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A microtiter enzyme-linked immunosorbent assay for protein tyrosine phosphatase
Biochimica etBiophysicaActa, 1157 (1993) 93-101 © 1993 Elsevier Science Publishers B.V. All rights reserved 0304-4165/93/$06.00
93
BBAGEN 23792
A microtiter enzyme-linked immunosorbent assay for protein tyrosine phosphatase Shrikant Mishra and Anne W. Hamburger University of Maryland Cancer Center, Department of Pathology, Baltimore, MD (USA) (Received 29 April 1992) (Revised manuscript received 25 November 1992)
Key words: Protein tyrosine phosphatase; ELISA; Signal transduction; Enzyme activity
We report the development of an enzyme-linked immunosorbent assay (ELISA) for protein tyrosine phosphatases (PTPases). PTPase activity, was monitored by quantitating the disappearance of O-phospho-L-tyrosine (P-Tyr) in an ELISA system using antigen capture followed by double antibody labelling. PTPase activity of agarose conjugated PTP-1B was demonstrated using the ELISA system. PTPase activity was sensitive to both PTB-1B concentrations and time of incubation. 1 mU of PTPase activity was defined as that amount of enzyme producing a rate of loss of 0.01 absorbance units/minute with a specific activity of 150 pmol P-Tyr/min per/zg protein based on the unit of PTPase activity from the conventional assay system. The PTP-1B activity was shown by the ELISA system to be completely inhibitable by Poly (Glu,Tyr)4 : 1 at 100/xg/ml. We used the ELISA system to detect PTPase activity in lysates of cultured cells. The PTPase activity of cell lysates of MDA-MB 468 breast carcinoma cells as obtained by the ELISA were compared with those obtained by a standard 32pi release assay using radio-labelled RaytideTM as PTPase substrate. The decrease in P-Tyr concentration was dependent on the time of incubation with the lysate and on lysate concentration and compared well with the release of 3ZPi in the radioactive assay system. Orthovanadate as well as heat denaturation inhibited the PTPase activity of the cell lysates in both the assay systems. The assay presented here is a simple immunological system capable of measuring activity of purified PTPases as well as PTPase levels in cell and tissue extracts.
Introduction Phosphorylation at protein tyrosyl residues is a major mechanism of cellular signal transduction and is involved in mitogenesis [1] as well as in neoplastic transformation [2]. Tyrosine phosphorylation is tightly regulated by the combined activities of specific protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). Previous studies have shown that many cellular growth factor receptors and cellular as well as viral oncogenic proteins are PTKs [3]. The growth factor PTKs are crucial in modulating cell growth. The
Correspondence to: A.W. Hamburger, University of Maryland Cancer Center, Department of Pathology 9-051, Bresslen Research Bldg., Baltimore, MD 21227, USA. Abbreviations: ELISA, enzyme linked immunoabsorbent assay; PTyr, O-phospho-L-tyrosine; PTKs, protein tyrosine kinases; PTPases, protein tyrosine phosphatases; PTP-1B, protein tyrosine phosphatase-lB, FBS, fetal bovine serum; Mops, 3-[N-morpholino]ethanesulfonic acid; EDTA, ethylenediamine tetracetic acid; PMSF, phenylmethylsulfonyl fluoride; pNPP, p-nitrophenyl phosphate; Hepes, N-[2-hydroxyether] piperazine-N'-[2 ethanesulfonic acid]; TCA, trichloro acetic acid; RCML, reduced carboxamidomethylated and maleylated lysozyme; MBP, myelin basic protein.
transduction of mitogenic signal is via phosphorylation of their tyrosyl residues [4]. The oncogenic PTKs, likewise, orchestrate uncontrolled cellular growth and tumorogenesis via phosphorylation of their crucial tyrosine side chains [5]. More recently, attention has been focused on the role of PTPases in controlling cellular tyrosine phosphorylation. Unlike PTKs, the PTPases constitute a distinct class of enzymes [6] of great structural diversity [7]. The localization of PTPase enzymes has been found to be both transmembrane as well as cytosolic [7,8] sites. It has been hypothesized that certain classes of PTPases may be mediators of specfic cellular signalling pathways, rather than having a nonspecific housekeeping function. Functionally the PTPases are involved in suppressing proliferative response to growth factors [9], exocytosis [10], contact inhibition of growth of fibroblasts [11], and cytokine mediated cellular signalling [12]. It has recently been hypothesized that PTPases may exhibit tumor supressor function [9] in lung and renal cell carcinomas [13]. Although a variety of PTPases have been cloned from human and non-primate cDNA libraries [8,12] the assay systems used to characterize these PTPases
94 have not been standardized. To date, radioactive [10,11,14-18] as well as non isotopic [19-21] assay systems for PTPases have been published. We present here a simple, reproducible, inexpensive, non-isotopic, and rapid ELISA system for measuring PTPase activity using purified PTP1-B coupled to agarose and P-Tyr as the enzyme substrate system. The PTPase activity was determined by monitoring the disappearance of P-Tyr, currently the substrate with the broadest spectrum of activity of known PTPases. The P-Tyr in the PTPase reaction mixture was captured on glutaraldehydecoated EIA plates, and quantitated using, as first and second antibodies, anti-P-Tyr mouse antibody and alkaline phosphatase coupled anti-mouse rabbit antibody. Materials and Methods
Protein tyrosine phosphatase 1B (PTP-1B), purified enzyme PTP-1B was purchased from Upstate Biotechnology Inc, Lake Placid, NY; as a glutathione-agarose conjugate (50 /xg/ml bead) with a manufacture specified specific activity of 2 nmol/min per /xg protein using the assay of dephosphorylation of tyrosine-phosphorylated proteins.
Cell culture MDA-MB-468 breast carcinoma cells (American Type Culture Collection HTB 132) were grown in Leibovitz's L-15 medium (GIBCO Laboratories, Life Tech. Inc., NY), at pH 7.4, supplemented with 10% fetal bovine serum (FBS) (Intergen, Purchase, NY), 10 units/ml penicillin, and 10 /zg/ml streptomycin, at 37°C. Cells were seeded at 3 x 105 cells/ml in 80 cm 2 flasks and grown to semi-confluency without change of media.
Preparation of total cell lysates and membrane fractions of MDA-MB-468 cells Unless otherwise mentioned, all the reagents were obtained from Sigma Chemical Co., St. Louis, MO. The semi-confluent cultures were washed with Hank's Balanced Salt Solution (HBSS), 10 ml/wash, to remove serum proteins. For preparation of total cell lysates, the cells were scraped from the 80 cm z flasks in 1 ml of buffer A (250 mM sucrose, 17 mM Mops, 25 mM EDTA, 1.0% Triton X-100, 0.2 ng/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM dithiothreitol(DTT), 5 mM MnC12, pH 6.0). The cell lysate was spun at 15 000 × g for 5 min at 4°C. The clear supernatant was kept at 4°C prior to the assay. For preparation of the membrane fraction, the cells were washed thrice with HBSS and incubated with 1 ml of buffer B (buffer A without Triton X-100 + 0.8 mg/ml digitonin) for two minutes to allow the
release of cytosolic material from the cells. Subsequently the medium was removed and the perforated monolayer was washed twice in 5 ml of buffer A. The perforated cells remained attached to the bottom of the flask. The cells were then scraped in 1 ml of buffer A and cell lysates were prepared as described before. The cell free extracts were then kept on ice. The protein concentration in the extracts were determined by using the Micro BCA protein assay reagent kit (Pierce, Rockford, IL).
Assay for tyrosine phosphatase activity Prior to the assay, 96-well enzyme immunoassay (EIA) plates (Costar, Cambridge, MA) were coated with 3% bovine serum albumin (BSA) (EIA grade) in 0.9% NaC1. The wells were completely filled to the top with this solution. The plates were incubated in a humidified atmosphere at room temperature for 2 h. To remove the unbound BSA, the wells were washed thrice with 0.9% NaCI. The BSA coated plates were incubated with 100 /xl/well of 2% glutaraldehyde in 0.9% NaC1 for 2 h in a humidified atmosphere at room temperature. As glutaraldehyde is known to cross-link proteins via binding to amine functional groups, this step allowed the glutaraldehyde to bind BSA amine groups on one end, leaving the other end vacant for subsequent binding of another amine group. After the glutaraldehyde incubation step, the 100 /zl of glutaraldehyde was removed by vaccum suction and the plate washed thrice with 0.9% NaCI to remove any unbound glutaraldehyde. The plates were stored filled with 0.9% NaC1 at 4°C prior to the assay. Prior to use, the glutathione-agarose conjugated PTP-1B was resuspended in PTPase assay buffer C (25 mM imidazol hydrochloride (pH 7.2), 1 mg/ml BSA, 0.1% v/v/3-mercaptoethanol) at 4°C. The beads were washed four times with the assay buffer to get rid of unbound PTP-IB. The PTPase assay was performed in a 150/zl volume. The PTPase assay mixture constituted of 125 /xl of P-Tyr (0 to 500 nM) and 25 /xl of an appropriate dilution of PTP-1B. The reaction was then allowed to proceed for 10 min at 25°C subjected to gentle rocking. The P-Tyr substrate was reduced to Pi and Tyr by PTP-1B. The reaction was terminated by centrifugation (10000 × g, 5 min at 4°C), resulting in the removal of the PTP-1B agarose conjugate from the aqueous phase. Supernatant, 100 /xl of the reaction, was then applied to glutaraldehyde coated E.I.A plate at 4°C for 4 h. This step ensured the binding of the amine group of the residual P-Tyr to glutaraldehyde. Binding at 4°C was determined to be a critical step for PTPase assay. At the end of the incubation, the wells were washed with 0.9% NaCI (100/xl/wash) and subsequently blocked with 100/zl of 1 mM L-lysine. This step allowed the unreacted glutaraldehyde to bind the amine group of L-Lysine. The L-Lysine blocking step (1
95 h at 25°C) and the subsequent steps were performed at room temperature. After this blocking step, the wells were washed four times with 0.9% NaC1 and then incubated with 100 izl of anti-P-Tyr antibody (Boehringer Mannheim, IN) (1 : 2,000 dilution in 0.9% NaC1) for 2 h in a humidified atmosphere at room temperature. The wells were washed four times with 0.9% NaC1 and subsequently incubated with an appropriate dilution of alkaline phosphatase coupled anti-mouse goat antibody (Biorad Laboratories, CA) as the second antibody, at a 1:20000 dilution, for 1 h at room temperature. The wells were washed twice with 0.9% NaCI and further washed twice with freshly prepared buffer D (1 M diethanolamine, 0.5 mM MgC12, pH 9.8). The plates were incubated with 100 /~1 of 1 mg/ml solution of p-nitrophenyl phosphate (pNPP), made in buffer D, for 30 min. The reaction was stopped by using 50 /zl of 1 N NaOH solution. The color developed was monitored at 405 nm using a 96-well plate reader (Dynatech). For performing the ELISA with HRP conjugated 2nd antibody, the wells were washed with 0.9% NaCI and then incubated, in dark, with 100 /~1 of O-phenylenediamine (OPD) in 0.1 M citrate buffer with 0.03% H 2 0 2. The reaction was terminated after 5 min using 50/zl of 2 N H2SO 4 and the color developed was monitored at 450 nm. The specificity of the PTPase assay was determined by co-incubating the reaction mixture with Poly (Glu, Tyr)l : 4 (PGT), (Sigma), a known inhibitor of PTP-1B. PTPase activity in the cell extracts were determined as follows. Dilutions of the cell extracts were performed in buffer A without Triton X-100. The PTPase assay was performed in a 100/xl reaction volume. 40/xl of an appropriate concentration of P-Tyr was incubated with 10 /xl of appropriately diluted cell extract for 0, 1, 2, 5, and 10 min at 37°C. The reaction was stopped by adding 50/xl of buffer E (50 mM NaC1, 5 mM EDTA, and 0.1 mM sodium orthovanadate) at 4°C. 100 /zl of PTPase reaction mixture/well was heated to 80°C for 10 rain, cooled on ice, and then added to the glutraldehyde coated EIA plates. The rest of the assay steps were identical as described before. Formula used to calculate activity of PTPase: A t = 10 - A t = 0 × t o t a l d i l u t i o n PTPase(mU/mg)
=
0.01 X c o n c . ( m g / m l )
where Zt=o is the EIA absorbance at 405 nm of the PTPase reaction mixture at 0 time, and Zt=10 is the EIA absorbance of the reaction mixture after 10 min of PTPase incubation. 1 mU of PTPase activity is defined as the amount of PTPase that can produce a decrease in the EIA absorbance of 0.01 min of PTPase reaction. In the radioactive assay, the 32Pi released in 10 min of reaction time gives the numbers of mUnit of PTPase present in the reaction mixture. 1 mU of PTPase
activity is defined as the amount of enzyme that releases one picomole of 32Pi/min [18]. Knowledge of the rate of PTPase reaction in terms of EIA absorbance as well as the number of mU of PTPase in the reaction (from the radioactive data) helped standardize the definition of unit of PTPase activity in the ELISA system. A factor of 10 in the denominator of the equation (time contribution) cancels a factor of 10 in that of the numerator (volume contribution).
Radioactive assay for PTPase activity The assay was performed essentially as described [18] except that the substrate protein, RaytideT M (Oncogene Science), was phosphorylated, at tyrosine residues by the recombinant p60 c-src instead of p43 T M as per manufacturer's recomendations. Briefly, p60 c-src stock solution was diluted to 1:20 in kinase dilution buffer (50 mM Hepes (pH 7.5), containing 0.1 mM EDTA, 0.015% Brij. 35, 0.1 mg/ml BSA, and 0.2% /3-mercaptoethanol). RaytideT M was reconstituted at 1 mg/ml in kinase assay buffer (kinase dilution buffer without the addition of BSA and /3-mercaptoethanol). 10 tzl of RaytideTM was mixed with 10 /xl of 1:20 diluted tyrosine kinase. The kinase reaction was initiated by adding 10/zl of ATP mixture (0.15 mM ATP, 30 mM MgC12, 200/zCi Ta2p-ATP/ml of kinase assay buffer). The reaction was carried on for 16 h at 37°C and terminated by adding 500/zl of 20% w / v TCA/20 mM NaHzPO 4 and 100 /.d of 0.1 mg/ml acetylated BSA (Life Tech). The precipitated reaction mixture was centrifuged at 30000 × g for 5 min and subsequently washed with 20% w / v TCA/20 mM NaHEPO 4 three times to remove the unreacted Ta2p-ATP and reconstituted in 100/zl of phosphatase assay buffer (50 mM Mops (pH 6.0), 1 mg/ml BSA, 0.5 mM DTT, 0.01% CHAPS). The specific activity of RaytideTM was determined to be 2 × 104 cpm//zg protein. The phosphatase reaction was performed in a 50/xl of reaction volume containing 5/zl of 32-p-RaytideZM (104 cpm), 5 /xl of appropriate dilution of the cell extract needed to exhibit measurable PTPase activity, and 40 Ixl of phosphatase assay buffer. The reaction was monitored at 37°C for 0, 2, 5, and 10 min and stopped by using 500 /zl of 20% w / v TCA/20 mM NaHEPO 4 with 100/.d of 0.1 mg/ml acetylated BSA. The precipitate was centrifuged at 30000 × g for 5 min. Radioactivity in 400/.d of supernatant was counted in 5 ml of Aquasol (NEN Research Product) using a Beckman LS 3801 liquid scintillation counter. Results
PTPase assay principle The PTPase assay flow chart is as shown in Fig. 1. This assay is based on the rationale that a PTPase reaction involving disappearance of phospho-tyrosine
96 (P-Tyr) can be monitored if the loss of P-Tyr can be quantitated. The rate of loss of P-Tyr is considered to be the rate of PTPase reaction (in the absence of interfering proteases). The PTPase reaction was performed with P-Tyr as the substrate prior to the determination of residual P-Tyr levels in the reaction mixture by the ELISA scheme. The level of P-Tyr in the PTPase reaction was quantitated by antigen capture of P-Tyr on glutaraldehyde (amine to amine cross-linker) coated plates using a double antibody enzyme linked immunosorbent assay (ELISA). The 1st antibody was a mouse monoclonal anti-P-Tyr antibody and the 2nd antibody was an anti-mouse goat antibody coupled to alkaline phosphatase. The sensitivity of the assay was compared for both alkaline phosphatase (AP) and horseradish peroxidase (HRP) coupled 2nd antibodies. Equal degrees of sensitivity were found (data not shown). As cells may possess endogenous peroxidase activity, the alkaline phosphatase method was chosen for further development.
E.I.A Plate
Coat With BSA
Glutaraldehyde Coupling
P-Tyr Coupling (PTPase Reaction Followed By Antigen capture)
Block With L-Lysine
Anti-P-Tyr Antibody
Alkaline Phosphatase Coupled 2~ antibody
Optimization of P-Tyr signal in EIA P-Tyr standards (0 to 5 /xM) were prepared in buffer C. Glutaraldehyde-treated plates were incubated overnight at 4°C with 100 ~1 of P-Tyr standards in six replicates. The specific immuno controls showing the specificity of anti-P-Tyr antibody to detect P-Tyr captured on glutaraldehyde coated EIA plates were performed (data not shown). Incubations with anti-PTyr antibody at a 1:2,000 dilution and the alkaline phosphatase coupled anti-mouse IgG at a 1:20000 were performed as described in the methods section. The EIA signal was optimal at the above mentioned 1st and 2nd antibody concentrations (data not shown). A P-Tyr concentration dependent linear increase in EIA signal was observed at 405 nm in the range of P-Tyr concentration of 0 to 5 /~M (Fig. 2). Beyond 5 /xM, the EIA absorbance reading was saturable. The sensitivity of the assay was determined using 2nd antibodies coupled to either alkaline phosphatase (AP) (Fig. 2) or horse radish peroxidase (HRP) (Fig. 2 inset) as the reporter enzymes. The AP and the HRP reactions were monitored at 405 nm and 450 nm respectively using standard protocols. Both the 2nd antibodies were equally capable of reporting changes in P-Tyr concentration in the same scale. We used AP coupled 2nd antibody as our reporter enzyme for the present assay. The efficiency of coupling of P-Tyr to glutaraldehyde was determined at different glutaraldehyde concentrations (0 to 5%). The EIA absorbance increased with increasing glutaraldehyde concentrations and was saturable at 2% glutaraldehyde (data not shown). Thus, the incubation of BSA coated EIA plates with 2% glutaraldehyde was efficient in trapping P-Tyr which was detected and quantitated by EIA in the range of P-Tyr concentration between 0 and 5 ~M.
Incubation With pNPP
Measure apsorbance at 405 nm
Determine Rate of PTPase Reaction Fig. 1. PTPase assay flow chart.
Validity of the EIA assay using purified PTP-1B The specificity of the ELISA system was tested using purified PTP-1B. The EIA assay was performed for different dilutions of PTP-1B (Fig. 3). The P-Tyr concentration was chosen at 10 nM which was in the linear portion of the standard curve. A linear decrease in absorbance over control was indicative of the disapperance of P-Tyr as a result of PTPase activity. The PTP-1B activity was measured from a 100/zl aliquot of a 150/zl PTPase reaction and was shown to be linear in the range of the enzyme concentration tested. A convenient scale for measuring PTPase activity was to define 1 milli unit of PTPase activity which produces a change of 0.01 Ab U / rai n in a reaction system containing 100 nM P-Tyr. In Fig. 3 it was determined that 1 ng of PTP-1B in the reaction mixture corresponded to a change of 0.1 Ab units in 10 min, i.e., 1 ng PTP-1B corresponds to 1 mU of PTPase activity. How this unit of PTPase activity compared with specific activity measured in terms of tools of P-Tyr disappearance/min per/zg protein was clear from the kinetics of enzymatic activity. The kinetics of PTP-1B reaction was performed (0 to 10 min) at 25°C and the residual P-Tyr in
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P-Tyr (nM) Fig. 2. Determination of the ability of glutaraidehyde coated EIA plates to bind P-Tyr. EIA wells were coated with 3% BSA prior to incubation with 2% glutaraldehyde. Wells were incubated with 5, 50, 500, and 5,000 nM P-Tyr, and EIA was performed as described in 'Materials and Methods'. The assay was performed using AP (405 nm) as well as HRP (450 nm) (indent) coupled 2nd antibodies. Values represent mean + S.D. of six replicate wells on a semilog plot.
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Enzyme (lag) Fig. 3. Modulation of PTPase activity with concentration of PTP-1B. The PTPase assay, in three replicates, was performed with agarose congugated PTP-1B as described in the 'Methods' section. The P-Tyr concentration in a 150-/zl reaction was fixed at 100 nM. The PTPase reaction was allowed to proceed for 10 min and the residual P-Tyr determined by EIA as % control absorbance as described in the 'Methods' section. Values represent mean_+S.D, of triplicate readings.
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Fig. 4. Kinetics of PTP-IB activity as measured by EIA. The kinetics of PTPase activity, in three replicates, was performed with agarose congugated PTP-1B (10 ng/ml) for the indicated times. The P-Tyr concentration in a 150-/zl reaction was fixed at 100 riM. The residual P-Tyr, presented of percent of absorbance for 100 nM P-Tyr (control absorbance) was determined by the ELISA method. Values represent mean -I-S.D. of triplicate readings.
98 the reaction determined using the E I A (Fig. 4). As seen in Fig. 4, the P-tyr concentration in the PTPase reaction decreased as a function of time. The rate of PTP-1B reaction was determined to be 0.01 Ab U / m i n with a c o r r e s p o n d i n g specfic activity of 150 p m o l s / m i n u t e / / ~ g protein. Thus combining the definitions of unit of PTPase activity from Fig. 3 and specific activity determined from the kinetics of PTP-1B reaction we can say that 1 mU of PTPase activity corresponds to a disappearance of 1.5 fmols of P-Tyr in 10 min of PTPase reaction. The kinetics of the PTPase reaction was found to be linear in the range of time incubation tested. While most PTPases are inhibited by micromolar concentrations of vanadate and molybdate, PTB-1B is the most sensitive to its specific inhibitor PGT [28]. We determined whether the inhibition of PTP-1B activity by P G T could be demonstratable by E.I.A. As shown in Fig. 5, P G T inhibited the PTP-1B activity in a dose-dependent manner. There was a 95% inhibition of PTPase activity of PTP-1B at a PGT concentration of 100 /zg/ml. Thus we conclude from the above data that PTPase reaction can be monitored using the above assay system presented.
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Fig. 6. Comparative PTPase kinetics of radioactive assay vs. ELISA. The radioactive assay and the ELISA for PTPase were performed as described in the 'Materials and Methods'. 0.1 mg/ml final concentration of total lysate (T) in six replicate wells were used in both the assays. The ELISA and the radioactive assay were performed as described in the methods section. The reaction kinetics were followed for 10 rain. The values were presented as mean±S.D, for DPM released and EIA absorbance (as % C) at 405 nm, respectively.
Quantitation of PTPase in cell lysates As many investigators are currently seeking to determine modulation of PTPase activity in cell culture systems, we measured the ability of the E I A to detect PTPase activity in cell lysates. PTPase activity was quantitated using total cell lysates and membrane
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Fig. 5. Inhibition of PTP-1B activity with Poly (Glu,Tyr)4:1 (PGT). PGT at 0 to 100 izg/ml final concentration was used in the PTPase reaction mixture. The definition of PTPase activitywas set as activity = [(Ab-Ab +)/Ab] x 100, where Ab and Ab + represent absorbance of 100 nM P-Tyr without and with 10 ng/ml PTP-1B, respectively. The PTPase activity without PGT was taken to be 100%. Values represent mean +_S.D. of triplicate readings.
preparations of M D A - M B 468 cells. PTPase activity by E L I S A was also compared with a standard radioactive assay system. The rate of PTPase reaction monitored at 405 nm, from the non-isotopic assay, and 32pi released, from the radioactive assay, were plotted as a function of time as a double Y plot. PTPase kinetics for total cell lysate was determined (Fig. 6). The radioactive assay monitors the appearance of the PTPase reaction product 32Pi, whereas the non-isotopic assay system determined the disappearance of the substrate P-Tyr. As shown in Figure 6, the kinetics of 32pi release varied inversely with the kinetics of P-Tyr disappearance during the total range of incubation times. The PTPase activities in total cell lysate (T) and the membrane fraction (M) were determined at different dilutions of the cell extracts (Fig. 7). From Fig. 7 and the definition of units of PTPase activity (i.e., 1 mU = 25% reduction of absorbance units over control) it is clear that the total cell lysate (T) and the membrane fraction (M) of M D A - M B 468 cells possessed 0.11 m U / / x g and 0.1 m U / i x g protein, respectively. The specificity of the PTPase assay for the cell lysates was performed with M D A - M B 468 total cell lysate. The PTPase assay was performed using the untreated lysates, lysates in presence of 100/~M orthovanadate and heat denatured (80°C for 15 min) lysates using both the assay systems. As shown in Fig. 8, heat denaturation of the lysate as well as the presence of orthovanadate in the PTPase reaction system significantly inhibited the PTPase activity to equal degree.
99 Discussion
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txg Protein Fig. 7. Changes of PTPase activity with dilution of MDA-MB 468 cell lysates. Starting with a stock of 5.0 mg/ml for (a) total cell lysate (T), and 2.1 mg/ml for (b) membrane fraction (M) PTPase activity were performed by the ELISA method with the indicated concentrations of cell lysates. P-Tyr was used at a concentration of 100 nM and the ELISA performed as described in Fig. 2. The ordinate denotes the % absorbance w.r.t, a control 10 min mock reaction containing 100 nM P-Tyr and no enzyme in a 100 /zl reaction. Values represent mean + S.D. of triplicate readings.
Thus the assay presented here is a simple immunological system capable of measuring purified PTPases as well as PTPase levels in cell and tissue extracts.
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Fig. 8. Inhibition of PTPase activity by orthovanadate and by heat denaturation. PTPase ELISA and the isotopic assays were performed as described in the experimental procedure. PTPase reactions were performed in appropriate buffers in both the assays in presence of untreated lysate (10/~g per reaction), lysate in presence of 100 /zM orthovanadate, or heat denaturated lysate (80°C for 15 min). DPM released (mean of triplicate counts) and E.I.A. absorbance (mean of six replicates) were plotted as shown.
PTPase enzymes have recently been determined to play a significant role in cell growth and oncogenesis by opposing the effects of a variety of PTKs. PTPases along with PTKs regulate total cellular phosphotyrosine levels. It has been determined that PTPases catalyze the dephosphorylation of the substrate proteins, typically PTKs, via a cysteine-phosphate intermediate [14]. A density dependent growth arrest of Swiss 3T3 cells was associated with increases in membrane PTPase activity [11] showing thereby that PTPases could play a role in cell proliferation and contact inhibition of cell growth. Several receptor associated PTPases have been found to have extracellular domains homologous to neural cell adhesion molecules [11] suggesting a role of PTPases in cell-cell interaction. PTPases has been implicated in regulating the action of M P F / p34 cdc2 [22], the mitotic control element, and thereby directly affecting the regulation of cell cycle. Further as many growth factor receptors and oncogenic proteins are PTKs, the PTPases could potentially function as antioncogenic or tumor suppressor proteins. For example, PTPy has been identified as a candidate tumor suppressor gene product in renal cell carcinoma (RCC) and lung carcinoma (LC) [13] and was shown to be mutated or absent in these cells. These findings have prompted a rapid increase in cloning, purification, and characterization of these PTPases [7,11,13,17,18,23]. Despite increased interest in PTPases, the assay systems used to characterize these enzymes have not been standardized. The activities of PTPases cloned thus far have been observed for a broad range of substrates. For example, the same PTPase can cleave phosphate groups from tyrosine phosphorylated substrates like reduced carboxamidomethylated and maleylated lysozyme (RCML), myelin basic protein (MBP), tyrosine phosphorylated casein, pp60 v-src, insulin receptor kinase, tyrosine phosphorylated epidermal growth factor receptor, tyrosine phosphorylated histone kinase, tyrosine phosphorylated Calpactin I, tyrosine phosphorylated src-peptide, and from P-Tyr. Although most PTPases exhibit high affinity for substrates with an associated high Vm~x [29], the K m and Vm~ values have been shown to change with the choice of substrates. For example, the kinetic parameters of receptor protein tyrosine phosphatase a (RPTPa) was determined using the radioactive assay system [18]. The assay was performed using two different substrate proteins, namely RCML, and MBP. The gins and VmaxS of RPTPot using RCML or MBP as substrate were entirely different. In fact PTPase activity determined using RCML as substrate was minimal. The radioactive assay systems typically use gamma- 32p-ATP and a suitable protein tyrosine kinase (like c-abl, v-abl, and c-src tyrosine kinases) to enzymatically transfer gamma-32p -
100 group to a suitable substrate protein (like MBP, RCML, Raytide, and bovine albumin) at its tyrosine residue and subsequently PTPase activity quantitated by measuring the amount of 32Pi released in to the supernatant of TCA precipitated PTPase reaction mixture. We used Raytide T M (Oncogene Science) as our substrate for comparison of our ELISA with the radioactive assay [10]. A major negative feature of the radioactive PTPase assay system however is that the response of the assay system depends to a large extent on the choice of the PTPase substrate [18]. A newly developed non isotopic assay system [20], which measures phosphotyrosine containing substrate protein levels by using anti-P-Tyr antibody, is more sensitive than its radioactive counterpart. However, it relies on a particle concentrator using a fluorescence immunoassay (Idexx Corp., Portland, ME). This is unavailable to many laboratories. The assay technique further demands the use of a non-linear dose response curve. A third method, a colorimetric assay [19], measures sub-nanomolar levels of inorganic phosphate, but may not be appropriate to detect PTPase activity due to posible contaminating inorganic phosphates in buffer systems. The undefined chemistry of the system used to measure P-Tyr levels may make the assay unsuitable to monitor PTPase activity in complex cellular systems. Although the PTPases never encounter the synthetic substrate P-Tyr in a physiological scenario, we chose P-Tyr as the PTPase substrate due to the following reasons. Firstly no kinases need to be used to prepare the phosphorylated form of the substrate. Secondly, the activities of different PTPases can be brought to scale by using P-Tyr as an universal substrate. Thirdly, the PTPase reaction can be followed by quantitating residual P-Tyr in the PTPase reaction mixture by using an antibody specific for the substrate. The PTPase assay flow chart is as shown in Fig. 1. The ELISA system was established using glutathione-agarose-coupled PTP-1B. The final assay conditions used for measuring PTPase activity were based on the following considerations. PTPase activity has been known to vary with pH, presence of divalent metal cations like Mn 2+, Zn 2+, as well as the choice of substrate [18]. The sample for the PTPase assay in our system was prepared in a buffer of pH 6.0 in presence of 10 mM Dq-T and 5 mM MnCI 2, as cellular PTPase activity is known to be maximal under these conditions [18]. The PTPase reaction as well as the EIA was performed under phosphate free conditions including the washing steps which were performed in 0.9% NaCI. The rationale for using phosphate free conditions was to monitor PTPase activity in the absence of any chemical that could act as a substrate or product analogue. The antigen capture of P-Tyr was performed using glutaraldehyde, which has long been known to act as a cross-linker of
amine groups [24]. We used glutaraldehyde to cross-link nonspecific amine groups of BSA to the specific amine groups of O-phospho-L-tyrosine. The EIA plates coated with 3% BSA needed to be cross-linked with glutaraldehyde at 2% for retention of P-Tyr up to a concentration of 5/zM. PTP-1B and PTPase in cellular lysates decreased the concentration of P-Tyr in a time and concentration dependent manner. This decrease in concentration of P-Tyr was measurable using the the double antibody EIA technique. The first antibody was a mouse monoclonal anti-P-Tyr antibody and the second antibody, an alkaline phosphatase coupled anti-mouse IgG. Alkaline phosphatase was chosen as the reporter enzyme as it is the most sensitive enzyme known for E.I.A. systems and not found in mammalian cells. The EIA performed with the use of H R P conjugated 2nd antibody led to identical results. 1 mU of PTPase activity has been previously defined as the release of 1 pmol of 32pi/min [18]. This was the definition by using a tyrosine phosphorylated synthetic peptide like Raytide TM as PTPase substrate. By knowing the percent change in EIA absorbance in 10 min the number of mU of PTPase in the reaction standardized the definition of 1 mU of PTPase activity as the amount of PTPase in the sample that could produce a rate of 0.01 Ab U / m i n as compared to a starting control absorbance for 100 nM P-Tyr captured on the EIA plate. The curve for disappearance of the substrate P-Tyr in the EIA assay compares well with the rate o f 32pi release monitored in the radioactive assay (Fig. 6). The PTPase ELISA responded well to lysate dilutions as expected (Fig. 7). Although orthovanadate is a specific PTPase inhibitor [26] we and others [27] have found that PTPase activity is not completely inhibited by the vanadium ion. As seen in Fig. 8, the radioactive assay data shows that there is a residual PTPase activity with orthovanadate treated lysates. The ELISA results of Fig. 8 showed an almost complete inhibition of PTPase activity with orthovanadate. The reason for this discrepancy could very well be due to differences in the substrates used for the radioactive assay (using Raytide T M as a synthetic peptide substrate) as compared with P-Tyr as a substrate used in the ELISA system. The ELISA system, however, was able to demonstrate the complete inhibition of PTPase activity of purified PTP-1B using P G T as its specific inhibitor (Fig. 5). In summary, the ELISA system is a highly reproducible PTPase assay system that is comparable to the radioactive assay system in monitoring the rate of PTPase reaction. The assay is a non-isotopic, rapid, sensitive, reproducible, and inexpensive system to monitor the PTPase activity in cell and tissue extracts.
101
Acknowledgements We would like to thank Dr. Bhanu P. Jena for advising us to use Poly (Glu,Tyr)4 : 1 as specific PTP-1B inhibitor and in other helpful discussions. We would like to thank Helen Spiker and Florence Wade for helping with the preparation of the manuscript. This work was funded by grants R01 CA 48193 and R01 HL 42069 from the National Institute of Health awarded to AWH.
References 1 Pelech, S,L., Sanghera, J.S. and Daya, M. (1990) Biochem. Cell Biol. 68, 1297-1330. 2 Marth, J., Peet, R., Krebs, E.G. and Perlmutter, R.M. (1985) Cell 43, 393-404. 3 Ullrich, A. and Schlessinger, J. (1990) Cell 61,203-212. 4 Hanks, S.K., Quinn, A.M. and Hunter, T.L. (1988) Science 241, 42-52. 5 Takeya, T. and Hanafusa, H. (1982) J. Virol. 44, 12-18. 6 Hunter, T. (1989) Cell 58, 1013-1016. 7 Krueger, N.X., Streuli, M. and Saito, H. (1990) EMBO J. 9, 3241-3252. 8 Jones, S.W., Erikson, R.L., Ingebristen, V.M. and Ingebritsen, T.S. (1989) J. Biol. Chem. 264, 7747-7753. 9 Cicirelli, M.F., Tonks, N.K., Diltz, C.D., Weiel, J.E., Fischer, E.H. and Krebs, E.G. (1990) Proc. Natl. Acad. Sci. USA 87, 5514-5516. 10 Jena, B.P., Padfield, P.J., Ingebristen, T.S. and Jamieson, J.D. (1991) J. Biol. Chem. 266, 17744-17746. 11 Pallen, Catherine, J. and Tong, P.H. (1991) Proc. Natl. Acd. Sci. U.S.A. 88, 6996-7000.
12 Mire, S., Anthoney, R. and Thorpe, R. (1991) J. Biol. Chem. 266, 18113-18118. 13 LaForgia, S., Morse, B., Levy, J., Barnea, G. and Cannizzaro, L.A. (1991) Proc. Natl. Acad. Sci. USA 88, 5036-5040. 14 Guan, K.L. and Dixon, J.E. (1991) J. Biol. Chem. 266, 1702617030. 15 Mei, Lin H. and Richard, L. (1991) J. Biol. Chem. 266, 1606316072. 16 Shriner, C.L. and Brautigan, D.L. (1984) J. Biol. Chem. 258, 11383-11390. 17 Roome, J., O'Hare, T., Pilch, P.F. and Brautigan, D.L. (1988) Biochem. J. 256, 493-500. 18 Daum, G., Zander, N.F., Morse, B., Hurwitz, D., Schlessinger, J. and Fischer, E.H. (1991)J. Biol. Chem. 266, 12211-12215. 19 Mustelin, T., Coggeshall, K.M. and Altman, A. (1989) Proc. Natl. Acad. Sci. USA 85, 6302-6306. 20 Babcook, J., Watts, J. Aebersold, R. and Ziltener, H.J. (1991) Analyt. Biochem. 196, 245-251. 21 Hoenig, M., Lee, R.J. and Ferguson, D.C. (1989) J. Biochem. Biophys. Methods 19, 249-252. 22 Gould, K.L. and Nurse, P. (1989) Nature (London) 342, 39-45. 23 Kaplan, R., Morse, B., Huebner, K,, Croce, C., Howk, R., et al. (1990) Proc. Natl. Acad. Sci. USA 87, 7000-7004. 24 Harlow, Ed., and Lane, D. (1988) Antibodies, A Laboratory Manual. Cold Spring Harbor Laboratories. 25 Hamburger, A.W., Mehta, D., Pinnamaneni, G, Chen, L.C. and Reid, Y. (1991) Pathobiology 59, 329-334. 26 Gordon, J.A. (1991) Methods Enzymol. 201, 477-482. 27 Cho, H., Ramer, S.E., Itoh, M., Winkler, D.G., Kitas, E., et al. (1991) Biochemistry 30, 6210-6216. 28 Tonks, N.K., Diltz, C.D. and Fischer, E.H. (1991) Methods Enzymol. 201, 427-442. 29 Tonks, N.K., Diltz, C.D. and Fischer, E.H. (1991) J. Biol. Chem. 263, 6731-6737.
93
BBAGEN 23792
A microtiter enzyme-linked immunosorbent assay for protein tyrosine phosphatase Shrikant Mishra and Anne W. Hamburger University of Maryland Cancer Center, Department of Pathology, Baltimore, MD (USA) (Received 29 April 1992) (Revised manuscript received 25 November 1992)
Key words: Protein tyrosine phosphatase; ELISA; Signal transduction; Enzyme activity
We report the development of an enzyme-linked immunosorbent assay (ELISA) for protein tyrosine phosphatases (PTPases). PTPase activity, was monitored by quantitating the disappearance of O-phospho-L-tyrosine (P-Tyr) in an ELISA system using antigen capture followed by double antibody labelling. PTPase activity of agarose conjugated PTP-1B was demonstrated using the ELISA system. PTPase activity was sensitive to both PTB-1B concentrations and time of incubation. 1 mU of PTPase activity was defined as that amount of enzyme producing a rate of loss of 0.01 absorbance units/minute with a specific activity of 150 pmol P-Tyr/min per/zg protein based on the unit of PTPase activity from the conventional assay system. The PTP-1B activity was shown by the ELISA system to be completely inhibitable by Poly (Glu,Tyr)4 : 1 at 100/xg/ml. We used the ELISA system to detect PTPase activity in lysates of cultured cells. The PTPase activity of cell lysates of MDA-MB 468 breast carcinoma cells as obtained by the ELISA were compared with those obtained by a standard 32pi release assay using radio-labelled RaytideTM as PTPase substrate. The decrease in P-Tyr concentration was dependent on the time of incubation with the lysate and on lysate concentration and compared well with the release of 3ZPi in the radioactive assay system. Orthovanadate as well as heat denaturation inhibited the PTPase activity of the cell lysates in both the assay systems. The assay presented here is a simple immunological system capable of measuring activity of purified PTPases as well as PTPase levels in cell and tissue extracts.
Introduction Phosphorylation at protein tyrosyl residues is a major mechanism of cellular signal transduction and is involved in mitogenesis [1] as well as in neoplastic transformation [2]. Tyrosine phosphorylation is tightly regulated by the combined activities of specific protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). Previous studies have shown that many cellular growth factor receptors and cellular as well as viral oncogenic proteins are PTKs [3]. The growth factor PTKs are crucial in modulating cell growth. The
Correspondence to: A.W. Hamburger, University of Maryland Cancer Center, Department of Pathology 9-051, Bresslen Research Bldg., Baltimore, MD 21227, USA. Abbreviations: ELISA, enzyme linked immunoabsorbent assay; PTyr, O-phospho-L-tyrosine; PTKs, protein tyrosine kinases; PTPases, protein tyrosine phosphatases; PTP-1B, protein tyrosine phosphatase-lB, FBS, fetal bovine serum; Mops, 3-[N-morpholino]ethanesulfonic acid; EDTA, ethylenediamine tetracetic acid; PMSF, phenylmethylsulfonyl fluoride; pNPP, p-nitrophenyl phosphate; Hepes, N-[2-hydroxyether] piperazine-N'-[2 ethanesulfonic acid]; TCA, trichloro acetic acid; RCML, reduced carboxamidomethylated and maleylated lysozyme; MBP, myelin basic protein.
transduction of mitogenic signal is via phosphorylation of their tyrosyl residues [4]. The oncogenic PTKs, likewise, orchestrate uncontrolled cellular growth and tumorogenesis via phosphorylation of their crucial tyrosine side chains [5]. More recently, attention has been focused on the role of PTPases in controlling cellular tyrosine phosphorylation. Unlike PTKs, the PTPases constitute a distinct class of enzymes [6] of great structural diversity [7]. The localization of PTPase enzymes has been found to be both transmembrane as well as cytosolic [7,8] sites. It has been hypothesized that certain classes of PTPases may be mediators of specfic cellular signalling pathways, rather than having a nonspecific housekeeping function. Functionally the PTPases are involved in suppressing proliferative response to growth factors [9], exocytosis [10], contact inhibition of growth of fibroblasts [11], and cytokine mediated cellular signalling [12]. It has recently been hypothesized that PTPases may exhibit tumor supressor function [9] in lung and renal cell carcinomas [13]. Although a variety of PTPases have been cloned from human and non-primate cDNA libraries [8,12] the assay systems used to characterize these PTPases
94 have not been standardized. To date, radioactive [10,11,14-18] as well as non isotopic [19-21] assay systems for PTPases have been published. We present here a simple, reproducible, inexpensive, non-isotopic, and rapid ELISA system for measuring PTPase activity using purified PTP1-B coupled to agarose and P-Tyr as the enzyme substrate system. The PTPase activity was determined by monitoring the disappearance of P-Tyr, currently the substrate with the broadest spectrum of activity of known PTPases. The P-Tyr in the PTPase reaction mixture was captured on glutaraldehydecoated EIA plates, and quantitated using, as first and second antibodies, anti-P-Tyr mouse antibody and alkaline phosphatase coupled anti-mouse rabbit antibody. Materials and Methods
Protein tyrosine phosphatase 1B (PTP-1B), purified enzyme PTP-1B was purchased from Upstate Biotechnology Inc, Lake Placid, NY; as a glutathione-agarose conjugate (50 /xg/ml bead) with a manufacture specified specific activity of 2 nmol/min per /xg protein using the assay of dephosphorylation of tyrosine-phosphorylated proteins.
Cell culture MDA-MB-468 breast carcinoma cells (American Type Culture Collection HTB 132) were grown in Leibovitz's L-15 medium (GIBCO Laboratories, Life Tech. Inc., NY), at pH 7.4, supplemented with 10% fetal bovine serum (FBS) (Intergen, Purchase, NY), 10 units/ml penicillin, and 10 /zg/ml streptomycin, at 37°C. Cells were seeded at 3 x 105 cells/ml in 80 cm 2 flasks and grown to semi-confluency without change of media.
Preparation of total cell lysates and membrane fractions of MDA-MB-468 cells Unless otherwise mentioned, all the reagents were obtained from Sigma Chemical Co., St. Louis, MO. The semi-confluent cultures were washed with Hank's Balanced Salt Solution (HBSS), 10 ml/wash, to remove serum proteins. For preparation of total cell lysates, the cells were scraped from the 80 cm z flasks in 1 ml of buffer A (250 mM sucrose, 17 mM Mops, 25 mM EDTA, 1.0% Triton X-100, 0.2 ng/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM dithiothreitol(DTT), 5 mM MnC12, pH 6.0). The cell lysate was spun at 15 000 × g for 5 min at 4°C. The clear supernatant was kept at 4°C prior to the assay. For preparation of the membrane fraction, the cells were washed thrice with HBSS and incubated with 1 ml of buffer B (buffer A without Triton X-100 + 0.8 mg/ml digitonin) for two minutes to allow the
release of cytosolic material from the cells. Subsequently the medium was removed and the perforated monolayer was washed twice in 5 ml of buffer A. The perforated cells remained attached to the bottom of the flask. The cells were then scraped in 1 ml of buffer A and cell lysates were prepared as described before. The cell free extracts were then kept on ice. The protein concentration in the extracts were determined by using the Micro BCA protein assay reagent kit (Pierce, Rockford, IL).
Assay for tyrosine phosphatase activity Prior to the assay, 96-well enzyme immunoassay (EIA) plates (Costar, Cambridge, MA) were coated with 3% bovine serum albumin (BSA) (EIA grade) in 0.9% NaC1. The wells were completely filled to the top with this solution. The plates were incubated in a humidified atmosphere at room temperature for 2 h. To remove the unbound BSA, the wells were washed thrice with 0.9% NaCI. The BSA coated plates were incubated with 100 /xl/well of 2% glutaraldehyde in 0.9% NaC1 for 2 h in a humidified atmosphere at room temperature. As glutaraldehyde is known to cross-link proteins via binding to amine functional groups, this step allowed the glutaraldehyde to bind BSA amine groups on one end, leaving the other end vacant for subsequent binding of another amine group. After the glutaraldehyde incubation step, the 100 /zl of glutaraldehyde was removed by vaccum suction and the plate washed thrice with 0.9% NaCI to remove any unbound glutaraldehyde. The plates were stored filled with 0.9% NaC1 at 4°C prior to the assay. Prior to use, the glutathione-agarose conjugated PTP-1B was resuspended in PTPase assay buffer C (25 mM imidazol hydrochloride (pH 7.2), 1 mg/ml BSA, 0.1% v/v/3-mercaptoethanol) at 4°C. The beads were washed four times with the assay buffer to get rid of unbound PTP-IB. The PTPase assay was performed in a 150/zl volume. The PTPase assay mixture constituted of 125 /xl of P-Tyr (0 to 500 nM) and 25 /xl of an appropriate dilution of PTP-1B. The reaction was then allowed to proceed for 10 min at 25°C subjected to gentle rocking. The P-Tyr substrate was reduced to Pi and Tyr by PTP-1B. The reaction was terminated by centrifugation (10000 × g, 5 min at 4°C), resulting in the removal of the PTP-1B agarose conjugate from the aqueous phase. Supernatant, 100 /xl of the reaction, was then applied to glutaraldehyde coated E.I.A plate at 4°C for 4 h. This step ensured the binding of the amine group of the residual P-Tyr to glutaraldehyde. Binding at 4°C was determined to be a critical step for PTPase assay. At the end of the incubation, the wells were washed with 0.9% NaCI (100/xl/wash) and subsequently blocked with 100/zl of 1 mM L-lysine. This step allowed the unreacted glutaraldehyde to bind the amine group of L-Lysine. The L-Lysine blocking step (1
95 h at 25°C) and the subsequent steps were performed at room temperature. After this blocking step, the wells were washed four times with 0.9% NaC1 and then incubated with 100 izl of anti-P-Tyr antibody (Boehringer Mannheim, IN) (1 : 2,000 dilution in 0.9% NaC1) for 2 h in a humidified atmosphere at room temperature. The wells were washed four times with 0.9% NaC1 and subsequently incubated with an appropriate dilution of alkaline phosphatase coupled anti-mouse goat antibody (Biorad Laboratories, CA) as the second antibody, at a 1:20000 dilution, for 1 h at room temperature. The wells were washed twice with 0.9% NaCI and further washed twice with freshly prepared buffer D (1 M diethanolamine, 0.5 mM MgC12, pH 9.8). The plates were incubated with 100 /~1 of 1 mg/ml solution of p-nitrophenyl phosphate (pNPP), made in buffer D, for 30 min. The reaction was stopped by using 50 /zl of 1 N NaOH solution. The color developed was monitored at 405 nm using a 96-well plate reader (Dynatech). For performing the ELISA with HRP conjugated 2nd antibody, the wells were washed with 0.9% NaCI and then incubated, in dark, with 100 /~1 of O-phenylenediamine (OPD) in 0.1 M citrate buffer with 0.03% H 2 0 2. The reaction was terminated after 5 min using 50/zl of 2 N H2SO 4 and the color developed was monitored at 450 nm. The specificity of the PTPase assay was determined by co-incubating the reaction mixture with Poly (Glu, Tyr)l : 4 (PGT), (Sigma), a known inhibitor of PTP-1B. PTPase activity in the cell extracts were determined as follows. Dilutions of the cell extracts were performed in buffer A without Triton X-100. The PTPase assay was performed in a 100/xl reaction volume. 40/xl of an appropriate concentration of P-Tyr was incubated with 10 /xl of appropriately diluted cell extract for 0, 1, 2, 5, and 10 min at 37°C. The reaction was stopped by adding 50/xl of buffer E (50 mM NaC1, 5 mM EDTA, and 0.1 mM sodium orthovanadate) at 4°C. 100 /zl of PTPase reaction mixture/well was heated to 80°C for 10 rain, cooled on ice, and then added to the glutraldehyde coated EIA plates. The rest of the assay steps were identical as described before. Formula used to calculate activity of PTPase: A t = 10 - A t = 0 × t o t a l d i l u t i o n PTPase(mU/mg)
=
0.01 X c o n c . ( m g / m l )
where Zt=o is the EIA absorbance at 405 nm of the PTPase reaction mixture at 0 time, and Zt=10 is the EIA absorbance of the reaction mixture after 10 min of PTPase incubation. 1 mU of PTPase activity is defined as the amount of PTPase that can produce a decrease in the EIA absorbance of 0.01 min of PTPase reaction. In the radioactive assay, the 32Pi released in 10 min of reaction time gives the numbers of mUnit of PTPase present in the reaction mixture. 1 mU of PTPase
activity is defined as the amount of enzyme that releases one picomole of 32Pi/min [18]. Knowledge of the rate of PTPase reaction in terms of EIA absorbance as well as the number of mU of PTPase in the reaction (from the radioactive data) helped standardize the definition of unit of PTPase activity in the ELISA system. A factor of 10 in the denominator of the equation (time contribution) cancels a factor of 10 in that of the numerator (volume contribution).
Radioactive assay for PTPase activity The assay was performed essentially as described [18] except that the substrate protein, RaytideT M (Oncogene Science), was phosphorylated, at tyrosine residues by the recombinant p60 c-src instead of p43 T M as per manufacturer's recomendations. Briefly, p60 c-src stock solution was diluted to 1:20 in kinase dilution buffer (50 mM Hepes (pH 7.5), containing 0.1 mM EDTA, 0.015% Brij. 35, 0.1 mg/ml BSA, and 0.2% /3-mercaptoethanol). RaytideT M was reconstituted at 1 mg/ml in kinase assay buffer (kinase dilution buffer without the addition of BSA and /3-mercaptoethanol). 10 tzl of RaytideTM was mixed with 10 /xl of 1:20 diluted tyrosine kinase. The kinase reaction was initiated by adding 10/zl of ATP mixture (0.15 mM ATP, 30 mM MgC12, 200/zCi Ta2p-ATP/ml of kinase assay buffer). The reaction was carried on for 16 h at 37°C and terminated by adding 500/zl of 20% w / v TCA/20 mM NaHzPO 4 and 100 /.d of 0.1 mg/ml acetylated BSA (Life Tech). The precipitated reaction mixture was centrifuged at 30000 × g for 5 min and subsequently washed with 20% w / v TCA/20 mM NaHEPO 4 three times to remove the unreacted Ta2p-ATP and reconstituted in 100/zl of phosphatase assay buffer (50 mM Mops (pH 6.0), 1 mg/ml BSA, 0.5 mM DTT, 0.01% CHAPS). The specific activity of RaytideTM was determined to be 2 × 104 cpm//zg protein. The phosphatase reaction was performed in a 50/xl of reaction volume containing 5/zl of 32-p-RaytideZM (104 cpm), 5 /xl of appropriate dilution of the cell extract needed to exhibit measurable PTPase activity, and 40 Ixl of phosphatase assay buffer. The reaction was monitored at 37°C for 0, 2, 5, and 10 min and stopped by using 500 /zl of 20% w / v TCA/20 mM NaHEPO 4 with 100/.d of 0.1 mg/ml acetylated BSA. The precipitate was centrifuged at 30000 × g for 5 min. Radioactivity in 400/.d of supernatant was counted in 5 ml of Aquasol (NEN Research Product) using a Beckman LS 3801 liquid scintillation counter. Results
PTPase assay principle The PTPase assay flow chart is as shown in Fig. 1. This assay is based on the rationale that a PTPase reaction involving disappearance of phospho-tyrosine
96 (P-Tyr) can be monitored if the loss of P-Tyr can be quantitated. The rate of loss of P-Tyr is considered to be the rate of PTPase reaction (in the absence of interfering proteases). The PTPase reaction was performed with P-Tyr as the substrate prior to the determination of residual P-Tyr levels in the reaction mixture by the ELISA scheme. The level of P-Tyr in the PTPase reaction was quantitated by antigen capture of P-Tyr on glutaraldehyde (amine to amine cross-linker) coated plates using a double antibody enzyme linked immunosorbent assay (ELISA). The 1st antibody was a mouse monoclonal anti-P-Tyr antibody and the 2nd antibody was an anti-mouse goat antibody coupled to alkaline phosphatase. The sensitivity of the assay was compared for both alkaline phosphatase (AP) and horseradish peroxidase (HRP) coupled 2nd antibodies. Equal degrees of sensitivity were found (data not shown). As cells may possess endogenous peroxidase activity, the alkaline phosphatase method was chosen for further development.
E.I.A Plate
Coat With BSA
Glutaraldehyde Coupling
P-Tyr Coupling (PTPase Reaction Followed By Antigen capture)
Block With L-Lysine
Anti-P-Tyr Antibody
Alkaline Phosphatase Coupled 2~ antibody
Optimization of P-Tyr signal in EIA P-Tyr standards (0 to 5 /xM) were prepared in buffer C. Glutaraldehyde-treated plates were incubated overnight at 4°C with 100 ~1 of P-Tyr standards in six replicates. The specific immuno controls showing the specificity of anti-P-Tyr antibody to detect P-Tyr captured on glutaraldehyde coated EIA plates were performed (data not shown). Incubations with anti-PTyr antibody at a 1:2,000 dilution and the alkaline phosphatase coupled anti-mouse IgG at a 1:20000 were performed as described in the methods section. The EIA signal was optimal at the above mentioned 1st and 2nd antibody concentrations (data not shown). A P-Tyr concentration dependent linear increase in EIA signal was observed at 405 nm in the range of P-Tyr concentration of 0 to 5 /~M (Fig. 2). Beyond 5 /xM, the EIA absorbance reading was saturable. The sensitivity of the assay was determined using 2nd antibodies coupled to either alkaline phosphatase (AP) (Fig. 2) or horse radish peroxidase (HRP) (Fig. 2 inset) as the reporter enzymes. The AP and the HRP reactions were monitored at 405 nm and 450 nm respectively using standard protocols. Both the 2nd antibodies were equally capable of reporting changes in P-Tyr concentration in the same scale. We used AP coupled 2nd antibody as our reporter enzyme for the present assay. The efficiency of coupling of P-Tyr to glutaraldehyde was determined at different glutaraldehyde concentrations (0 to 5%). The EIA absorbance increased with increasing glutaraldehyde concentrations and was saturable at 2% glutaraldehyde (data not shown). Thus, the incubation of BSA coated EIA plates with 2% glutaraldehyde was efficient in trapping P-Tyr which was detected and quantitated by EIA in the range of P-Tyr concentration between 0 and 5 ~M.
Incubation With pNPP
Measure apsorbance at 405 nm
Determine Rate of PTPase Reaction Fig. 1. PTPase assay flow chart.
Validity of the EIA assay using purified PTP-1B The specificity of the ELISA system was tested using purified PTP-1B. The EIA assay was performed for different dilutions of PTP-1B (Fig. 3). The P-Tyr concentration was chosen at 10 nM which was in the linear portion of the standard curve. A linear decrease in absorbance over control was indicative of the disapperance of P-Tyr as a result of PTPase activity. The PTP-1B activity was measured from a 100/zl aliquot of a 150/zl PTPase reaction and was shown to be linear in the range of the enzyme concentration tested. A convenient scale for measuring PTPase activity was to define 1 milli unit of PTPase activity which produces a change of 0.01 Ab U / rai n in a reaction system containing 100 nM P-Tyr. In Fig. 3 it was determined that 1 ng of PTP-1B in the reaction mixture corresponded to a change of 0.1 Ab units in 10 min, i.e., 1 ng PTP-1B corresponds to 1 mU of PTPase activity. How this unit of PTPase activity compared with specific activity measured in terms of tools of P-Tyr disappearance/min per/zg protein was clear from the kinetics of enzymatic activity. The kinetics of PTP-1B reaction was performed (0 to 10 min) at 25°C and the residual P-Tyr in
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P-Tyr (nM) Fig. 2. Determination of the ability of glutaraidehyde coated EIA plates to bind P-Tyr. EIA wells were coated with 3% BSA prior to incubation with 2% glutaraldehyde. Wells were incubated with 5, 50, 500, and 5,000 nM P-Tyr, and EIA was performed as described in 'Materials and Methods'. The assay was performed using AP (405 nm) as well as HRP (450 nm) (indent) coupled 2nd antibodies. Values represent mean + S.D. of six replicate wells on a semilog plot.
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Enzyme (lag) Fig. 3. Modulation of PTPase activity with concentration of PTP-1B. The PTPase assay, in three replicates, was performed with agarose congugated PTP-1B as described in the 'Methods' section. The P-Tyr concentration in a 150-/zl reaction was fixed at 100 nM. The PTPase reaction was allowed to proceed for 10 min and the residual P-Tyr determined by EIA as % control absorbance as described in the 'Methods' section. Values represent mean_+S.D, of triplicate readings.
55
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Fig. 4. Kinetics of PTP-IB activity as measured by EIA. The kinetics of PTPase activity, in three replicates, was performed with agarose congugated PTP-1B (10 ng/ml) for the indicated times. The P-Tyr concentration in a 150-/zl reaction was fixed at 100 riM. The residual P-Tyr, presented of percent of absorbance for 100 nM P-Tyr (control absorbance) was determined by the ELISA method. Values represent mean -I-S.D. of triplicate readings.
98 the reaction determined using the E I A (Fig. 4). As seen in Fig. 4, the P-tyr concentration in the PTPase reaction decreased as a function of time. The rate of PTP-1B reaction was determined to be 0.01 Ab U / m i n with a c o r r e s p o n d i n g specfic activity of 150 p m o l s / m i n u t e / / ~ g protein. Thus combining the definitions of unit of PTPase activity from Fig. 3 and specific activity determined from the kinetics of PTP-1B reaction we can say that 1 mU of PTPase activity corresponds to a disappearance of 1.5 fmols of P-Tyr in 10 min of PTPase reaction. The kinetics of the PTPase reaction was found to be linear in the range of time incubation tested. While most PTPases are inhibited by micromolar concentrations of vanadate and molybdate, PTB-1B is the most sensitive to its specific inhibitor PGT [28]. We determined whether the inhibition of PTP-1B activity by P G T could be demonstratable by E.I.A. As shown in Fig. 5, P G T inhibited the PTP-1B activity in a dose-dependent manner. There was a 95% inhibition of PTPase activity of PTP-1B at a PGT concentration of 100 /zg/ml. Thus we conclude from the above data that PTPase reaction can be monitored using the above assay system presented.
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Fig. 6. Comparative PTPase kinetics of radioactive assay vs. ELISA. The radioactive assay and the ELISA for PTPase were performed as described in the 'Materials and Methods'. 0.1 mg/ml final concentration of total lysate (T) in six replicate wells were used in both the assays. The ELISA and the radioactive assay were performed as described in the methods section. The reaction kinetics were followed for 10 rain. The values were presented as mean±S.D, for DPM released and EIA absorbance (as % C) at 405 nm, respectively.
Quantitation of PTPase in cell lysates As many investigators are currently seeking to determine modulation of PTPase activity in cell culture systems, we measured the ability of the E I A to detect PTPase activity in cell lysates. PTPase activity was quantitated using total cell lysates and membrane
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Fig. 5. Inhibition of PTP-1B activity with Poly (Glu,Tyr)4:1 (PGT). PGT at 0 to 100 izg/ml final concentration was used in the PTPase reaction mixture. The definition of PTPase activitywas set as activity = [(Ab-Ab +)/Ab] x 100, where Ab and Ab + represent absorbance of 100 nM P-Tyr without and with 10 ng/ml PTP-1B, respectively. The PTPase activity without PGT was taken to be 100%. Values represent mean +_S.D. of triplicate readings.
preparations of M D A - M B 468 cells. PTPase activity by E L I S A was also compared with a standard radioactive assay system. The rate of PTPase reaction monitored at 405 nm, from the non-isotopic assay, and 32pi released, from the radioactive assay, were plotted as a function of time as a double Y plot. PTPase kinetics for total cell lysate was determined (Fig. 6). The radioactive assay monitors the appearance of the PTPase reaction product 32Pi, whereas the non-isotopic assay system determined the disappearance of the substrate P-Tyr. As shown in Figure 6, the kinetics of 32pi release varied inversely with the kinetics of P-Tyr disappearance during the total range of incubation times. The PTPase activities in total cell lysate (T) and the membrane fraction (M) were determined at different dilutions of the cell extracts (Fig. 7). From Fig. 7 and the definition of units of PTPase activity (i.e., 1 mU = 25% reduction of absorbance units over control) it is clear that the total cell lysate (T) and the membrane fraction (M) of M D A - M B 468 cells possessed 0.11 m U / / x g and 0.1 m U / i x g protein, respectively. The specificity of the PTPase assay for the cell lysates was performed with M D A - M B 468 total cell lysate. The PTPase assay was performed using the untreated lysates, lysates in presence of 100/~M orthovanadate and heat denatured (80°C for 15 min) lysates using both the assay systems. As shown in Fig. 8, heat denaturation of the lysate as well as the presence of orthovanadate in the PTPase reaction system significantly inhibited the PTPase activity to equal degree.
99 Discussion
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txg Protein Fig. 7. Changes of PTPase activity with dilution of MDA-MB 468 cell lysates. Starting with a stock of 5.0 mg/ml for (a) total cell lysate (T), and 2.1 mg/ml for (b) membrane fraction (M) PTPase activity were performed by the ELISA method with the indicated concentrations of cell lysates. P-Tyr was used at a concentration of 100 nM and the ELISA performed as described in Fig. 2. The ordinate denotes the % absorbance w.r.t, a control 10 min mock reaction containing 100 nM P-Tyr and no enzyme in a 100 /zl reaction. Values represent mean + S.D. of triplicate readings.
Thus the assay presented here is a simple immunological system capable of measuring purified PTPases as well as PTPase levels in cell and tissue extracts.
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Fig. 8. Inhibition of PTPase activity by orthovanadate and by heat denaturation. PTPase ELISA and the isotopic assays were performed as described in the experimental procedure. PTPase reactions were performed in appropriate buffers in both the assays in presence of untreated lysate (10/~g per reaction), lysate in presence of 100 /zM orthovanadate, or heat denaturated lysate (80°C for 15 min). DPM released (mean of triplicate counts) and E.I.A. absorbance (mean of six replicates) were plotted as shown.
PTPase enzymes have recently been determined to play a significant role in cell growth and oncogenesis by opposing the effects of a variety of PTKs. PTPases along with PTKs regulate total cellular phosphotyrosine levels. It has been determined that PTPases catalyze the dephosphorylation of the substrate proteins, typically PTKs, via a cysteine-phosphate intermediate [14]. A density dependent growth arrest of Swiss 3T3 cells was associated with increases in membrane PTPase activity [11] showing thereby that PTPases could play a role in cell proliferation and contact inhibition of cell growth. Several receptor associated PTPases have been found to have extracellular domains homologous to neural cell adhesion molecules [11] suggesting a role of PTPases in cell-cell interaction. PTPases has been implicated in regulating the action of M P F / p34 cdc2 [22], the mitotic control element, and thereby directly affecting the regulation of cell cycle. Further as many growth factor receptors and oncogenic proteins are PTKs, the PTPases could potentially function as antioncogenic or tumor suppressor proteins. For example, PTPy has been identified as a candidate tumor suppressor gene product in renal cell carcinoma (RCC) and lung carcinoma (LC) [13] and was shown to be mutated or absent in these cells. These findings have prompted a rapid increase in cloning, purification, and characterization of these PTPases [7,11,13,17,18,23]. Despite increased interest in PTPases, the assay systems used to characterize these enzymes have not been standardized. The activities of PTPases cloned thus far have been observed for a broad range of substrates. For example, the same PTPase can cleave phosphate groups from tyrosine phosphorylated substrates like reduced carboxamidomethylated and maleylated lysozyme (RCML), myelin basic protein (MBP), tyrosine phosphorylated casein, pp60 v-src, insulin receptor kinase, tyrosine phosphorylated epidermal growth factor receptor, tyrosine phosphorylated histone kinase, tyrosine phosphorylated Calpactin I, tyrosine phosphorylated src-peptide, and from P-Tyr. Although most PTPases exhibit high affinity for substrates with an associated high Vm~x [29], the K m and Vm~ values have been shown to change with the choice of substrates. For example, the kinetic parameters of receptor protein tyrosine phosphatase a (RPTPa) was determined using the radioactive assay system [18]. The assay was performed using two different substrate proteins, namely RCML, and MBP. The gins and VmaxS of RPTPot using RCML or MBP as substrate were entirely different. In fact PTPase activity determined using RCML as substrate was minimal. The radioactive assay systems typically use gamma- 32p-ATP and a suitable protein tyrosine kinase (like c-abl, v-abl, and c-src tyrosine kinases) to enzymatically transfer gamma-32p -
100 group to a suitable substrate protein (like MBP, RCML, Raytide, and bovine albumin) at its tyrosine residue and subsequently PTPase activity quantitated by measuring the amount of 32Pi released in to the supernatant of TCA precipitated PTPase reaction mixture. We used Raytide T M (Oncogene Science) as our substrate for comparison of our ELISA with the radioactive assay [10]. A major negative feature of the radioactive PTPase assay system however is that the response of the assay system depends to a large extent on the choice of the PTPase substrate [18]. A newly developed non isotopic assay system [20], which measures phosphotyrosine containing substrate protein levels by using anti-P-Tyr antibody, is more sensitive than its radioactive counterpart. However, it relies on a particle concentrator using a fluorescence immunoassay (Idexx Corp., Portland, ME). This is unavailable to many laboratories. The assay technique further demands the use of a non-linear dose response curve. A third method, a colorimetric assay [19], measures sub-nanomolar levels of inorganic phosphate, but may not be appropriate to detect PTPase activity due to posible contaminating inorganic phosphates in buffer systems. The undefined chemistry of the system used to measure P-Tyr levels may make the assay unsuitable to monitor PTPase activity in complex cellular systems. Although the PTPases never encounter the synthetic substrate P-Tyr in a physiological scenario, we chose P-Tyr as the PTPase substrate due to the following reasons. Firstly no kinases need to be used to prepare the phosphorylated form of the substrate. Secondly, the activities of different PTPases can be brought to scale by using P-Tyr as an universal substrate. Thirdly, the PTPase reaction can be followed by quantitating residual P-Tyr in the PTPase reaction mixture by using an antibody specific for the substrate. The PTPase assay flow chart is as shown in Fig. 1. The ELISA system was established using glutathione-agarose-coupled PTP-1B. The final assay conditions used for measuring PTPase activity were based on the following considerations. PTPase activity has been known to vary with pH, presence of divalent metal cations like Mn 2+, Zn 2+, as well as the choice of substrate [18]. The sample for the PTPase assay in our system was prepared in a buffer of pH 6.0 in presence of 10 mM Dq-T and 5 mM MnCI 2, as cellular PTPase activity is known to be maximal under these conditions [18]. The PTPase reaction as well as the EIA was performed under phosphate free conditions including the washing steps which were performed in 0.9% NaCI. The rationale for using phosphate free conditions was to monitor PTPase activity in the absence of any chemical that could act as a substrate or product analogue. The antigen capture of P-Tyr was performed using glutaraldehyde, which has long been known to act as a cross-linker of
amine groups [24]. We used glutaraldehyde to cross-link nonspecific amine groups of BSA to the specific amine groups of O-phospho-L-tyrosine. The EIA plates coated with 3% BSA needed to be cross-linked with glutaraldehyde at 2% for retention of P-Tyr up to a concentration of 5/zM. PTP-1B and PTPase in cellular lysates decreased the concentration of P-Tyr in a time and concentration dependent manner. This decrease in concentration of P-Tyr was measurable using the the double antibody EIA technique. The first antibody was a mouse monoclonal anti-P-Tyr antibody and the second antibody, an alkaline phosphatase coupled anti-mouse IgG. Alkaline phosphatase was chosen as the reporter enzyme as it is the most sensitive enzyme known for E.I.A. systems and not found in mammalian cells. The EIA performed with the use of H R P conjugated 2nd antibody led to identical results. 1 mU of PTPase activity has been previously defined as the release of 1 pmol of 32pi/min [18]. This was the definition by using a tyrosine phosphorylated synthetic peptide like Raytide TM as PTPase substrate. By knowing the percent change in EIA absorbance in 10 min the number of mU of PTPase in the reaction standardized the definition of 1 mU of PTPase activity as the amount of PTPase in the sample that could produce a rate of 0.01 Ab U / m i n as compared to a starting control absorbance for 100 nM P-Tyr captured on the EIA plate. The curve for disappearance of the substrate P-Tyr in the EIA assay compares well with the rate o f 32pi release monitored in the radioactive assay (Fig. 6). The PTPase ELISA responded well to lysate dilutions as expected (Fig. 7). Although orthovanadate is a specific PTPase inhibitor [26] we and others [27] have found that PTPase activity is not completely inhibited by the vanadium ion. As seen in Fig. 8, the radioactive assay data shows that there is a residual PTPase activity with orthovanadate treated lysates. The ELISA results of Fig. 8 showed an almost complete inhibition of PTPase activity with orthovanadate. The reason for this discrepancy could very well be due to differences in the substrates used for the radioactive assay (using Raytide T M as a synthetic peptide substrate) as compared with P-Tyr as a substrate used in the ELISA system. The ELISA system, however, was able to demonstrate the complete inhibition of PTPase activity of purified PTP-1B using P G T as its specific inhibitor (Fig. 5). In summary, the ELISA system is a highly reproducible PTPase assay system that is comparable to the radioactive assay system in monitoring the rate of PTPase reaction. The assay is a non-isotopic, rapid, sensitive, reproducible, and inexpensive system to monitor the PTPase activity in cell and tissue extracts.
101
Acknowledgements We would like to thank Dr. Bhanu P. Jena for advising us to use Poly (Glu,Tyr)4 : 1 as specific PTP-1B inhibitor and in other helpful discussions. We would like to thank Helen Spiker and Florence Wade for helping with the preparation of the manuscript. This work was funded by grants R01 CA 48193 and R01 HL 42069 from the National Institute of Health awarded to AWH.
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