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Methodologies for the detection and nonradioactive assay of protein-tyrosine kinase activities in human brain and gliomas
NEUROPROTOCOLS: A Companion to Methods Vol. 1, No. 3, December, pp. 201-206, 1992
in Neurosciences
Methodologies for the Detection and Nonradioactive Assay of Protein-Tyrosine Activities in Human Brain and Gliomas
Kinase
Gert Rijksen and Gerard E. J. Staal Department
of Hematology,
Laboratory
of Medical
Enzymology,
University
A procedure for an enzyme-linked immunoadsorbent assay for the determination of protein-tyrosine kinase (PTK) activity from various sources, including normal brain and brain tumors, is described. The general PTK substrate poly(GluNa,Tyr) (4: 1) is coated to the wells of a microtiter plate. After incubation with PTK sample and ATP, the number of phosphorylated tyrosyl residues is quantitated with phosphotyrosine-specific antibodies and a secondary peroxidase-labeled antibody. The assay is optimized with respect to coating and phosphorylation conditions. It is linear with phosphorylation time and with sample protein concentrations in a sufficiently wide range. PTK activities measured with this assay correlate well with those of a nonradioactive dot-blot assay (Rijksen et a/., 1989, Anal. Biochem. 182,98102) and with conventional radioactive assays in which [“P]ATP is used as the substrate. Compared to the latter assays, it appears to be more sensitive and far easier to perform. o 1992 Academic Press, Inc.
Over the past decade intensive studies have been directed toward understanding cellular protein tyrosine phosphorylation. It now appears that protein-tyrosine kinases (PTK) and protein-tyrosine phosphatases (PTPase) regulate such critical cellular functions as signal transduction, growth, and differentiation (l-3). Protein-tyrosine kinases can be broadly classified into two groups, the receptor kinases and the nonreceptor kinases, with most members of the latter group belonging to the src family of tyrosine kinases. Human gliomas and glioma cell lines often show an increased expression of-among othersplatelet-derived growth factor (PDGF) receptor, epiderma1 growth factor (EGF) receptor, and insulin-like growth factor receptor-l (4,5). These receptors all exhibit intracellular tyrosine kinase activity upon stimulation with their respective ligands. In normal human brain, high levels of the src protein are expressed (6, 7). Also srcrelated PTKs, such as the products of the c-fyn and c-yes genes, are known to exist in normal brain (8, 9). These findings indicate the importance of the determination and 1058.6741/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Hospital
litrecht,
The Netherlands
characterization of PTKs in normal brain and the comparison with those in brain tumors. Therefore, there is a need for simple and reproducible assay methods for determining PTK activity. In general, PTK activity is measured using [32P]ATP and macromolecular PTK substrates. The incorporation of [32P]phosphate is measured by precipitating the polypeptide substrate on filter paper with TCA, extensive washing, and counting for radioactivity (10, 11). More recently, a nonradioactive dot-blot assay, based on the detection of phosphorylated tyrosyl residues with monoclonal antibodies to phosphotyrosine, was described (12). This principle was applied to a liquid assay system for quantitative determinations as well as to a solid-phase variant, which appeared to be particularly useful for rapid semi-quantitative screening of, e.g., elution fractions obtained in purification procedures (13). The results of both methods correlated well with those of the conventional radioactive assay. Later, solid-phase ELISAs based on the same principle were reported (14,15): the polypeptide PTK substrate is coated to the wells of a microtiter plate. After incubation with the PTK sample and ATP, the phosphorylated tyrosyl residues are probed with phosphotyrosine-specific antibody. After incubation with peroxidase-labeled second antibody, the extent of phosphorylation is quantified by using a suitable peroxidase substrate. In these methods random copolymers of tyrosine and glutamic acid have generally been used as artificial substrates. This substrate has been reported as a good one for most PTKs (11). The procedures for the dotblot assays are described in detail elsewhere (13). In this paper we give a detailed description of an ELISA method and compare its results with those of the dot-blot method. Some results of the PTK activity in human brain and gliomas are also included.
DESCRIPTION OF METHODS Materials All chemicals used in buffers, poly(glutamic acidtyrosine, 4:l; PGT), fatty acid-free bovine serum albumin 201
Inc. reserved.
202
RIJKSEN AND STAAL
(BSA), 3,3’,5,5’-tetramethylbenzidine (TMB), and aprotinin were obtained from Sigma (St. Louis, MO). Microtiter plates were from Greiner (The Netherlands). Antiphosphotyrosine IG2 was from Amersham (Buckinghamshire, UK); rabbit anti-mouse peroxidase was from Dakopatts (Denmark). Phenylmethylsulfonyl fluoride (PMSF) was supplied by Merck (Darmstadt, Germany). Biopsies from human astrocytomas and oligodendrogliomas were obtained by surgery at the Department of Neurosurgery, University Hospital Utrecht. Furthermore, biopsy specimens that were obtained in temporal or frontal lobectomies performed on epileptic patients were examined and considered to represent normal brain tissue. The tissues were pulverized in the frozen state using a dismembrator and stored at -80°C until use. In some experiments involving the development of the ELISA, cytosolic extracts from breast cancer specimens or rat spleen were used. Sample Preparation All procedures were performed at 4°C. The dismembrated tissues were thawed and resuspended in 3 vol of extraction buffer containing 20 mM Hepes (pH 7.2), 0.25 M sucrose, 1 mM MgClz, 1 mM EDTA, 10 mM dithiothreitol, 1 mM PMSF, and 5 pg/ml aprotinin. Cell debris and nuclei were removed by centrifugation at 8OOgfor 10 min. The supernatant was centrifuged at 48,000g for 1 h. The 48,000g supernatant was used for the assay of cytosolic PTK activity. PTK Assay by the Dot-Blot
Procedure
The procedure has been described in detail previously (13). In this method the PTK sample is incubated with the artificial substrate PGT and unlabeled ATP. After termination of the phosphorylation reaction by addition
of an excess of EDTA/EGTA, an aliquot of the reaction mixture is transferred to a polyvinylidene difluoride (PVDF) membrane. The extent of tyrosine phosphorylation is measured by probing the membrane with antiphosphotyrosine antibody followed by detection by the immunogold silver staining method. The signal is quantified by densitometry. The PTK activity is calculated using a standard of phosphorylated PGT (see below) and is expressed as picomoles of tyrosyl residues phosphorylated in PGT per minute per milligram of protein. The phosphorylated PGT standard was prepared using a cytosolic extract of a grade I/II astrocytoma as described (13). Phosphate incorporated in the PGT was quantitated by performing a microscale experiment in parallel with [32P]ATP under identical conditions and appeared to be 1.35 pmol of phosphate/pg of PGT. From the mean molecular weight of 28,500 Da, constituting 36 tyrosyl residues per PGT molecule, the incorporation of the standard was calculated to be 1.1 mmol/mol tyrosyl residues. Protein contents of cytosolic extracts were determined according to Lowry et al. (16). If contamination with blood was expected, the protein content was corrected by spectrophotometric determination of the hemoglobin concentration (17). We thereby assumed that the contribution of PTK activity from red blood cells was nonsignificant. Determination
of PTK Activity
by ELISA
Coating of microtiter plates. The substrate PGT was coated to the wells of a 96-well microtiter plate by adding 125 gl of PGT in phosphate-buffered saline (PBS: 0.14 M NaCl, 8.93 mM Na2HP04 * 12Hz0, 1.28 mM NaHzP04. 2Hz0). Plates were covered with Elisa plate sealer tape to prevent evaporation. After incubation for 16 h at 37°C the wells were washed once with 250 ~1 of washing buffer (0.1% Tween 20 in PBS). After the plates were emptied, they were dried for 2-3 h at 37°C. Coated plates
o-1 0
0.25
PGT (mg/ml) FIG. 1. Optimization of the PGT concentration. ELISA plates were coated with increasing concentrations of PGT in PBS. Phosphorylation was performed with 33 (O), 67 (V), and 125 (0) ng of sample protein from a cytosolic rat spleen extract. Phosphorylation and detection were performed under standard assay conditions.
600
ATP hM) FIG. 2. Influence of ATP concentration. PTK activities of breast cancer (0) and rat spleen (W cytosolic extracts were measured by ELISA using increasing concentrations of ATP. Phosphorylation and detection were performed under standard assay conditions.
NONRADIOACTIVE -_____
ASSAYS
FOR
PROTEIN-TYROSINE
were stored at 4°C in plastic bags. They could be stored for at least 3 months without any appreciable loss of performance. To determine the optimal concentration of PGT, plates were coated with increasing amounts of PGT. Saturating conditions were reached at a PGT concentration of 0.25 mg/ml (Fig. 1). In further experiments a concentration of 0.25 mg/ml PGT was used throughout. To quantitate the actual amount of bound PGT per well, we used the 32P-labeled PGT standard (see above) and coated it under standard conditions, assuming that the binding of [32P]PGT is comparable to that of PGT. From these experiments it appeared that under saturating conditions 0.75 pg PGT per well was bound. Blocking of residual binding sites with BSA appeared unnecessary, showing again that PGT is a good binding substrate for microtiter plates. Phosphorylation. The ELISA plates were allowed to reach room temperature before being opened. Immediately before use, samples were diluted in incubation buffer containing 50 mM Hepes, pH 7.2,lO mM MgC&, 2 InM MnC12, 0.1 mM EDTA, 0.4 mM EGTA, 0.5 mM dithiothreitol, 2.5 mM NaF, and 0.02% BSA; 50 ~1 was added in duplicate or triplicate to the wells of an ELISA plate. The plate was preincubated at 37°C for some minutes before phosphorylation was initiated by adding 50 ~1 of prewarmed ATP in 50 mM Hepes, pH 7.2, containing 10 mM MgClz, 2 mM MnClz and 200 pM Na,VO,. The optimal ATP con-
KINASE
ACTIVITY.~_~~. ___
203
centration was determined by constructing a substrate saturation curve, as shown in Fig. 2. KM values were in the range of 30-50 pM for PTKs from both breast cancer and rat spleen extracts; no substrate inhibition occurred at ATP concentrations > 1 mM. In further experiments a final concentration of 1 mM ATP was used throughout. After initiation of the phosphorylation reaction, the plates were covered and incubated at 37°C in an incubator. The reaction was terminated by emptying the plates and washing four times with 250 ~1 washing buffer. The reaction appeared to be linear up to at least 20-50 min, depending on the PTK activity (Fig. 3). In further experiments a phosphorylation time of 15 min was used throughout. Blank reactions from which either the PTK sample or the ATP was omitted were included. Optical density from either control was always ~0.1. Detection. After washing, the wells were incubated for 1 h at room temperature with 100 ~1 antiphosphotyrosine (dilution to be determined for each new batch) in PBS including 0.1% BSA. The plates were consecutively emptied carefully by patting them vigorously upside down on paper towels. The wells were washed four times with washing buffer. Next, 100 ~1 rabbit anti-mouse peroxidase conjugate, diluted l/300 in PBS and including 0.1% BSA and 1% gelatin, was added. After incubation for 1 h at room temperature the wells were washed four times with washing buffer, again each time carefully emptying the plates by patting them vigorously on paper towels. Signal development was initiated by adding 100 ~1 of color-developing reagent consisting of 0.51 mM TMB, 1.2% dimethyl sulfoxide, 0.004% HzOz, and 110 mM sodium acetate, pH 5.5. After 10 min the color development was stopped by the addition of 100 ~12 M H2S04. The optical density at 450 nm was measured with an ELISA reader. The specificity of the reaction was confirmed in control experiments in which the antiphosphotyrosine antibody
no interruption after
phosphorylation in wash buffer with 1st antibody
after 1st antibody in wash buffer with 2nd antibody after 2nd antibody in wash buffer
0
10
20
30
40
50
60
Time (min.) FIG. 3. Time course was performed with spleen extract as the minated by emptying were according to the
of the phosphorylation reaction. The PTK ELBA 10 (O), 33 (0), and 67 (V) ng of a cytosolic rat sample. The phosphorylation reaction was terthe wells at varying time points. Other conditions standard protocol.
L ~-.
O.D.
450 nrll
FIG. 4. Overnight interruption of ELISA procedure. The procedure was interrupted at the stages indicated and the plates were stored overnight at 4’C. The next morning the procedure was continued according to the standard protocol. The error bars indicate the SD of a triplicate assay.
204
RIJKSEN
was preincubated with 10 mM phenyl phosphate. This treatment completely abolished the signal, proving that it was specific for phosphotyrosyl residues. The procedure can be interrupted overnight at various stages, as shown in Fig. 4. First, the plates can be kept in the refrigerator in washing buffer after the phosphorylation step. Alternatively, the incubation with either the first or the second antibody can be prolonged overnight at 4°C without any loss of signal. However, storage in washing buffer after incubation with first or second antibody appeared to be detrimental.
RESULTS The ELISA procedure was used to measure PTK activity in crude cytosolic extracts of gliomas, breast cancers, and rat spleen. Variation of the amount of sample protein showed that the assay was linear with sample protein up to 100-200 ng per well for gliomas, to 200 ng for breast cancer, and to 50 ng for rat spleen extracts (Fig. 5). At higher amounts of sample the signal started to level off. The optical density at which deviation from linearity occurred varied among the various tissue types, but was rather consistent within samples of one tissue type. It appeared that the limitations of the assay system were not involved, but rather down-regulation of PTK activity
AND
STAAL
after initiation of the phosphorylation reaction with ATP (results not shown). Standard deviations of triplicate assays were <5%. Next, we compared the ELISA with a nonradioactive dot-blot assay, which we developed earlier (12, 13). Cytosolic fractions from benign breast tumors and malignant breast carcinomas were prepared and assayed for PTK activity both by ELISA and by dot-blot assay. The comparison of both assays is shown in Fig. 6. A linear correlation was obtained with a correlation coefficient of 0.92. Earlier, we showed that the dot-blot assay correlated well with the conventional radioactive filter-paper assay (12). The latter assay was used to measure PTK activities of normal human brain and gliomas (Table 1). Astrocytomas were divided into low-grade (grades I and II) and highgrade (grades III and IV) tumors. Low-grade astrocytomas showed, on average, higher activities (range, 163-812 pmol/min/mg) than normal adult brain (range, 48-285 pmol/min/mg). Also in the group of oligodendrogliomas, increased activities were found (range, 167-935 pmol/ min/mg). This group of gliomas consisted mainly of highgrade oligodendrogliomas (grades C and D). Remarkably, most PTK activities in the high-grade astrocytomas (range, 58-353 pmol/min/mg) were in the same range as those of normal brain (see also Ref. (18)).
DISCUSSION The present ELISA is a further development of the use of monoclonal antibodies to phosphotyrosyl residues to
OF 0 sample
protein
(rig/well)
FIG. 5. Variation of sample protein. The PTK ELISA was performed with increasing amounts of sample protein from cytosolic extracts from rat spleen (O), human gliomas (0, V), and breast cancer (*). Phosphorylation and detection were performed under standard assay conditions
/ 100
200 Dot-Blot
300 assay
400
500
600
(pmol/min.mg)
FIG. 6. Correlation between dot-blot activities of 15 cytosolic extracts of breast by dot-blot assay and ELISA. PTK pm01 * mini’ * mg-’ and as relative activity, coefficient is 0.92.
assay and ELISA. The PTK cancer biopsies were measured activities are expressed in respectively. The correlation
NONRADIOACTIVE
ASSAYS FOR PROTEIN-TYROSINE ______ KINASE ACTIVITY
determine PTK activity, providing a simple and nonlaborious alternative for existing methods. Previously we developed a nonradioactive dot-blot procedure based on the same principle (12). In this method phosphorylation of the PGT substrate occurs in the liquid phase. After termination of the reaction, an aliquot of the reaction mixture is transferred to a PVDF membrane. The extent of tyrosyl phosphorylation is measured by probing the membrane with antiphosphotyrosine antibody followed by detection with the immunogold silver staining procedure (13). This method appeared to be quantitative and at least as sensitive as the conventional assays using [“‘PIATP. The results of both methods correlated well. The main difference of the present ELISA from the dotblot method is the immobilization of the PGT substrate during the phosphorylation reaction. It is therefore hardly possible to compare the kinetics of the reaction in both systems. In the ELISA the PGT concentration cannot be optimized in terms of enzyme affinity, as the available well surface is limiting. Nevertheless, the KM for ATP is more or less the same in both systems. Remarkably, despite the limitation with respect to the PGT concentration, the ELISA is more sensitive than the dot-blot assay. The detection limit is four to five times lower in the former. This might be explained by the probability that all phosphorylated tyrosyl residues in the well are available for the antibody, whereas in the dot-blot assay the PGT is immobilized after phosphorylation, which means that part of the phosphotyrosine may be masked by an incorrect spatial orientation at the membrane surface. The present method is apparently also much more sensitive than earlier described ELISAs based on the same principle (14, 15), although comparison is rather difficult because of the use of purified enzyme preparations in the latter reports. The reason for this difference in sensitivity is not quite clear. However, one particular difference in the coating conditions with respect to the method of Farley et al. (15) appeared to be of critical importance. In Farley’s protocol 0.02% sodium azide was present during coating. In our hands the presence of azide was most det-
TABLE
~~
205
rimental to the ultimate result (not shown). In addition, the ATP concentration used by Farley et al. was far from optimal in our procedure. With respect, to the method of Lhzaro et al. (14), we needed more PGT to optimize the coating. Comparison, however, is not possible because these authors do not provide data about the actual amount of bound PGT. The correlation obtained between results from the ELISA and the dot-blot assay on the one hand (present study) and from the dot-blot assay and conventional radioactive filter-paper assay (12) on the other suggests that all three methods are interchangeable. However, the applicability of the ELISA is somewhat limited because the incorporation of phosphate cannot be quantitated directly and only relative activities are measured in one ELISA plate. The method is very suitable, however, for investigations involving enzyme regulation, screening for inhibitors, purification procedures, growth factor action, etc. Its use for other applications, which demand absolute and reproducible PTK activities, awaits the development of internal and external standards and controls (work in progress). For this reason, until now we have used the dot-blot assay to measure PTK activities in tumors and normal tissues. In oligodendrogliomas and low-grade astrocytomas, cytosolic tyrosine kinase activities were increased in accordance with findings in other kinds of tumors (19). Remarkably, the activity in high-grade astrocytomas was not different from that in normal brain, probably as a result of necrosis present in most of these tumors (18). Further studies of this kind will be facilit,ated tremendously by the development of simple PTK assays like the one presented here, provided that the day-to-day variation in signal development can be accounted for and the actual incorporation of phosphate can be quantitated directly.
ACKNOWLEDGMENTS The authors gratefully of S. S. Adriaansen-Slot.
acknowledge the expert technical assistance B. A. van Oirschot. and H. Schraag.
1
REFERENCES
PTK Activity in Cystosolic Extracts of Normal Human Brain and Gliomas
1. Cantley, L. C., Auger, A., Kapeller, R., and
PTK activity (pmol/min/mg protein)
2. Tonks,
N. K.,
K. R., Carpenter, Soltoff, S. (1991)
and Charbonneau,
C., Duckworth, B.. Grazrani, Cell 64, 2X1-302.
H. (1989)
‘I’ronds
Hioch~m.
Sci.
14,497-500. Adult human brain Oligodrendroglioma Low-grade astrocytoma High-grade astrocytoma
Median
Range
163 356 501 207
48-285 167-935 163-812 58-353
(n) (14) (7) (7) (11)
3. Fischer, E. H., Charbonneau, 253, 401-406. 4. Heldin, 487-496.
C.-H.,
and
Westermark,
H., and
Tonks,
B. (1989)
N. K. (1991) Eur.
,/. Riochrm.
Scirvwe
184,
5. Tuzi, N. L., Venter, D. J., Kumar, Y., Staddon, S. L., Lemoine. N. R.. and Gullick, W. d. (1991) Rr. d. (hncer 6.1, 227-233.
206
RIJKSEN
6. Brugge, J. S., Cotton, P. C., Qearal, D., and Keane, R. W. (1985) Nature 7. Okada,
M.,
and
Nakagawa,
H.
A. E., Barrett,
J. N.,
Nonner,
316, 554-557.
(1988)
J. Biochen.
(Tokyo)
104,
297-305. 8. Kypta,
R. M., Hemming,
A., and Courtneidge,
S. A. (1988)
EMBO
J. 7,3837-3844. 9. Sudol, 10. Sahal,
M., and Hanafusa,
H. (1986)
D., and Fujita-Yamaguchi,
Mol.
Cell. Bid.
Y. (1987)
Anal.
6, 2839-2846. Biochem.
167,
23-30. 11. Braun, S., Raymond, 259, 2051-2054. 12. Rijksen, Biochem.
G., Van
W. E., and Racker, Oirschot,
182,98-102.
B. A., and
E. (1984) Staal,
J. Biol.
Chem.
G. E. J. (1989)
Anal.
AND
STAAL
13. Rijksen, G., Van Oirschot, B. A., and Staal, G. E. J. (1991) Methods Enzymol. 200,98-107. 14. Lizaro, I., Gonzalez, M., Roy, G., Villar, L. M., and Gonzalez-PorquC, P. (1991) Anal. Biochem. 192, 257-261. 15. Farley, K., Mett, H., McGlynn, E., Murray, B., and Lydon, N. B. (1992) Anal. Biochem. 203, 151-157. 16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 139, 265-275. 17. Dacey, J. V., and Lewis, S. M. (1975) Practical Hematology, p. 180, Churchill Livingstone, New York. 18. Van Erp, H. E., Rijksen, G., Van Veelen, C. W. M., Van der Heijden, J. H. W., Maarschalkerweerd, B. N. B., and Staal, G. E. J. (1992) J. Neurochem. 58, 554-561. 19. Hennipman, A., Van Oirschot, B. A., Smits, J., Rijksen, G., and Staal, G. E. J. (1989) Cancer Res. 49, 516-521.
in Neurosciences
Methodologies for the Detection and Nonradioactive Assay of Protein-Tyrosine Activities in Human Brain and Gliomas
Kinase
Gert Rijksen and Gerard E. J. Staal Department
of Hematology,
Laboratory
of Medical
Enzymology,
University
A procedure for an enzyme-linked immunoadsorbent assay for the determination of protein-tyrosine kinase (PTK) activity from various sources, including normal brain and brain tumors, is described. The general PTK substrate poly(GluNa,Tyr) (4: 1) is coated to the wells of a microtiter plate. After incubation with PTK sample and ATP, the number of phosphorylated tyrosyl residues is quantitated with phosphotyrosine-specific antibodies and a secondary peroxidase-labeled antibody. The assay is optimized with respect to coating and phosphorylation conditions. It is linear with phosphorylation time and with sample protein concentrations in a sufficiently wide range. PTK activities measured with this assay correlate well with those of a nonradioactive dot-blot assay (Rijksen et a/., 1989, Anal. Biochem. 182,98102) and with conventional radioactive assays in which [“P]ATP is used as the substrate. Compared to the latter assays, it appears to be more sensitive and far easier to perform. o 1992 Academic Press, Inc.
Over the past decade intensive studies have been directed toward understanding cellular protein tyrosine phosphorylation. It now appears that protein-tyrosine kinases (PTK) and protein-tyrosine phosphatases (PTPase) regulate such critical cellular functions as signal transduction, growth, and differentiation (l-3). Protein-tyrosine kinases can be broadly classified into two groups, the receptor kinases and the nonreceptor kinases, with most members of the latter group belonging to the src family of tyrosine kinases. Human gliomas and glioma cell lines often show an increased expression of-among othersplatelet-derived growth factor (PDGF) receptor, epiderma1 growth factor (EGF) receptor, and insulin-like growth factor receptor-l (4,5). These receptors all exhibit intracellular tyrosine kinase activity upon stimulation with their respective ligands. In normal human brain, high levels of the src protein are expressed (6, 7). Also srcrelated PTKs, such as the products of the c-fyn and c-yes genes, are known to exist in normal brain (8, 9). These findings indicate the importance of the determination and 1058.6741/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Hospital
litrecht,
The Netherlands
characterization of PTKs in normal brain and the comparison with those in brain tumors. Therefore, there is a need for simple and reproducible assay methods for determining PTK activity. In general, PTK activity is measured using [32P]ATP and macromolecular PTK substrates. The incorporation of [32P]phosphate is measured by precipitating the polypeptide substrate on filter paper with TCA, extensive washing, and counting for radioactivity (10, 11). More recently, a nonradioactive dot-blot assay, based on the detection of phosphorylated tyrosyl residues with monoclonal antibodies to phosphotyrosine, was described (12). This principle was applied to a liquid assay system for quantitative determinations as well as to a solid-phase variant, which appeared to be particularly useful for rapid semi-quantitative screening of, e.g., elution fractions obtained in purification procedures (13). The results of both methods correlated well with those of the conventional radioactive assay. Later, solid-phase ELISAs based on the same principle were reported (14,15): the polypeptide PTK substrate is coated to the wells of a microtiter plate. After incubation with the PTK sample and ATP, the phosphorylated tyrosyl residues are probed with phosphotyrosine-specific antibody. After incubation with peroxidase-labeled second antibody, the extent of phosphorylation is quantified by using a suitable peroxidase substrate. In these methods random copolymers of tyrosine and glutamic acid have generally been used as artificial substrates. This substrate has been reported as a good one for most PTKs (11). The procedures for the dotblot assays are described in detail elsewhere (13). In this paper we give a detailed description of an ELISA method and compare its results with those of the dot-blot method. Some results of the PTK activity in human brain and gliomas are also included.
DESCRIPTION OF METHODS Materials All chemicals used in buffers, poly(glutamic acidtyrosine, 4:l; PGT), fatty acid-free bovine serum albumin 201
Inc. reserved.
202
RIJKSEN AND STAAL
(BSA), 3,3’,5,5’-tetramethylbenzidine (TMB), and aprotinin were obtained from Sigma (St. Louis, MO). Microtiter plates were from Greiner (The Netherlands). Antiphosphotyrosine IG2 was from Amersham (Buckinghamshire, UK); rabbit anti-mouse peroxidase was from Dakopatts (Denmark). Phenylmethylsulfonyl fluoride (PMSF) was supplied by Merck (Darmstadt, Germany). Biopsies from human astrocytomas and oligodendrogliomas were obtained by surgery at the Department of Neurosurgery, University Hospital Utrecht. Furthermore, biopsy specimens that were obtained in temporal or frontal lobectomies performed on epileptic patients were examined and considered to represent normal brain tissue. The tissues were pulverized in the frozen state using a dismembrator and stored at -80°C until use. In some experiments involving the development of the ELISA, cytosolic extracts from breast cancer specimens or rat spleen were used. Sample Preparation All procedures were performed at 4°C. The dismembrated tissues were thawed and resuspended in 3 vol of extraction buffer containing 20 mM Hepes (pH 7.2), 0.25 M sucrose, 1 mM MgClz, 1 mM EDTA, 10 mM dithiothreitol, 1 mM PMSF, and 5 pg/ml aprotinin. Cell debris and nuclei were removed by centrifugation at 8OOgfor 10 min. The supernatant was centrifuged at 48,000g for 1 h. The 48,000g supernatant was used for the assay of cytosolic PTK activity. PTK Assay by the Dot-Blot
Procedure
The procedure has been described in detail previously (13). In this method the PTK sample is incubated with the artificial substrate PGT and unlabeled ATP. After termination of the phosphorylation reaction by addition
of an excess of EDTA/EGTA, an aliquot of the reaction mixture is transferred to a polyvinylidene difluoride (PVDF) membrane. The extent of tyrosine phosphorylation is measured by probing the membrane with antiphosphotyrosine antibody followed by detection by the immunogold silver staining method. The signal is quantified by densitometry. The PTK activity is calculated using a standard of phosphorylated PGT (see below) and is expressed as picomoles of tyrosyl residues phosphorylated in PGT per minute per milligram of protein. The phosphorylated PGT standard was prepared using a cytosolic extract of a grade I/II astrocytoma as described (13). Phosphate incorporated in the PGT was quantitated by performing a microscale experiment in parallel with [32P]ATP under identical conditions and appeared to be 1.35 pmol of phosphate/pg of PGT. From the mean molecular weight of 28,500 Da, constituting 36 tyrosyl residues per PGT molecule, the incorporation of the standard was calculated to be 1.1 mmol/mol tyrosyl residues. Protein contents of cytosolic extracts were determined according to Lowry et al. (16). If contamination with blood was expected, the protein content was corrected by spectrophotometric determination of the hemoglobin concentration (17). We thereby assumed that the contribution of PTK activity from red blood cells was nonsignificant. Determination
of PTK Activity
by ELISA
Coating of microtiter plates. The substrate PGT was coated to the wells of a 96-well microtiter plate by adding 125 gl of PGT in phosphate-buffered saline (PBS: 0.14 M NaCl, 8.93 mM Na2HP04 * 12Hz0, 1.28 mM NaHzP04. 2Hz0). Plates were covered with Elisa plate sealer tape to prevent evaporation. After incubation for 16 h at 37°C the wells were washed once with 250 ~1 of washing buffer (0.1% Tween 20 in PBS). After the plates were emptied, they were dried for 2-3 h at 37°C. Coated plates
o-1 0
0.25
PGT (mg/ml) FIG. 1. Optimization of the PGT concentration. ELISA plates were coated with increasing concentrations of PGT in PBS. Phosphorylation was performed with 33 (O), 67 (V), and 125 (0) ng of sample protein from a cytosolic rat spleen extract. Phosphorylation and detection were performed under standard assay conditions.
600
ATP hM) FIG. 2. Influence of ATP concentration. PTK activities of breast cancer (0) and rat spleen (W cytosolic extracts were measured by ELISA using increasing concentrations of ATP. Phosphorylation and detection were performed under standard assay conditions.
NONRADIOACTIVE -_____
ASSAYS
FOR
PROTEIN-TYROSINE
were stored at 4°C in plastic bags. They could be stored for at least 3 months without any appreciable loss of performance. To determine the optimal concentration of PGT, plates were coated with increasing amounts of PGT. Saturating conditions were reached at a PGT concentration of 0.25 mg/ml (Fig. 1). In further experiments a concentration of 0.25 mg/ml PGT was used throughout. To quantitate the actual amount of bound PGT per well, we used the 32P-labeled PGT standard (see above) and coated it under standard conditions, assuming that the binding of [32P]PGT is comparable to that of PGT. From these experiments it appeared that under saturating conditions 0.75 pg PGT per well was bound. Blocking of residual binding sites with BSA appeared unnecessary, showing again that PGT is a good binding substrate for microtiter plates. Phosphorylation. The ELISA plates were allowed to reach room temperature before being opened. Immediately before use, samples were diluted in incubation buffer containing 50 mM Hepes, pH 7.2,lO mM MgC&, 2 InM MnC12, 0.1 mM EDTA, 0.4 mM EGTA, 0.5 mM dithiothreitol, 2.5 mM NaF, and 0.02% BSA; 50 ~1 was added in duplicate or triplicate to the wells of an ELISA plate. The plate was preincubated at 37°C for some minutes before phosphorylation was initiated by adding 50 ~1 of prewarmed ATP in 50 mM Hepes, pH 7.2, containing 10 mM MgClz, 2 mM MnClz and 200 pM Na,VO,. The optimal ATP con-
KINASE
ACTIVITY.~_~~. ___
203
centration was determined by constructing a substrate saturation curve, as shown in Fig. 2. KM values were in the range of 30-50 pM for PTKs from both breast cancer and rat spleen extracts; no substrate inhibition occurred at ATP concentrations > 1 mM. In further experiments a final concentration of 1 mM ATP was used throughout. After initiation of the phosphorylation reaction, the plates were covered and incubated at 37°C in an incubator. The reaction was terminated by emptying the plates and washing four times with 250 ~1 washing buffer. The reaction appeared to be linear up to at least 20-50 min, depending on the PTK activity (Fig. 3). In further experiments a phosphorylation time of 15 min was used throughout. Blank reactions from which either the PTK sample or the ATP was omitted were included. Optical density from either control was always ~0.1. Detection. After washing, the wells were incubated for 1 h at room temperature with 100 ~1 antiphosphotyrosine (dilution to be determined for each new batch) in PBS including 0.1% BSA. The plates were consecutively emptied carefully by patting them vigorously upside down on paper towels. The wells were washed four times with washing buffer. Next, 100 ~1 rabbit anti-mouse peroxidase conjugate, diluted l/300 in PBS and including 0.1% BSA and 1% gelatin, was added. After incubation for 1 h at room temperature the wells were washed four times with washing buffer, again each time carefully emptying the plates by patting them vigorously on paper towels. Signal development was initiated by adding 100 ~1 of color-developing reagent consisting of 0.51 mM TMB, 1.2% dimethyl sulfoxide, 0.004% HzOz, and 110 mM sodium acetate, pH 5.5. After 10 min the color development was stopped by the addition of 100 ~12 M H2S04. The optical density at 450 nm was measured with an ELISA reader. The specificity of the reaction was confirmed in control experiments in which the antiphosphotyrosine antibody
no interruption after
phosphorylation in wash buffer with 1st antibody
after 1st antibody in wash buffer with 2nd antibody after 2nd antibody in wash buffer
0
10
20
30
40
50
60
Time (min.) FIG. 3. Time course was performed with spleen extract as the minated by emptying were according to the
of the phosphorylation reaction. The PTK ELBA 10 (O), 33 (0), and 67 (V) ng of a cytosolic rat sample. The phosphorylation reaction was terthe wells at varying time points. Other conditions standard protocol.
L ~-.
O.D.
450 nrll
FIG. 4. Overnight interruption of ELISA procedure. The procedure was interrupted at the stages indicated and the plates were stored overnight at 4’C. The next morning the procedure was continued according to the standard protocol. The error bars indicate the SD of a triplicate assay.
204
RIJKSEN
was preincubated with 10 mM phenyl phosphate. This treatment completely abolished the signal, proving that it was specific for phosphotyrosyl residues. The procedure can be interrupted overnight at various stages, as shown in Fig. 4. First, the plates can be kept in the refrigerator in washing buffer after the phosphorylation step. Alternatively, the incubation with either the first or the second antibody can be prolonged overnight at 4°C without any loss of signal. However, storage in washing buffer after incubation with first or second antibody appeared to be detrimental.
RESULTS The ELISA procedure was used to measure PTK activity in crude cytosolic extracts of gliomas, breast cancers, and rat spleen. Variation of the amount of sample protein showed that the assay was linear with sample protein up to 100-200 ng per well for gliomas, to 200 ng for breast cancer, and to 50 ng for rat spleen extracts (Fig. 5). At higher amounts of sample the signal started to level off. The optical density at which deviation from linearity occurred varied among the various tissue types, but was rather consistent within samples of one tissue type. It appeared that the limitations of the assay system were not involved, but rather down-regulation of PTK activity
AND
STAAL
after initiation of the phosphorylation reaction with ATP (results not shown). Standard deviations of triplicate assays were <5%. Next, we compared the ELISA with a nonradioactive dot-blot assay, which we developed earlier (12, 13). Cytosolic fractions from benign breast tumors and malignant breast carcinomas were prepared and assayed for PTK activity both by ELISA and by dot-blot assay. The comparison of both assays is shown in Fig. 6. A linear correlation was obtained with a correlation coefficient of 0.92. Earlier, we showed that the dot-blot assay correlated well with the conventional radioactive filter-paper assay (12). The latter assay was used to measure PTK activities of normal human brain and gliomas (Table 1). Astrocytomas were divided into low-grade (grades I and II) and highgrade (grades III and IV) tumors. Low-grade astrocytomas showed, on average, higher activities (range, 163-812 pmol/min/mg) than normal adult brain (range, 48-285 pmol/min/mg). Also in the group of oligodendrogliomas, increased activities were found (range, 167-935 pmol/ min/mg). This group of gliomas consisted mainly of highgrade oligodendrogliomas (grades C and D). Remarkably, most PTK activities in the high-grade astrocytomas (range, 58-353 pmol/min/mg) were in the same range as those of normal brain (see also Ref. (18)).
DISCUSSION The present ELISA is a further development of the use of monoclonal antibodies to phosphotyrosyl residues to
OF 0 sample
protein
(rig/well)
FIG. 5. Variation of sample protein. The PTK ELISA was performed with increasing amounts of sample protein from cytosolic extracts from rat spleen (O), human gliomas (0, V), and breast cancer (*). Phosphorylation and detection were performed under standard assay conditions
/ 100
200 Dot-Blot
300 assay
400
500
600
(pmol/min.mg)
FIG. 6. Correlation between dot-blot activities of 15 cytosolic extracts of breast by dot-blot assay and ELISA. PTK pm01 * mini’ * mg-’ and as relative activity, coefficient is 0.92.
assay and ELISA. The PTK cancer biopsies were measured activities are expressed in respectively. The correlation
NONRADIOACTIVE
ASSAYS FOR PROTEIN-TYROSINE ______ KINASE ACTIVITY
determine PTK activity, providing a simple and nonlaborious alternative for existing methods. Previously we developed a nonradioactive dot-blot procedure based on the same principle (12). In this method phosphorylation of the PGT substrate occurs in the liquid phase. After termination of the reaction, an aliquot of the reaction mixture is transferred to a PVDF membrane. The extent of tyrosyl phosphorylation is measured by probing the membrane with antiphosphotyrosine antibody followed by detection with the immunogold silver staining procedure (13). This method appeared to be quantitative and at least as sensitive as the conventional assays using [“‘PIATP. The results of both methods correlated well. The main difference of the present ELISA from the dotblot method is the immobilization of the PGT substrate during the phosphorylation reaction. It is therefore hardly possible to compare the kinetics of the reaction in both systems. In the ELISA the PGT concentration cannot be optimized in terms of enzyme affinity, as the available well surface is limiting. Nevertheless, the KM for ATP is more or less the same in both systems. Remarkably, despite the limitation with respect to the PGT concentration, the ELISA is more sensitive than the dot-blot assay. The detection limit is four to five times lower in the former. This might be explained by the probability that all phosphorylated tyrosyl residues in the well are available for the antibody, whereas in the dot-blot assay the PGT is immobilized after phosphorylation, which means that part of the phosphotyrosine may be masked by an incorrect spatial orientation at the membrane surface. The present method is apparently also much more sensitive than earlier described ELISAs based on the same principle (14, 15), although comparison is rather difficult because of the use of purified enzyme preparations in the latter reports. The reason for this difference in sensitivity is not quite clear. However, one particular difference in the coating conditions with respect to the method of Farley et al. (15) appeared to be of critical importance. In Farley’s protocol 0.02% sodium azide was present during coating. In our hands the presence of azide was most det-
TABLE
~~
205
rimental to the ultimate result (not shown). In addition, the ATP concentration used by Farley et al. was far from optimal in our procedure. With respect, to the method of Lhzaro et al. (14), we needed more PGT to optimize the coating. Comparison, however, is not possible because these authors do not provide data about the actual amount of bound PGT. The correlation obtained between results from the ELISA and the dot-blot assay on the one hand (present study) and from the dot-blot assay and conventional radioactive filter-paper assay (12) on the other suggests that all three methods are interchangeable. However, the applicability of the ELISA is somewhat limited because the incorporation of phosphate cannot be quantitated directly and only relative activities are measured in one ELISA plate. The method is very suitable, however, for investigations involving enzyme regulation, screening for inhibitors, purification procedures, growth factor action, etc. Its use for other applications, which demand absolute and reproducible PTK activities, awaits the development of internal and external standards and controls (work in progress). For this reason, until now we have used the dot-blot assay to measure PTK activities in tumors and normal tissues. In oligodendrogliomas and low-grade astrocytomas, cytosolic tyrosine kinase activities were increased in accordance with findings in other kinds of tumors (19). Remarkably, the activity in high-grade astrocytomas was not different from that in normal brain, probably as a result of necrosis present in most of these tumors (18). Further studies of this kind will be facilit,ated tremendously by the development of simple PTK assays like the one presented here, provided that the day-to-day variation in signal development can be accounted for and the actual incorporation of phosphate can be quantitated directly.
ACKNOWLEDGMENTS The authors gratefully of S. S. Adriaansen-Slot.
acknowledge the expert technical assistance B. A. van Oirschot. and H. Schraag.
1
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PTK activity (pmol/min/mg protein)
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Median
Range
163 356 501 207
48-285 167-935 163-812 58-353
(n) (14) (7) (7) (11)
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