ANALYTICAL

BIOCHEMISTRY

202,361-366

(19%)

Continuous Spectrophotometric Assay, of Protein Tyrosine Phosphatase Using Phosphotyrosine’ Zhizhuang Zhao, *p2 Norbert F. Zander,* Dean A. Malencik,? Sonia R. Anderson,? and Edmond H. Fischer* *Department of Biochemistry, University of Washington, Seattle, Biophysics, Oregon State Uniuersity, Corvallis, Oregon 97331

Received

December

98195; and TDepartment

of Biochemistry1

9, 1991

A continuous activity assay for protein tyrosine phosphatases (PTPs), employing phosphotyrosine (P-Tyr) as a substrate, has been developed and applied to measure the activities of two purified enzymes, namely, the full length T-cell protein tyrosine phosphatase (TC PTP) and its truncated form (TCACll PTP). The reaction was followed by changes in ultraviolet absorption and fluorescence resulting from the dephdsphorylation of P-Tyr. Both enzymes obey Michaelis-Menten kinetics, with K,,, = 304 pM, V,,,, = 62,000 unitslmg for TC PTP and Km = 194 pM, V,, = 73,000 units/mg for TCAC 11 PTP. The D- and L-forms of P-Tyr are equally effective as substrates. The optimum pH for both enzymes is 4.75. The known effecters of PTPs have the predicted effects on Catalytic activity. 0 1992 Academic Press,

Washington

Inc.

Protein tyrosine phosphorylation plays a crucial role in the regulation of cell proliferation and differentiation (1,2). Progress in recent years has been directed toward an understanding of the structures and physiological functions of the protein tyrosine phosphatases (PTPs)~ (3). Following the isolation and characterization of PTP 1B from human placenta (4), the leukocyte common an-

r This work was supported in part by NIH Grants DK07902, DK13912, and GM42508 and a grant from Muscular Dystrophy Association. N. F. Zander is the recipient of a research stipend of the Deutsche Forschungsgemeinschaft (Germany). ’ To whom correspondence should be addressed. 3 Abbreviations used: PTP, protein tyrosine phosphatase; P-Tyr, 0-phosphotyrosine; Mops, morpholinepropanesulfonic acid; Mes, 2-(N-morpholino)ethanesulfonic acid; RCM-lysozyme, reduced, carboxamidomethylated, and maleylated lysozyme; MBP, myelin basic protein; TC PTP, human T-cell PTP; TCACll PTP, truncated form of TC PTP lacking the 11-kDa C-terminal sequence; RPTPo, receptor-linked PTP alpha; EGF, epidermal growth factor. 0003-2697192 $3.00 Copyright 0 1992 hy Academic Press, All rights of reproduction in any form

tigen CD45 structurally homologous to PTP 1B was shown to possessPTP activity (5). Since then, a number of receptor-linked and nonreceptor PTPs have been identified. Among these, a full-length human T-cell PTP (TC PTP) and its truncated form lacking the llkDa C-terminal sequence (TCACll PTP) and a human recombinant receptor-linked PTP (RPTPa) were obtained from baculovirus expression systems (6,7). Another leukocyte antigen-related PTP and its soluble domain containing the catalytic site were purified from an Escherichia cob expression system (8,9). Finally, a higher-molecular-weight form of PTP 1B was also isolated from human placenta (10). The catalytic activities of the PTPs are generally determined using phosphorylated proteins or peptides as substrates. Physiologically relevant substrates such as the insulin receptor and the EGF receptor (11,6) are available only in small quantities. Artificial substrates, such as reduced carboxamidomethylated, and maleylated lysozyme (RCM-lysozyme) (4), myelin basic protein (MBP) (12), bovine serum albumin (13), histone H2b (ll), casein (14), poly(Glu,Tyr) (Xi), and synthetic peptides corresponding to the autophosphorylation sites of the insulin receptor, EGF receptor (9), and src family kinases (10) also have been employed in the activity measurements. These materials are phosphorylated in a reaction mixtures that include [T-~‘P]ATP and partially or totally purified tyrosine kinases. However, since the extent of phosphate incorporated is often low, the presence of unphosphorylated materials may result in product inhibition of the PTPs. In addition, due to the short life time of 32P, these substrates need to be prepared at regular intervals. Recently, a fluorescence immunoassay was developed for protein tyrosine kinases and phosphatases using an anti-phosphotyrosine monoclonal antibody (16). However, nonlinearity in the dose-response limits its applicability to enzyme kinetics studies. 361

Inc. reserved.

362

ZHAO

Phosphotyrosine (P-Tyr) has been used as a substrate in assays for acid phosphatase (17) and for CD45 (18). In both instances, the activity was measured by determining the release of inorganic phosphate in a discontinuous calorimetric procedure. With a phosphate group attached to the phenolic hydroxyl, P-Tyr displays markedly different absorption and fluorescence properties from tyrosine. A shift in the absorption maximum and a ca. twofold reduction in maximum absorbance for P-Tyr at pH 7.0 were reported (19). An even greater blue shift and reduction in absorbance were observed at low pH (20). Phosphorylation of tyrosine also eliminates its susceptibility to collisional quenching, one of the factors contributing to the fluorescence of tyrosine (19). This article describes the development of a continuous spectrophotometric assay for the PTPs based on the aforementioned spectral differences. It includes the application of this assay system to eight different protein phosphatases, determination of Km and V,,, for both TC PTP and TCACll PTP, pH optima, and action of effecters. MATERIALS

ET

AL.

250

Tyrosine and P-Tyr in both L- and D-forms were purchased from Sigma Chemical Co. TCACll PTP, TC PTP, and RPTPcx were purified from Sf9 cells after recombinant baculovirus infection (6,7). CD45 was purified from human spleen (5) and protein phosphatase 1 and 2A from rabbit muscle (21). Both alkaline phosphatase from bovine intestinal mucosa and acid phosphatase from potato were purchased from Sigma Chemical Co. Methods The absorbance and fluorescence spectra were measured with a Perkin-Elmer X 3B uv/vis Spectrophotometer and a Perkin-Elmer LS50 fluorometer, respectively. The concentrations of tyrosine and P-Tyr were determined by weight. The former was verified by ultraviolet absorbance measurements, employing a molar extinction coefficient 1420 M-’ cm-’ at 274.6 nm, and the latter by inorganic phosphate determination after acid hydrolysis (22). Three buffer systems used for the activity assays were 25 mM Mops-NaOH (pH 6.5-7.5), 25 mM Mes-NaOH (pH 5.5-6.5), and 25 mM sodium acetate (pH 3.0-5.5). The assay media also contained 1.0 mM EDTA, 1.0 mM dithiothreitol, and the indicated concentrations of P-Tyr. Unless otherwise specified, the L-form of P-Tyr was employed. The reaction was started by the addition of a few microliters of the diluted enzyme, usually giving a final concentration of 0.05 to 0.5 pg/ml of PTP. The course of the reaction was moni-

280 270 Wave Length (nm)

290

300

, , , , -.---‘:“-. --..-.-; -.___.. 1

AND METHODS

Materials

260

2090 300

320 310 Wave Length

330 (nm)

340

350

FIG. 1. (A) Absorption spectra. Solid line, 0.5 mM tyrosine; dashed line, 0.5 mM P-Tyr. (B) Fluorescence emission spectra following excitation at 285 nm. Solid line, 10 PM tyrosine; dashed line, 50 PM P-Tyr. The buffer in both cases contained 25 mM sodium acetate (pH 4.75).

tored by measurement of either the ultraviolet absorbance at 280 nm (Beckman DU-6 spectrophotometer equipped with a six position cuvette holder, allowing simultaneous analyses of up to six samples) or the fluorescence intensity at 305 nm following excitation at 285 nm, with light path of 1.0 cm for both measurements. The sample volume was 0.5 to 1.0 ml for the absorbance measurements and 2.0 ml for the fluorescence measurements. A data processor built into the spectrophotometer automatically gave the initial reaction rates. All reactions were performed at room temperature (21-23’C). RESULTS

Absorbance and Fluorescence Spectra of Tyrosine and P-Tyr Figure 1A shows the ultraviolet absorbance spectra of 0.5 mM tyrosine and 0.5 mM P-Tyr measured at pH 4.75. P-Tyr shows a distinctively lower absorbance than tyrosine and a blue shift in the absorption maximum. At 280 nm, the ratio of A&A,-, is 10.8. Even greater differences can be found in the fluorescence emission spectra. Figure 1B shows the corresponding emission spectra of

SPECTROPHOTOMETRIC

ASSAY

OF

PROTEIN

TYROSINE

363

PHOSPHATASES

fluorescence, respectively. At 0.5 mM P-Tyr, the initial linear phase lasts for nearly 5 min (Fig. 2A). Since this is a simple reaction involving two spectral components, the extent of dephosphorylation (f) is readily calculated from the observed change in absorbance and the difference between the absorbencies of tyrosine and P-Tyr, according to

f = Wotm- 4-~,AWr,rr - &Tyr). 0'

0

J 5

10

15

20

25

30

Time (minutes)

VI

We thus calculated that the extent of reaction achieved after 30 min of incubation with 0.36 pg/ml of TCACll PTP, as shown in Fig. 2A, is 78% of the maximum. Doubling the amount of enzyme leads to a corresponding doubling in initial reaction rate as determined by either method. pH Dependence of PTP Activity

"0

5

IO

15

20

Time (minutes) FIG. 2. Time course of the PTP-catalyzed dephosphorylation of P-Tyr. (A) Absorbance measured at 280 nm. Reactions were performed with 0.5 mM P-Tyr and either 0.36 fig/ml (solid line) or 0.18 rglml (dashed line) TCAll PTP. (B) Fluorescence monitored at 305 nm with an excitation wavelength of 285 nm. The assays were performed with 50 FM P-Tyr plus either 0.28 rglml (solid line) or 0.14 pglml (dashed line) of AC PTP. All reactions were carried out at pH 4.75.

10 PM tyrosine and 50 pM P-Tyr, obtained upon excitation at 285 nm. At a fixed emission wavelength of 305 nm, a ratro FTyllFP-Tyr of 20.5 was determined upon comparison of equal concentrations (10 PM) of tyrosine and P-Tyr. Variations in pH over the range examined (pH 3 to pH 9) had noticeable effects on the absorbance and fluorescence of P-Tyr, corresponding to a pK, of ca. 5.8 (not shown), but little effects on those of tyrosine. Note that the estimated pK, value for P-Tyr is consistent with the secondary dissociation constant of phenolic phosphate reported (20). At pH 7.0, absorbance and fluorescence ratios of A&A,.,, = 4.2 and FtiIF,-,, = 10.2, respectively, were obtained.

Time Courses of the PTP-Catalyzed of P-Tyr

Dephosphorylation

Figures 2A and 2B illustrate the time course of the dephosphorylation of P-Tyr catalyzed by TCACll PTP as monitored by changes in ultraviolet absorbance and

Figure 3 shows the pH dependence of the PTP activities. The optimum pH for both TCACll PTP and TC PTP is 4.75. The assays were performed in three buffers according to their pH ranges. The composition of the buffer per se was shown to have no effect on the activity of the enzymes. The effect of pH on the absorbance of P-Tyr itself was normalized to the values obtained for A,, and A,-,, at different pH. Kinetics of PTPs

As illustrated by the double-reciprocal plots in Fig. 4, both phosphatases obey Michaelis-Menten kinetics with P-Tyr as a substrate. Comparison of the kinetic constants (Table 1) with the values reported for the two commonly used substrates, RCM-lysozyme and MBP, reveals large differences in K, values-which range from the submicromolar to the micromolar in the former cases (6) to 200-350 PM for P-Tyr. However, the

3 FIG. 3. pH Dependence with 0.5 mM P-Tyr under and Methods. 0, TC PTP;

4

5 PH

6

of PTP activity. Assays the conditions described 0, TCACll PTP.

7 were under

performed Materials

ZHAO

ET

AL.

.75

TABLE Substrate

2

Stereospecificity”

.60 PTPs

5 T

.45

TCACll

z

.30

TC PTP

.15 0

0

.Ol l/[P-Tyr]

.02 (I/bM)

.03

FIG. 4. Lineweaver-Burk plots. Assays were performed with 0.25 pg/ml enzyme at pH 4.75. The rate was determined by measuring increase in absorbance (see Eq. [l]). 0, TC PTP; 0, TCACll PTP.

V,, for P-Tyr is considerably higher than the values obtained with the two phosphoprotein substrates. In contrast to the differing specificities of two phosphatases determined with MBP and RCM-lysozyme, both enzymes give similar values of K,,, and V,, when P-Tyr serves as the substrate. To illustrate the effect of pH on activity, the kinetic data obtained at pH 7.0 for TCACll PTP also are presented in Table 1. Note that pH variation has a greater effect on V,, than on K,. When the pH is increased from 4.75 to 7.0, V,, decreases by more than sixfold while K, increases by less than twofold.

PTP

P-Tyr

Rate6

L-P-Tyr D-P-Tyr L-P-Tyr D-P-Tyr

1.0 0.98 1.0 1.04

’ Assay conditions: 0.5 fig/ml enzyme, 4.75. ’ The relative initial rate. ’ Extent of substrate dephosphorylation

Comparison

% Reacted 90 92 a7 88

0.5 mM of D- or L-P-Tyr,

after

with Other Protein

30 min

pH

incubation.

Phosphatases

Table 3 summarizes the results of ultraviolet absorbance assays performed with different tyrosine and serine-threonine protein phosphatases at pH 4.75 and 7.0. Like TC PTP and TCACll PTP, two other tyrosinespecific phosphatases, including the transmembrane CD45 and RPTPa, also prefer the lower pH. Their specific activities determined are comparable to those obtained with MBP or RCM-lysozyme as substrate (12,7). As expected, protein phosphatase type 1, type 2A, and alkaline phosphatase show little or no activity toward P-Tyr at either pH. However, the broad specificity potato acid phosphatase exhibits good activity at lower pH. Effecters of PTPs

Substrate

Stereospecificity

The dephosphorylation of the D-form of P-Tyr was also studied employing the same assay system. Surprisingly, D-P-Tyr and L-P-Tyr are equally effective as substrates. Table ‘2 summarizes the relative initial reaction rates and the completeness of reaction determined after 30 min of incubation for both TCACll PTP and TC PTP. The similarity between the two sets of results indicates that the activity of the enzymes is independent of the stereo configuration of P-Tyr. For simplicity, Table 2 shows only the results obtained at pH 4.75. Similar results were obtained at pH 7.0.

A variety of effecters was shown to influence the activity of the PTPs (6,23). Table 4 demonstrates the responsiveness of the assay method to known effecters of TCACll PTP and TC PTP. As observed with RCM-lysozyme and MBP, submillimolar concentration of vanadate and micromolar concentrations of heparin almost totally abolish PTP activity toward P-Tyr. However, in contrast to the activation of the TC PTP activity ob-

TABLE Comparison Phosphatases

TABLE

Phosphotyrosine PH 4.75 4.75 7.06 D One unit ‘K,,, and

PTPs TCACll TC PTP TCACll

1

as a Substrate of PTPs K,, (PM)

PTP PTP

194 304 357

V,,

(unitsjmg) 73,000 62,000 10,000

is equivalent to 1 nmol phosphate released per minute. Vk for TC PTP at pH 7.0 were not determined.

TCACll PTP TC PTP CD45 RPTPa Acid phosphatase Alkaline.phosphatase Phosphatase 1 Phosphatase 2ab

of Protein

3 Phosphatase

Activities”

pH 4.75

pH 7.0

71,000 60,500 3,400 700 13,000 12 0 30

9500 5400 500 200 1800 45 0 21

a Data represent the specific activity (units/mg) of each phosphatase measured with 0.5 mM L-P-Tyr at the indicated pH. ’ Catalytic subunit.

SPECTROPHOTOMETRIC TABLE

Effecters Effecters

a Assays were performed pH 4.75. Data represent

of PTPs” PTP 100 0 0 63 83 88 100

with relative

OF

4

TCACll

Control 0.1 mM VO3p0.5 pM Heparin 50 mM NaF 1 mM zn2+ 0.4 mM Tyr 1 mM Spermine

ASSAY

0.2 rg/ml activities.

TC PTP 100 I 3 63 81 95 86

enzyme,

0.5 mM L-P-Tyr

at

served with RCM-lysozyme as substrate, spermine has little effect on the rate of hydrolysis of P-Tyr. Moreover, Zn2+ becomes a much less effective inhibitor of the PTPs, whereas the classic protein phosphatase inhibitor sodium fluoride, which is a mild activator of human placenta PTP 1B with RCM-lysozyme as substrate (23), is slightly inhibitory. Note that product inhibition by tyrosine is minimal, especially for TC PTP. DISCUSSION

Two continuous spectrophotometric assays were developed for the determination of PTP activity, utilizing the increases in fluorescence and/or absorbance that occur when the phosphate group of P-Tyr is replaced with the phenolic proton of tyrosine. The magnitude of the changes depends on assay conditions and on the monitoring wavelength. However, the background fluorescence or absorbance of P-Tyr is small in all casesallowing accurate and sensitive measurements. At pH 4.75, for example, the molar extinction coefficients for tyrosine and P-Tyr are 1200 M-’ cm-’ and 111 M-’ cm-‘, respectively. Hence, an increase in absorbance of 1.089 corresponds to the production of 1.0 mM tyrosine. A spectrophotometer capable of detecting 0.001 absorbance differences could be employed to measure concentration changes ranging upward from 1 @M. The observed differences in fluorescence, which are the net result of alterations in both absorption and emission, could be employed in assays carried out at still lower concentrations. However, the high Km values listed in Table 1 require the assays be performed in the concentration range of 0.1-1.0 mM P-Tyr. The corresponding absorbance values fall within the range where percent absorption (and hence total fluorescence emission) is no longer proportional to absorbance. Thus, without reducing the cuvette path length or increasing the excitation wavelengths to still higher values, fluorescence assays are nonlinear with respect to substrate concentration in this range. The experiments summarized in Figs. 3 and 4 and Tables l-4 were conducted

PROTEIN

TYROSINE

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PHOSPHATASES

using the absorption spectrophotometer rather than the fluorometer. All the PTPs tested showed greater activity at pH 4.75 than at pH 7.0 (Table 3). Similar pH preference was observed with human placenta PTP 1A and 1B when p-nitrophenyl phosphate served as substrate (23) and RPTPa with protein substrates (7). Possible protonation of the carboxyl group of P-Tyr does not appear to be involved in the reaction since TCACll PTP and TC PTP show no stereospecificity toward P-Tyr. The truncation of TC PTP into TCACll PTP markedly raises its activity toward RCM-lysozyme but reduces its activity toward MBP (6). With P-Tyr as a substrate, however, the two enzyme forms display comparable Km and V,,, values and similar responsiveness to effecters (Table 4). Possibly the effecters influence the interactions of the protein substrates with binding sites outside the catalytic segment. Spectrophotometric monitoring of the hydrolysis of P-Tyr has the distinct advantages of a continuous assay: the ability to acquire results rapidly, to observe directly the progress of a reaction, and to determine initial reaction rates. In addition, P-Tyr exhibits high selectivity for tyrosine phosphatases-with the exception of the broad specificity potato acid phosphatase (Table 3). Although p-nitrophenyl phosphate has been extensively used as a substrate in phosphatase assays (23), it does not allow a distinction to be made between tyrosine and serine-threonine phosphatases. A limitation of the present assay results from the fact that measurements are performed at a wavelength where proteins absorb and fluorescence. Crude extracts possessing low PTP activity need to be assayed at concentrations giving large background signals. In such cases, discontinuous determination of inorganic phosphate release employing Malachite green as an indicator (24) remains the methods of choice. Although synthetic peptides with defined amino acid sequences have been used to study the specificities of protein kinase (25,26), no specific sequences have been recognized for PTP substrates. However, the availability of a novel solid phase method for the synthesis of phosphotyrosyl-containing peptides (27) should allow this strategy to be applied to the phosphatases. The spectrophotometric methods that are described here for P-Tyr can be applied just as well to phosphotyrosylcontaining peptides, enabling a better understanding of PTP action. Likewise, it could be used to follow the phosphorylation of tyrosine-containing peptides catalyzed by protein tyrosine kinases.

ACKNOWLEDGMENTS We thank Dr. Giinter of the enzymes.

Daum

and Curtis

D. Diltz

for supplying

some

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