green fluorescent protein, N-cadherin was amplified by PCR using a 5′ primer containing a BglII site and 3′ primer that excludes the stop codon and contains aXmaI site. The digested PCR product was cloned into theBglII/XmaI sites of the pEGF-N1 plasmid (Clontech). To prepare N-cadherinΔ884–891-GFP, the KpnI/ApaI fragment in N-cadherin was replaced with the equivalent fragment that encompasses the deletion. Catalytically inactive GST-PTP1B (C215S) was produced inEscherichia coli TKB1 cells (Stratagene, La Jolla, CA) that constitutively express a tyrosine kinase. GST-β-catenin was produced in H5α cells. Cultures were induced with 0.4 mmisopropyl-1-thio-β-d-galactopyranoside and allowed to express GST fusions for 3 h. The cultures were harvested by centrifugation at 3000 × g for 10 min, and the pellets were stored at −80 °C until use. Protein was extracted using B-PER (Pierce) containing 1% protease inhibitor mixture (Sigma) and 1 mm sodium orthovanadate (Sigma), according to the manufacturer's instructions. GST fusion proteins were purified on glutathione-Sepharose 4B (Amersham Biosciences) and confirmed by biotinylation and blotting with HRP-avidin (Fig. 1 A,top). Phosphorylation of GST-PTP1B on tyrosine residues was confirmed by immunoblot using the PY20 antibody (Fig.1 B). The following peptides mimicking the PTP1B binding site on N-cadherin were synthesized and purified to more than 90% by HPLC (BIO-SYNTHESIS, Lewisville, TX): Peptide 1 (P1), TAGSLSSL (872–879); Peptide 2 (P2), SLNSSSSG (878–885); Peptide 3 (P3), SGGEQDVD (884–891). Peptide 3R (P3R) corresponds to the reverse sequence of P3 and was used as a control. Peptide 4 (P4) (VFDYEGSG) mimics an 8-amino acid sequence within the β-catenin-binding region and was also synthesized and used as a control. Peptides corresponding to amino acids 878–891 (AP2/3) and 884–891 (AP3) of the PTP1B binding region of N-cadherin were synthesized covalently attached to the Antennapedia cell permeation sequence (Genemed, San Francisco, CA). An antennapedia sequence fused to the reverse of P3 was also prepared and used as control. All peptides were dissolved in sterile, deionized water and stored in small aliquots at −80 °C for future use. Purified GST-N-cadherin deletion constructs were biotinylated using EZ-link Sulfo-NHS-LC-Biotin (Pierce), and biotinylation was confirmed by immunoblot using streptavidin-HRP (Fig. 1 A). Binding assays were carried out as previously described (19Rhee J. Lilien J. Balsamo J. J. Biol. Chem. 2001; 276: 6640-6644Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Biotinylated GST-N-cadherin deletion constructs (3 μg/well in 50 μl of PBS) were applied to NeutrAvidin-coated 96-well plates (Pierce) and incubated at room temperature for 1 h. After blocking with 2% BSA (Sigma) in PBS for 1 h at room temperature and washing three times in PBS, purified GST-PTP1B or GST-β-catenin (in 50 μl of 0.5% BSA/PBS) was added to the wells and incubated at room temperature for another 1 h. The wells were then washed with PBS and incubated with anti-PTP1B or anti-β-catenin (0.1 μg/well) for 1 h at room temperature. The wells were washed three times with TBST (50 mm Tris, 150 mm NaCl, 0.1% Tween 20) over a period of 30 min and incubated with anti-mouse HRP antibody (0.2 μg/ml in 0.5% bovine serum albumin in TBST) for 1 h at room temperature. After washing the wells five times with TBST, bound PT1PB was determined using 3,3′,5,5′-tetramethylbenzidine (Sigma) as a substrate for HRP, and absorbance was measured at 492 nm. To determine competition for binding to N-cadherin, increasing concentrations of peptides mimicking the PTP1B binding sequence or GST-β-catenin were added to the wells simultaneously with PTP1B, and the amount of PTP1B or β-catenin bound was determined as above, using the appropriate antibodies. 20 μg of GST-N-cadherin was immobilized on 300 μl of glutathione-Sepharose 4B and washed in CKII kinase buffer (25 mm Tris-HCl, pH 7.4, 10 mm MgCl2, 200 mm NaCl, 0.1 mm ATP) or GSK-3β kinase buffer (20 mmTris-HCl, pH 7.5, 10 mm MgCl2, 5 mmdithiothreitol, and 0.2 mm ATP). The proteins were phosphorylated in a total volume of 500 μl for 30–60 min at 30 °C with 10 units of casein kinase II (Promega, Madison, WI) or 1 unit of glycogen synthase kinase-3β (Calbiochem). The beads were then washed with PBS, and bound protein was eluted with 10 mm reduced glutathione in 50 mm Tris-HCl, pH 8.0, containing 1% protease inhibitor mixture and 1 mm NaF. Eluted protein was biotinylated with Sulfo-NHS-LC-Biotin for immobilization, and the presence of phosphorylated serine residues was determined by immunoblots using anti-phosphoserine antibody. Mouse L cells were used for both stable and transient transfections with N-cadherin constructs. Cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 5% fetal calf serum (Invitrogen) and 1% penicillin-streptomycin (Invitrogen) and transfected with pCMV-Δ872–891, pCMV-Δ878–891, or pCMV-Δ884–891. Stable clones were selected with 500 μg/ml G418. L cells transfected with empty pCMV-FLAG-4a were used as control. Expression of N-cadherin was analyzed by immunoblots using NCD-2 or anti-FLAG antibody. L cells were transfected with full-length N-cadherin or N-cadherin deletion constructs using LipofectAMINE (Invitrogen), incubated in Dulbecco's modified Eagle's medium with 5% fetal bovine serum for 24 h, and lysed in ice-cold lysis buffer (2% Nonidet P-40 and 1% protease inhibitor mixture in PBS). Lysates were cleared by centrifugation at 15,000 × g for 10 min, and aliquots containing equivalent amounts of protein were incubated overnight with 5 μl of NCD-2 (1 mg/ml) at 4 °C. 50 μl of goat anti-rat IgG magnetic beads were added, and the mixture was incubated at 4 °C with mixing for 1 h. The magnetic beads were collected using a magnetic stand. For the secondary immunoprecipitation, 5 μl of polyclonal anti-β-catenin was added to the supernatant from the previous step and incubated at 4 °C for 4 h with rotation. Magnetic beads conjugated to goat anti-rabbit IgG were added, incubated at 4 °C with mixing for 1 h, and collected as above. The beads were washed four times with lysis buffer and one time with TBS, resuspended in SDS sample buffer, fractionated by SDS-PAGE, and transferred to PVDF membranes. The membranes were immunoblotted with the antibody indicated in the figure. Cell surface proteins were labeled with the cell membrane-impermeable reagent, NHS-LC-Biotin (Pierce) at room temperature for 30 min. Cells were washed three times with ice-cold PBS (pH 8.0) to remove any remaining biotinylation reagent and lysed as described above. Cell lysates were immunoprecipitated with NCD-2 as described above, fractionated by SDS-PAGE, and immunoblotted with HRP-conjugated streptavidin. Cell adhesion assays were carried out as described previously (14Balsamo J. Leung T.-C. Ernst H. Zanin M.K.B. Hoffman S. Lilien J. J. Cell Biol. 1996; 134: 801-813Crossref PubMed Scopus (194) Google Scholar). To prepare the substrate for adhesion, purified N-cadherin-Fc (23Lambert M. Padilla F. Mege R.M. J. Cell Sci. 2000; 113: 2207-2219Crossref PubMed Google Scholar) was immobilized on 96-well plates precoated with anti-mouse IgG (BD Biocoat; BD Biosciences, Bedford, MA) or poly-l-lysine (Sigma). Plates were washed and blocked with 2% BSA for 1 h. Approximately 104 cells were added to each well in 50 μl of HBSGKCa, in the presence of the indicated peptide at 4 μg/ml. After 1 h at 37 °C, nonadherent cells were removed, the wells were washed gently four times with HBSGKCa, and adherent cells were quantified by staining with crystal violet. Substrates for neurite growth were prepared by coating eight-well slides with polylysine followed by N-cadherin-Fc or laminin, followed by washing and blocking with 2% BSA. The presence of neurites was quantitatively assessed in sparse single cell cultures of E8 chick neural retina. Peptides were added at 8 μm, 2 h after plating. After overnight culture in Dulbecco's modified Eagle's medium containing 1% insulin/transferring/selenium (Invitrogen) cells were fixed in 4% p-formaldehyde, and ∼200 cells were evaluated for the presence of neurites. Neurite growth was visualized using phase optics. PTP1B is ubiquitous in the cell and can interact with many different partners. We have previously shown that PTP1B binds directly to the N-cadherin cytoplasmic domain, controlling the association of N-cadherin with β-catenin and therefore its function (13Balsamo J. Arregui C. Leung T.-C. Lilien J. J. Cell Biol. 1998; 143: 523-532Crossref PubMed Scopus (143) Google Scholar, 14Balsamo J. Leung T.-C. Ernst H. Zanin M.K.B. Hoffman S. Lilien J. J. Cell Biol. 1996; 134: 801-813Crossref PubMed Scopus (194) Google Scholar). Furthermore, binding is dependent on phosphorylation of tyrosine 152 of PTP1B (19Rhee J. Lilien J. Balsamo J. J. Biol. Chem. 2001; 276: 6640-6644Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). To be able to specifically perturb the cadherin-PTP1B association, we sought to identify the sequence in the cytoplasmic domain of N-cadherin that is necessary for binding of PTP1B. The full-length cytoplasmic domain of N-cadherin and several deletion mutants were expressed as GST fusion proteins (Fig. 1 A), and the purified fusion proteins were biotinylated and immobilized on streptavidin-coated wells. Catalytically inactive GST-PTP1B, phosphorylated on tyrosine residues (Fig. 1 B), was then added, and binding was measured using anti-PTP1B antibody in an enzyme-linked immunosorbent assay. We began with two constructs, C2 (amino acids 752–878) and C3 (amino acids 872–912), which overlap by 7 amino acids. Binding of PTP1B to either construct shows approximately the same dose dependence as binding to the full-length cytoplasmic domain (C1; Fig. 1 C), suggesting that the binding site for PTP1B encompasses the COOH terminus of C2 and the NH2terminus of C3. A deletion of 8 amino acids from the COOH terminus of C2 (C4; amino acids 752–870) eliminates binding to PTP1B (Fig.1 C), mapping the NH2 terminus of the binding domain between amino acid residues 871 and 878. Sequential deletions from the NH2 terminus of C3 set the COOH terminus of the binding region at amino acid residues 887–891; construct C5 (amino acids 887–912) is positive for binding, whereas C6 (amino acids 891–912) is not (Fig. 1 C). These results establish the boundaries of the PTP1B binding region at amino acids 872 and 891 on the cytoplasmic domain of N-cadherin. Construct C7 containing just this sequence (amino acids 872–891) binds PTP1B as well as the full-length cytoplasmic domain C1 (Fig. 1 C), whereas C8, consisting of the cytoplasmic domain of cadherin lacking this core sequence (C8; Δ872–891) does not bind (Fig. 1 C). The same cadherin constructs used above to evaluate PTP1B binding were also evaluated for β-catenin binding (not shown). The results are consistent with prior studies by Simcha et al. (24Simcha I. Kirkpatrick C. Sadot E. Shtutman M. Polevoy G. Geiger B. Peifer M. Ben-Ze'ev A. Mol. Biol. Cell. 2001; 12: 1177-1188Crossref PubMed Scopus (45) Google Scholar) (see also Refs. 25Arregui C. Pathre P. Lilien J. Balsamo J. J. Cell Biol. 2000; 149: 1263-1273Crossref PubMed Scopus (106) Google Scholar, 26Jou T.-S. Stewart D.B. Stappert J. Nelson W.J. Marrs J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5067-5071Crossref PubMed Scopus (303) Google Scholar, 27Stappert J. Kemler R. Cell Adhes. Commun. 1994; 2: 319-327Crossref PubMed Scopus (199) Google Scholar) placing the PTP1B binding region NH2-terminal to the β-catenin binding region, with an overlap of about 7 amino acids. To better understand the relationship between these two binding domains, we used synthetic peptides as competitors for the in vitrobinding between PTP1B and N-cadherin and between β-catenin and N-cadherin. Each peptide mimics an 8-amino acid stretch of the PTP1B binding sequence, from the NH2 to the COOH terminus, overlapping with the next sequence by 2 amino acids (see diagram in Figs. 2 A and 9). Binding of PTP1B to the cytoplasmic domain of N-cadherin is reduced to background levels at a concentration of 0.8–1.0 μg/well of peptides 2 or 3, corresponding to the COOH-terminal two-thirds of the PTP1B binding sequence (Fig. 2 B). The reverse sequence of peptide 3, P3R, has no effect on binding. At similar concentrations, peptide 1, corresponding to the NH2 terminus of the PTP1B binding sequence and the portion overlapping the β-catenin-binding domain, inhibits only about 20–30% of PTP1B binding to cadherin (Fig.2 B). In contrast, peptide 1 is an effective competitor of the binding of β-catenin to N-cadherin at a concentration of 0.8 μg/well (Fig. 2 C), whereas peptides 2 and 3 and the control peptide 3R, at this same concentration, have no effect on the β-catenin-N-cadherin interaction (Fig. 2 C). Peptide P4, corresponding to the sequence NH2-terminal to the putative PTP1B-binding domain and well within the reported β-catenin binding region, does not compete for PTP1B binding to N-cadherin but completely abolishes β-catenin binding to N-cadherin (Fig. 2, B andC).Figure 9Diagramatic representation of the β-catenin and PTP1B target region of the cytoplasmic domain of N-cadherin, the peptides used to analyze in vitrobinding (P1, P2, P3, and P4), and those used as antennapedia fusions for introduction into embryonic chick neural retina cells (AP2/3 and AP3). Shown is a diagram of the deletion constructs used for analysis of cell surface expression of N-cadherin following transfection into L cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) These results suggest that, although the sequence corresponding to amino acids 872–878 in the cytoplasmic tail of N-cadherin has the potential to interact with both β-catenin and PTP1B, the interaction with PTP1B is of lower affinity. This is confirmed by comparing the time course of binding of PTP1B to deletion construct C2, containing the region of overlap between the PTP1B and β-catenin binding sites, with the full-length cytoplasmic domain of cadherin (C1), the full putative PTP1B-binding domain (C7), or the deletion constructs C3 and C5. PTP1B binds to C2 at a slower rate than to C1 or C7 or to the COOH-terminal region of the PTP1B binding domain (C3 and C5): 25% of control values after 30 min for C2, as compared with about 60% for C1, C3, C5, and C7 (Fig. 2 D). In addition, β-catenin and PTP1B do not compete for binding to C1 (Fig.3 A). However, β-catenin does compete for binding of PTP1B to the NH2-terminal or overlapping portion of PTP1B binding region (C2) (Fig.3 B). There are 11 serine and threonine residues in the combined β-catenin/PTP1B binding site in N-cadherin and 7 in the PTP1B site alone. This suggests the possibility that serine/threonine phosphorylation could modulate the binding of either effector. We do see an increase in binding of β-catenin to the full-length cytoplasmic domain of N-cadherin after in vitrophosphorylation of serine residues as reported (24Simcha I. Kirkpatrick C. Sadot E. Shtutman M. Polevoy G. Geiger B. Peifer M. Ben-Ze'ev A. Mol. Biol. Cell. 2001; 12: 1177-1188Crossref PubMed Scopus (45) Google Scholar, 27Stappert J. Kemler R. Cell Adhes. Commun. 1994; 2: 319-327Crossref PubMed Scopus (199) Google Scholar) but no effect in the binding of PTP1B (Fig. 4,A and B). To be able to perturb the interaction between N-cadherin and PTP1B in cells that express endogenous N-cadherin, we designed two cell membrane-permeable peptides containing the sequences between amino acids 878–891 and 884–891 (AP2/3 and AP3, respectively; see Figs. 2 A and 9) of N-cadherin covalently linked to the antennapedia "Trojan" peptide (28Derossi D. Chassaing G. Prochiantz A. Trends Cell Biol. 1998; 8: 84-88Abstract Full Text PDF PubMed Scopus (659) Google Scholar, 29Prochiantz A. Curr. Opin. Neurobiol. 1996; 6: 629-634Crossref PubMed Scopus (196) Google Scholar). These peptides m
Referência(s)