ANALYTICAL
BIOCHEMISTRY
196,245-251
(1991)
Automated Nonisotopic Assay for Protein-Tyrosine Kinase and Protein-Tyrosine Phosphatase Activities’ John Babcook,
Julian
Watts,
Ruedi Aebersold,
and Hermann
The Biomedical Research Centre, 2222 Health Sciences Mall, Vancouver, British Columbia V6T 123, Canada
Received
January
University
J. Ziltener’ of British
Columbia,
22,199l
A sensitive, automated, and nonisotopic assay for protein-tyrosine kinases and phosphatases has been developed. The assay uses commercially available antiphosphotyrosine monoclonal antibodies and the recently developed particle concentration immunofluorescence immunoassay technology. The assay is specific for phosphotyrosine residues, can be performed faster, and is at least loo-fold more sensitive than the current standard filter type radioassay. Myelin basic protein and a synthetic peptide corresponding to the autophosphorylation site of ~56~“’ performed equally well in the Myelin basic protein detection of p56 I& kinase activity. phosphorylated on tyrosine residues by ~56~~ was successfully used as substrate in the detection of phosphatase activity and vanadate or molybdate were shown to inhibit the phosphatase activity. The assay is particularly useful for the rapid detection of enzyme activities in column fractions from biochemical procedures steps and also for screening of large numbers of potential inhibitors or activators of protein-tyrosine kinases and phosphatases. 0 1991 Academic Press, Inc.
The control of cellular activation, growth and differentiation by mitogens and other cytokines is often achieved via alterations in the tyrosine phosphorylation states of regulatory proteins (1). Several mitogens such as epidermal growth factor (2), macrophage colony stimulating factor-l (3), and the steel locus product (4) all bind to cell surface receptors that are protein-tyrosine
1 This work was supported in part by the Science and Technology Development Fund of British Columbia, the Biomedical Research Centre and by Operating Grant MT-10868 from the Medical Research Council (MRC) of Canada to R.A. R.A. was the recipient of an MRC of Canada Scholarship. ‘To whom correspondence and reprint requests should be addressed. 0003-X97/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
enzymes specified by proto-oncogenes. However, for the vast majority of proto-oncogene-encoded receptor-like protein-tyrosine kinases, the ligands remain to be identified. Activation of the oncogenic potential of these and nonreceptor protein-tyrosine kinases such as ~60”” (5) and p561ck(6) results from mutations which lead to a high level of constitutive phosphorylating activity. Recent studies have also demonstrated the occurrence of receptor-like protein-tyrosine phosphatases, such as CD45 (7), LAR (8), and LRP (9), for which the interacting surface molecules are still not known. The rate of identification of new protein-tyrosine kinases and phosphatases has greatly overtaken their enzymological characterization. This reflects, in part, the low levels of expression of these regulatory enzymes, which necessitates the development of highly sensitive techniques for their assay. In the present paper, we describe a sensitive, rapid, and automated method for the detection of protein-tyrosine kinases and protein-tyrosine phosphatases. The method is based on the detection of phosphotyrosine residues using commercially available anti-phosphotyrosine monoclonal antibodies instead of radioisotopes as currently widely used. MATERIAL
AND
METHODS
Reagents and Buffers Bovine brain myelin basic protein (MBP): 2-[Nmorpholinolethanesulfonic acid (MES), sodium ortho3 Abbreviations used: AP, alkaline phosphatase; EDC, l-ethyl-3-(3dimethylaminopropyl)carbodiimide; MBP, myelin basic protein (bovine brain); PAP, potato acid phosphatase; PCFIA, particle concentration fluorescence immunoassay; mab, monoclonal antibody; MES, 2-[N-morpholinolethanesulfonic acid, BSA, bovine serum albumin; MOPS, 3-[hJ-morpholinolpropanesulfonic acid; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; NBS, newborn bovine serum; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinebis(ethanesulfonic acid); FITC, fluorescein isothiocyanate; RFU, relative fluorescence units.
245
246
BABCOOK ET AL.
vanadate (Na,VO,) , sodium molybdate (NqMO,) , polyglutamic acid-tyrosine random copolymer (4:1), and trisma base (Tris) were purchased from Sigma, (St. Louis, MO). ATP, bovine serum albumin (BSA), 3-[Nmorpholinolpropanesulfonic acid (MOPS), dithiothreito1 (DTT), phenylmethylsulfonyl fluoride (PMSF), potato acid phosphatase (PAP, 60 U/ml), and alkaline phosphatase (AP, 1000 U/ml) were obtained from Boehringer Mannheim (Laval, Quebec). Ethylenediaminetetraacetic acid (EDTA), Nonidet-P40 (NP-40), NaN,, NaCl, MnCl, , glycerol, and phosphoric acid were purchased from BDH (Toronto, Ontario). Newborn bovine serum (NBS) and 10X phosphate-buffered saline (PBS) were from Gibco (Grand Island, NY). 1,4-Piperazinebis(ethanesulfonic acid) (PIPES) was from Aldrich (Milwaukee, WI). 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide-HCl (EDC) was from Pierce (Rockford, IL). [Y-~~P]ATP (4500 Ci/mmol, 10 mCi/ml) and Ecolume scintillation fluid were from ICN (Mississauga, Ontario). Polyclonal goat anti-mouse FITC-conjugated antibodies were obtained from Calbiochem (San Diego, CA). Anti-phosphotyrosine monoclonal antibodies were purchased from ICN (Mississauga, Ontario) (PY20) (lo), Upstate Biotechnology Inc., (Lake Placid, NY) (4GlO) (ll), or Boehringer Mannheim, (Laval, Quebec) (lG2). The ~56’“~ used was a DEAE-Sepharose fraction of either sf9 cell lysates overexpressing recombinant ~56’“’ using a baculovirus expression system, or LSTRA cell membranes (a mouse cell line known to overexpress ~56’“~ (6)) (Watts et al., manuscript in preparation). The enzyme was eluted in 25 mM Tris HCl, pH 7.5, 0.1 M glycerol, 1 mM EDTA, 1 mM PMSF, 0.1% NP-40 at about 350 mM NaCl. Buffers were as follows: Buffer
A
Buffer
B
Buffer
C
Buffer Buffer
D E
20
mM Tris HCl (pH 7.5), 0.2 mM DTT, 0.5% BSA, 4 mM Na,VO, 1X PBS (pH 7.3), 2% NBS (0.2 pm filtered), 0.2% (w/v) NaN, 20 mM MOPS (pH 7.5), 0.2 mM DTT, 10 mM MnCl,, 0.1 mM Na,VO,, 100 mM NaCl, 5% glycerol 20 mM PIPES (pH 6.0), 0.5% BSA 20 mM Tris (pH 9.6), 0.5% BSA
Instrumentation
and Mode of Operation
Protein-tyrosine kinase and phosphatase assays were performed by particle concentration fluorescence immunoassay (PCFIA) using a screen machine manufactured by Idexx Corp. (Portland, ME). The assays were performed in filtration plates that have a standard 96-well microtiter format. The wells are conical and have a 0.2pm filter at their base. Below the filter is a sump to which vacuum can be applied. Substrate proteins or peptides were immobilized on 0.8 pm diameter polysty-
rene particles which were added to the plates. Prepared plates were then applied to the machine, where unbound reagents were washed through the filter and drained to waste. Washing cycles and the addition of reagents such as antibodies labeled with fluorochrome were automatically performed on all 96 samples in parallel. Fluorescent intensity was measured in each well. Up to 10 96-well plates could be assayed in one run using this system.
Substrate Immobilization
to Assay Particles
Fluoricon 0.8 pm diameter carboxyl-activatedpolystyrene particles (Idexx, Portland) were coupled with MBP or a peptide (lck peptide) containing the sequence KKGGRLIEDEYTARQGGKRLIEDEYTARQ. The peptide contained a tandem repeat of the site of the ~56’“’ autophosphorylation site (12) (residue 394) and two terminal lysine residues for coupling via NH, groups in a carbodiimide (EDC)-mediated coupling reaction. The peptide was synthesized using an Applied Biosystems Model 430A peptide synthesizer purified by reversephase HPLC (13) by Dr. Ian Clark-Lewis. Coupling was performed by mixing 8 ml of 0.1 M MES (pH 4.5), 1 ml of 0.8 pm Fluoricon carboxyl-activated polystyrene particles 5% (w/v), 1 mg of MBP or ~56’“~ peptide, and 5 mg of EDC. The contents were vortexed and then incubated at room temperature overnight. The particles were sedimented by centrifugation at 6500 rpm (6000g) for 10 min in a Sorval5C-5B centrifuge. The supernatant was aspirated and the pelleted particles were washed with 20 ml of buffer A. The particles were centrifuged and resuspended in 40 ml buffer A to a final concentration of 25 pg protein/ml and 0.125% (w/v) particles assuming 100% coupling yield. The washed particles were stored in buffer A containing 0.2% NaN, at 4°C. NaN, was removed by one washing step prior to use of the beads. Coupling of peptide or MBP via carboxyl groups was performed by using Fluoricon 0.8 pm diameter aminoactivated polystyrene particles with the above coupling protocol.
Protein-Tyrosine
Kinase Assay by PCFIA
Stock solutions of 0.1 M ATP and 1.0 M MnCl, in 20 Tris HCl (pH 7.5) were thawed daily. Two milliliters of substrate-coated particles was mixed with 20 ~1 of 0.1 M ATP and 40 ~1 of 1.0 M MnCl, stock so that the final concentration after addition of kinase sample was 0.5 mM ATP and 10 mM MnCl,. Twenty microliters of this substrate/particle suspension was added to each of the 96 wells of the filtration plate, either manually or automatically by the screen machine. To test protein-tyrosine kinase activity in sample fractions containing the protein-tyrosine kinase ~56~~‘, 20 ~1 of fractions or dilutions were transferred into the wells of the filtration plate containing assay particles, mM
ASSAY
FOR
PROTEIN-TYROSINE
KINASES
ATP and MnCl,. The plates were incubated at 37°C for 15 min (maximal phosphorylation was observed after 2 min, data not shown). The plates were then placed in the screen machine and the following sequential steps were performed automatically: (i) the wells were drained and washed with buffer B to remove kinase; (ii) 20 ~1 of anti-phosphotyrosine mab PY20 or 4GlO was added at 1 pg/ml in buffer A; (iii) plates were incubated for 10 min at room temperature; (iv) wells were drained and washed with buffer B; (v) 20 ~1 of polyclonal FITC conjugated goat anti-mouse antibody was added at 4 PugI ml in buffer B and incubated for 10 min; (vi) wells were drained and washed twice in buffer B and the amount of fluorescence in each well was then determined using excitation at 485 nm and emission at 535 nm and values were recorded as relative fluorescence units (RFU). To compare the sensitivity of the PCFIA kinase assay to the standard [y-32P]ATP-based kinase assay (filtertype assay), PCFIA conditions were used to model conditions of the radioassay. First, buffer A was replaced with a buffer that is used in the [32P]phosphate incorporation filter assay (buffer C), with the exception that glycerol was omitted as it was found to clog the 0.2-pm filter membrane in the filtration plate. Furthermore, the ATP concentration was altered to 50 pM final. Otherwise assays were performed as the standard PCFIA kinase assay.
[y-32P]ATP
Protein-Tyrosine
Kinase Assay
Radioactive [T-~~P]ATP based kinase assays were performed as previously described (14), with minor modifications to facilitate comparison with the PCFIA kinase assay. Under standard conditions assays were performed in buffer C at a final volume of 25 ~1. Serial dilutions of purified recombinant, ~56’“~ were transferred into microfuge tubes to give the same final concentrations as used in the PCFIA kinase method. To perform the assay, 15 ~1 of 1.67 mg/ml MBP (370 pglml final) was added in microfuge tubes on ice with 5 ~1 of p561ck sample and 5 ~1 of 250 pM ATP containing 1 j&i of [T-~~P]ATP. The mixture was incubated for 15 min at 37°C. The assay was terminated by spotting 20-~1 aliquots of the reaction mixture onto 1.5-cm2 pieces of Whatman P81 phosphocellulose paper. Substrate was allowed to absorb onto filters for 30 s and nonincorporated radioactivity was removed by 10 washes in 1% phosphoric acid over 2 h. The wet filters were transferred into 6-ml plastic scintillation vials containing 2 ml of Ecolume scintillation fluid and radioactivity was measured in a Packard scintillation counter. In modified versions of this assay different concentrations of MBP and specific activities of [T-~~P]ATP were used as indicated in the text.
AND
PROTEIN-TYROSINE
Protein-Tyrosine
247
PHOSPHATASES
Phosphatase
Assay by PCFIA
MBP-coated particles were phosphorylated in vitro by mixing 10 ml of MBP-coated particles at 25 pg MBP/ ml, 0.125% (w/v) beads in buffer A, 0.5 mM ATP, 10 mM MnCl,, and approximately 70 ng of purified recombinant ~56’“~. The mixture was incubated at 37°C for 30 min, centrifuged at 6000 g for 10 min, and then the supernatant aspirated. The pellet was washed twice in 15 ml of buffer D to remove residual ~56’“~ and was finally resuspended in 10 ml of buffer D (0.125% beads). PAP was diluted in buffer D to 10 U/ml (5 U/ml final in assay) and serially diluted in two-fold steps in a 96well plate. Twenty microliters of PAP sample was transferred to the filtration plate wells containing 20 ~1 of phosphorylated MBP particles and incubated at 37°C for 15 min. The assay was then completed as described for the PCFIA kinase assay after incubation with kinase. AP assays were performed similarly; AP was diluted to 10 U/ml (5 U/ml final) and serially diluted in buffer E, and the assay was continued as above for PAP.
Phosphatase
Inhibition
Sodium orthovanadate or sodium molybdate were diluted to 40 mM (20 mM final in assay) in buffer D containing 2 U/ml PAP. Inhibitor was serially diluted threefold in buffer D containing 2 U/ml PAP in a 96well plate. Twenty microliters of titrated inhibitors were then transferred to filtration plate wells containing 20 ~1 of p56’ck-phosphorylated MBP-coated particles followed by a 15-min incubation at 37°C. The plate was further processed as described in the PCFIA kinase method and the fluorescence in each well measured.
RESULTS
Protein-Tyrosine Kinase Assay by PCFIA MBP as a Substrate
and with
Anti-phosphotyrosine antibodies are widely used in the study of protein-tyrosine phosphorylation events. Here we examined whether these antibodies could be exploited for the measurement of protein-tyrosine kinase or phosphatase activities. Phosphorylation or dephosphorylation of tyrosine on immobilized substrates was measured by an immunological technique using the particle concentration fluorescence immunoassay (PCFIA). The enzyme sample was directly added into wells of the filtration plate and substrate (together with optimal concentrations of ATP (0.5 mM) and MnCl, (10 mM) was then added. Concentrations of ATP in the range from 2.5 to 0.05 mM and of MnCl, in the range from 25 to 2.5 mM were found to give satisfactory results (data not shown). Enzyme was removed from the substrate by an automatically performed filtration step and
248
BABCOOK
6000
ET
AL.
zB 6000
6000
01 o4
DILUTION
10
100
IO6
10°C
DILUTION
CFP~~‘~~(-FOLD)
104
Background
CFP~~‘~~(-FOLD)
FIG. 1. Assay for protein-tyrosine kinase. A: Serial dilutions of partially purified ~56~~ preparation were tested for protein-tyrosine kinase activity using the PCFIA and mab PY20 (open symbols) or the “P-incorporation filter assay (closed symbols). Parameters used were either optimized for PCFIA (U, n ) or optimized for the filter-type assay (0,O). B: Amounts of the p56”k-catalyzed immunoreactive phosphotyrosine residues were measured using either anti-phosphotyrosine mab PY20 (0) or mab 4GlO (O), under optimized PCFIA conditions.
the level of phosphotyrosine was quantitated using anti-phosphotyrosine mab and fluoresceinated second antibody. To assess the sensitivity of the assay, serial dilutions of a partially purified preparation of the protein-tyrosine kinase ~56 lck from LSTRA cells were tested in a kinase assay using PCFIA and the results compared to results obtained in the standard filter based assay that uses [32P]phosphate incorporation. Figure 1 shows the typical dose response curves of p56”’ kinase activity obtained by the two methods, each performed under conditions optimized for the respective method. The doseresponse curve obtained in the standard filter type assay was linear whereas the dose response of the PCFIA was nonlinear reflecting the complexity of this phosphotyrosine detection system. Using identical samples, the sensitivity of the PCFIA method was approximately loo-fold greater than the sensitivity of the standard filter-type radioassay. PCFIA was also performed using the conditions optimal for the filter-type assay and vice versa. There was about a 50% reduced signal but a similar titration endpoint when the PCFIA was performed with the conditions used for the isotopic assay. No signal was seen in the filter-type assay using PCFIA conditions (Fig. la). Two commercially available anti-phosphotyrosine antibodies PY20 and 4GlO resulted in very similar signals in the assay (Fig. lb) whereas a third mab lG2 was ineffective (data not shown).
Protein-Tyrosine Substrate A synthetic dues followed
Kinase Assay with a Synthetic peptide with two N-terminal lysine resiby two glycine residues and two repeated
sequences corresponding to the autophosphorylation site of ~56’“~ (tyrosine 394) was coupled to carboxyl activated microsphere particles using EDC as cross-linker. Figure 2 shows a comparison of PCFIA signal obtained, after incubation with titrated amounts of ~56”~ kinase, with substrate beads coated either with the ~56~” peptide or with MBP. Both substrates were coupled to carboxy- as well as to amino-activated beads. The signal obtained with peptide coupled to carboxy-activated beads was identical with signals obtained with either type of MBP beads, whereas ~56’~~ peptide coupled to amino-activated beads yielded an approximately 70% reduced signal.
2500-
1500
-I
‘p..-$...-a* ..q b3
i 500
' 100
.. .
1000
1 o4
DILUTION
...a
IO5
4
E.CKGlXUND
OF ~56’~
FIG. 2. Serial dilutions of ~56”’ were tested for protein-tyrosine kinase activity using: (i) the substrate MBP (solid line) coupled either to amino-activated microsphere particles via peptide carboxyl groups (0) or to carboxy-activated particles via peptide amino groups (0); (ii) a synthetic peptide corresponding to the p56*’ autophosphorylation site (Tyr 394) (dotted line) coupled either via peptide amino groups to carboxy-activated particles (0) or via peptide carboxyl groups to amino-activated microsphere particles (a).
ASSAY
Detection
FOR
PROTEIN-TYROSINE
Limits for Phosphotyrosine
KINASES
Residues by PCFIA
To determine the detection limit for protein-tyrosine residues by the PCFIA system, assay particles coupled with MBP were phosphorylated with [T-~~P]ATP of known specific activity using recombinant ~56’“~ and then serially diluted in nonphosphorylated MBPcoated beads. Twenty-microliter aliquots of the serial dilutions were either counted in a ,&scintillation counter or transferred into a filtration plate and phosphotyrosine residues measured by PCFIA. The last dilution point giving a PCFIA signal that was two standard deviations above background corresponded to less than lo-l5 mol of phosphotyrosine (data not shown).
AND
PROTEIN-TYROSINE 80000
249
PHOSPHATASES
A
LSTRA cells l/5 dilution
9 d ::
60000
0 0
20
1500
40
60
100
60
120
LSTRA cells l/l 50 dilution
B w
1000
Detection of ~56’~ Kinase Activity in Column Fractions Following Ion-Exchange Chromatography Protein-tyrosine kinase activity was detected in column fractions obtained after standard ion-exchange chromatography using PCFIA or the standard isotope filter assay. Extracts of 10’ LSTRA cells or lo8 YAC cell membranes were applied to DEAE-Sepharose ion-exchange columns and proteins eluted by an increasing salt gradient. Figure 3a shows the protein-tyrosine kinase activity profile obtained with 1:5 dilutions of column fractions of LSTRA cell extract measured by the standard 32P-incorporation filter-type assay, using the p561ckpeptide as substrate. Figure 3b shows the kinase profile obtained by testing a 1:150 dilution of the above fractions using the PCFIA and the same ~56’“~ peptide substrate. The profiles obtained by both methods matched very well. Fractions of the YAC cell extract separated by the above chromatographic method were also tested by both kinase assays. Whereas an identical kinase elution profile was obtained with the PCFIA method using a 1:5 dilution of the column fractions (Fig. 3c), no 32P-incorporation could be detected with the standard filter type assay (data not shown) even using undiluted column fractions. Protein-Tyrosine
Phosphatase Assay
MBP-coated particles phosphorylated on tyrosine by ~56”~ were used as substrates for the detection of phosphatases. Serially diluted amounts of phosphatases were incubated with phosphotyrosine MBP beads and residual phosphotyrosine residues measured by PCFIA. Potato acid phosphatase at 5 U/ml catalyzed the complete removal of phosphate from phosphotyrosine residues, whereas alkaline phosphatase was not able to cleave phosphate residues from this substrate (Fig. 4a). The phosphatase inhibitors molybdate and vanadate were then tested in the PCFIA phosphatase assay by incubating serial dilutions of each of the two inhibitors with a constant amount of potato acid phosphatase and phosphotyrosine MBP beads. Both inhibitors com-
0
k
.~~,...,.,~,.~‘,I~.,~.~, 0
20
1000
40
60
80
100
YAC Ceils l/5 dilution
C
600
0
T
I
,.,I
0
I
20
,.I,,
,I
40
I,,
60
120
I,
80
I,
I,,
100
/
120
FRACTION NUMBER FIG. 3. Protein-tyrosine kinase activity of column fractions obtained after ion-exchange chromatography of cell extracts obtained from LSTRA cells (A and B) or YAC cells (C). A: A 115 dilution of fractions obtained from 10’ LSTRA cells were tested using the [32P] filter assay. B: A l/150 dilution of the above fractions from LSTRA cells were tested using the PCFIA technique. C: A l/5 dilution of fractions obtained from 10’ YAC cells were tested using the PCFIA technique.
pletely inhibited the phosphatase (1U PAP/ml) at 20 mM concentration and had a similar dose-response down to 0.2 PM (Fig. 4b). DISCUSSION The present study describes the use of anti-phosphotyrosine antibodies and the PCFIA technique for the measurement of protein-tyrosine kinase and proteintyrosine phosphatase activities. Comparison with the standard 32Pmethod (Fig. 1) revealed a loo-fold higher sensitivity of the PCFIA technique. The dose-response curve was nonlinear but the calculation of absolute amounts of incorporated phosphate residues could be obtained from standard curves run in parallel in the same assay. This makes the PCFIA method less conve-
250
BABCOOK
ET
AL.
E3 A I
0 s
;: lNHl!3lTOR
FIG. 4.
A: Serial dilutions of alkaline using as a substrate MBP phosphorylated plate wells containing 1 U/ml of potato phosphatase activity.
-
P A
CONCENTRATION
(mM)
phosphatase (O), or potato acid phosphatase (Cl) were tested for protein-tyrosine phosphatase activity on tyrosine by p56 ‘lr . B: Serial dilutions of vanadate (0) or molybdate (0) were added to filtration acid phosphatase and as substrate phosphorylated MBP and were tested for inhibition of potato acid
nient for enzyme kinetics performed on purified kinases or phosphatases. The nonlinearity of the dose-response of the PCFIA is likely due to the nature of the detection system that employs polyclonal fluoresceinated antimouse antibodies that bind to the monoclonal antiphosphotyrosine antibody and to the potential quenching of the fluorescent signal that increases with increasing fluorescence present in the well. Because of this nonlinear dose-response, samples of unknown kinase activities, such as column fractions from biochemical purifications, should therefore always be tested at several (two or three) different dilutions. This will help to better estimate the relative amounts of kinase activities present in the different peaks. Three commercially available anti-phosphotyrosine mab’s were tested, of which PY20 and 4GlO were found to be nearly equal in performance (Fig. lb) whereas a third (lG2) did not result in a significant signal (data not shown). Free phosphotyrosine but not free phosphoserine or phosphothreonine was able to block binding of PY20 to phosphorylated MBP, indicating specificity of the assay for phosphotyrosine residues (data not shown). It is conceivable that the anti-phosphotyrosine antibodies might also recognize phosphoserine or phosphothreonine under certain circumstances, depending on the local environment of the phosphoamino acid. However the PCFIA is still more specific than the filter assay, particularly when protein substrates like MBP, casein, etc. are used and only partially purified tyrosine kinases are being tested. PCFIA-based protein-tyrosine kinase assay was also established with a synthetic peptide as substrate for the detection of p561ck. The substrate peptide corresponds to the autophosphorylation site of ~56’~~. Preliminary PCFIA experiments with a 15-amino acid residue pep-
tide gave poor signals (data not shown). A peptide was therefore specially designed for the PCFIA that had two N-terminal lysine residues allowing high efficiency coupling to microsphere particles, this was followed by two glycine residues that served as spacers, the next 11 amino acids corresponded to the p561ck autophosphorylation site followed by two glycine residues and a repeat of the p561ck autophosphorylation site. This peptide coupled via free amino groups with carboxy-activated microsphere particles yielded signals identical with the MBP-coated beads, whereas amino-activated microsphere beads coupled with the peptide via free carboxyl groups had a 70% reduced signal (Fig. 2). This indicates that peptides can be used successfully in this type of assay and opens the way for use of specific peptide sequences that are recognized by individual kinases or phosphatases. The fact that the orientation in which the peptide was coupled to microsphere particles affected the signal indicates that the assays will have to be assessed and optimized for each individual peptide substrate. Microsphere beads coupled with a polyglutamic acidtyrosine random copolymer as substrate were also used in some p561ck assays. Although the test worked well the signal was about 2- to 3-fold reduced compared to the ~56’“~ peptide coupled via the N-terminus, thus indicating that general substrates can also be used with this method (data not shown). Application of the PCFIA technique in the analysis of cell extracts separated by ion-exchange chromatography indicated that PCFIA compared favorably with the filter-type assay (Fig. 3) with respect to sensitivity and selection. LSTRA cells overexpress p561ck kinase activity and the presence of this enzyme is easily detected in the column fractions obtained from 10’ cells by PCFIA
ASSAY
FOR
PROTEIN-TYROSINE
KINASES
or the filter assay using synthetic peptide as substrate. PCFIA resolved the peak seen at fractions 76-90 into two peaks that is less well seen with the filter assay. These two peaks might reflect the presence of isoenzymes or differentially phosphorylated forms of ~56’“~. However, a small peak observed at positions 35-38 with the filter assay could not be seen with the PCFIA technique, this activity was likely due to the presence of a serineithreonine kinase in the extracts that was able to phosphorylate the threonine residues present in the peptide substrate used in the assay. YAC cells do not have elevated ~56’“~ levels and the protein-tyrosine kinase activity in column fractions obtained from 10’ YAC cells (lo-fold less than used in the above experiment with LSTRA cell extract) could not be detected with the filter type assay but was still easily detected with PCFIA (Fig. 3~). The increased sensitivity of this assay might therefore greatly facilitate the detection and analysis of novel kinases. Protein-tyrosine phosphatases are currently being discovered at an increasing rate, largely by recombinant techniques on the basis of a phosphatase consensus sequence (15). The PCFIA technique used for the detection of kinases was easily adapted to the measurements of phosphatases as shown with the example of phosphorylated MBP used as substrate and potato acid phosphatase, an enzyme that is known to have a very broad specificity. No activity for alkaline phosphatase, an enzyme with more restricted specificity than potato acid phosphatase, could be detected under the conditions used (Fig. 4a). These results demonstrate the feasibility of phosphatase assays using the PCFIA technique. The present data however do not allow one to draw a conclusion on the sensitivity of the method. Again it will be more complicated to perform accurate measurements of enzyme kinetics, because of the nonlinear dose-response curve and the fact that this method measures a decrease in fluorescence signal, whereas the standard 32P assay monitors release of radioisotopes. Molybdate and vanadate both inhibitors of phosphatases were able to block the activity of potato acid phosphatase with similar dose-response curves (Fig. 3b). The use of the PCFIA for the screening of potential inhibitors or activators of kinases and phosphatases might well be one of the biggest assets of this method. Since 10 plates (960 sample wells) can be automatically screened per machine run in approximately 4 h, the rapid screening of large numbers of substances that might specifically interact with these enzymes can be performed. Tyrosine phosphorylation and dephosphorylation have been identified as key events in many cellular signaling steps. Numerous methods for the measurement of the kinases and phosphatases involved have been proposed and are used in the different laboratories. The
AND
PROTEIN-TYROSINE
251
PHOSPHATASES
assay described in this paper is easily applicable for measurements of protein-tyrosine kinases and phosphatases. The primary limitations of the assay are the nonlinear dose-response curves and the fact that the machine processing the filtration plates currently does not allow accurate temperature control which both make measurements of enzyme kinetics more difficult and necessitate the inclusion of proper kinase and phosphatase standard preparations in the PCFIA runs. Nevertheless it has the advantage over conventional assays, of nearly fully automated performance, rapidity, at least loo-fold increased sensitivity and does not involve the use of radioisotopes. The increased sensitivity should allow the detection and facilitate purification of novel kinases and phosphatases, and the high volume capacity of this method should allow the identification of substances that might inhibit or stimulate these enzymes. ACKNOWLEDGMENTS The authors thank Dr. Steve Pelech for the critical reading of the manuscript. We thank Dr. Ian Clark-Lewis for the synthesis and purification of the peptide as well as his advise in its design. We further acknowledge the technical assistance of Carol Kubanek, Philip Owen, and Jan Scheuer for assistance in preparation of the manuscript.
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N. Engl. Sci.
14,
404-407. 13. Clark-Lewis, I., Aebersold, R., Ziltener, H. J., Schrader, J. W., Hood, L. E., and Kent, S. B. H. (1986) Science 231,134-139. 14. Sanghera, J. S., Paddon, H. B., Bader, (1990) J. Biol. Chem. 265, 52-57. 15. Jirik, F. R., Janzen, N. M., Melhado, (1990) FEBS Lett. 273, 239-242.
S. A., and Pelech, I. G., and Harder,
S. L. K. W.
BIOCHEMISTRY
196,245-251
(1991)
Automated Nonisotopic Assay for Protein-Tyrosine Kinase and Protein-Tyrosine Phosphatase Activities’ John Babcook,
Julian
Watts,
Ruedi Aebersold,
and Hermann
The Biomedical Research Centre, 2222 Health Sciences Mall, Vancouver, British Columbia V6T 123, Canada
Received
January
University
J. Ziltener’ of British
Columbia,
22,199l
A sensitive, automated, and nonisotopic assay for protein-tyrosine kinases and phosphatases has been developed. The assay uses commercially available antiphosphotyrosine monoclonal antibodies and the recently developed particle concentration immunofluorescence immunoassay technology. The assay is specific for phosphotyrosine residues, can be performed faster, and is at least loo-fold more sensitive than the current standard filter type radioassay. Myelin basic protein and a synthetic peptide corresponding to the autophosphorylation site of ~56~“’ performed equally well in the Myelin basic protein detection of p56 I& kinase activity. phosphorylated on tyrosine residues by ~56~~ was successfully used as substrate in the detection of phosphatase activity and vanadate or molybdate were shown to inhibit the phosphatase activity. The assay is particularly useful for the rapid detection of enzyme activities in column fractions from biochemical procedures steps and also for screening of large numbers of potential inhibitors or activators of protein-tyrosine kinases and phosphatases. 0 1991 Academic Press, Inc.
The control of cellular activation, growth and differentiation by mitogens and other cytokines is often achieved via alterations in the tyrosine phosphorylation states of regulatory proteins (1). Several mitogens such as epidermal growth factor (2), macrophage colony stimulating factor-l (3), and the steel locus product (4) all bind to cell surface receptors that are protein-tyrosine
1 This work was supported in part by the Science and Technology Development Fund of British Columbia, the Biomedical Research Centre and by Operating Grant MT-10868 from the Medical Research Council (MRC) of Canada to R.A. R.A. was the recipient of an MRC of Canada Scholarship. ‘To whom correspondence and reprint requests should be addressed. 0003-X97/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
enzymes specified by proto-oncogenes. However, for the vast majority of proto-oncogene-encoded receptor-like protein-tyrosine kinases, the ligands remain to be identified. Activation of the oncogenic potential of these and nonreceptor protein-tyrosine kinases such as ~60”” (5) and p561ck(6) results from mutations which lead to a high level of constitutive phosphorylating activity. Recent studies have also demonstrated the occurrence of receptor-like protein-tyrosine phosphatases, such as CD45 (7), LAR (8), and LRP (9), for which the interacting surface molecules are still not known. The rate of identification of new protein-tyrosine kinases and phosphatases has greatly overtaken their enzymological characterization. This reflects, in part, the low levels of expression of these regulatory enzymes, which necessitates the development of highly sensitive techniques for their assay. In the present paper, we describe a sensitive, rapid, and automated method for the detection of protein-tyrosine kinases and protein-tyrosine phosphatases. The method is based on the detection of phosphotyrosine residues using commercially available anti-phosphotyrosine monoclonal antibodies instead of radioisotopes as currently widely used. MATERIAL
AND
METHODS
Reagents and Buffers Bovine brain myelin basic protein (MBP): 2-[Nmorpholinolethanesulfonic acid (MES), sodium ortho3 Abbreviations used: AP, alkaline phosphatase; EDC, l-ethyl-3-(3dimethylaminopropyl)carbodiimide; MBP, myelin basic protein (bovine brain); PAP, potato acid phosphatase; PCFIA, particle concentration fluorescence immunoassay; mab, monoclonal antibody; MES, 2-[N-morpholinolethanesulfonic acid, BSA, bovine serum albumin; MOPS, 3-[hJ-morpholinolpropanesulfonic acid; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; NBS, newborn bovine serum; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinebis(ethanesulfonic acid); FITC, fluorescein isothiocyanate; RFU, relative fluorescence units.
245
246
BABCOOK ET AL.
vanadate (Na,VO,) , sodium molybdate (NqMO,) , polyglutamic acid-tyrosine random copolymer (4:1), and trisma base (Tris) were purchased from Sigma, (St. Louis, MO). ATP, bovine serum albumin (BSA), 3-[Nmorpholinolpropanesulfonic acid (MOPS), dithiothreito1 (DTT), phenylmethylsulfonyl fluoride (PMSF), potato acid phosphatase (PAP, 60 U/ml), and alkaline phosphatase (AP, 1000 U/ml) were obtained from Boehringer Mannheim (Laval, Quebec). Ethylenediaminetetraacetic acid (EDTA), Nonidet-P40 (NP-40), NaN,, NaCl, MnCl, , glycerol, and phosphoric acid were purchased from BDH (Toronto, Ontario). Newborn bovine serum (NBS) and 10X phosphate-buffered saline (PBS) were from Gibco (Grand Island, NY). 1,4-Piperazinebis(ethanesulfonic acid) (PIPES) was from Aldrich (Milwaukee, WI). 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide-HCl (EDC) was from Pierce (Rockford, IL). [Y-~~P]ATP (4500 Ci/mmol, 10 mCi/ml) and Ecolume scintillation fluid were from ICN (Mississauga, Ontario). Polyclonal goat anti-mouse FITC-conjugated antibodies were obtained from Calbiochem (San Diego, CA). Anti-phosphotyrosine monoclonal antibodies were purchased from ICN (Mississauga, Ontario) (PY20) (lo), Upstate Biotechnology Inc., (Lake Placid, NY) (4GlO) (ll), or Boehringer Mannheim, (Laval, Quebec) (lG2). The ~56’“~ used was a DEAE-Sepharose fraction of either sf9 cell lysates overexpressing recombinant ~56’“’ using a baculovirus expression system, or LSTRA cell membranes (a mouse cell line known to overexpress ~56’“~ (6)) (Watts et al., manuscript in preparation). The enzyme was eluted in 25 mM Tris HCl, pH 7.5, 0.1 M glycerol, 1 mM EDTA, 1 mM PMSF, 0.1% NP-40 at about 350 mM NaCl. Buffers were as follows: Buffer
A
Buffer
B
Buffer
C
Buffer Buffer
D E
20
mM Tris HCl (pH 7.5), 0.2 mM DTT, 0.5% BSA, 4 mM Na,VO, 1X PBS (pH 7.3), 2% NBS (0.2 pm filtered), 0.2% (w/v) NaN, 20 mM MOPS (pH 7.5), 0.2 mM DTT, 10 mM MnCl,, 0.1 mM Na,VO,, 100 mM NaCl, 5% glycerol 20 mM PIPES (pH 6.0), 0.5% BSA 20 mM Tris (pH 9.6), 0.5% BSA
Instrumentation
and Mode of Operation
Protein-tyrosine kinase and phosphatase assays were performed by particle concentration fluorescence immunoassay (PCFIA) using a screen machine manufactured by Idexx Corp. (Portland, ME). The assays were performed in filtration plates that have a standard 96-well microtiter format. The wells are conical and have a 0.2pm filter at their base. Below the filter is a sump to which vacuum can be applied. Substrate proteins or peptides were immobilized on 0.8 pm diameter polysty-
rene particles which were added to the plates. Prepared plates were then applied to the machine, where unbound reagents were washed through the filter and drained to waste. Washing cycles and the addition of reagents such as antibodies labeled with fluorochrome were automatically performed on all 96 samples in parallel. Fluorescent intensity was measured in each well. Up to 10 96-well plates could be assayed in one run using this system.
Substrate Immobilization
to Assay Particles
Fluoricon 0.8 pm diameter carboxyl-activatedpolystyrene particles (Idexx, Portland) were coupled with MBP or a peptide (lck peptide) containing the sequence KKGGRLIEDEYTARQGGKRLIEDEYTARQ. The peptide contained a tandem repeat of the site of the ~56’“’ autophosphorylation site (12) (residue 394) and two terminal lysine residues for coupling via NH, groups in a carbodiimide (EDC)-mediated coupling reaction. The peptide was synthesized using an Applied Biosystems Model 430A peptide synthesizer purified by reversephase HPLC (13) by Dr. Ian Clark-Lewis. Coupling was performed by mixing 8 ml of 0.1 M MES (pH 4.5), 1 ml of 0.8 pm Fluoricon carboxyl-activated polystyrene particles 5% (w/v), 1 mg of MBP or ~56’“~ peptide, and 5 mg of EDC. The contents were vortexed and then incubated at room temperature overnight. The particles were sedimented by centrifugation at 6500 rpm (6000g) for 10 min in a Sorval5C-5B centrifuge. The supernatant was aspirated and the pelleted particles were washed with 20 ml of buffer A. The particles were centrifuged and resuspended in 40 ml buffer A to a final concentration of 25 pg protein/ml and 0.125% (w/v) particles assuming 100% coupling yield. The washed particles were stored in buffer A containing 0.2% NaN, at 4°C. NaN, was removed by one washing step prior to use of the beads. Coupling of peptide or MBP via carboxyl groups was performed by using Fluoricon 0.8 pm diameter aminoactivated polystyrene particles with the above coupling protocol.
Protein-Tyrosine
Kinase Assay by PCFIA
Stock solutions of 0.1 M ATP and 1.0 M MnCl, in 20 Tris HCl (pH 7.5) were thawed daily. Two milliliters of substrate-coated particles was mixed with 20 ~1 of 0.1 M ATP and 40 ~1 of 1.0 M MnCl, stock so that the final concentration after addition of kinase sample was 0.5 mM ATP and 10 mM MnCl,. Twenty microliters of this substrate/particle suspension was added to each of the 96 wells of the filtration plate, either manually or automatically by the screen machine. To test protein-tyrosine kinase activity in sample fractions containing the protein-tyrosine kinase ~56~~‘, 20 ~1 of fractions or dilutions were transferred into the wells of the filtration plate containing assay particles, mM
ASSAY
FOR
PROTEIN-TYROSINE
KINASES
ATP and MnCl,. The plates were incubated at 37°C for 15 min (maximal phosphorylation was observed after 2 min, data not shown). The plates were then placed in the screen machine and the following sequential steps were performed automatically: (i) the wells were drained and washed with buffer B to remove kinase; (ii) 20 ~1 of anti-phosphotyrosine mab PY20 or 4GlO was added at 1 pg/ml in buffer A; (iii) plates were incubated for 10 min at room temperature; (iv) wells were drained and washed with buffer B; (v) 20 ~1 of polyclonal FITC conjugated goat anti-mouse antibody was added at 4 PugI ml in buffer B and incubated for 10 min; (vi) wells were drained and washed twice in buffer B and the amount of fluorescence in each well was then determined using excitation at 485 nm and emission at 535 nm and values were recorded as relative fluorescence units (RFU). To compare the sensitivity of the PCFIA kinase assay to the standard [y-32P]ATP-based kinase assay (filtertype assay), PCFIA conditions were used to model conditions of the radioassay. First, buffer A was replaced with a buffer that is used in the [32P]phosphate incorporation filter assay (buffer C), with the exception that glycerol was omitted as it was found to clog the 0.2-pm filter membrane in the filtration plate. Furthermore, the ATP concentration was altered to 50 pM final. Otherwise assays were performed as the standard PCFIA kinase assay.
[y-32P]ATP
Protein-Tyrosine
Kinase Assay
Radioactive [T-~~P]ATP based kinase assays were performed as previously described (14), with minor modifications to facilitate comparison with the PCFIA kinase assay. Under standard conditions assays were performed in buffer C at a final volume of 25 ~1. Serial dilutions of purified recombinant, ~56’“~ were transferred into microfuge tubes to give the same final concentrations as used in the PCFIA kinase method. To perform the assay, 15 ~1 of 1.67 mg/ml MBP (370 pglml final) was added in microfuge tubes on ice with 5 ~1 of p561ck sample and 5 ~1 of 250 pM ATP containing 1 j&i of [T-~~P]ATP. The mixture was incubated for 15 min at 37°C. The assay was terminated by spotting 20-~1 aliquots of the reaction mixture onto 1.5-cm2 pieces of Whatman P81 phosphocellulose paper. Substrate was allowed to absorb onto filters for 30 s and nonincorporated radioactivity was removed by 10 washes in 1% phosphoric acid over 2 h. The wet filters were transferred into 6-ml plastic scintillation vials containing 2 ml of Ecolume scintillation fluid and radioactivity was measured in a Packard scintillation counter. In modified versions of this assay different concentrations of MBP and specific activities of [T-~~P]ATP were used as indicated in the text.
AND
PROTEIN-TYROSINE
Protein-Tyrosine
247
PHOSPHATASES
Phosphatase
Assay by PCFIA
MBP-coated particles were phosphorylated in vitro by mixing 10 ml of MBP-coated particles at 25 pg MBP/ ml, 0.125% (w/v) beads in buffer A, 0.5 mM ATP, 10 mM MnCl,, and approximately 70 ng of purified recombinant ~56’“~. The mixture was incubated at 37°C for 30 min, centrifuged at 6000 g for 10 min, and then the supernatant aspirated. The pellet was washed twice in 15 ml of buffer D to remove residual ~56’“~ and was finally resuspended in 10 ml of buffer D (0.125% beads). PAP was diluted in buffer D to 10 U/ml (5 U/ml final in assay) and serially diluted in two-fold steps in a 96well plate. Twenty microliters of PAP sample was transferred to the filtration plate wells containing 20 ~1 of phosphorylated MBP particles and incubated at 37°C for 15 min. The assay was then completed as described for the PCFIA kinase assay after incubation with kinase. AP assays were performed similarly; AP was diluted to 10 U/ml (5 U/ml final) and serially diluted in buffer E, and the assay was continued as above for PAP.
Phosphatase
Inhibition
Sodium orthovanadate or sodium molybdate were diluted to 40 mM (20 mM final in assay) in buffer D containing 2 U/ml PAP. Inhibitor was serially diluted threefold in buffer D containing 2 U/ml PAP in a 96well plate. Twenty microliters of titrated inhibitors were then transferred to filtration plate wells containing 20 ~1 of p56’ck-phosphorylated MBP-coated particles followed by a 15-min incubation at 37°C. The plate was further processed as described in the PCFIA kinase method and the fluorescence in each well measured.
RESULTS
Protein-Tyrosine Kinase Assay by PCFIA MBP as a Substrate
and with
Anti-phosphotyrosine antibodies are widely used in the study of protein-tyrosine phosphorylation events. Here we examined whether these antibodies could be exploited for the measurement of protein-tyrosine kinase or phosphatase activities. Phosphorylation or dephosphorylation of tyrosine on immobilized substrates was measured by an immunological technique using the particle concentration fluorescence immunoassay (PCFIA). The enzyme sample was directly added into wells of the filtration plate and substrate (together with optimal concentrations of ATP (0.5 mM) and MnCl, (10 mM) was then added. Concentrations of ATP in the range from 2.5 to 0.05 mM and of MnCl, in the range from 25 to 2.5 mM were found to give satisfactory results (data not shown). Enzyme was removed from the substrate by an automatically performed filtration step and
248
BABCOOK
6000
ET
AL.
zB 6000
6000
01 o4
DILUTION
10
100
IO6
10°C
DILUTION
CFP~~‘~~(-FOLD)
104
Background
CFP~~‘~~(-FOLD)
FIG. 1. Assay for protein-tyrosine kinase. A: Serial dilutions of partially purified ~56~~ preparation were tested for protein-tyrosine kinase activity using the PCFIA and mab PY20 (open symbols) or the “P-incorporation filter assay (closed symbols). Parameters used were either optimized for PCFIA (U, n ) or optimized for the filter-type assay (0,O). B: Amounts of the p56”k-catalyzed immunoreactive phosphotyrosine residues were measured using either anti-phosphotyrosine mab PY20 (0) or mab 4GlO (O), under optimized PCFIA conditions.
the level of phosphotyrosine was quantitated using anti-phosphotyrosine mab and fluoresceinated second antibody. To assess the sensitivity of the assay, serial dilutions of a partially purified preparation of the protein-tyrosine kinase ~56 lck from LSTRA cells were tested in a kinase assay using PCFIA and the results compared to results obtained in the standard filter based assay that uses [32P]phosphate incorporation. Figure 1 shows the typical dose response curves of p56”’ kinase activity obtained by the two methods, each performed under conditions optimized for the respective method. The doseresponse curve obtained in the standard filter type assay was linear whereas the dose response of the PCFIA was nonlinear reflecting the complexity of this phosphotyrosine detection system. Using identical samples, the sensitivity of the PCFIA method was approximately loo-fold greater than the sensitivity of the standard filter-type radioassay. PCFIA was also performed using the conditions optimal for the filter-type assay and vice versa. There was about a 50% reduced signal but a similar titration endpoint when the PCFIA was performed with the conditions used for the isotopic assay. No signal was seen in the filter-type assay using PCFIA conditions (Fig. la). Two commercially available anti-phosphotyrosine antibodies PY20 and 4GlO resulted in very similar signals in the assay (Fig. lb) whereas a third mab lG2 was ineffective (data not shown).
Protein-Tyrosine Substrate A synthetic dues followed
Kinase Assay with a Synthetic peptide with two N-terminal lysine resiby two glycine residues and two repeated
sequences corresponding to the autophosphorylation site of ~56’“~ (tyrosine 394) was coupled to carboxyl activated microsphere particles using EDC as cross-linker. Figure 2 shows a comparison of PCFIA signal obtained, after incubation with titrated amounts of ~56”~ kinase, with substrate beads coated either with the ~56~” peptide or with MBP. Both substrates were coupled to carboxy- as well as to amino-activated beads. The signal obtained with peptide coupled to carboxy-activated beads was identical with signals obtained with either type of MBP beads, whereas ~56’~~ peptide coupled to amino-activated beads yielded an approximately 70% reduced signal.
2500-
1500
-I
‘p..-$...-a* ..q b3
i 500
' 100
.. .
1000
1 o4
DILUTION
...a
IO5
4
E.CKGlXUND
OF ~56’~
FIG. 2. Serial dilutions of ~56”’ were tested for protein-tyrosine kinase activity using: (i) the substrate MBP (solid line) coupled either to amino-activated microsphere particles via peptide carboxyl groups (0) or to carboxy-activated particles via peptide amino groups (0); (ii) a synthetic peptide corresponding to the p56*’ autophosphorylation site (Tyr 394) (dotted line) coupled either via peptide amino groups to carboxy-activated particles (0) or via peptide carboxyl groups to amino-activated microsphere particles (a).
ASSAY
Detection
FOR
PROTEIN-TYROSINE
Limits for Phosphotyrosine
KINASES
Residues by PCFIA
To determine the detection limit for protein-tyrosine residues by the PCFIA system, assay particles coupled with MBP were phosphorylated with [T-~~P]ATP of known specific activity using recombinant ~56’“~ and then serially diluted in nonphosphorylated MBPcoated beads. Twenty-microliter aliquots of the serial dilutions were either counted in a ,&scintillation counter or transferred into a filtration plate and phosphotyrosine residues measured by PCFIA. The last dilution point giving a PCFIA signal that was two standard deviations above background corresponded to less than lo-l5 mol of phosphotyrosine (data not shown).
AND
PROTEIN-TYROSINE 80000
249
PHOSPHATASES
A
LSTRA cells l/5 dilution
9 d ::
60000
0 0
20
1500
40
60
100
60
120
LSTRA cells l/l 50 dilution
B w
1000
Detection of ~56’~ Kinase Activity in Column Fractions Following Ion-Exchange Chromatography Protein-tyrosine kinase activity was detected in column fractions obtained after standard ion-exchange chromatography using PCFIA or the standard isotope filter assay. Extracts of 10’ LSTRA cells or lo8 YAC cell membranes were applied to DEAE-Sepharose ion-exchange columns and proteins eluted by an increasing salt gradient. Figure 3a shows the protein-tyrosine kinase activity profile obtained with 1:5 dilutions of column fractions of LSTRA cell extract measured by the standard 32P-incorporation filter-type assay, using the p561ckpeptide as substrate. Figure 3b shows the kinase profile obtained by testing a 1:150 dilution of the above fractions using the PCFIA and the same ~56’“~ peptide substrate. The profiles obtained by both methods matched very well. Fractions of the YAC cell extract separated by the above chromatographic method were also tested by both kinase assays. Whereas an identical kinase elution profile was obtained with the PCFIA method using a 1:5 dilution of the column fractions (Fig. 3c), no 32P-incorporation could be detected with the standard filter type assay (data not shown) even using undiluted column fractions. Protein-Tyrosine
Phosphatase Assay
MBP-coated particles phosphorylated on tyrosine by ~56”~ were used as substrates for the detection of phosphatases. Serially diluted amounts of phosphatases were incubated with phosphotyrosine MBP beads and residual phosphotyrosine residues measured by PCFIA. Potato acid phosphatase at 5 U/ml catalyzed the complete removal of phosphate from phosphotyrosine residues, whereas alkaline phosphatase was not able to cleave phosphate residues from this substrate (Fig. 4a). The phosphatase inhibitors molybdate and vanadate were then tested in the PCFIA phosphatase assay by incubating serial dilutions of each of the two inhibitors with a constant amount of potato acid phosphatase and phosphotyrosine MBP beads. Both inhibitors com-
0
k
.~~,...,.,~,.~‘,I~.,~.~, 0
20
1000
40
60
80
100
YAC Ceils l/5 dilution
C
600
0
T
I
,.,I
0
I
20
,.I,,
,I
40
I,,
60
120
I,
80
I,
I,,
100
/
120
FRACTION NUMBER FIG. 3. Protein-tyrosine kinase activity of column fractions obtained after ion-exchange chromatography of cell extracts obtained from LSTRA cells (A and B) or YAC cells (C). A: A 115 dilution of fractions obtained from 10’ LSTRA cells were tested using the [32P] filter assay. B: A l/150 dilution of the above fractions from LSTRA cells were tested using the PCFIA technique. C: A l/5 dilution of fractions obtained from 10’ YAC cells were tested using the PCFIA technique.
pletely inhibited the phosphatase (1U PAP/ml) at 20 mM concentration and had a similar dose-response down to 0.2 PM (Fig. 4b). DISCUSSION The present study describes the use of anti-phosphotyrosine antibodies and the PCFIA technique for the measurement of protein-tyrosine kinase and proteintyrosine phosphatase activities. Comparison with the standard 32Pmethod (Fig. 1) revealed a loo-fold higher sensitivity of the PCFIA technique. The dose-response curve was nonlinear but the calculation of absolute amounts of incorporated phosphate residues could be obtained from standard curves run in parallel in the same assay. This makes the PCFIA method less conve-
250
BABCOOK
ET
AL.
E3 A I
0 s
;: lNHl!3lTOR
FIG. 4.
A: Serial dilutions of alkaline using as a substrate MBP phosphorylated plate wells containing 1 U/ml of potato phosphatase activity.
-
P A
CONCENTRATION
(mM)
phosphatase (O), or potato acid phosphatase (Cl) were tested for protein-tyrosine phosphatase activity on tyrosine by p56 ‘lr . B: Serial dilutions of vanadate (0) or molybdate (0) were added to filtration acid phosphatase and as substrate phosphorylated MBP and were tested for inhibition of potato acid
nient for enzyme kinetics performed on purified kinases or phosphatases. The nonlinearity of the dose-response of the PCFIA is likely due to the nature of the detection system that employs polyclonal fluoresceinated antimouse antibodies that bind to the monoclonal antiphosphotyrosine antibody and to the potential quenching of the fluorescent signal that increases with increasing fluorescence present in the well. Because of this nonlinear dose-response, samples of unknown kinase activities, such as column fractions from biochemical purifications, should therefore always be tested at several (two or three) different dilutions. This will help to better estimate the relative amounts of kinase activities present in the different peaks. Three commercially available anti-phosphotyrosine mab’s were tested, of which PY20 and 4GlO were found to be nearly equal in performance (Fig. lb) whereas a third (lG2) did not result in a significant signal (data not shown). Free phosphotyrosine but not free phosphoserine or phosphothreonine was able to block binding of PY20 to phosphorylated MBP, indicating specificity of the assay for phosphotyrosine residues (data not shown). It is conceivable that the anti-phosphotyrosine antibodies might also recognize phosphoserine or phosphothreonine under certain circumstances, depending on the local environment of the phosphoamino acid. However the PCFIA is still more specific than the filter assay, particularly when protein substrates like MBP, casein, etc. are used and only partially purified tyrosine kinases are being tested. PCFIA-based protein-tyrosine kinase assay was also established with a synthetic peptide as substrate for the detection of p561ck. The substrate peptide corresponds to the autophosphorylation site of ~56’~~. Preliminary PCFIA experiments with a 15-amino acid residue pep-
tide gave poor signals (data not shown). A peptide was therefore specially designed for the PCFIA that had two N-terminal lysine residues allowing high efficiency coupling to microsphere particles, this was followed by two glycine residues that served as spacers, the next 11 amino acids corresponded to the p561ck autophosphorylation site followed by two glycine residues and a repeat of the p561ck autophosphorylation site. This peptide coupled via free amino groups with carboxy-activated microsphere particles yielded signals identical with the MBP-coated beads, whereas amino-activated microsphere beads coupled with the peptide via free carboxyl groups had a 70% reduced signal (Fig. 2). This indicates that peptides can be used successfully in this type of assay and opens the way for use of specific peptide sequences that are recognized by individual kinases or phosphatases. The fact that the orientation in which the peptide was coupled to microsphere particles affected the signal indicates that the assays will have to be assessed and optimized for each individual peptide substrate. Microsphere beads coupled with a polyglutamic acidtyrosine random copolymer as substrate were also used in some p561ck assays. Although the test worked well the signal was about 2- to 3-fold reduced compared to the ~56’“~ peptide coupled via the N-terminus, thus indicating that general substrates can also be used with this method (data not shown). Application of the PCFIA technique in the analysis of cell extracts separated by ion-exchange chromatography indicated that PCFIA compared favorably with the filter-type assay (Fig. 3) with respect to sensitivity and selection. LSTRA cells overexpress p561ck kinase activity and the presence of this enzyme is easily detected in the column fractions obtained from 10’ cells by PCFIA
ASSAY
FOR
PROTEIN-TYROSINE
KINASES
or the filter assay using synthetic peptide as substrate. PCFIA resolved the peak seen at fractions 76-90 into two peaks that is less well seen with the filter assay. These two peaks might reflect the presence of isoenzymes or differentially phosphorylated forms of ~56’“~. However, a small peak observed at positions 35-38 with the filter assay could not be seen with the PCFIA technique, this activity was likely due to the presence of a serineithreonine kinase in the extracts that was able to phosphorylate the threonine residues present in the peptide substrate used in the assay. YAC cells do not have elevated ~56’“~ levels and the protein-tyrosine kinase activity in column fractions obtained from 10’ YAC cells (lo-fold less than used in the above experiment with LSTRA cell extract) could not be detected with the filter type assay but was still easily detected with PCFIA (Fig. 3~). The increased sensitivity of this assay might therefore greatly facilitate the detection and analysis of novel kinases. Protein-tyrosine phosphatases are currently being discovered at an increasing rate, largely by recombinant techniques on the basis of a phosphatase consensus sequence (15). The PCFIA technique used for the detection of kinases was easily adapted to the measurements of phosphatases as shown with the example of phosphorylated MBP used as substrate and potato acid phosphatase, an enzyme that is known to have a very broad specificity. No activity for alkaline phosphatase, an enzyme with more restricted specificity than potato acid phosphatase, could be detected under the conditions used (Fig. 4a). These results demonstrate the feasibility of phosphatase assays using the PCFIA technique. The present data however do not allow one to draw a conclusion on the sensitivity of the method. Again it will be more complicated to perform accurate measurements of enzyme kinetics, because of the nonlinear dose-response curve and the fact that this method measures a decrease in fluorescence signal, whereas the standard 32P assay monitors release of radioisotopes. Molybdate and vanadate both inhibitors of phosphatases were able to block the activity of potato acid phosphatase with similar dose-response curves (Fig. 3b). The use of the PCFIA for the screening of potential inhibitors or activators of kinases and phosphatases might well be one of the biggest assets of this method. Since 10 plates (960 sample wells) can be automatically screened per machine run in approximately 4 h, the rapid screening of large numbers of substances that might specifically interact with these enzymes can be performed. Tyrosine phosphorylation and dephosphorylation have been identified as key events in many cellular signaling steps. Numerous methods for the measurement of the kinases and phosphatases involved have been proposed and are used in the different laboratories. The
AND
PROTEIN-TYROSINE
251
PHOSPHATASES
assay described in this paper is easily applicable for measurements of protein-tyrosine kinases and phosphatases. The primary limitations of the assay are the nonlinear dose-response curves and the fact that the machine processing the filtration plates currently does not allow accurate temperature control which both make measurements of enzyme kinetics more difficult and necessitate the inclusion of proper kinase and phosphatase standard preparations in the PCFIA runs. Nevertheless it has the advantage over conventional assays, of nearly fully automated performance, rapidity, at least loo-fold increased sensitivity and does not involve the use of radioisotopes. The increased sensitivity should allow the detection and facilitate purification of novel kinases and phosphatases, and the high volume capacity of this method should allow the identification of substances that might inhibit or stimulate these enzymes. ACKNOWLEDGMENTS The authors thank Dr. Steve Pelech for the critical reading of the manuscript. We thank Dr. Ian Clark-Lewis for the synthesis and purification of the peptide as well as his advise in its design. We further acknowledge the technical assistance of Carol Kubanek, Philip Owen, and Jan Scheuer for assistance in preparation of the manuscript.
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H. (1982)
6. Marth,
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12-18. R. M.
(1985)
Cell43,393-404. 7. Tonks, Walsh,
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