THE JOURNAL BIOLOGICAL No. of 25, PP. 22771-22776, Q 1993 … · 1999-01-15 · THE JOURNAL of BIOLOGICAL CHEMISTRY Q 1993 by The American Society for Biochemistry and Molecular Biology,

THE JOURNAL of BIOLOGICAL CHEMISTRY Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol . 268, No. Issue of October 25, PP. 22771-22776, 1993 Printed in U.S.A.

Expression, Purification, and Characterization of SH2-containing Protein Tyrosine Phosphatase, SH-PTP2*

(Received for publication, April 23, 1993, and in revised form, June 16, 1993)

Seiji Sugimoto, Robert J. Lechleider*§n, Steve E. Shoelsonll, Benjamin G. NeelS**, and Christopher T. WalshSS From the Department of Biological Chemistry and Molecular Pharmacology and the IlJoslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 02115 the $Molecular Medicine Unit, Beth Israel Hospital, and the §Division of Pulmonary Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02215

Ahuman protein tyrosine phosphatase containing two src homology 2 (SH2) domains (SH-PTP2) was expressed in Escherichia coli under T7 promoter control and purified to near homogeneity. The purified protein, with molecular mass of 68 kDa on SDS-polyacrylamide gel elec- trophoresis, was identified as SH-PTP2 by its protein tyrosine phosphatase activity and N-terminal amino acid sequence analysis. Its protein tyrosine phosphatase activity was sensitive to pH and salt concentration. Whereas its optimum pH for the low molecular weight substrate para-nitrophenyl phosphate is 5.6, the pH op- tima for peptide substrates were shifted toward neutral. With the artificial protein substrate reduced, carboxyamidomethylated, and maleylated lysozyme, it displays 2000-fold lower K, (1.7 px) and 2.4-fold higher kcet (0.11 s-l) than with para-nitrophenyl phosphate. Among the phosphopeptides from autophosphorylation sites of receptors for epidermal growth factor and platelet-derived growth factor, SH-PTP2 displayed high activity toward phosphopeptides corresponding to pY992 of the epidermal growth factor receptor and pY1009 and pY1021 of the platelet-derived growth factor receptor. In further enzymatic studies with phosphopeptides corresponding to pY1009, SH-PTP2 showed nonlinear Line- weaver-Burk double-reciprocal plots, suggesting that the phosphopeptide corresponding to pY1009 may have a substrate and allosteric effect.

Protein tyrosine phosphorylation, regulated by the interplay between protein tyrosine kinases and protein tyrosine phosphatases, is an important mechanism for the control of cell proliferation and differentiation (1-5). Although the structure, function, and regulation of protein tyrosine kinases have been rather well established (6, 7), those of the protein tyrosine phosphatases remain obscure (8-10). So far, more than 30 transmembrane and nontransmembrane protein tyrosine phosphatases have been identified (11-13). Among the nontrans-

49152 (to B. G. N.) and GM20011 (to C. T. W.), funds from F. Hoff- * This work was supported by National Institutes of Health Grants

mann-La Roche Ltd. (to B. G. N. and C. T. W.) and the National Science Foundation (to S. E. S.), and by Joslin Diabetes Center National Insti- tutes of Health DERC Grant 36836. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Fund Postdoctoral Fellowship DRG-062. ll Supported by Damon Runyon-Walter Winchell Cancer Research

** Supported in part by a Junior Faculty Research Award from the American Cancer Society.

Chemistry and Molecular Pharmacology, Harvard Medical School, 240 $$ To whom correspondence should be addressed: Dept. of Biological

Longwood Ave., Boston, MA 02115. Tel.: 617-432-1715; Fax: 617-432- 2452.

membrane protein tyrosine phosphatases, we have cloned SH- PTPl(14), also known as PTPlC (151, HCP (16), SHP (17), and PTPN6 (18) and SH-PTP2 (19), also known as SH-PTP3 (201, PTPlD (21), and PTP2C (221, both of which contain two SH2 domains upstream from the conserved catalytic domain. Mouse Syp is reported as a homologue of SH-PTP2 based on the high similarity of the cDNA sequence (23). Rat PTPLl (24) may be also a homologue of SH-PTP2, based on the high similarity in reported partial amino acid sequences of PTPLl with SH- PTP2.

SH2l domains are also found in several other types of sig- naling proteins, such as src family protein tyrosine kinases, GTPase-activating protein, phospholipase C-y, and p85, the regulatory subunit of phosphatidylinositol 3-kinase (25). SH2 domains bind to tyrosine-phosphorylated sequences in proteins and peptides, thereby facilitating inter- and intramolecular protein-protein interactions, including enzyme-substrate interactions (26). Recently, phosphotyrosine-independent binding to SH2 domains has also been reported (27). The finding of protein tyrosine phosphatases with SH2 domains that would, presum- ably, target these enzymes to specific phosphotyrosine-containing protein substrates is of distinct physiological interest.

Although SH-PTP1 is predominantly expressed in hemato- poietic cells (14, 16, 17), SH-PTP2 is ubiquitously expressed (19, 20, 22). Based on its sequence similarity and comparable expression pattern, SH-PTP2 may be the homologue of the Drosophila corkscrew gene product (Csw) (19,281. Genetic epis- tasis experiments indicate that Csw functions in the terminal class signal transduction pathway in concert with the Dro- sophila c-Raf homologue (l(1) polehill gene product (29) or D- Raf), to positively transduce signals generated by the torso receptor protein tyrosine kinase (30, 311, a PDGF receptor (PDGFR) homologue.

We and others (21, 23, 32) have shown that SH-PTP2 is tyrosyl-phosphorylated in vivo upon activation of the EGF receptor (EGFR) or the PDGFR, although this has not been shown to be a direct effect of the receptor kinase. Moreover, we have found that SH-PTP2 is directly bound to EGFR and PDGFR via its N-terminal SH2 domain following ligand activation (32). Since the EGFR (33-35) and the PDGFR (3640) are protein tyrosine kinases and are autophosphorylated within the cytoplasmic domain, it seems likely that SH2 domains in SH-PTP2 will play a crucial role in binding to the EGFR and PDGFR via their autophosphorylation sites and for further signal transduction. SH-PTP2 binds to phosphorylated

The abbreviations used are: SH2, src homology 2; EGF, epidermal growth factor; EGFR, EGF receptor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; p-NPP, para-nitrophenyl phosphate; RCM-lysozyme, reduced, carboxyamidomethylated, and maleylated ly-

fonate; DTT, dithiothreitol; BSA, bovine serum albumin. sozyme; pY, phosphotyrosyl residue MES, 2-(N-morpholino)ethanesul-

22771

22772 Expression, Purification, and Characterization of SH-PTP2

PDGFR via C-terminal tyrosine 1009.2 The effects of phosphorylation of SH-PTP2 and binding of SH-PTPP SH2 domain to receptor autophosphorylation site(s) on SH-PTP2 enzymatic activity have not been determined.

In this paper, we describe the expression, purification, and enzymatic properties of recombinant SH-PTP2 derived from Escherichia coli, including its interactions with phosphopeptides corresponding to autophosphorylation sites of EGFR and PDGFR as protein tyrosine phosphatase substrates and effectors.

EXPERIMENTAL PROCEDURES Materials-Restriction endonucleases NdeI and SalI were purchased

from New England Biolabs and ThaI from Life Technologies, 1nc.p-NPP was from Sigma. Reduced, carboxyamidomethylated, and maleylated (RCM) lysozyme was from Life Technologies, Inc. v-Ab1 was from On- cogene Science. The expression vector PET-SHPTP1 (41) was kindly donated by D. Pei (Harvard Medical School). Oligonucleotide adapters

Medical School). Phosphopeptide EGFR pY1173 was kindly donated by and sequencing primers were synthesized by A. Nussbaum (Harvard

H. Cho (Harvard Medical School). Plasmid Construction-A 1.95-kilobase pair ThaI-Sal1 fragment en-

coding amino acid residues 4-593 of SH-PTP2 was isolated from plasmid pBS FB 21-7, which is a cloning vector pBluescript KS (-1 containing the entire cDNA of SH-PTP2 (19). The N-terminal 3 amino acids were re-introduced using two oligonucleotide adapters: 5'-TAT- GACATCG-3' (10-mer) and 5'-ACTGTAGC-3' (8-mer), to facilitate sub- cloning. The 10- and 8-mer adapters and the 1.95-kilobase pair ThaI- SalI fragment were ligated into NdeI-SaZI-linearized PET-SH-PTP1 to generate plasmid PET-SHPTP2. The sequence from the Shine-Dalgano sequence to 15th amino acid from the N terminus was confirmed by dideoxy sequencing using the T7 promoter primer, 5"TAATACGACT- CACTATAGGG-3' (20-mer).

Expression and Purification of SH-PTPZ-E. coli strain BL21 (DE3) transformed with plasmid PET-SHPTP2 was grown in 4 liters of LB medium containing 50 pg/ml ampicillin at 37 "C to an absorbance at 595 nm of 0.8 and induced for 3 h a t 30 "C with 0.4 mM isopropyl-l-thio-a- D-galactopyranoside. Cells were harvested by centrifugation and resus- pended in 100 ml of 10 mM Tris-HC1, pH 7.8, containing 1 mM EDTA and 10 mM 2-mercaptoethanol (buffer A) supplemented with protease inhibi- tors (0.5 mM ortho-phenanthroline, 0.64 mM benzamidine, 0.29 mM phenylmethylsulfonyl fluoride, 20 pg/ml soybean trypsin inhibitor, 20 pg/ml aprotinin, 20 pg/ml leupeptin, and 20 pg/ml pepstatin). The cells were disrupted by French Press, and the crude lysate was centrifuged at 15,000 rpm for 15 min in a Sorvall SS-34 rotor. The supernatant was loaded onto a Q-Sepharose Fast Flow (Sigma) column (11.2 x 2.5 cm) equilibrated with buffer A. The column was washed with 200 ml of buffer A, and 0-500 mM NaCl concentration gradient was developed in 400 ml of buffer A at 0.5 mumin. Activity, recovered in the flow-through and very early part of gradient elution, was precipitated by 60% saturated ammonium sulfate, dissolved in buffer A containing 20% saturated ammonium sulfate, and loaded onto a phenyl-Sepharose (Sigma) column (13.2 x 2.5 cm) equilibrated with buffer A containing 20% saturated ammonium sulfate. The column was washed with the same buffer, and activity was eluted using an ammonium sulfate concentration gradient, 20 to 0%, in 400 ml of buffer A a t 0.5 mumin. The active fractions, eluted at approximately 8% saturated ammonium sulfate, were dia- lyzed against 10 mM MES, pH 5.7, containing 10 mM 2-mercaptoethanol (buffer B), and divided into four aliquots of equal volume. Each aliquot was loaded onto Mono S HR 10/10 (Pharmacia LKB Biotechnology Inc.) column equilibrated with buffer B. The column was developed with a gradient of 0-250 mM NaCl in 500 ml of buffer B a t 2.5 ml/min. Pooled fractions, eluting from 125 to 150 mM NaC1, were concentrated using a Centriprep-10 (Amicon) and stored a t -80 "C in the presence of 33% (v/v) glycerol.

Assay for Protein Qrosine Phosphatase Actiuity-Withp-NPP as substrate, typically 10 mMp-NPP was incubated with 87 pg/ml SH-PTP2 a t 24 "C for 130 min in 50 pl of 50 mM 3,3-dimethylglutarate, pH 5.6, containing 50 mM NaCl, 10 mM DTT, and 2 mM EDTA. The reaction was quenched with 950 pl of 1 M NaOH, and the absorbance of p-nitrophenolate at 405 nm was measured. The amount ofp-nitrophenol released were calculated by comparison with a standard curve obtained with p-nitrophenolate (Sigma).

R. J. Lechleider, S. Sugimoto, S. E. Shoelson, J. Cooper, C. T. Walsh, and B. G. Neel, manuscript in preparation.

To assay the dephosphorylation of phosphopeptides, the release of Pi was measured by malachite green assay (4244). Typically, 500 PM phosphopeptide was incubated with 87 pg/ml SH-PTP2 a t 20 "C for 12 min in 50 pl of 50 mM HEPES, pH 7.1, containing 150 mM NaCI, 10 mM DTT, and 2 mM EDTA. The reaction was quenched with 950 pl of malachite green reagent, and the absorbance a t 650 nm was measured. The amount of released Pi was calculated with a standard curve. Phospho- peptides were synthesized as described in Piccione et al. (45) using the methodology of Kitas et al. (46). Sequence of phosphopeptides used here were as follows: EGFR pY992, DADEpYLIPQQGFF; EGFR pY1068, WEpYINQSVPK, EGFR pY1086, NVPpYHNGPLNP; EGFR pY1148, NPDpYQQDFFPK EGFR pY1173, TAENAEpYLRVA, PDGFR pY740, DGGpYMDMSKDE; PDGFR pY751, SVDpYVPMLDMK PDGFR pY771, S S N p W Y D N Y ; PDGFR pY1009, SVLpYTAVQPNE; PDGFR pY1021, DNDpYIIPLPDPK, with pY indicating the phosphorylated tyrosine.

To assay the dephosphorylation of a protein substrate, phosphorylated RCM-lysozyme was prepared essentially by the method of Tonks et al. (47), but using v-Ab1 as the kinase. Typical specific activity obtained was 13 pCi/mol. Assay conditions were essentially the same as those of Tonks (47). The indicated concentration of phosphorylated RCM-lysozyme was incubated with 320 ng/ml SH-PTP2 at 30 "C for 5 min in 60 pl of reaction buffer consisting of 25 mM HEPES, pH 7.2, containing 1 mg/ml bovine serum albumin (BSA), 5 lll~ EDTA, and 10 mM DTT. At 1 and 5 min, 25 pl of reaction mixture was transferred to a suspension of activated charcoal. From the counts in the supernatant, dephosphorylation velocities were calculated.

Other Methods-The concentration of protein was determined by Bradford assay (Bio-Rad) using BSA as standard (48). SDS-PAGE was carried out as described by Laemmli (49). Gel filtration was carried out with Superose 12 (10/30, Pharmacia) equilibrated with 25 mM HEPES, pH 7.2, containing 200 mM NaCl, a t a flow rate of 0.5 mumin using aldolase ( M , = 158,000), BSA ( M , = 67,000), and ovalbumin (M, = 43,000) as standards. N-terminal sequencing was performed by the Edman method (50) using an automated gas-phase sequenator (Applied Biosystems).

RESULTS

Expression and Purification of SH-P!l'P2-Previously we ob- served that SH-PTP1, a protein highly similar to SH-PTP2, was highly expressed in E. coli and that when expressed in bacterial cells, SH-PTP1 accumulated partially as a soluble protein (41). Therefore, we used the same expression vector with the coding region of SH-PTP2. The expression level of SH-PTP2 was low, approximately 1% of total E. coli cell protein compared with about 10% for SH-PTP1. However, most of the activity was recovered in the soluble fraction (data not shown).

Soluble bacterially expressed SH-PTP2 was purified accord- ing to the scheme summarized in Table I. During the purification,p-NPP was used as substrate. E. coli alkaline phosphatase also reacts with p-NPP. During the first three steps, SH-PTP2 is likely contaminated with alkaline phosphatase, suggesting that the yield for the last three steps is higher than the values in Table I. Using this scheme, SH-PTP2 was purified to greater than 90% purity based on SDS-PAGE analysis (Fig. 1). The molecular mass of the purified protein was 68 kDa, consistent with the 593-amino acid sequence and similar to the in vivo molecular mass of 68-70 kDa (21,23,32). The purified enzyme behaved as a monomer on gel filtration (data not shown). The N-terminal sequence of the purified protein was analyzed up to the 10th residue and matched the predicted sequence from residue 2 on, indicating processing of the initiating N-terminal methionine.

Biochemical Characterization of SH-PTP2 Phosphatase Ac- tivity toward p-NPP-We elucidated the optimum pH, salt concentration, and temperature for SH-PTP2 activity initially US-

ing the low molecular weight substratep-NPP. As shown in Fig. 2, like SH-PTP1 (41) and other protein tyrosine phosphatases (51), SH-PTP2 showed an acidic pH optimum with this substrate, pH 5.6. As determined by the NaCl concentration profile for SH-PTP2, the optimal salt concentration for SH-PTP2 activity towardp-NPP was 50 mM a t pH 5.6 with only 9% activity

Expression, Purification, and Characterization of SH-PTP2 22773

TABLE I Summary of SH-PTP2 purification

steps Volume Protein Activity Specific activity -Fold Yield

ml mglml unitslml unitslmg % 125 Crude lysate 31.0 24 0.77 1 100

Q-Sepharose 200 9.4 12 1.28 1.7 80 Ammonium sulfate Precipitation 50 19 37 1.95 2.5 62 Phenyl-Sepharose 112 0.36 2.7 7.5 9.7 10

60 Mono S 0.05 1.5 30 39 3 Centriprep 10 2.25 1.33 40 30 39 3

kDa ,”> 1. - - -

200

97.4

68.0 - - 43.0

29.0

10.4

M 1 FIG. 1. SDSPAGE of purified SH-PTP2. Lane M, protein marker;

lane 1, SH-FTP2. Molecular masws of the standard protein makers are shown on the left.

3 4 5 6 7 8 9 1 0

PH FIG. 2. pH dependence of SH-FTPP. Substrates are 10 mM p-NPP

(closed circle), 500 p~ EGFR pY1173 (open circle), and 500 p~ EGFR pY992 (open triangle). See text for details of receptor phosphopeptides. Assays were done under the same conditions as described under “Ex- perimental Procedures,” except for buffers and pH. For p-NPP, 50 mM acetate was used a t pH 4.3-5.6, 50 mM succinate a t pH 5.66.3, and 50 mM 1,3-bis[tris(hydroxymethyl)methylaminolpropane at pH 6.3-9.1. For pY peptides, 50 mM 3,3-dimethylglutarate was used at pH 4.0-6.1 and 50 mM 1,3-bis[tris(hydroxymethyl)methylaminolpropane at pH 6.1-9.1.

at high salt (300 mM) (data not shown). SH-PTP2 required long reaction times because of its low

activity toward p-NPP. To assess the stability of SH-PTP2 under such reaction conditions, we analyzed temperature dependence by Arrhenius analysis. Fig. 3 shows that SH-PTP2 was stable below 24 “C at pH 5.6, even at long reaction times (130 min 1.

Kinetics of SH-PTP2 toward p-NPP and Phosphotyrosyl- RCM-lysozyme-As a p-nitrophenylphosphatase, SH-PTP2 is a slow enzyme (kcat 0.046 s’), even under optimum pH and ionic strength conditions (Table 11). However, assay with phospho-

.” 0.0031 0.0032 0.0033 0.0034 0.0035

1IK FIG. 3. Arrhenius plot of SH-PTP2 using p-NPP as substrate.

Assays were done under the same conditions as described under Ex- perimental Procedures,” except for temperature. Natural log of velocities in micromolar/min a t 18.5, 21, 24, 30, 37, and 46 “C were plotted uersus reciprocals of temperature in Kelvin.

tyrosyl-RCM-lysozyme (Table 111) revealed a 2.4-fold higher catalytic activity (kcat 0.11 s-l). Concomitantly, K, drops 2000- fold fromp-NPP (3.6 m ~ ) to RCM-lysozyme (1.7 PM). Thus, the use ofp-NPP severely underestimates (by some 5000-fold) the catalytic efficiency (kcaJKm = 13 M - ~ s-l versus 6.5 x lo4 M-’ s-l) compared with a protein substrate.

Properties of SH-PTP2 toward Phosphopeptide /?om the EGFR and the PDGFR Autophosphorylation Sites-Given the in vivo evidence for specific binding of SH-PTP2 to EGFR and PDGFR (32), we have evaluated the properties of autophosphorylation site peptides from the cytoplasmic domains of each of these transmembrane growth factor receptors as substrates of and/or effectors for SH-PTP2. An initial screen utilized the synthetic 11-13-amino acid phosphopeptides corresponding to each known autophosphorylation site (33-40).

As indicated in Fig. 4A, on incubation at pH 5.6 (the pH optimum determined for p-NPP (see Fig. 2)), the EGFR pY992 was dramatically better as a substrate than the other EGFR autophosphorylation site peptides. When the EGFR pY992 and EGFR pY1173 phosphopeptides were compared (“good” and “bad” substrates, respectively), EGFR pY1173 showed a pH optimum at pH 6.1 (Fig.2), but the EGFR pY992 substrate showed a pH optimum in the physiological pH range (Fig. 2). Thus, subsequent characterization was done under physiological conditions. At neutral pH, the EGFR phosphopeptides show a similar profile to that at acidic pH (Fig. 4, B versus A ) with EGFR pY992 showing >lO-fold preferential substrate activity for SH-PTPB.

In comparison with EGFR peptides, the distinction between the five phosphopeptides from the PDGFR at pH 5.6 was less (Fig. 4A); PDGFR pY1021 was chosen as was PDGFR pY1009 for subsequent kinetic evaluation. At physiological pH, PDGFR pY1009 is now preferred about 2-fold over PDGFR pY1021 and 4-10-fold over the other sites (Fig. 4B).


Kinetic constants for FTPases for D-NPP TABLE I1

Enzymes kcat Km k,.dK, Conditions Ref.

5-1

YOP51 1234 PTPU323 YPTPl

48

rIAR LARD1 V H 1 cdc25 SH-PTP1 Full length 155

1.6 6.1 4.1 0.3 0.033

SH-PTP1 SH2 domains deleted 110

SH-PTP1 (PTPlC) Full length 37.4

SH-PTP2 Full length 0.046

SH-PTP2 (PTP2C) SH2 domains deleted 4ga

m M x 1 0 1 ~ ~ ~ 2.40 514 0.23 209 1.18 1.36 0.42 14.5 1.73 2.37 7.7 0.039 50

148 0.00066 1.0

24-38 2.9-4.6

1.5 25

3.6 0.013

NDa

pH 5.0, 100 mM acetate, I = 0.15 M, 30 "C Same as above pH 5.5, 100 mM acetate, I = 0.15 M, 30 "C pH 5.0,40 mM MES, 30 "C pH 6.0, 100 m~ MES, 25 "C pH 5.5, 100 m~ acetate, I = 0.15 M, 25 "C pH 8.2,lOO mM Tris, 250 m~ NaCl, 37 "C pH 5.5, 100 mM MES, 150 mM NaCl, 10 mM DTl', 1 mM

EDTA. 23 "C pH 6.3, '100 mM MES, 150 mM NaCl, 10 mM DTT, 1 mM

EDTA, 23 "C DH 5.0. 25 mM acetate. 20% dvcerol. 1 mM DTT. 1 mM

I" I

EDTA, 22 oc pH 5.6, 50 mM 3,3-dimethlyglutarate. 50 mM NaCl, 10 mM

DIT, 2 mM EDTA, 24 OC- - pH 5.0, 25 mM acetate, 20% glycerol, 1 mM D m , 1 mM

EDTA, 22 "C

51 51 51 53 54 51 55 41

41

56

This WOI

22

"K,,, not determined. 49 s-l estimated from reported velocity a t a fured concentration of 10 mM p-NPP (22).

TABLE I11 Kinetic constants for SH-FTPs toward RCM-lvsozvme

5-I pM X 101 M - I 5-I

PTPlC Full length 0.14 1.9 73 pH 7.0, 25 mM imidazole HC1, 1 mM DTl', 1 mM EDTA, 1 56

SH-PTP2 Full length 0.11 1.7 65 pH 7.2,25 mM HEPES, 10 mM DTT, 5 mM EDTA, 1 mg/ml This work

PTP2C" SH2 domains deleted 0.35" ND" pH 7.0, 25 mM imidazole HC1, 1 mM D m , 1 mM EDTA, 1 22

mg/ml BSA, 30 "C

BSA, 30 "C

mg/ml BSA, 30 "C

a K,,, not determined. 0.35 s-l estimated from reported velocity a t a fixed concentration of 1 PM RCM-lysozyme (22).

As mentioned above, our unpublished data indicate that SH- PTP2 binds to phosphorylated PDGFR via C-terminal tyrosine 1009,2 suggesting that PDGFR pY1009 may both regulate and act as substrate for the protein tyrosine phosphatase activity of SH-PTPS. Therefore, we chose PDGFR pY1009 for further K , and kcat analysis with pure SH-PTP2. Deviations from simple Michaelis-Menten hyperbolic saturation behavior were de- tected. Fig. 5 shows a Lineweaver-Burk plot of PDGFR pY1009 which reproducibly shows curvature. A simple linear analysis from data at low substrate concentration would yield a y axis crossing in the negative region, which precludes calculation of the K , and kcat. However, for data at higher substrate concentration, the slope approaches linear behavior, and an extrapo- lated line gave a V,,, estimate of 20 pdmin (inset of Fig. 5). It may be that this peptide shows both allosteric and substrate effects.

DISCUSSION

In this work, the full-length human SH2 domain-containing protein tyrosine phosphatase, SH-PTP2, was expressed in E. coli and highly purified. Basic enzymatic properties have been assessed with a low molecular weight substrate p-NPP and subsequently with 11-13 residue phosphopeptides and tyrosine-phosphorylated RCM-lysozyme.

Protein tyrosine phosphatase activity of SH-PTP2 toward p-NPP shows an acidic pH optimum and sensitivity to ionic strength, similar to SH-PTP1 (41). From the Arrhenius plot (Fig. 3), SH-PTP2 was stable for 130 min at pH 5.6 below 24 "C, but was labile above 24 "C. Although assays with RCM-lysozyme were done at 30 "C for 5 min, our data were within linear regions of Pi release versus time, indicating no inactivation during the reaction.

In Table 11, K , and kcat values for p-NPP are compared with those reported for other protein tyrosine phosphatases (22,41,

51, 53-56). As a p-nitrophenylphosphatase, SH-PTP2 is dramatically slow, even under optimum pH and ionic strength conditions. It has 30,000-fold lower kcat than the Yersinia protein tyrosine phosphatase Yop (51). The kcat of SH-PTP2 as a p-nitrophenylphosphatase is lower than that of VH1 (51) and slightly higher than that of cdc25 (55) . At 10 mM p-NPP, SH- PTP2 has about 2% the catalytic activity of the highly similar, E. coli-expressed, SH-PTP1 (41 and data not shown). To deter- mine that SH-PTP2 expressed was not largely inactive, we compared the activity of SH-PTP2 toward phosphotyrosyl- RCM-lysozyme with that of SH-PTP1 (PTPlC) (56) and with the value just reported for SH2 domain-deleted SH-PTP2 mutant (ASH2-PTP2C) (22) (Table 111). Kinetic data for SH-PTPZ are almost the same as those of SH-PTP1 (PTPlC) (561, and the kcat of SH-PTP2 is about one-third the value of the SH2 domain-deleted SH-PTPZ mutant (22). We also compared the activity of SH-PTP2 toward the phosphotyrosyl peptides EGFR pY992 and EGFR pY1173 with SH-PTP1 expressed in E. coli (41). SH-PTP2 is only 5-8-fold slower than SH-mPl under the same assay conditions (data not shown). These comparisons are consistent with the view that the purified SH-FTP2 is correctly folded full-length enzyme. It may be worthwhile to express full-length SH-PTP2 in a eukaryotic overproduction system (e.g. baculovirus) to compare catalytic efficiency.

Since SH-PTP2 has SH2 domains and protein tyrosine phosphatase domain, it is possible that phosphotyrosyl proteins serve as both substrates and SHZ domain targets. Using phosphotyrosyl peptide substrates from the EGFR (33-35) and PDGFR (36-40) cytoplasmic domains, SH-PTP2 shows high activity for three of the 10 autophosphorylation site peptides (Fig. 4). For PDGFR pY1009 which has been examined care- fully, the anomalous dependence of velocity on pY peptide concentration may reflect both allosteric and active site binding.

Songyang et al. (57) selected the sequence motif of pY-ViI-X-V

Expression, Purification, and Characterization of SH-PTP2 22775

A

992 1068 1086 1148 1173 740 751 771 1009 1021

EGFR PDGFR

pY peptidm

992 1068 1086 1148 1173 740 751 771 1009 1021

EGFR pY peptides

PDGFR

FIG. 4. Regioselectivity of recognition by SH-FTP2 of phosphotyrosine from autophosphorylation sites of EGF receptor and PDGF receptor at pH 5.6 (A) and pH 7.1 (B) . See text for details of receptor phosphopeptides. Assays were done under the same conditions as described under "Experimental Procedures" ( B ), but 50 mM succinate buffer, pH 5.6, was used instead of 50 mM HEPES buffer (A).

00 0.00s 0.010 0.01s 0

MSI (1II"

!O

FIG. 5. Lineweaver-Burk plot of SH-PTP2 using PDGFFt pYlOOS as substrate. Assays were done under the same conditions as described under "Experimental Procedures," at 29.5 pg/ml of SH-PTP2. Three separate experiments are represented by open circles, closed circles, and closed triangles. In the inset, the high concentration region of substrate is shown in detail.

from a synthetic peptide library as an enriched ligand for the N-terminal SH2 domain of SH-PTP2. PDGFR pY1009 has the sequence pYTAV, approximating the enriched sequence. More- over, we recently found that pY1009 is the binding site in PDGFR for glutathione S-transferase fusion proteins with SH2 domains derived from SH-PTP2.' Initial binding studies of PDGFR pY1009 to the isolated SH2 domains of SH-PTP2 show

binding with differential affinity3 These results support the assumption that either one or both of the SH2 domains is the allosteric site for PDGFR pY1009.

For further characterization of the allosteric effect of PDGFR pY1009 phosphotyrosyl substrate on SH-PTP2, the assay system we present in this work is complicated because PDGFR pY1009 acts as substrate and effector. Therefore, we are trying to establish a system to separate substrate and effector. Re- cently we find that addition of PDGFR pY1009, but not the other nine phosphotyrosine peptides of Fig. 4, substantially stimulates enzymatic activity toward dephosphorylation of phosphotyrosyl-RCM-lysozyme, which by itself has no allosteric effect on enzymatic activity, at least in the range of concentration used in this work.' These results suggest that occu- pation of the SH2 domains may serve to up-regulate phosphatase activity.

SH-PTP2 demonstrates substantial protein tyrosine phosphatase activity toward peptides EGFR pY992 and PDGFR pY1021 in addition to PDGFR pY1009 (Fig. 4B). In contrast to PDGFR pY1009, EGFR pY992 and PDGFR pY1021 have some common sequence properties, such as more than 2 acidic amino acid residues N-terminal to the phosphotyrosine, aliphatic amino acid residues at both +1 and +2 position, and Pro at +3 position. By these properties, EGFR pY992 and PDGFR pY1021 are distinct from other phosphopeptides derived from autophosphorylation sites of the EGFR receptor and PDGFR, suggesting that these peptides might provide good phosphotyrosyl substrates for SH-PTPZ.

Zhang et al. (58) have determined the specificity of the catalytic domains of protein tyrosine phosphatase from human and Yersinia, toward phosphotyrosyl peptides and acidic amino acid residues at positions -1, -2, and -4 and Pro at position +3 are important for high activity. If the rules suggested by Zhang et al. (58) are applicable to SH-PTP2, EGFR pY992 corresponds to the best substrate derived from EGFR autophosphorylation sites, and PDGFR pY1021 also matches the sequence determi- nants for a good substrate. Interestingly, both of these two sites are also PLC-7 binding site (34, 59); however, the biological significance of this finding is not clear.

Comparing kinetic data of SH-PTP2 (full length) with SH2 domain-deleted SH-PTP2 (ASH2-PTP2C) (221, the velocity reported towardp-NPP of ASH2-PTP2C (49 s-') is 103-fold higher than kcat of full-length SH-PTP2 (0.046 s-l) (Table 11). Toward RCM-lysozyme, the velocity of ASH2-PTP2C (0.35 s-')(22) is only %fold higher than full-length SH-PTPZ (0.H s-l) (Table 111). We recently (41) reported that deletion of SH2 domains from SH-PTP1 increases the catalytic efficiency toward p-NPP, but only by 3-5-fold, from 1.0 x lo3 M - ~ ssl to 2.9-4.6 x lo3 s-l (Table 11). These comparisons of kinetic data between full- length and SH2 domain-deleted SH-PTP1 and SH-PTP2 imply negative regulation of 5-1000-fold of protein tyrosine phosphatase activity by unoccupied SH2 domains.

Limited tryptic cleavage of the 68-kDa intact SH-PTP2 to yield a 65-kDa fragment also increases protein tyrosine phosphatase activity by f~ur- fo ld .~ Similarly, after partial tryptic digestion resulting in cleavage of the C-terminal 5-kDa fragment, SH-PTP1 (PTPlC) enzymatic activity increased about 20-fold (56). We have not confirmed the cleavage site yet for SH-PTP2, but if tryptic digestion of SH-PTP2 also removes the C-terminal region, then this would be an indication that the C-terminal tail of SH-PTP2, as in SH-PTP1, also has negative autoregulatory effect.

So far we have noted the stimulatory or inhibitory effects of phosphopeptides from receptor kinases and SH2 domains and the C-terminal regions of SH-PTPs. It is important, we believe,

S. Sugimoto and C. T. Walsh, unpublished results.


to clarify the relationship of these effects. "0 resolve these issues, we plan to compare the properties of SH2 and C-terminal domain-deleted or -substituted mutants and examine the contribution of each domain to both binding and catalytic ac- tivities using phosphorylated and nonphosphorylated peptides or proteins.

During the preparation of this manuscript, two papers have appeared, one describing the in vivo characterization of SH- PTP2 in a transient expression system (21) and another char- acterizing the murine homologue of SH-PTP2 (23). In the for- mer paper, SH-PTP2 (named PTPlD by these authors) was co-transfected into 293 cells with receptor chimeras composed of the extracellular domain of the EGFR and cytoplasmic domain of the PDGFR. Immunoprecipitated SH-PTP2 (PTPlD) from ~-[~~S]Met-labeled 293 cell transfectants was analyzed with or without stimulation by EGF. On EGF stimulation, the phosphorylation level of SH-PTP2 was elevated and protein tyrosine phosphatase activity was increased slightly (1.2-fold). The contribution of phosphorylation and SH2 domain binding to this increase in phosphatase activity is unclear. Experiments are currently under way in our labs to clarify these issues.

Acknowledgments-We thank H. Cho for phosphopeptide preparation, D. Pei for expression plasmid, and the Dana Farber Cancer Insti- tute and Harvard Microchemistry Facility for N-terminal amino acid sequencing analysis.

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