Dimerization In Vivo and Inhibition of the Nonreceptor Form of Protein Tyrosine Phosphatase Epsilon

cyt-PTP ${varepsilon}$ is a naturally occurring nonreceptor form of the receptor-typeprotein tyrosine phosphatase (PTP) epsilon. As such, cyt-PTP ${varepsilon}$ enables analysis of phosphatase regulation in the absence ofextracellular domains, which participate in dimerization andinactivation of the receptor-type phosphatases receptor-typeprotein tyrosine phosphatase alpha (RPTP ${alpha}$ ) and CD45. Using immunoprecipitationand gel filtration, we show that cyt-PTP ${varepsilon}$ forms dimers and higher-orderassociations in vivo, the first such demonstration among nonreceptorphosphatases. Although cyt-PTP ${varepsilon}$ readily dimerizes in the absenceof exogenous stabilization, dimerization is increased by oxidativestress. Epidermal growth factor receptor stimulation can affectcyt-PTP ${varepsilon}$ dimerization and tyrosine phosphorylation in eitherdirection, suggesting that cell surface receptors can relayextracellular signals to cyt-PTP ${varepsilon}$ , which lacks extracellulardomains of its own. The inactive, membrane-distal (D2) phosphatasedomain of cyt-PTP ${varepsilon}$ is a major contributor to intermolecular bindingand strongly interacts in a homotypic manner; the presence ofD2 and the interactions that it mediates inhibit cyt-PTP ${varepsilon}$ activity.Intermolecular binding is inhibited by the extreme C and N terminiof D2. cyt-PTP ${varepsilon}$ lacking these regions constitutively dimerizes,and its activities in vitro towards para-nitrophenylphosphateand in vivo towards the Kv2.1 potassium channel are markedlyreduced. We conclude that physiological signals can regulatedimerization and phosphorylation of cyt-PTP ${varepsilon}$ in the absence ofdirect interaction between the PTP and extracellular molecules.Furthermore, dimerization can be mediated by the D2 domain anddoes not strictly require the presence of PTP extracellulardomains.

INTRODUCTION

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Introduction

Phosphorylation of tyrosine residues in proteins is a centralmechanism for protein regulation and is well established asa master regulator of physiological processes. Tyrosine phosphorylationis reversible and is controlled by the generically opposingactivities of protein tyrosine kinases and protein tyrosinephosphatases (PTPs) (25). Receptor-type PTPs (RPTPs), whichare a major structural subfamily of the PTP superfamily, areintegral membrane proteins which possess extracellular domainsof various lengths and typically two cytosolic phosphatase domains(2, 12, 49). Although their importance in regulating biologicalprocesses is well established, relatively little is known abouthow activities of RPTPs are regulated.

Extracellular molecules have been suggested to bind RPTPs andto influence PTP activity or function, much as binding by pleiotrophininhibits activity of PTPß/ ${zeta}$ (36). However, little isknown about how such binding affects signal transduction acrosscell membranes or RPTP activity (5, 41, 42, 53). Structuralstudies have suggested that ligand-induced dimerization inhibitsactivity of RPTPs by stabilizing homodimeric structures, inwhich the helix-turn-helix "wedge" domain of one molecule preventsaccess of substrates to the catalytic domain of its dimerizationpartner (6, 34, 51). Dimerization by native RPTPs has been difficultto observe and in several cases could be examined only afteradditional stabilization. For example, dimerization of RPTP ${alpha}$ by a disulfide link introduced into its extracellular domainor of an epidermal growth factor receptor (EGFR)-CD45 chimericprotein following EGF treatment reduced phosphatase activity(13, 27, 35). In parallel, destabilization of the wedge domainincreased activity of CD45 in vivo (35). The critical role ofextracellular domains is also evident in that isoforms of CD45,which differ in the structure and glycosylation of their extracellulardomains, homodimerize and are inhibited to different extents(52). Yet, recent data have suggested that interactions betweencytosolic regions of RPTP ${alpha}$ or of CD45 are not limited to thewedge domain (7, 8, 17, 23, 26) and that homodimerization maynot occur among all RPTPs (24, 40). These findings raise thequestion of whether intermolecular interactions among RPTPsdepend on input from membrane-spanning and extracellular domains.

Prominent candidates for mediating intermolecular interactionsare the membrane-distal (D2) PTP domains present in most RPTPs.Although typically inactive, structures and sequences of D2domains are often very similar to those of active, membrane-proximal(D1) domains to the point where activities of the D2 domainsof RPTP ${varepsilon}$ , RPTP ${alpha}$ , or LAR could be significantly increased by asmall number of specific point mutations (10, 31-33, 40). Evidencefor a regulatory role for D2 domains has come from studies inwhich D2 of CD45 bound D1 of the phosphatase (17, 23) and theD2 domains of RPTP ${alpha}$ , RPTP ${sigma}$ , LAR, RPTP ${delta}$ , and RPTPµ bound RPTP ${alpha}$ (7). The effect of D2-D1 binding is context dependent—itmay inhibit PTP activity, as in the case of PTP ${sigma}$ (50), or canhelp achieve optimal enzymatic activity, as in CD45 (18). Bindingby the RPTP ${alpha}$ D2 domain was increased by oxidative stress, UVirradiation, or heat shock but not by tetradecanoyl phorbolacetate, serum, or EGF treatment (8), suggesting that distinctphysiological signals can affect dimerization. In all, the highdegree of conservation of D2 domains and their participationin molecular interactions strongly indicate that these domainshave significant regulatory roles, although many details ofhow this is achieved are missing.

PTP epsilon (PTP ${varepsilon}$ ) exists as a small group of four proteins producedby the single PTP ${varepsilon}$ gene. The two most prevalent are the receptor-type(RPTP ${varepsilon}$ ) and non-receptor-type (cyt-PTP ${varepsilon}$ ) forms, each the productof a distinct PTP ${varepsilon}$ mRNA species. p67 and p65 PTP ${varepsilon}$ are shortermolecules that are expressed together with either RPTP ${varepsilon}$ or cyt-PTP ${varepsilon}$ and whose production is regulated at the levels of translationand posttranslational processing (16, 20, 21, 29, 39, 45). RPTP ${varepsilon}$ supports the transformed phenotype of Neu-induced mammary epithelialtumor cells, most likely by activating Src (14, 15, 19). Thesame PTP ${varepsilon}$ isoform can also down-regulate insulin receptor signalingin BHK cells (1, 38). cyt-PTP ${varepsilon}$ dephosphorylates and inactivatesthe Kv1.5 and Kv2.1 voltage-gated potassium channels in Schwanncells in vivo, in correlation with reduced myelination of sciaticnerves in young PTP ${varepsilon}$ -deficient mice (43). Other roles for PTP ${varepsilon}$ include inhibition of JAK-STAT signaling in M1 leukemia cells(46, 47), suppression of endothelial cell proliferation (48),and ensuring proper functioning of mouse macrophages (44).

Structural differences between RPTP ${varepsilon}$ and cyt-PTP ${varepsilon}$ are limitedto their N termini; the extracellular and transmembranal domainsof RPTP ${varepsilon}$ are replaced in cyt-PTP ${varepsilon}$ by a stretch of 12 hydrophilicamino acid residues. Downstream sequences, including the twotandem PTP domains, are identical in both molecules. As a resultRPTP ${varepsilon}$ is an integral membrane protein and cyt-PTP ${varepsilon}$ is predominantlycytosolic, although some cyt-PTP ${varepsilon}$ molecules can be found in associationwith the cell membrane and in the nucleus (16, 21, 28). cyt-PTP ${varepsilon}$ is therefore an unusual naturally occurring non-receptor-typePTP with two catalytic domains. As such it enables us to addressthe question of whether membrane-spanning and extracellulardomains and direct input from extracellular events are in factrequired for dimerization in vivo, in the context of a naturallyoccurring molecule.

In this study we use coimmunoprecipitation and gel filtrationto show that cyt-PTP ${varepsilon}$ very readily forms homotypic dimers andhigher-order associations in vivo. This significant basal self-associationcan be further affected by EGFR activation or increased oxidativestress, indicating that dimerization can be up- or down-regulatedby physiological events. These findings also indicate that extracellularevents can influence cyt-PTP ${varepsilon}$ dimerization indirectly, by mediationof cell surface molecules such as the EGFR. We demonstrate thatthe inactive cyt-PTP ${varepsilon}$ D2 domain is crucial in mediating interactionsbetween cyt-PTP ${varepsilon}$ molecules and that this domain strongly interactsin a homotypic manner and inhibits cyt-PTP ${varepsilon}$ activity. We alsoshow that, in the context of the entire cyt-PTP ${varepsilon}$ molecule, theextreme N and C termini of the D2 domain act to inhibit dimerization.Accordingly, cyt-PTP ${varepsilon}$ lacking either or both D2 termini constitutivelydimerizes and exhibits significantly reduced activity in vitrotowards para-nitrophenylphosphate (PNPP) and in vivo towardsthe Kv2.1 potassium channel in cells. We conclude that physiologicalevents can regulate dimerization and phosphorylation of cyt-PTP ${varepsilon}$ by a mechanism involving D2 and without requiring direct inputfrom extracellular molecules.

MATERIALS AND METHODS

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Materials and Methods

DNA constructs. cDNAs of cyt-PTP ${varepsilon}$ , RPTP ${varepsilon}$ , Kv2.1, and Y527F Src in the pcDNA3 plasmid(Invitrogen) were described previously (21, 43). The followingcDNAs were derived from mouse cyt-PTP ${varepsilon}$ by PCR and site-directedmutagenesis and cloned into pcDNA3: D1 + 12 (amino acid residues1 to 394, numbering as in the sequence with accession numberU36758), D2 + 12 (residues 1 to 12 linked to residues 360 to642), D2 (residues 360 to 642), D2-N (residues 381 to 642),D2-C (residues 360 to 620), D2-N,C (residues 381 to 620), ${Delta}$ N(full-length cyt-PTP ${varepsilon}$ [residues 1 to 642] from which residues360 to 380 had been deleted), ${Delta}$ C (residues 1 to 620), and ${Delta}$ N,C(residues 1 to 620, from which residues 360 to 380 had beendeleted). Constructs were tagged with either FLAG or hemagglutinin(HA) at their C termini and were sequenced prior to use. EGFRcDNA was a generous gift of Yosef Yarden.

Cell culture. NIH 3T3 and 293 human embryonic kidney cells were grown in Dulbeccomodified Eagle medium (Gibco-BRL), supplemented with 10% fetalcalf serum (Gibco-BRL), 2 mM glutamine, 50 U of penicillin/ml,and 50 µg of streptomycin/ml. Transfections were doneby the calcium phosphate technique (11) or with the Lipofectaminereagent (Invitrogen). 293 cells stably expressing Kv2.1 weregenerated by transfection of cells with Kv2.1 cDNA followedby selection in 1.5 mg of G-418/ml. Ras-transformed NIH 3T3cells were produced by transfecting NIH 3T3 cells with EJ-RascDNA. Cells were maintained in medium containing 0.5% fetalcalf serum until foci of transformed cells arose, which werethen pooled. In some studies 293 cells were transfected with1 µg of EGFR cDNA, grown in 0.1% serum for 24 h, and thenstimulated with 100 ng of EGF (Sigma)/ml as indicated in Fig.2. When used, 10 mM N-acetylcysteine was added to medium containing0.1% serum during the last 12 to 14 h of incubation prior toEGF stimulation. In other cases cells in regular growth mediumwere treated with 1 mM H₂O₂ for 10 min and lysed and processedimmediately.

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FIG. 2. EGFR signaling and oxidative stress affect cyt-PTP ${varepsilon}$ dimerization. (A) EGFR stimulation affects cyt-PTP ${varepsilon}$ phosphorylation and dimerization. 293 cells transfected with the EGFR and cyt-PTP ${varepsilon}$ were treated with EGF for the times indicated. (Top) Dimerization of cyt-PTP ${varepsilon}$ following EGF treatment, assayed as for Fig. 1B; (second from top) cyt-PTP ${varepsilon}$ tyrosine phosphorylation; (third and fourth from top) immunoprecipitated FLAG-cyt-PTP ${varepsilon}$ and expression of HA-cyt-PTP ${varepsilon}$ , respectively; (fifth from top) phosphorylation of cell proteins—mostly EGFR autophosphorylation—following EGF treatment; (bottom) expression levels of the EGFR in transfected cells. M, mock-transfected cells. (B) Bar diagram showing dimerization of cyt-PTP ${varepsilon}$ at 5 and 60 min following application of EGF, relative to 0 min; results are from four and six independent experiments, respectively. Coprecipitated HA-cyt-PTP ${varepsilon}$ amounts were normalized to amounts of FLAG-cyt-PTP ${varepsilon}$ actually precipitated. *, P = 0.0286, and **, P = 0.0022, both by the Mann-Whitney test. (C) EGF treatment affects cyt-PTP ${varepsilon}$ dimerization in NIH 3T3 cells. The experiment was performed as described for panel A with NIH 3T3 cells transiently expressing cyt-PTP ${varepsilon}$ . The asterisk denotes a nonspecific band. Note reduced dimerization at 5 min and increased dimerization at 60 min. (D) Dimerization and tyrosine phosphorylation of cyt-PTP ${varepsilon}$ following treatment of 293 cells with 1 mM H₂O₂. Subpanels shown are similar to those for panel A. Qualitatively similar phosphorylation results were obtained in cells expressing only FLAG-tagged cyt-PTP ${varepsilon}$ (data not shown). Size markers are in kilodaltons. (E) Effect of EGFR stimulation on cyt-PTP ${varepsilon}$ is not mediated by H₂O₂. The experiment presented in panel A was repeated with cells exposed to 10 mM N-acetylcysteine (NAC) for 12 to 14 h immediately preceding EGF stimulation. A reduction in dimerization at 5 min and an increase at 60 min post-EGF stimulation were detected in this experiment as well. Shown is an experiment representative of two performed.

Protein analysis. Cells were washed with phosphate-buffered saline and lysed incold buffer A (50 mM Tris-Cl [pH 7.5], 100 mM NaCl, 1% NonidetP-40), containing protease inhibitors [1 mM N-( ${alpha}$ -aminoethyl)benzene-sulfonylfluoride, 40 µM bestatin, 15 µM E64, 20 µMleupeptin, 15 µM pepstatin; Sigma]. For analysis afterEGF and H₂O₂ treatments buffer contained also 0.5 mM sodiumpervanadate. For immunoprecipitation 0.5 to 1 mg of total cellprotein was mixed with anti-FLAG M2 affinity beads (Sigma) orwith 3 µl of antiphosphotyrosine antibodies (TransductionLaboratories) coupled to protein A-Sepharose beads for 2 to4 h at 4°C, followed by four extensive washes with radioimmunoprecipitationassay buffer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and blotting were performed as described previously(20). In control experiments where known, graded amounts ofprotein were subjected to SDS-PAGE and blotting, the intensitiesof signals obtained were proportional in a linear fashion tothe different amounts of antigen loaded on the gel. Primaryantibodies used in this study included polyclonal anti-PTP ${varepsilon}$ (15),anti-Kv2.1 (Alomone), anti-HA (Santa Cruz), monoclonal anti-v-Src(Calbiochem), antiphosphotyrosine (Transduction Laboratories,clone PY20, or Santa Cruz, clone PY99), anti-FLAG (M2, Sigma),and anti-EGFR (Sigma).

Analysis of cyt-PTP ${varepsilon}$ by gel filtration. Cells were lysed in buffer A, after which lysates were centrifugedfor 10 min at 13,000 x g and for 60 min at 100,000 x g to removeall insoluble material. One milligram of protein was fractionatedat 4°C on a Superdex 200 gel-filtration column (Pharmacia),previously equilibrated with running buffer (phosphate-bufferedsaline supplemented with 5% glycerol and protease inhibitors).The column was run at a constant flow rate of 0.5 ml/min, andfractions of 0.3 ml were collected until all loaded proteinhad flowed through. Equal portions of each fraction were resolvedby SDS-PAGE, blotted, and probed with anti-PTP ${varepsilon}$ antibodies todetermine the distribution of cyt-PTP ${varepsilon}$ among the fractions. Molecularmass standards (listed in the legend to Fig. 3) were fractionatedunder the same conditions, enabling estimation of molecularmass of the various fractions by semilogarithmic curve fitting.

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FIG. 3. Analysis of complexes formed by cyt-PTP ${varepsilon}$ by gel filtration. (A) Ras-transformed NIH 3T3 cells were lysed and fractionated on a Superdex 200 FPLC system. Shown is the distribution of endogenous cyt-PTP ${varepsilon}$ among column fractions in untreated cells (top) or in cells exposed to 1 mM H₂O₂ for 10 min (bottom). Fraction 1 immediately follows the column void volume. (B) Graph depicting relative amounts of cyt-PTP ${varepsilon}$ in fractions of panel A. Arrows denote elution of molecular mass standards. Standards (Sigma) used were the following: dextran blue (I), bovine thyroglobulin (II), horse spleen apoferritin (III), ß-amylase (IV), alcohol dehydrogenase (V), bovine serum albumin (VI), and carbonic anhydrase (not shown), at 2,000, 669, 443, 200, 150, 66, and 29 kDa, respectively. (C) cyt-PTP ${varepsilon}$ forms homodimers in vivo. 293 cells were transfected with HA- and FLAG-tagged cyt-PTP ${varepsilon}$ , and following gel filtration, FLAG-cyt-PTP ${varepsilon}$ was precipitated. (Top) Coprecipitated HA-cyt-PTP ${varepsilon}$ ; (bottom) precipitated FLAG-cyt-PTP ${varepsilon}$ .

Purification of PTP ${varepsilon}$ and activity assay. FLAG-tagged cyt-PTP ${varepsilon}$ was expressed in 293 cells. Following anti-FLAGprecipitation, beads underwent three extensive washes in bufferB (20 mM HEPES [pH 7.6], 100 mM KCl, 0.5 mM EDTA, 0.4% NonidetP-40, 20% [vol/vol] glycerol) and two rinses in buffer 54K (50mM Tris-Cl [pH 7.9], 150 mM NaCl, 0.5% Triton X-100). Boundmaterial was specifically eluted by incubating the beads at30°C for 3 min in an equal volume of TBS buffer (20 mM Tris-Cl[pH 7.35], 150 mM NaCl) containing 1 mg of FLAG peptide (Sigma)/mland 0.1 mM EGTA. Phosphatase activity of the eluted materialwas assayed in 96-well plates in reaction mixtures containingequal amounts of PTP ${varepsilon}$ in 5 to 15 µl of eluate and 200 µlof assay buffer (50 mM morpholineethanesulfonic acid [pH 7.0],0.5 mg of bovine serum albumin/ml, 10 mM PNPP). Activity wasmeasured by monitoring the increase in absorption at 405 nmfor an hour at room temperature, during which absorption waslinear with time. Negative controls included reaction mixturescontaining eluates prepared from mock-transfected cells or reactionmixtures containing cyt-PTP ${varepsilon}$ to which 0.5 mM sodium pervanadatehad been added; phosphatase activity in these controls was negligible.Activity measured was normalized to the relative amount of PTP ${varepsilon}$ actually present in each eluate as determined by protein blotting.

RESULTS

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Results

RPTP ${varepsilon}$ and cyt-PTP ${varepsilon}$ form homotypic interactions in vivo. In order to determine whether RPTP ${varepsilon}$ or cyt-PTP ${varepsilon}$ can form homotypicinteractions in vivo, we expressed HA- and FLAG-tagged versionsof either protein in 293 cells. These and other constructs usedthroughout this study are shown schematically in Fig. 1A. Followingimmune precipitation of FLAG-tagged molecules, the presenceof coprecipitated HA-tagged molecules was determined by blottingthe precipitate with anti-HA antibodies. As seen in Fig. 1B,HA-RPTP ${varepsilon}$ readily coprecipitated with FLAG-RPTP ${varepsilon}$ , and similar resultswere obtained with FLAG- and HA-tagged cyt-PTP ${varepsilon}$ . This resultindicates that the divergent N termini of both molecules donot prevent intermolecular homotypic associations from beingformed. Similar results were obtained when the antibodies werereversed, i.e., by immunoprecipitation with HA and probing withFLAG antibodies (data not shown). RPTP ${varepsilon}$ and cyt-PTP ${varepsilon}$ could becoprecipitated with each other (data not shown), indicatingthat formation of heterotypic interactions among PTP ${varepsilon}$ moleculesis not dependent upon the two molecules having identical N termini.This last result is primarily of mechanistic importance, sincethe expression patterns of RPTP ${varepsilon}$ and cyt-PTP ${varepsilon}$ in cells and tissuesrarely overlap (16). In separate experiments comparison of thefraction of HA-cyt-PTP ${varepsilon}$ molecules that coprecipitated with agiven fraction of precipitated FLAG-cyt-PTP ${varepsilon}$ molecules indicatedthat 12 to 25% of cyt-PTP ${varepsilon}$ molecules were involved in homotypicassociations in unstimulated cells. In contrast with RPTP ${alpha}$ (8,27), PTP ${varepsilon}$ coimmunoprecipitation was detected with ease; chemicalcross-linkers or engineered disulfide links were not requiredthroughout these studies.

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FIG. 1. cyt-PTP ${varepsilon}$ and RPTP ${varepsilon}$ form homodimers in vivo. (A) Schematic diagrams of PTP ${varepsilon}$ proteins used in this study. Indicated are the 12 N-terminal residues unique to cyt-PTP ${varepsilon}$ (N), the D1 and D2 PTP domains, and the site of HA or FLAG tag addition (T). Dashed lines denote deleted sequences. (B) 293 cells were transfected with HA- and FLAG-tagged RPTP ${varepsilon}$ (R), after which FLAG-tagged RPTP ${varepsilon}$ was precipitated. (Left) (top) Coprecipitated HA-RPTP ${varepsilon}$ ; (middle) precipitated FLAG-RPTP ${varepsilon}$ ; (bottom) HA-RPTP ${varepsilon}$ present in cell lysates. Right panels show similar experiments for cyt-PTP ${varepsilon}$ (cyt). NS, nonspecific bands; Ig, immunoglobulin. Size markers are in kilodaltons.

cyt-PTP ${varepsilon}$ dimerization is affected by EGFR signaling and oxidative stress. In order to examine whether cellular signaling processes canaffect the degree of dimerization of cyt-PTP ${varepsilon}$ , we examined cyt-PTP ${varepsilon}$ following EGF stimulation in 293 cells. Basal levels of cyt-PTP ${varepsilon}$ dimers—evident as coprecipitation of HA- and FLAG-taggedcyt-PTP ${varepsilon}$ —were observed in the cells prior to EGF stimulation(Fig. 2A). Massive autophosphorylation of the EGFR was evidentwithin the first 5 min following stimulation, attesting to activationof the receptor by EGF (Fig. 2A). In parallel, a clear and reproducible39% decrease in cyt-PTP ${varepsilon}$ dimerization was noted 5 min followingEGF treatment; dimerization levels rose to prestimulation values10 min following application of EGF and rose to 310% of controlvalues at 60 min poststimulation (Fig. 2A and B). Similar resultswere also obtained in NIH 3T3 cells, in which exogenous cyt-PTP ${varepsilon}$ was expressed at lower levels (Fig. 2C). Decrease in dimerizationat 5 min and its increase at 60 min were detected also in 293cells that had been pretreated with the reactive oxygen speciesscavenger N-acetylcysteine, suggesting that effects of EGFRstimulation in this system are most likely not mediated by reactiveoxygen species (Fig. 2E). Interestingly, stimulation of theEGFR also affected cyt-PTP ${varepsilon}$ phosphorylation (Fig. 2A). Phosphorylationwas virtually undetectable before stimulation; 5 min after applicationof EGF, when dimerization was at its lowest, cyt-PTP ${varepsilon}$ phosphorylationwas significantly increased. At 60 min, when dimerization wasmaximal, phosphorylation levels had significantly decreasedalthough they were still slightly higher than baseline levels.We conclude that extracellular events can indirectly influencecyt-PTP ${varepsilon}$ dimerization through mediation of receptor-type molecules,such as the EGFR. Furthermore, EGFR stimulation can affect bothdimerization and phosphorylation of cyt-PTP ${varepsilon}$ ; analysis of functionalinteractions between cyt-PTP ${varepsilon}$ and the EGFR is beyond the scopeof the present study and will be presented elsewhere.

Previous studies have shown that oxidative stress promotes dimerizationof the related receptor-type RPTP ${alpha}$ by inducing a switch in theconformation of the molecule, and coimmunoprecipitation experimentswith RPTP ${alpha}$ were unsuccessful without prior treatment of cellswith H₂O₂ (8). As noted above, dimers of cyt-PTP ${varepsilon}$ are readilydetected in the absence of H₂O₂. Nonetheless, treatment of cellsexpressing PTP ${varepsilon}$ with H₂O₂ significantly increased interactionsbetween PTP ${varepsilon}$ molecules (Fig. 2D). Interestingly, while H₂O₂ treatmentsignificantly increased overall cellular tyrosine phosphorylation,a parallel increase in tyrosine phosphorylation of cyt-PTP ${varepsilon}$ wasnot detected (Fig. 2D). This finding is in agreement with thedifferent trends for phosphorylation versus dimerization observedfollowing EGFR stimulation. In separate experiments lysing cellsin the presence of dithiothreitol or performing SDS-PAGE analysisof precipitated material or of crude cell lysates in the absenceof ß-mercaptoethanol did not change the ability ofcyt-PTP ${varepsilon}$ to dimerize (data not shown). This indicates that intermoleculardisulfide bonds are not major stabilizers of cyt-PTP ${varepsilon}$ dimerization,although it does not strictly rule out participation of intramoleculardisulfide bonds in this process. In all, we conclude that cyt-PTP ${varepsilon}$ molecules can form dimers without relying on the presence ofmembrane-spanning and extracellular domains and that cellularsignaling events and the degree of oxidative stress to whichcells are exposed can affect the degree of dimerization.

Analysis of cyt-PTP ${varepsilon}$ complexes by gel filtration. We next examined interactions of cyt-PTP ${varepsilon}$ by gel filtration.This approach enabled us to examine complexes that include endogenous,untagged cyt-PTP ${varepsilon}$ from untransfected cells, as well as to confirmimmunoprecipitation results by a second, unrelated experimentaltechnique. Lysates of Ras-transformed NIH 3T3 cells, which expresssignificant amounts of endogenous cyt-PTP ${varepsilon}$ (H. Toledano-Katchalski,J. Kraut, and A. Elson, unpublished data), were fractionatedon a Superdex-200 fast protein liquid chromatography (FPLC)system. Cells were fractionated either with or without priortreatment with H₂O₂, and molecular masses of the various columnfractions were estimated by separation of molecular mass standardson the same column. Amounts of cyt-PTP ${varepsilon}$ in each fraction wereestimated by protein blotting with anti-PTP ${varepsilon}$ antibodies, whichrevealed the presence of full-length cyt-PTP ${varepsilon}$ ( $~$ 70-kDa) and ofthe smaller p67 and p65 PTP ${varepsilon}$ proteins (21). cyt-PTP ${varepsilon}$ showed abroad elution profile starting approximately at the mass ofthe cyt-PTP ${varepsilon}$ monomer and extending towards higher molecular masses(Fig. 3A and B, nontreated samples). Most of the protein wasfound in a single peak (fractions 27 to 29), in agreement withthe size of the cyt-PTP ${varepsilon}$ monomer (70 kDa). About a third as muchPTP ${varepsilon}$ fractionated as a peak of higher mass (fraction 24), inagreement with the expected size of cyt-PTP ${varepsilon}$ dimers ( $~$ 140 kDa).Some cyt-PTP ${varepsilon}$ protein was recovered in the range of $~$ 1,200 kDa(fractions 7 and 8), beyond the optimal range of separationby the column system used. Low levels of cyt-PTP ${varepsilon}$ were detectedalso in fractions of intermediate mass. To rule out the possibilitythat this broad separation profile resulted from nonspecificinteractions with the column matrix, we separated similar cellextracts on a glycerol gradient and obtained a very similarseparation pattern (data not shown). The separation profiletherefore most likely reflects the dynamic nature of homo- orheterotypic interactions in which cyt-PTP ${varepsilon}$ participates.

Treatment of cells with H₂O₂ prior to lysis and fractionationcaused several changes in the separation profile. Peaks at $~$ 130to 140 kDa (fractions 24 to 26) and at $~$ 1,200 kDa (fractions5 to 8) were significantly increased in intensity at the expenseof the cyt-PTP ${varepsilon}$ monomer peak, which nearly disappeared (fractions27 to 29, $~$ 70 kDa) (Fig. 3A and B, H₂O₂-treated samples). Thisresult indicated that H₂O₂ treatment significantly increasedintermolecular interactions involving cyt-PTP ${varepsilon}$ , in agreementwith the increase in coimmunoprecipitation of tagged cyt-PTP ${varepsilon}$ molecules observed in Fig. 2D.

The presence of cyt-PTP ${varepsilon}$ in fractions of higher molecular massmay indicate its participation in homotypic and/or heterotypicinteractions. In order to determine whether the separation profileof cyt-PTP ${varepsilon}$ was due at least in part to homotypic interactions,we transfected 293 cells with FLAG- and with HA-tagged cyt-PTP ${varepsilon}$ and fractionated lysates of these cells by FPLC. Column separationprofiles in these experiments were very similar to those ofendogenous cyt-PTP ${varepsilon}$ from NIH 3T3 cells, and a similar shift tolarger molecular masses was encountered following H₂O₂ treatmentin these experiments as well (data not shown). In order to determinewhether the various column fractions contained cyt-PTP ${varepsilon}$ moleculesbound to one another, coimmunoprecipitation experiments werecarried out in fractions 5 and 6 for high-molecular-mass complexes,fractions 23 to 26 for putative dimers, and fractions 27 and28 for cyt-PTP ${varepsilon}$ monomers (Fig. 3C). Coprecipitation of HA-taggedcyt-PTP ${varepsilon}$ was detected in fractions 24 to 26, indicating thatcyt-PTP ${varepsilon}$ dimers were indeed present in these fractions (Fig.3C). As expected, amounts of coprecipitating HA-cyt-PTP ${varepsilon}$ weresignificantly reduced in fractions 27 and 28, which includedmolecules similar in mass to monomeric cyt-PTP ${varepsilon}$ , despite highlevels of precipitating FLAG-tagged cyt-PTP ${varepsilon}$ in these fractions.Importantly, this last result indicated that coprecipitationof HA- and FLAG-tagged cyt-PTP ${varepsilon}$ in fractions 24 to 26 was dueto interactions present in cells and did not reflect associationsformed in column fractions following separation. Very littleHA-cyt-PTP ${varepsilon}$ was coprecipitated from fractions 5 and 6, probablydue in part to the small amounts of FLAG-cyt-PTP ${varepsilon}$ present inthese fractions; large amounts of HA-cyt-PTP ${varepsilon}$ were coprecipitatedfrom these fractions from cells treated with H₂O₂ (data notshown). In all, these results independently confirm that cyt-PTP ${varepsilon}$ dimers exist in cells in vivo and that increased oxidative stresswithin cells can induce significant association between cyt-PTP ${varepsilon}$ molecules.

The D2 domain is an important mediator of binding between cyt-PTP ${varepsilon}$ molecules. In order to identify sequences that mediate homotypic binding,we examined cyt-PTP ${varepsilon}$ molecules in which either the D2 or theD1 PTP domain was removed (D1 + 12 and D2 + 12 proteins, respectively,"+12" indicating the presence of the 12 N-terminal-most residuesunique to cyt-PTP ${varepsilon}$ ; Fig. 1A). Binding of each of these proteinsto itself, to the other deletion mutant, or to full-length cyt-PTP ${varepsilon}$ was examined by coprecipitation (Fig. 4). The D2 + 12 proteinbound its counterparts more strongly than did the D1 + 12 protein;in particular, D2 + 12 molecules were capable of strong homotypicbinding, while only very weak binding of D1 + 12 molecules toeach other was detected. Binding between D1 and D2 domains wasalso detected in these experiments (Fig. 4).

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FIG. 4. Strong homotypic binding of the D2 domain. 293 cells were transfected with HA- and FLAG-tagged cyt-PTP ${varepsilon}$ constructs as indicated, after which FLAG-PTP ${varepsilon}$ was precipitated. (Top) Coprecipitated HA-PTP ${varepsilon}$ ; (middle) precipitated FLAG-PTP ${varepsilon}$ ; (bottom) HA-PTP ${varepsilon}$ present in cell lysates. Size markers are in kilodaltons.

Further analysis revealed that the interaction between D2 +12 molecules could not be further enhanced by treatment withH₂O₂ (Fig. 5A). In contrast, binding between D2 domains lackingthe 12 N-terminal residues was observed only in cells that hadbeen treated with H₂O₂ (Fig. 5A). Interestingly, replacing the12 N-terminal residues of D2 + 12 with the D1 domain of PTP ${varepsilon}$ did not reduce strong constitutive homotypic binding (data notshown), indicating that the precise sequence of the proteinsegment added to the N terminus of the D2 domain is not critical.Together, these results indicated that the potential for homotypicbinding exists in D2 domains but is apparently masked by intramolecularinteractions within D2. Presumably, conformational changes inD2 caused by oxidation by H₂O₂, as in the case of RPTP ${alpha}$ (8),or by addition of N-terminal residues, as done here, unmaskthe binding ability of D2 domains. As the full-length cyt-PTP ${varepsilon}$ molecule contains sequences N-terminal to the D2 domain, thesedata strongly suggest that the D2 domain is not "closed" andis capable of binding in the context of full-length cyt-PTP ${varepsilon}$ .

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FIG. 5. Regulation of D2-mediated binding. (A) Addition of H₂O₂ or of N-terminal sequences unmasks the binding ability of the D2 domain. 293 cells were transfected with HA- and FLAG-tagged cyt-PTP ${varepsilon}$ constructs as indicated. Some cell cultures were treated with 1 mM H₂O₂ immediately prior to lysis, after which FLAG-PTP ${varepsilon}$ was precipitated. (Top) Coprecipitated HA-PTP ${varepsilon}$ ; (middle) precipitated FLAG-PTP ${varepsilon}$ ; (bottom) HA-PTP ${varepsilon}$ present in cell lysates. (B) Regulation of D2-mediated binding by the C- and N-terminal regions of D2. 293 cells were transfected with full-length HA-cyt-PTP ${varepsilon}$ and with FLAG-tagged D2 constructs as indicated, after which FLAG-PTP ${varepsilon}$ was precipitated. (Top) Coprecipitated HA-PTP ${varepsilon}$ ; (middle) precipitated FLAG-D2; (bottom) HA-cyt-PTP ${varepsilon}$ present in cell lysates. Note the higher mobility of D2-N. Size markers are in kilodaltons.

Sequences at the termini of D2 control intermolecular binding and activity of cyt-PTP ${varepsilon}$ In order to identify subdomains in D2 that regulate intermolecularbinding, we examined the effects of deleting sequences at theN or C terminus of this domain on its binding ability. Sequencesdeleted were the 21 N-terminal or the 22 C-terminal residuesof the D2 domain (residues 360 to 380 and 621 to 642 in full-lengthcyt-PTP ${varepsilon}$ , respectively). Isolated D2 proteins lacking one orboth of these regions were examined in coprecipitation experimentstogether with full-length cyt-PTP ${varepsilon}$ (Fig. 5B). Consistent withresults of Fig. 5A, the D2 protein did not precipitate full-lengthcyt-PTP ${varepsilon}$ (Fig. 5B). However, removal of the N-terminal regioncaused significant binding to occur between this protein (D2-N)and full-length cyt-PTP ${varepsilon}$ , implying that the N-terminal regionof D2 had prevented this domain from binding cyt-PTP ${varepsilon}$ . In contrast,the D2 C terminus appeared to affect binding by D2 mainly positively.Removal of the 22 C-terminal-most residues from D2 domains alreadylacking their N terminus (construct D2-N,C) significantly reducedbinding to cyt-PTP ${varepsilon}$ , suggesting that the C-terminal region ofD2 assists binding in trans to other cyt-PTP ${varepsilon}$ molecules. In agreement,D2 lacking only the C-terminal region (D2-C) bound cyt-PTP ${varepsilon}$ significantlymore weakly than did D2-N (Fig. 5B). However, binding of D2-Cwas still stronger than that of D2; this implies that the Cterminus of D2 also has a role in inhibiting binding in thecontext of the D2 molecule. Removal of the 21 N-terminal residuesfrom D2 or from the entire cyt-PTP ${varepsilon}$ molecule significantly alteredtheir electrophoretic mobility (Fig. 5A and 6A). The cause forthis is not clear but may be related to the high proportionof charged residues (9 of 21) in this region.

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FIG. 6. Deletion of the D2 domain N- and/or C-terminal residues from full-length cyt-PTP ${varepsilon}$ induces homotypic binding and inhibits activity of cyt-PTP ${varepsilon}$ . (A) 293 cells were transfected with HA- and FLAG-tagged versions of full-length cyt-PTP ${varepsilon}$ (FL) or mutants lacking the C terminus ( ${Delta}$ C), N terminus ( ${Delta}$ N), or both termini ( ${Delta}$ NC) of D2 as indicated, followed by anti-FLAG immunoprecipitation. (Top) Coprecipitated HA-PTP ${varepsilon}$ ; (middle) precipitated FLAG-PTP ${varepsilon}$ ; (bottom) HA-PTP ${varepsilon}$ present in cell lysates. (B) Homotypic binding of mutants is not further increased by exposure to H₂O₂. Cells indicated were treated with 1 mM H₂O₂ for 10 min prior to lysis. Shown are results obtained for the ${Delta}$ N mutant. (C) In vitro PTP activity of full-length cyt-PTP ${varepsilon}$ and of the three deletion mutants. cyt-PTP ${varepsilon}$ proteins were purified from 293 cells by immunoprecipitation and elution, and activities of equal amounts of protein towards PNPP were measured and normalized as detailed in Materials and Methods. Results (means ± standard errors) are from one experiment (in triplicate) and are representative of three similar experiments performed. *, P < 0.0001 by Student's t test. (D) Deletion mutants of PTP ${varepsilon}$ are severely impaired in their ability to dephosphorylate the Kv2.1 potassium channel in cells. 293 cells stably expressing Kv2.1 were transfected with constitutively active Y527F Src and with full-length (FL) or ${Delta}$ N, ${Delta}$ C, or ${Delta}$ N,C cyt-PTP ${varepsilon}$ as indicated. (Top) Precipitation of the cells by tyrosine-phosphorylated Kv2.1; (middle and bottom) expression levels of Kv2.1 and PTP ${varepsilon}$ and of Src in the lysates used, respectively. Shown is one experiment representative of two performed. Note the increased mobility of cyt-PTP ${varepsilon}$ lacking the D2 N terminus. Size markers are in kilodaltons.

We next examined the consequences of removing the N and C terminiof D2 from full-length cyt-PTP ${varepsilon}$ . Removal of either or both ofthese sequences resulted in similarly strong constitutive binding(Fig. 6A), which was not increased further by H₂O₂ (Fig. 6B).We next examined activities of cyt-PTP ${varepsilon}$ molecules purified fromcells towards PNPP. Activities of cyt-PTP ${varepsilon}$ molecules lackingeither or both termini were significantly reduced, despite thefact that the sequence of the D1 domain, which is the only activePTP domain in PTP ${varepsilon}$ (32), was not altered (Fig. 6C). Similar resultswere obtained when activities of these mutant proteins towardsthe voltage-gated potassium channel Kv2.1, a physiological substrateof cyt-PTP ${varepsilon}$ (43), were examined in cells. In agreement with previousresults, Kv2.1 was clearly tyrosine phosphorylated when expressedtogether with activated Y527F Src; Kv2.1 phosphorylation wasreduced by 94% when cyt-PTP ${varepsilon}$ was coexpressed (Fig. 6D) (43).In contrast, expression of cyt-PTP ${varepsilon}$ molecules lacking eitheror both the N and C termini of D2 reduced Kv2.1 phosphorylationonly mildly (Fig. 6D), indicating that all three deletion mutantswere significantly less active than full-length cyt-PTP ${varepsilon}$ towardsKv2.1. Importantly, confocal microscopy revealed that all threemutants exhibited subcellular localization patterns similarto those of full-length cyt-PTP ${varepsilon}$ , indicating that their increasedassociation and reduced activity were not caused by widespreadaggregation or altered subcellular localization (data not shown).In all, these results indicate that in the context of the full-lengthcyt-PTP ${varepsilon}$ molecule, the N- and C-terminal sequences of D2 negativelyregulate intermolecular binding and that removal of these sequencesresults in constitutive dimerization and in reduction in enzymeactivity.

Presence of D2 reduces cyt-PTP ${varepsilon}$ catalytic activity. We next examined the effect of the presence of the D2 domainon cyt-PTP ${varepsilon}$ activity. It should be stressed that the D2 domainof cyt-PTP ${varepsilon}$ is catalytically inactive (32) and did not resultin detection of background PTP activity when expressed on itsown (Fig. 7). Here, too, activity was measured in vitro by usingpurified PTP ${varepsilon}$ molecules. Activity of full-length cyt-PTP ${varepsilon}$ wasreadily detected in this system, while deletion of the D2 domain,as in the D1 + 12 protein, resulted in doubling of PTP ${varepsilon}$ activity.On the other hand, adding similar amounts of inactive D2 + 12protein to the active D1 + 12 protein significantly reducedactivity of D1 + 12, although it was still higher than thatof full-length cyt-PTP ${varepsilon}$ (Fig. 7). Incomplete reduction of activityto cyt-PTP ${varepsilon}$ levels is probably due to inhibition being causedin this case by a bimolecular reaction, rather than by a moreefficient unimolecular reaction as in full-length cyt-PTP ${varepsilon}$ . Takentogether, these results indicate that the net effect of theD2 domain on cyt-PTP ${varepsilon}$ activity is inhibitory. Both results areconsistent with D2 mediating inhibitory interactions betweencyt-PTP ${varepsilon}$ molecules and with its ability to bind various domainsof cyt-PTP ${varepsilon}$ , as shown in several experiments with multiple PTP ${varepsilon}$ proteins (Fig. 3 to 6). Note that inhibition by D2 was demonstratedwithout treatment with hydrogen peroxide, i.e., it reflectsan inherent property of this domain. These results also indicatethat D1 and D2 domains located on the same or on separate moleculescan interact with each other.

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FIG. 7. D2 inhibits cyt-PTP ${varepsilon}$ activity. Full-length (F.L.), D1 + 12, and D2 + 12 cyt-PTP ${varepsilon}$ proteins were expressed in 293 cells as indicated. cyt-PTP ${varepsilon}$ proteins were then purified by immunoprecipitation via an N-terminal FLAG tag followed by elution, and their activities in vitro towards PNPP were determined. Results (means ± standard errors) are from one experiment (in triplicate) and are representative of three similar experiments performed. **, P < 0.0042, versus full-length cyt-PTP ${varepsilon}$ . Reduced activity of D1 + 12 in the presence of D2 + 12 protein is significant also versus D1 + 12 protein alone (P = 0.0184).

DISCUSSION

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Discussion

cyt-PTP ${varepsilon}$ is rather unique among PTPs since it is a naturallyoccurring, non-receptor-type isoform of RPTP ${varepsilon}$ and, therefore,in a manner atypical of non-receptor-type PTPs, possesses twoPTP domains; these domains are identical to those of the receptor-typeform of PTP ${varepsilon}$ . As such this molecule enables examination of therelative roles of various cytosolic domains in promoting self-associationin the absence of extracellular domains. The present study elaboratesand expands on results obtained previously with the relatedRPTP ${alpha}$ (8). Points unique to this study include the demonstrationthat cyt-PTP ${varepsilon}$ molecules can form dimers and possibly higher-orderassociations in vivo in a manner that is independent of membrane-spanningand extracellular domains. Furthermore, we show that associationsof cyt-PTP ${varepsilon}$ are readily detected, including those in cells thatexpress endogenous cyt-PTP ${varepsilon}$ , without the need for additionalstabilization. The ability to form homotypic associations istherefore an inherent property of the cytosolic domains of PTP ${varepsilon}$ .We also establish that, despite lack of direct contact withextracellular molecules, cyt-PTP ${varepsilon}$ can be influenced indirectlyby extracellular events through mediation of other receptors,such as the EGFR. The significant basal interaction among cyt-PTP ${varepsilon}$ molecules allowed us to demonstrate that physiological signalingcan down-regulate cyt-PTP ${varepsilon}$ dimerization, as seen during the firstfew minutes following stimulation of the EGFR. The data do notexclude the possibility that cell surface molecules, such asthe EGFR, can serve as sensors of extracellular events alsofor RPTPs that have extracellular domains of their own. Thispossibility is in fact somewhat appealing in light of the ongoingdifficulties in defining specific roles for such domains indirect transduction of signals across cell membranes. At themolecular level we identify a critical role for the D2 domainin mediating dimerization of cyt-PTP ${varepsilon}$ . We show that binding bythe D2 domain of cyt-PTP ${varepsilon}$ is regulated by sequences at its endsand that removal of one or of both these sequences from full-lengthcyt-PTP ${varepsilon}$ promotes dimerization and significantly reduces cyt-PTP ${varepsilon}$ activity. We also show that the "closed," inactive conformationof D2 can be relaxed by addition of sequences at its N terminus,suggesting that D2 is somewhat relaxed and accessible for bindingin the context of the full-length cyt-PTP ${varepsilon}$ . Finally, we demonstratethat the net effect of D2 on cyt-PTP ${varepsilon}$ activity is inhibitory.Inhibition is inherent to D2 and can be demonstrated in theabsence of oxidation by H₂O₂. In all, while this study doesnot rule out a role for extracellular domains in RPTP dimerformation, it shows that other mechanisms for achieving thisend exist.

Experiments presented here establish a link between a mainstreammechanism of cellular regulation, namely EGFR stimulation, anddimerization of cyt-PTP ${varepsilon}$ . The effects of EGFR stimulation oncyt-PTP ${varepsilon}$ are complex. In the first few minutes following applicationof EGF, dimerization of cyt-PTP ${varepsilon}$ is reduced and its tyrosinephosphorylation is increased. These changes in cyt-PTP ${varepsilon}$ are thenamong the first events which follow EGFR activation, leadingone to speculate that EGFR activation leads directly or indirectlyto cyt-PTP ${varepsilon}$ phosphorylation, resulting in reduced dimerizationof the PTP. Further studies are required to determine whethercyt-PTP ${varepsilon}$ is a direct target of the EGFR and whether phosphorylationand dimerization of cyt-PTP ${varepsilon}$ affect each other. With time cyt-PTP ${varepsilon}$ phosphorylation is reduced and dimerization is increased, suggestingthat these changes are caused by events further downstream fromthe receptor itself. It is noteworthy that EGF stimulation didnot result in detectable changes in RPTP ${alpha}$ dimerization (8). FurtherEGF-induced reduction in the already low basal levels of RPTP ${alpha}$ dimers may not have been detectable in the system used in thatstudy.

Increased dimerization of cyt-PTP ${varepsilon}$ following direct treatmentof cells with H₂O₂ agrees with previous studies, in which additionof H₂O₂ to growth media at concentrations similar to those usedhere increased dimerization and inhibition of RPTP ${alpha}$ (8) and resultedin oxidation of cellular PTPs in general (37). H₂O₂ treatmentof RPTP ${alpha}$ and PTEN resulted in direct oxidation of specific cysteineresidues, which, in the case of RPTP ${alpha}$ , mediated self-association(8, 30). We therefore believe that the effect of H₂O₂ seen inthis study results from direct oxidation of cyt-PTP ${varepsilon}$ molecules.Activation of growth factor receptors, such as those for EGF(3) or platelet-derived growth factor (4, 37), can generateH₂O₂. However, H₂O₂ produced by the EGFR probably does not affectthe cyt-PTP ${varepsilon}$ dimerization observed here since changes in cyt-PTP ${varepsilon}$ dimerization and phosphorylation following EGF treatment weredetected also in the presence of the reactive oxygen speciesscavenger N-acetylcysteine. This is in agreement with detectionof reduced dimerization of cyt-PTP ${varepsilon}$ 5 min after EGF stimulation,the opposite of what would have been expected if the stimulationeffects were mediated by H₂O₂.

A major factor in the ability of cyt-PTP ${varepsilon}$ molecules to bind eachother is the D2 domain, which is capable of strong self-associationand of binding D1 domains in vivo. Data shown here are consistentwith the extreme N and C termini of D2 (residues 360 to 380and 621 to 643 of cyt-PTP ${varepsilon}$ , respectively) interacting with eachother in a way that prevents the D2 domain from binding otherPTP ${varepsilon}$ molecules. Accordingly, removal of either or of both D2termini from full-length cyt-PTP ${varepsilon}$ increases dimerization andinhibits activity of cyt-PTP ${varepsilon}$ . Binding of the N and C terminihas been described previously in the case of RPTP ${alpha}$ (8) and mayoccur in other PTPs as well as a mechanism for regulating thebinding abilities of D2 domains. It is of interest to note thatthe N terminus of D2 contains a 4-amino-acid insertion in severalRPTPs, which is missing from the analogous region of D1 domainsof the same RPTPs (40). This insert, located in the loop betweenthe ${alpha}$ 1' and ${alpha}$ 2' helices in the D2 domain as noted for the LARprotein (40), exists in PTP ${varepsilon}$ , although its sequence is markedlydivergent from that of LAR or of RPTP ${alpha}$ . The roles of this insertand of the loop of which it is part are not known at present,although it is tempting to speculate that they may be relatedto the N-C binding within D2. The roles of the D2 C terminusdiffer somewhat in the more limited context of an isolated D2domain, where it appears to play roles both in promoting andin inhibiting binding. Reduced abilities of the D2 C terminusto promote binding in the context of full-length cyt-PTP ${varepsilon}$ areconsistent with modification of the binding abilities of theD2 domain by addition of protein sequences at its N terminus(Fig. 5A).

Our data are consistent with D2-D2 and D1-D2 interactions stabilizingbinding of cyt-PTP ${varepsilon}$ molecules and producing inactive dimers (Fig.8A). As each full-length cyt-PTP ${varepsilon}$ molecule contains a singleD2 domain, associations between D2 domains can occur only indimers. An attractive feature of the dimer model of Fig. 8Ais that it allows for the D1 wedge structures to block accessto and inhibit activity of the D1 domains of their dimerizationpartner. Involvement of the wedge structure in regulation ofRPTP ${alpha}$ activity is believed to be significant (26, 27), and theresidues of the RPTP ${alpha}$ wedge domain are conserved in cyt-PTP ${varepsilon}$ .Furthermore, the dimer model also accounts for the strong D2-D2interactions and for dimerization of cyt-PTP ${varepsilon}$ , both of whichhave been documented here. Although less consistent with ourdata, we cannot rule out the possibility of D1-D2 interactionsoccurring within the same molecule, in effect stabilizing itin a "closed," inactive conformation (Fig. 8B) or assistingin binding cyt-PTP ${varepsilon}$ dimers (Fig. 8A). As immunoprecipitationand gel filtration techniques used in this study tend to focuson intermolecular interactions, further studies are requiredto clarify the existence and role of intramolecular interactionswithin cyt-PTP ${varepsilon}$ .

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FIG. 8. Inter- and intramolecular associations of cyt-PTP ${varepsilon}$ . (A) Intermolecular binding is mediated by interactions between D2 domains of adjacent molecules, which provide opportunity for the wedge domains (black triangles), located at the N termini of the D1 domains, to interact and inhibit PTP ${varepsilon}$ activity. Binding between D1 (hatched) and D2 (open) domains within a single cyt-PTP ${varepsilon}$ molecule is also consistent with this model. (B) The less favored possibility of intramolecular binding between D1 and D2 domains within a single cyt-PTP ${varepsilon}$ molecule, which inhibits cyt-PTP ${varepsilon}$ activity. Interactions can be affected by input from other signaling events, such as EGFR signaling, shown in this study.

How general are the results presented here? Our data raise thepossibility of D2-D2 interactions occurring between moleculesof other PTPs and promoting their homodimerization or heterodimerization,possibly acting as a general regulatory mechanism in PTPs thatcontain two PTP domains. Individual D2 domains have differentabilities to bind D1 domains or full-length RPTP molecules (9,50) and can affect PTP activities (e.g., reference 22). It istherefore reasonable to suggest that the D2 domain of a givenRPTP can interact with D2 domains of other full-length RPTPs,but the strength of this effect and its effects on PTP activityshould vary from case to case.

ACKNOWLEDGMENTS

We thank Liat Yakir and Yosef Yarden for the EGFR cDNA and antibodiesand Galit Shohat, Opher Gileadi, and Ghil Jona for helpful discussions.We also thank the anonymous reviewers of the manuscript forhelpful comments.

This study was supported by The Israel Science Foundation. A.E.is the incumbent of the Adolfo and Evelyn Blum Career DevelopmentChair of Cancer Research at The Weizmann Institute.

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel. Phone: 972-8-934-2331. Fax: 972-8-934-4108. E-mail: ari.elson{at}weizmann.ac.il.

REFERENCES

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