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A solid-phase assay for the phosphorylation of proteins blotted on nitrocellulose membrane filters
ANALYTICAL
BIOCHEMISTRY
158,
130- 137 ( 1986)
A Solid-Phase Assay for the Phosphorylation of Proteins Blotted on Nitrocellulose Membrane Filters’ FLAVIA VALTORTA,**~ WERNER SCHIEBLER,* REINHARD JAHN,* BRUNOCECCARELLI,~.ANDPAULGREENGARD* *Laboratory of Molecular and Cellular Neuroscience, TDepartment of Medical Pharmacology, CNR of Peripheral Neuropathies and Neuromuscular
The Rockefeller University, New York, New York 10021, Center of Cytopharmacology, and Center for the Study Diseases, University of Milano, 20129 Milano. Italy
and
Received February 20. 1986 A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters, and the blotted polypeptides are phosphorylated with the catalytic subunit of cyclic AMP (adenosine 3’:5’-monophosphate)-dependent protein kinase. The method was developed for the assay of dephosphosynapsin I, but it has also proven suitable for the phosphorylation of other proteins. The patterns of phosphorylation of tissue samples phosphorylated using the new method are similar to those obtained using the conventional test tube assay.Once phosphorylated, the adsorbed proteins can be digested with proteases and subjected to phosphopeptide mapping. The phosphorylated blotted proteins can also be analyzed by overlay techniques for the immunological detection of polypeptides. 0 1986 Academic press. hc. KEY WORDS: protein phosphorylation; protein blotting; nitrocellulose; protein kinases; neurochemistry; synapsin I.
An increasing body of experimental evidence indicates that both in neural and in nonneural cells protein phosphorylation plays an important role in the modulation of physiological processes. Responses to a large variety of extracellular signals are mediated by second messengers (Ca’+, cyclic nucleotides) which activate specific protein kinases, which in turn regulate the state of phosphorylation of various substrate proteins (1,2). Two different experimental approaches for the study of protein phosphorylation are currently available: (i) Tracing the incorporation of 32P from prelabeled endogenous ATP into proteins (in vivo labeling). With this technique the incorporation of phosphate into proteins in intact cell preparations can be compared under different physiological conditions. It is not suitable,
however, for measuring the degree of phosphorylation of proteins, since neither the specific radioactivity of endogenous ATP nor the turnover rates of the different components participating in the process can be established. (ii) Studying the state of protein phosphorylation by measuring the amount of radioactive phosphate that can be incorporated into extracted proteins in the presence of exogenous kinases and radioactive ATP of known specific activity (back phosphorylation). A major limitation of this technique derives from the extraction procedure, which must have the characteristics of inactivating endogenous kinases and phosphatases, without interfering with the exogenous kinases added subsequently to the extract. In an attempt to circumvent this limitation, we have developed a novel technique for the phosphorylation of proteins, based on their immobilization on nitrocellulose after electrophoretic separation. This technique was developed to assay the state of phosphorylation
’ This work was supported by the United States Environmental Protection Agency under Assistance Agreement CR-8 10608-01 (P.G.) and by a M.D.A. grant to B.C. 0003-2697186 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights oi reproduction in any form reserved.
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of synapsin I, a neuron-specific phosphoprotein (3,4). The same technique has proven suitable for the phosphorylation of other proteins as well. MATERIALS
AND
METHODS
Materials. Nitrocellulose membranes (pore size 0.22 pm) were purchased from Schleicher and Schuell (Keene, New Hampshire), and cellulose thin-layer chromatography sheets were from Eastman Kodak (Rochester, New York). [T-~~P]ATP (2900 Ci/mmol) was obtained from New England Nuclear (Boston, Mass.), horseradish peroxidase-conjugated goat anti-rabbit IgG from Cappel (Cochranville, Pa.). a-Chymotrypsin was from Worthington Biochemical Corporation (Freehold, N.J.). Trypsin I, ATP, DTE,’ and SDS were from Sigma Chemical Company (St. Louis, MO.). Purified catalytic subunit of CAMP-dependent protein kinase and purified histone f2B were kindly provided by A. C. Nairn and A. Horiuchi of this laboratory. Composition of solutions. Solution A: 20% methanol/l25 tIIM T&96 mM glycine, pH 8.3. Solution B: 200 mM NaCl/SO mM TrisHCl, pH 7.4. Solution C: 10% (v/v) acetic acid/ 1% (v/v) pyridine, pH 3.5. Solution D: 37.5% n-butanol/25% pyridine/7.5% acetic acid (v/v). Solution E: 150 mM NaCl/lO mM sodium phosphate/0.05% Tween 20, pH 7.4. Purification of synapsin Ifrom bovine brain.
Synapsin I was prepared from bovine brain using the procedure of Schiebler et al. (5). Synapsin I prepared by this procedure is virtually entirely in the dephosphorylated form: Treatment of Synapsin I with various protein phosphatases prior to phosphorylation by catalytic subunit of CAMP-dependent protein kinase in the presence of [Y-~~P]ATP yielded no additional phosphate incorporation into Synapsin I. * Abbreviations used: CAMP, adenosine 3’:5’-monophosphate; SDS, sodium dodecylsulphate; EDTA, ethylenediaminetetraacetate; EGTA, ethylene glycol bis(Paminoethyl ether) N,N,N’,N’-tetraacetic acid; DTE. dithioerythritol.
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131
Preparation of acid extracts from rat striaturn. Male Sprague-Dawley rats were decap-
itated, the brain was quickly removed, and the caudatoputamen (about 100 mg tissue, wet weight) was excised and homogenized in 900 ~1 of 0.32 M sucrose. The homogenate was added to 5 ml of cold 5 IIIM zinc acetate and centrifuged at 4000g for 20 min. The pellet was resuspended in 5 ml of a cold solution containing 10 mM citric acid and 0.1% (v/v) Nonidet-P40 (pH 3.0) kept on ice for 15 min and centrifuged for 15 min in a Beckman Microfuge. The supernatant, referred to as the “acid extract,” was removed and adjusted to pH6.5 byadding 1/10volof0.5MNa2HP04. Polyacrylamide gel electrophoresis. Protein samples, solubilized in “SDS-stop solution” (3% SDS/2% (v/v) P-mercaptoethanol/ 1 mM EDTA/8% (wt/v) sucrose/62 mM Tris HCI (pH 6.7)) were boiled for 2 min and resolved on either 10% or 7.5% polyacrylamide slab gels using the discontinuous system described by Laemmli (6). Purified [32P]phosphosynapsin I or [32P]histone f2B (phosphorylated as described below) were included in some gels as markers. Electrophoretic transfer. After electrophoresis, the gels were applied to a sheet of nitrocellulose membrane (0.22 pm pore size) and the proteins transferred by the method of Towbin et al. (7), with the modification that electrophoretic blotting was performed with solution A at 150 mA for 20-24 h. The 32Plabeled synapsin 1 or histone f2B bands were identified by autoradiography and used to localize the nonradioactive corresponding regions in the different lanes. These regions were cut out from the nitrocellulose sheet and stored dry before performing the back phosphorylation assay. For the phosphorylation assay of the rat striatum acid extracts, the strips of the whole blotted lanes were used. Phosphorylation ofprotein blots. Prior to the phosphorylation assay, the membrane filters containing the proteins of interest were preincubated for 1 h in a “blocking solution” containing 0.4% (wt/v) Ficoll400 dissolved in solution B supplemented with 0.1% (v/v) Triton
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X 100. This preincubation was found to be very effective in reducing the background radioactivity. Nitrocellulose pieces containing the proteins of interest were then incubated in a solution (pH 7.4) whose composition was: NaCl, 25 mM/MgClz , 10 mM/Hepes, 50 mM/EGTA, 1 mM/P-mercaptoethanol, 0.1 mM/O. 1- 1 PM catalytic subunit of CAMP-dependent protein kinase/ 10 nM- 10 pM [Y-~*P]ATP (5-600 &i/ nmol)/O. 1% Triton X 100. The phosphorylation reaction was carried out at room temperature for 90 min in a final volume of 0.5-3 ml. The reaction was terminated by extensive washing (3 h to overnight) in solution B supplemented with 0.1% (v/v) Tween 20. The phosphorylated proteins were examined by autoradiography. In some experiments, radioactive phosphate incorporation was determined by liquid scintillation counting of the nitrocellulose pieces. Back phosphorylation of proteins in test tubes. Test tube back phosphorylation assays were performed by incubating the protein samples (either tissue extracts or purified bovine synapsin I or histone f2B) in 0.1 ml of a solution (pH 7.4) whose final composition was: Hepes, 50 mM/MgC12, 10 mM/EDTA, 1 mM/ EGTA, 1 mM/O. l- 1 pM purified catalytic subunit of CAMP-dependent protein kinase/ 10 nM-10 PM [y-32P]ATP (5-600 &i/nmol). After a 30-min incubation at 30°C the reaction was terminated by the addition of 25 ~1 of a fivefold concentrated “SDS-stop solution,” followed by immediate boiling for 2 min. The phosphorylated proteins were subsequently separated on SDS-polyacrylamide slab gels and transferred to nitrocellulose filters. Nonradioactive phosphosynapsin I was prepared by phosphorylating 300 ng of dephosphosynapsin I in the same reaction mixture as described above, except that 50 PM unlabeled ATP was used. Tryptic-chymotryptic fingerprinting. Tryptic-chymotryptic fingerprinting of phosphorylated synapsin I was performed by a modification of the procedure of Huttner and Greengard (4). Nitrocellulose or gel pieces
ET
AL.
containing phosphorylated synapsin I were counted by Cerenkov radiation. The pieces were soaked overnight in distilled water and then incubated for 24 h at 37°C in 1.5 ml of a solution containing 25 mM ammonium bicarbonate/l mM DTE/75 pg/ml trypsin I/50 pg/ml chymotrypsin. The nitrocellulose or gel pieces were removed and the efficiency of the extraction was monitored by Cerenkov radiation counting. Fresh trypsin I (25 pg/ml) plus chymotrypsin (25 ,ug/ml) were added to the eluate and the incubation was continued for an additional 6 h. The eluate was then frozen with liquid nitrogen, lyophilized, resuspended in 1 ml distilled water and lyophilized again. The dry residue was dissolved in 50 ~1 of solution C and a trace amount of pyronin Y was added. Ten-microliter aliquots were spotted on cellulose thin-layer chromatography sheets (Eastman) and the phosphopeptides were separated by electrophoresis at 25 mA (3-4 h) in the first dimension, and ascending chromatography in solution D in the second dimension. The plates were dried and analyzed by autoradiography. Immunoperoxidase staining of gel transfers. In some experiments, after the phosphorylation assay nitrocellulose pieces containing the synapsin I bands were processed for synapsin I immunoreactivity as follows: (a) incubation for 15 min in solution E; (b) incubation for 1 h in a 1:200 dilution of affinity purified antisynapsin I rabbit immunoglobulins in solution E; (c) washing with five changes of solution E over a total period of 30 min; (d) incubation for 1 h in a 1: 1000 dilution of horseradish per oxidase-conjugated goat anti-rabbit IgGs in solution E; (e) washing with several changes of solution E over a total period of 30 min followed by two changes of solution E without detergent; (f) incubation for about 30 min with peroxidase substrate solution (5 ml of solution E without detergent/ 1 ml of 3 mg/ml4-chloroI-napthol in methanol/6 ~1 of 30% hydrogen peroxide). The reaction was terminated by dilution in solution E without detergent, followed by washing in distilled water. The developed sheets were stored in the dark.
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ASSAY
Other procedures. Proteins were determined by the method of Bradford (8), using the BioRad protein assay reagent and bovine serum albumin as a standard. Autoradiography was performed with Kodak-X-OmatAR films at room temperature or at -70°C with a DuPont Cronex Lightning Plus intensifying screen. RESULTS Phosphorylation of bovine synapsin I. Figure 1 shows the results obtained by applying our method of phosphorylation of blotted proteins to bovine synapsin I. Different amounts of purified bovine dephosphosynapsin I were subjected to SDS-polyacrylamide gel electrophoresis, transferred electrophoretically onto sheets of nitrocellulose, and used as substrate for the purified catalytic subunit of CAMP-dependent protein kinase. Incubation of the pieces of nitrocellulose with the enzyme led to incorporation of 32P from [T-~~P]ATP into the synapsin I bands. The amount of phosphorylated synapsin I detected by autoradiography was proportional to the amount of loaded protein, as shown in Fig. 2. The sensitivity of the assay could be increased by increasing the specific activity of [T-~~P]ATP to 1.9 @/pmol (final concentration of ATP:76 nM); 400 pg of synapsin I could be measured under these conditions (not shown). When nitrocellulose filters containing identical amounts of dephosphosynapsin I and nonradioactive phosphosynapsin I were in-
-. ng
Synl
0
10 20
40
80
160 320
FIG. 1. Phosphorylation of bovine Synapsin I blotted on nitrocellulose. The indicated amounts of purified bovine synapsin I were subjected to SDS-lo% polyacrylamide gel electrophoresis, transferred to a nitrocellulose filter and subsequently pbospborylated by 200 nM purified catalytic subunit of CAMP-dependent protein kinase. The phosphorylation reaction was carried out at room temperature for 90 min in the presence of 17 nM [y-‘*P]ATP (600 rCi/ nmol). Phosphorylated synapsin I was visualized by autoradiography. The upper and lower bands are synapsin Ia and Ib, respectively.
Jd, 40
320
160
D
ng Syn I FIG. 2. Proportionality between 32P incorporation and the amount of substrate protein. The individual synapsin I bands from the experiment in Fig. 1 were cut from the nitrocellulose filter using the autoradiogram as a guide and the radioactivity was determined by liquid scintillation counting of the intact nitrocellulose pieces. The results are presented as phosphate incorporation after subtraction of the blank (radioactivity incorporated in the absence of synapsin I).
cubated with purified catalytic subunit and radioactive ATP, the dephosphosynapsin I became phosphorylated, whereas virtually no radioactivity was incorporated into prephosphorylated synapsin I. An autoradiogram from one of these experiments is shown in Fig. 3a. The total amount of synapsin I, as shown by immunoperoxidase labeling of the same pieces of nitrocellulose, was similar for the two samples (Fig. 3b). In some experiments, various concentrations of the enzyme were tested. Concentrations higher than 100 nM seemed to increase not only the incorporation of phosphate into the synapsin I bands, but also the background radioactivity (data not shown). This phenomenon is probably due to adherence of autophosphorylated catalytic subunit to nitrocellulose filters. Some experiments were performed to compare the incorporation of 32P from [T-~~P]ATP into synapsin I in acid extracts phosphorylated by the new procedure with the incorporation obtained by the conventional test tube assay. Various amounts of tissue extracts from rat
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FIG. 3. (a) Comparison of phosphorylation on nitrocellulose of dephospho- and phosphosynapsin I. Thirtysix nanograms of dephosphosynapsin 1(left lane) or phosphosynapsin I (synapsin I prephosphorylated by 200 nM catalytic subunit of CAMP-dependent protein kinase with 50 fiM unlabeled ATP; right lane) were transferred from a SDS-lo% polyacrylamide gel to a nitrocellulose filter and phosphorylated by 200 nM catalytic subunit in the presence of 12 nM [Y-‘~P]ATP (300 &i/nmol). (b) The same samples were then analyzed for synapsin I immunoreactivity using an immunoperoxidase labeling technique.
brain were phosphorylated either prior to or after electrophoresis and blotting on nitrocellulose. With both methods, a linear relationship existed between the radioactivity incorporated into the synapsin I bands and the total amount of protein present in the tissue extract. Figure 4 shows the results of one such experiment, using acid extracts of rat striatum. Similar results were obtained with extracts from each of several other brain regions examined (not shown). Tryptic-chymotrypticfingerprinting ofphosphorylated synapsin I. Under certain in vitro conditions, protein kinases are able to catalyze incorporation of phosphate into unphysiological substrates and into unphysiological sites of physiological substrates. It seemed necessary therefore to determine the specificity of the phosphorylation reaction carried out with our new technique. For this purpose we analyzed phosphopeptide maps of phosphorylated bovine synapsin I. Synapsin I was digested by incubating the blot with proteases (see Materials and Methods). By this procedure 95% of the radioactive counts were removed from the
ET AL.
filters. Thus elution of the digested peptides from nitrocellulose is highly efficient. The resulting peptides were subjected to two dimensional separation on a cellulose sheet. The phosphopeptide map shown in Fig. 5a was obtained from Synapsin I proteolyzed after phosphorylation on the blot by the catalytic subunit of CAMP-dependent protein kinase, whereas the peptide map shown in Fig. 5b was obtained from bovine synapsin I phosphorylated by the same enzyme in a test tube, subjected to SDS-polyacrylamide gel electrophoresis and then eluted from the gel. In both cases the phosphorylation of synapsin I occurred at one site, which can be identified with the “site 1” described by Huttner and Greengard (4). This site, located in the collagenase-insensitive portion of the molecule, is specifically phosphorylated by CAMP-dependent protein kinase. (The minor spot seen in Fig. 5a is probably a breakdown product of the major spot (unpublished results)).
jtg protein
FIG. 4. Comparison of radioactive phosphate incorporation into synapsin I in rat striatum acid extracts phosphorylated by catalytic subunit of CAMP-dependent protein kinase in a test tube and on a nitrocellulose filter. Different amounts of rat striatum acid extracts (ranging fropm 0.72 to 5.76 rg protein) were phosphorylated either prior to (0) or after (0) electrophoresis on a SDS-IO% polyacrylamide gel and blotting on a nitrocellulose filter. Following autoradiography, the synapsin I bands were cut from the nitrocellulose filters and the incorporation of 3zP was quantitated by liquid scintillation counting. (Catalytic subunit: 200 nM; [y-“P]ATP: 10 pM (5.3 &i/nmol)).
SOLID-PHASE
PHOSPHORYLATION
b
L 0
*
Q
FIG. 5. Autoradiographs showing tryptic-chymotryptic fingerprints of purified bovine synapsin I phosphorylated by 100 nM catalytic subunit of CAMP-dependent protein kinase. (a) Bovine synapsin I was phosphorylated on nitrocellulose as described in the legend to Fig. 1 and subsequently proteolyzed by trypsin and chymotrypsin. Phosphopeptides were spotted on cellulose plates and separated by horizontal electrophoresis in the first dimension (negative pole, left; positive pole, right) and by ascending chromatography in the second dimension. (b) Phosphopeptide map of bovine synapsin I phosphorylated in a test tube, digested as described under Materials and Methods and subjected to fingerprinting as described for (a). 0. origin.
Phosphorylation of other substrates for the catalytic subunit of CAMP-dependent protein kinase. Experiments were performed to determine whether our procedure could be applied to the phosphorylation of other substrates for the catalytic subunit of CAMP-dependent protein kinase. For this purpose, histone f2B was used as a substrate. The autoradiogram in Fig. 6 compares the phosphorylation on nitrocellulose of 1 pg of histone f2B with the phosphorylation of the same amount of protein substrate in the test tube. Further experiments were performed to compare the phosphoprotein pattern of tissue samples phosphorylated by the new procedure with the pattern obtained by a conventional
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135
test tube assay. Parallel aliquots of rat striatum acid extracts were phosphorylated either after or prior to electrophoretic separation and blotting on nitrocellulose (Figs. 7a and b, respectively). The concentrations of the catalytic subunit and of [T-~~P]ATP were the same for the two samples. The protein phosphorylation patterns were qualitatively similar for the two methods, although two additional prominent low molecular weight bands were present in the samples phosphorylated on nitrocellulose. DISCUSSION
Protein blotting has been used previously for the detection of proteins as specific antigens by immuno-overlay techniques, but a number of other applications are being developed (for recent reviews, see (9-l 1)). The data presented in this report indicate that proteins immobilized on nitrocellulose membranes can be efa
b
-
FIG. 6. Phosphorylation of histone f2B. Histone f2B (1 pg) was phosphorylated either after (left lane) or prior to (right lane) electrophoresis on a SDS- 10% polyacrylamide gel and blotting on a nitrocellulose membrane (catalytic subunit: 100 nM; [Y-~~P]ATP: 12 nM (250 pCi/nmol)).
FIG. 7. Comparison of phosphoprotein patterns in rat striatum acid extracts phosphorylated on nitrocellulose filters (a) and in a test tube (b). Aliquots (100 pg protein) of rat striatum acidic extracts were resolved on a SDS10% polyacrylamide gel and transferred to nitrocellulose. The samples were phosphorylated by catalytic subunit of CAMP-dependent protein kinase either after blotting on nitrocellulose (a) or prior to electrophoresis (b) (catalytic subunit: 100 nM; [y-“P]ATP:17 nM (350 ~Ci/nmol)).
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ficiently phosphorylated. Successful phosphorylation of proteins on membrane filters provides an additional proof of the versatility of protein blotting, which is becoming one of the most widely used techniques for the immunochemical and biochemical analysis of proteins. The procedure described here provides an estimate of the dephosphorylated forms of phosphoproteins, as does the classical test tube back phosphorylation assay. Resolution and reproducibility achieved by this method are similar to those obtained with the test tube back phosphorylation. The procedure is suitable for quantitative studies, since the amount of 32P incorporated increases linearly with the amount of substrate protein. The specificity of the phosphorylation reaction is indicated by the results of the trypticchymotryptic map of synapsin I and by the fact that, when synapsin I was prephosphorylated in a test tube by the catalytic subunit with unlabeled ATP, no 32P became incorporated into the molecule during the solidphase phosphorylation assay. The minimum amount of substrate protein detected by the new procedure was 400 pg (5 fmol). The sensitivity of the assay depends mainly on the specific activity of [T-~~P]ATP and on the level of the background (which is very low if the samples are preincubated in “blocking solution” and a low concentration of catalytic subunit is used). The technique also proved to be applicable to the phosphorylation of proteins other than synapsin I, as shown by the experiments with histone f2B (one of the most effective substrates for the catalytic subunit, often used as the standard substrate for CAMP-dependent protein kinase) and with proteins present in acid extracts from rat striatum. In the striatal acid extracts, virtually all the bands present in the sample phosphorylated in the test tube were also observed in the solid-phase phosphorylation assay. However, in the blot phosphorylation assay the radioactive bands were more sharply defined and two additional low molecular weight bands were observed. These
ET
AL.
differences might be explained by the fact that the interaction between the enzyme and the substrates is different in the two assays: on the nitrocellulose, proteins are immobilized and highly concentrated on a very thin layer and are therefore accessible to the enzyme in bulk. This might allow the detection of low affinity substrates not seen by test tube back phosphorylation. The solid-phase phosphorylation assay circumvents several problems associated with the test tube back phosphorylation assay, as follows: (i) the method of phosphorylation on blots is particularly suitable for the analysis of proteins extracted in the presence of detergents (e.g., SDS) or of other agents that cause inactivation of exogenous kinases; (ii) no interference by endogenous kinases or phosphatases is possible; (iii) substrates with low affinity, that cannot be detected by the test tube back phosphorylation assay, can be detected by the solid-phase assay, both because of concentrating the substrates on the nitrocellulose and because of separating them from competitive substrates; (iv) some phosphorylation sites can be masked by protein-protein interactions in test tube back phosphorylation assays. The solid-phase technique may therefore be useful not only for the study of the state of phosphorylation of known substrates, but also for the detection of additional substrates for protein kinases. The solid-phase phosphorylation assay has several other advantages: (i) adsorbed proteins may be stored for periods of months before performing the phosphorylation assay; (ii) the process of autoradiography is very easy, since no gel drying is required; (iii) after phosphorylation, immobilized proteins may be used for additional labeling procedures, such as immunolabeling. Certain possible limitations of the solidphase phosphorylation assay should be mentioned. Adsorbance of autophosphorylated protein kinases to the nitrocellulose filters might cause a high background. This problem was largely circumvented by occupying unbound sites of the filter by preincubation with
SOLID-PHASE
PHOSPHORYLATION
a blocking solution prior to the phosphorylation assay (see Methods). Preincubating the enzyme with unlabeled ATP further lowered the background deriving from adherence of the autophosphorylated protein kinase (data not shown). Limitations to the applicability of the technique might also arise from other factors affecting the overall efficiency of the blotting procedure. For example, it is well known that blots give a biased representation of gel patterns, as the efficiency of the elution of proteins from polyacrylamide gels is inversely related to the peptide molecular weight (9,12,13). In addition, under certain conditions, low molecular weight-proteins are not very well retained by nitrdcellulose filters (12,13). In principle, these problems could be circumvented by using large pore polyacrylamide gels and long transfer times for high molecular weight proteins, whereas the use of small pore size nitrocellulose membranes or of other matrices could be considered for low molecular weight proteins (13- 15). REFERENCES 1. Nestler, E. J., and Greengard, P. (1984) Protein Phosphorylation in the Nervous System, Wiley, New York. 2. Rosen, 0. M., and Krebs, E. G. (eds.) (1981) Protein Phosphotylation, Cold Spring Harbor Conf. Cell Prolif., Vol. 8, Cold Spring Harbor, New York. 3. Ueda, T., and Greengard, P. (1977) Adenosine 3’:5’monophosphate-regulated phosphoprotein system of neuronal membranes. I. Solubilization, purification, and some properties of an endogenous phosphoprotein. J. Biol. Chem. 252,5 155-5 163. 4. Huttner, W. B., and Greengard, P. (1979) Multiple phosphorylation sites in Protein I and their differ-
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ential regulation by cyclic AMP and calcium. Proc. Natl. Acad. Sci. USA 76, 5402-5406. 5. Schiebler, W., Jahn, R., Doucet, J. P., Rothlein, J., and Greengard, P. ( 1986) Characterization of synapsin I binding to small synaptic vesicles. J. Biol. Chem. 261,8383-8390. 6. Laemmli, U. K. ( 1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. 7. Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76,4350-4354.
Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72,248-254. 9. Gershoni, J. M., and Palade, G. E. (1983) Protein blotting: Principle and applications. Anal. Biochem. 131, 1-15. 10. Gershoni, J. M. ( 1985) Protein blotting: Development and perspectives. Trends Biochem. Sci. (Vol. 10) 8.
3, 103-106.
Il. Towbin, H., and Gordon, J. (1984) Immunoblotting and dot immunobinding: Current status and outlook. J. Immunol. Methods 72, 3 13-340. 12. Bumette, W. N. (198 1) Western blotting: Electrophoretie transfer of proteins from sodium dodecylsulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated Protein A. Anal. Biochem. 112, 195203.
13. Lin, W., and Kasamatsu, H. (1983) On the electrotransfer of polypeptides from gels to nitrocellulose membranes. Anal. Biochem. 128, 303-3 11. 14. Bittner, M., Kupferer, P., and Morris, C. F. (1980) Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzyloxymethyl celhtlose or nitrocellulose sheets.Anal. Biochem. 102,459471. 15.
Gershoni, J. M., and Palade, G. E. (1982) Electrophoretic transfer of proteins from sodium dodecylsulfate-polyacrylamide gels to a positively charged membrane filter. Anal. Biochem. 124,396405.
BIOCHEMISTRY
158,
130- 137 ( 1986)
A Solid-Phase Assay for the Phosphorylation of Proteins Blotted on Nitrocellulose Membrane Filters’ FLAVIA VALTORTA,**~ WERNER SCHIEBLER,* REINHARD JAHN,* BRUNOCECCARELLI,~.ANDPAULGREENGARD* *Laboratory of Molecular and Cellular Neuroscience, TDepartment of Medical Pharmacology, CNR of Peripheral Neuropathies and Neuromuscular
The Rockefeller University, New York, New York 10021, Center of Cytopharmacology, and Center for the Study Diseases, University of Milano, 20129 Milano. Italy
and
Received February 20. 1986 A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters, and the blotted polypeptides are phosphorylated with the catalytic subunit of cyclic AMP (adenosine 3’:5’-monophosphate)-dependent protein kinase. The method was developed for the assay of dephosphosynapsin I, but it has also proven suitable for the phosphorylation of other proteins. The patterns of phosphorylation of tissue samples phosphorylated using the new method are similar to those obtained using the conventional test tube assay.Once phosphorylated, the adsorbed proteins can be digested with proteases and subjected to phosphopeptide mapping. The phosphorylated blotted proteins can also be analyzed by overlay techniques for the immunological detection of polypeptides. 0 1986 Academic press. hc. KEY WORDS: protein phosphorylation; protein blotting; nitrocellulose; protein kinases; neurochemistry; synapsin I.
An increasing body of experimental evidence indicates that both in neural and in nonneural cells protein phosphorylation plays an important role in the modulation of physiological processes. Responses to a large variety of extracellular signals are mediated by second messengers (Ca’+, cyclic nucleotides) which activate specific protein kinases, which in turn regulate the state of phosphorylation of various substrate proteins (1,2). Two different experimental approaches for the study of protein phosphorylation are currently available: (i) Tracing the incorporation of 32P from prelabeled endogenous ATP into proteins (in vivo labeling). With this technique the incorporation of phosphate into proteins in intact cell preparations can be compared under different physiological conditions. It is not suitable,
however, for measuring the degree of phosphorylation of proteins, since neither the specific radioactivity of endogenous ATP nor the turnover rates of the different components participating in the process can be established. (ii) Studying the state of protein phosphorylation by measuring the amount of radioactive phosphate that can be incorporated into extracted proteins in the presence of exogenous kinases and radioactive ATP of known specific activity (back phosphorylation). A major limitation of this technique derives from the extraction procedure, which must have the characteristics of inactivating endogenous kinases and phosphatases, without interfering with the exogenous kinases added subsequently to the extract. In an attempt to circumvent this limitation, we have developed a novel technique for the phosphorylation of proteins, based on their immobilization on nitrocellulose after electrophoretic separation. This technique was developed to assay the state of phosphorylation
’ This work was supported by the United States Environmental Protection Agency under Assistance Agreement CR-8 10608-01 (P.G.) and by a M.D.A. grant to B.C. 0003-2697186 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights oi reproduction in any form reserved.
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of synapsin I, a neuron-specific phosphoprotein (3,4). The same technique has proven suitable for the phosphorylation of other proteins as well. MATERIALS
AND
METHODS
Materials. Nitrocellulose membranes (pore size 0.22 pm) were purchased from Schleicher and Schuell (Keene, New Hampshire), and cellulose thin-layer chromatography sheets were from Eastman Kodak (Rochester, New York). [T-~~P]ATP (2900 Ci/mmol) was obtained from New England Nuclear (Boston, Mass.), horseradish peroxidase-conjugated goat anti-rabbit IgG from Cappel (Cochranville, Pa.). a-Chymotrypsin was from Worthington Biochemical Corporation (Freehold, N.J.). Trypsin I, ATP, DTE,’ and SDS were from Sigma Chemical Company (St. Louis, MO.). Purified catalytic subunit of CAMP-dependent protein kinase and purified histone f2B were kindly provided by A. C. Nairn and A. Horiuchi of this laboratory. Composition of solutions. Solution A: 20% methanol/l25 tIIM T&96 mM glycine, pH 8.3. Solution B: 200 mM NaCl/SO mM TrisHCl, pH 7.4. Solution C: 10% (v/v) acetic acid/ 1% (v/v) pyridine, pH 3.5. Solution D: 37.5% n-butanol/25% pyridine/7.5% acetic acid (v/v). Solution E: 150 mM NaCl/lO mM sodium phosphate/0.05% Tween 20, pH 7.4. Purification of synapsin Ifrom bovine brain.
Synapsin I was prepared from bovine brain using the procedure of Schiebler et al. (5). Synapsin I prepared by this procedure is virtually entirely in the dephosphorylated form: Treatment of Synapsin I with various protein phosphatases prior to phosphorylation by catalytic subunit of CAMP-dependent protein kinase in the presence of [Y-~~P]ATP yielded no additional phosphate incorporation into Synapsin I. * Abbreviations used: CAMP, adenosine 3’:5’-monophosphate; SDS, sodium dodecylsulphate; EDTA, ethylenediaminetetraacetate; EGTA, ethylene glycol bis(Paminoethyl ether) N,N,N’,N’-tetraacetic acid; DTE. dithioerythritol.
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Preparation of acid extracts from rat striaturn. Male Sprague-Dawley rats were decap-
itated, the brain was quickly removed, and the caudatoputamen (about 100 mg tissue, wet weight) was excised and homogenized in 900 ~1 of 0.32 M sucrose. The homogenate was added to 5 ml of cold 5 IIIM zinc acetate and centrifuged at 4000g for 20 min. The pellet was resuspended in 5 ml of a cold solution containing 10 mM citric acid and 0.1% (v/v) Nonidet-P40 (pH 3.0) kept on ice for 15 min and centrifuged for 15 min in a Beckman Microfuge. The supernatant, referred to as the “acid extract,” was removed and adjusted to pH6.5 byadding 1/10volof0.5MNa2HP04. Polyacrylamide gel electrophoresis. Protein samples, solubilized in “SDS-stop solution” (3% SDS/2% (v/v) P-mercaptoethanol/ 1 mM EDTA/8% (wt/v) sucrose/62 mM Tris HCI (pH 6.7)) were boiled for 2 min and resolved on either 10% or 7.5% polyacrylamide slab gels using the discontinuous system described by Laemmli (6). Purified [32P]phosphosynapsin I or [32P]histone f2B (phosphorylated as described below) were included in some gels as markers. Electrophoretic transfer. After electrophoresis, the gels were applied to a sheet of nitrocellulose membrane (0.22 pm pore size) and the proteins transferred by the method of Towbin et al. (7), with the modification that electrophoretic blotting was performed with solution A at 150 mA for 20-24 h. The 32Plabeled synapsin 1 or histone f2B bands were identified by autoradiography and used to localize the nonradioactive corresponding regions in the different lanes. These regions were cut out from the nitrocellulose sheet and stored dry before performing the back phosphorylation assay. For the phosphorylation assay of the rat striatum acid extracts, the strips of the whole blotted lanes were used. Phosphorylation ofprotein blots. Prior to the phosphorylation assay, the membrane filters containing the proteins of interest were preincubated for 1 h in a “blocking solution” containing 0.4% (wt/v) Ficoll400 dissolved in solution B supplemented with 0.1% (v/v) Triton
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X 100. This preincubation was found to be very effective in reducing the background radioactivity. Nitrocellulose pieces containing the proteins of interest were then incubated in a solution (pH 7.4) whose composition was: NaCl, 25 mM/MgClz , 10 mM/Hepes, 50 mM/EGTA, 1 mM/P-mercaptoethanol, 0.1 mM/O. 1- 1 PM catalytic subunit of CAMP-dependent protein kinase/ 10 nM- 10 pM [Y-~*P]ATP (5-600 &i/ nmol)/O. 1% Triton X 100. The phosphorylation reaction was carried out at room temperature for 90 min in a final volume of 0.5-3 ml. The reaction was terminated by extensive washing (3 h to overnight) in solution B supplemented with 0.1% (v/v) Tween 20. The phosphorylated proteins were examined by autoradiography. In some experiments, radioactive phosphate incorporation was determined by liquid scintillation counting of the nitrocellulose pieces. Back phosphorylation of proteins in test tubes. Test tube back phosphorylation assays were performed by incubating the protein samples (either tissue extracts or purified bovine synapsin I or histone f2B) in 0.1 ml of a solution (pH 7.4) whose final composition was: Hepes, 50 mM/MgC12, 10 mM/EDTA, 1 mM/ EGTA, 1 mM/O. l- 1 pM purified catalytic subunit of CAMP-dependent protein kinase/ 10 nM-10 PM [y-32P]ATP (5-600 &i/nmol). After a 30-min incubation at 30°C the reaction was terminated by the addition of 25 ~1 of a fivefold concentrated “SDS-stop solution,” followed by immediate boiling for 2 min. The phosphorylated proteins were subsequently separated on SDS-polyacrylamide slab gels and transferred to nitrocellulose filters. Nonradioactive phosphosynapsin I was prepared by phosphorylating 300 ng of dephosphosynapsin I in the same reaction mixture as described above, except that 50 PM unlabeled ATP was used. Tryptic-chymotryptic fingerprinting. Tryptic-chymotryptic fingerprinting of phosphorylated synapsin I was performed by a modification of the procedure of Huttner and Greengard (4). Nitrocellulose or gel pieces
ET
AL.
containing phosphorylated synapsin I were counted by Cerenkov radiation. The pieces were soaked overnight in distilled water and then incubated for 24 h at 37°C in 1.5 ml of a solution containing 25 mM ammonium bicarbonate/l mM DTE/75 pg/ml trypsin I/50 pg/ml chymotrypsin. The nitrocellulose or gel pieces were removed and the efficiency of the extraction was monitored by Cerenkov radiation counting. Fresh trypsin I (25 pg/ml) plus chymotrypsin (25 ,ug/ml) were added to the eluate and the incubation was continued for an additional 6 h. The eluate was then frozen with liquid nitrogen, lyophilized, resuspended in 1 ml distilled water and lyophilized again. The dry residue was dissolved in 50 ~1 of solution C and a trace amount of pyronin Y was added. Ten-microliter aliquots were spotted on cellulose thin-layer chromatography sheets (Eastman) and the phosphopeptides were separated by electrophoresis at 25 mA (3-4 h) in the first dimension, and ascending chromatography in solution D in the second dimension. The plates were dried and analyzed by autoradiography. Immunoperoxidase staining of gel transfers. In some experiments, after the phosphorylation assay nitrocellulose pieces containing the synapsin I bands were processed for synapsin I immunoreactivity as follows: (a) incubation for 15 min in solution E; (b) incubation for 1 h in a 1:200 dilution of affinity purified antisynapsin I rabbit immunoglobulins in solution E; (c) washing with five changes of solution E over a total period of 30 min; (d) incubation for 1 h in a 1: 1000 dilution of horseradish per oxidase-conjugated goat anti-rabbit IgGs in solution E; (e) washing with several changes of solution E over a total period of 30 min followed by two changes of solution E without detergent; (f) incubation for about 30 min with peroxidase substrate solution (5 ml of solution E without detergent/ 1 ml of 3 mg/ml4-chloroI-napthol in methanol/6 ~1 of 30% hydrogen peroxide). The reaction was terminated by dilution in solution E without detergent, followed by washing in distilled water. The developed sheets were stored in the dark.
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133
ASSAY
Other procedures. Proteins were determined by the method of Bradford (8), using the BioRad protein assay reagent and bovine serum albumin as a standard. Autoradiography was performed with Kodak-X-OmatAR films at room temperature or at -70°C with a DuPont Cronex Lightning Plus intensifying screen. RESULTS Phosphorylation of bovine synapsin I. Figure 1 shows the results obtained by applying our method of phosphorylation of blotted proteins to bovine synapsin I. Different amounts of purified bovine dephosphosynapsin I were subjected to SDS-polyacrylamide gel electrophoresis, transferred electrophoretically onto sheets of nitrocellulose, and used as substrate for the purified catalytic subunit of CAMP-dependent protein kinase. Incubation of the pieces of nitrocellulose with the enzyme led to incorporation of 32P from [T-~~P]ATP into the synapsin I bands. The amount of phosphorylated synapsin I detected by autoradiography was proportional to the amount of loaded protein, as shown in Fig. 2. The sensitivity of the assay could be increased by increasing the specific activity of [T-~~P]ATP to 1.9 @/pmol (final concentration of ATP:76 nM); 400 pg of synapsin I could be measured under these conditions (not shown). When nitrocellulose filters containing identical amounts of dephosphosynapsin I and nonradioactive phosphosynapsin I were in-
-. ng
Synl
0
10 20
40
80
160 320
FIG. 1. Phosphorylation of bovine Synapsin I blotted on nitrocellulose. The indicated amounts of purified bovine synapsin I were subjected to SDS-lo% polyacrylamide gel electrophoresis, transferred to a nitrocellulose filter and subsequently pbospborylated by 200 nM purified catalytic subunit of CAMP-dependent protein kinase. The phosphorylation reaction was carried out at room temperature for 90 min in the presence of 17 nM [y-‘*P]ATP (600 rCi/ nmol). Phosphorylated synapsin I was visualized by autoradiography. The upper and lower bands are synapsin Ia and Ib, respectively.
Jd, 40
320
160
D
ng Syn I FIG. 2. Proportionality between 32P incorporation and the amount of substrate protein. The individual synapsin I bands from the experiment in Fig. 1 were cut from the nitrocellulose filter using the autoradiogram as a guide and the radioactivity was determined by liquid scintillation counting of the intact nitrocellulose pieces. The results are presented as phosphate incorporation after subtraction of the blank (radioactivity incorporated in the absence of synapsin I).
cubated with purified catalytic subunit and radioactive ATP, the dephosphosynapsin I became phosphorylated, whereas virtually no radioactivity was incorporated into prephosphorylated synapsin I. An autoradiogram from one of these experiments is shown in Fig. 3a. The total amount of synapsin I, as shown by immunoperoxidase labeling of the same pieces of nitrocellulose, was similar for the two samples (Fig. 3b). In some experiments, various concentrations of the enzyme were tested. Concentrations higher than 100 nM seemed to increase not only the incorporation of phosphate into the synapsin I bands, but also the background radioactivity (data not shown). This phenomenon is probably due to adherence of autophosphorylated catalytic subunit to nitrocellulose filters. Some experiments were performed to compare the incorporation of 32P from [T-~~P]ATP into synapsin I in acid extracts phosphorylated by the new procedure with the incorporation obtained by the conventional test tube assay. Various amounts of tissue extracts from rat
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FIG. 3. (a) Comparison of phosphorylation on nitrocellulose of dephospho- and phosphosynapsin I. Thirtysix nanograms of dephosphosynapsin 1(left lane) or phosphosynapsin I (synapsin I prephosphorylated by 200 nM catalytic subunit of CAMP-dependent protein kinase with 50 fiM unlabeled ATP; right lane) were transferred from a SDS-lo% polyacrylamide gel to a nitrocellulose filter and phosphorylated by 200 nM catalytic subunit in the presence of 12 nM [Y-‘~P]ATP (300 &i/nmol). (b) The same samples were then analyzed for synapsin I immunoreactivity using an immunoperoxidase labeling technique.
brain were phosphorylated either prior to or after electrophoresis and blotting on nitrocellulose. With both methods, a linear relationship existed between the radioactivity incorporated into the synapsin I bands and the total amount of protein present in the tissue extract. Figure 4 shows the results of one such experiment, using acid extracts of rat striatum. Similar results were obtained with extracts from each of several other brain regions examined (not shown). Tryptic-chymotrypticfingerprinting ofphosphorylated synapsin I. Under certain in vitro conditions, protein kinases are able to catalyze incorporation of phosphate into unphysiological substrates and into unphysiological sites of physiological substrates. It seemed necessary therefore to determine the specificity of the phosphorylation reaction carried out with our new technique. For this purpose we analyzed phosphopeptide maps of phosphorylated bovine synapsin I. Synapsin I was digested by incubating the blot with proteases (see Materials and Methods). By this procedure 95% of the radioactive counts were removed from the
ET AL.
filters. Thus elution of the digested peptides from nitrocellulose is highly efficient. The resulting peptides were subjected to two dimensional separation on a cellulose sheet. The phosphopeptide map shown in Fig. 5a was obtained from Synapsin I proteolyzed after phosphorylation on the blot by the catalytic subunit of CAMP-dependent protein kinase, whereas the peptide map shown in Fig. 5b was obtained from bovine synapsin I phosphorylated by the same enzyme in a test tube, subjected to SDS-polyacrylamide gel electrophoresis and then eluted from the gel. In both cases the phosphorylation of synapsin I occurred at one site, which can be identified with the “site 1” described by Huttner and Greengard (4). This site, located in the collagenase-insensitive portion of the molecule, is specifically phosphorylated by CAMP-dependent protein kinase. (The minor spot seen in Fig. 5a is probably a breakdown product of the major spot (unpublished results)).
jtg protein
FIG. 4. Comparison of radioactive phosphate incorporation into synapsin I in rat striatum acid extracts phosphorylated by catalytic subunit of CAMP-dependent protein kinase in a test tube and on a nitrocellulose filter. Different amounts of rat striatum acid extracts (ranging fropm 0.72 to 5.76 rg protein) were phosphorylated either prior to (0) or after (0) electrophoresis on a SDS-IO% polyacrylamide gel and blotting on a nitrocellulose filter. Following autoradiography, the synapsin I bands were cut from the nitrocellulose filters and the incorporation of 3zP was quantitated by liquid scintillation counting. (Catalytic subunit: 200 nM; [y-“P]ATP: 10 pM (5.3 &i/nmol)).
SOLID-PHASE
PHOSPHORYLATION
b
L 0
*
Q
FIG. 5. Autoradiographs showing tryptic-chymotryptic fingerprints of purified bovine synapsin I phosphorylated by 100 nM catalytic subunit of CAMP-dependent protein kinase. (a) Bovine synapsin I was phosphorylated on nitrocellulose as described in the legend to Fig. 1 and subsequently proteolyzed by trypsin and chymotrypsin. Phosphopeptides were spotted on cellulose plates and separated by horizontal electrophoresis in the first dimension (negative pole, left; positive pole, right) and by ascending chromatography in the second dimension. (b) Phosphopeptide map of bovine synapsin I phosphorylated in a test tube, digested as described under Materials and Methods and subjected to fingerprinting as described for (a). 0. origin.
Phosphorylation of other substrates for the catalytic subunit of CAMP-dependent protein kinase. Experiments were performed to determine whether our procedure could be applied to the phosphorylation of other substrates for the catalytic subunit of CAMP-dependent protein kinase. For this purpose, histone f2B was used as a substrate. The autoradiogram in Fig. 6 compares the phosphorylation on nitrocellulose of 1 pg of histone f2B with the phosphorylation of the same amount of protein substrate in the test tube. Further experiments were performed to compare the phosphoprotein pattern of tissue samples phosphorylated by the new procedure with the pattern obtained by a conventional
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135
test tube assay. Parallel aliquots of rat striatum acid extracts were phosphorylated either after or prior to electrophoretic separation and blotting on nitrocellulose (Figs. 7a and b, respectively). The concentrations of the catalytic subunit and of [T-~~P]ATP were the same for the two samples. The protein phosphorylation patterns were qualitatively similar for the two methods, although two additional prominent low molecular weight bands were present in the samples phosphorylated on nitrocellulose. DISCUSSION
Protein blotting has been used previously for the detection of proteins as specific antigens by immuno-overlay techniques, but a number of other applications are being developed (for recent reviews, see (9-l 1)). The data presented in this report indicate that proteins immobilized on nitrocellulose membranes can be efa
b
-
FIG. 6. Phosphorylation of histone f2B. Histone f2B (1 pg) was phosphorylated either after (left lane) or prior to (right lane) electrophoresis on a SDS- 10% polyacrylamide gel and blotting on a nitrocellulose membrane (catalytic subunit: 100 nM; [Y-~~P]ATP: 12 nM (250 pCi/nmol)).
FIG. 7. Comparison of phosphoprotein patterns in rat striatum acid extracts phosphorylated on nitrocellulose filters (a) and in a test tube (b). Aliquots (100 pg protein) of rat striatum acidic extracts were resolved on a SDS10% polyacrylamide gel and transferred to nitrocellulose. The samples were phosphorylated by catalytic subunit of CAMP-dependent protein kinase either after blotting on nitrocellulose (a) or prior to electrophoresis (b) (catalytic subunit: 100 nM; [y-“P]ATP:17 nM (350 ~Ci/nmol)).
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VALTORTA
ficiently phosphorylated. Successful phosphorylation of proteins on membrane filters provides an additional proof of the versatility of protein blotting, which is becoming one of the most widely used techniques for the immunochemical and biochemical analysis of proteins. The procedure described here provides an estimate of the dephosphorylated forms of phosphoproteins, as does the classical test tube back phosphorylation assay. Resolution and reproducibility achieved by this method are similar to those obtained with the test tube back phosphorylation. The procedure is suitable for quantitative studies, since the amount of 32P incorporated increases linearly with the amount of substrate protein. The specificity of the phosphorylation reaction is indicated by the results of the trypticchymotryptic map of synapsin I and by the fact that, when synapsin I was prephosphorylated in a test tube by the catalytic subunit with unlabeled ATP, no 32P became incorporated into the molecule during the solidphase phosphorylation assay. The minimum amount of substrate protein detected by the new procedure was 400 pg (5 fmol). The sensitivity of the assay depends mainly on the specific activity of [T-~~P]ATP and on the level of the background (which is very low if the samples are preincubated in “blocking solution” and a low concentration of catalytic subunit is used). The technique also proved to be applicable to the phosphorylation of proteins other than synapsin I, as shown by the experiments with histone f2B (one of the most effective substrates for the catalytic subunit, often used as the standard substrate for CAMP-dependent protein kinase) and with proteins present in acid extracts from rat striatum. In the striatal acid extracts, virtually all the bands present in the sample phosphorylated in the test tube were also observed in the solid-phase phosphorylation assay. However, in the blot phosphorylation assay the radioactive bands were more sharply defined and two additional low molecular weight bands were observed. These
ET
AL.
differences might be explained by the fact that the interaction between the enzyme and the substrates is different in the two assays: on the nitrocellulose, proteins are immobilized and highly concentrated on a very thin layer and are therefore accessible to the enzyme in bulk. This might allow the detection of low affinity substrates not seen by test tube back phosphorylation. The solid-phase phosphorylation assay circumvents several problems associated with the test tube back phosphorylation assay, as follows: (i) the method of phosphorylation on blots is particularly suitable for the analysis of proteins extracted in the presence of detergents (e.g., SDS) or of other agents that cause inactivation of exogenous kinases; (ii) no interference by endogenous kinases or phosphatases is possible; (iii) substrates with low affinity, that cannot be detected by the test tube back phosphorylation assay, can be detected by the solid-phase assay, both because of concentrating the substrates on the nitrocellulose and because of separating them from competitive substrates; (iv) some phosphorylation sites can be masked by protein-protein interactions in test tube back phosphorylation assays. The solid-phase technique may therefore be useful not only for the study of the state of phosphorylation of known substrates, but also for the detection of additional substrates for protein kinases. The solid-phase phosphorylation assay has several other advantages: (i) adsorbed proteins may be stored for periods of months before performing the phosphorylation assay; (ii) the process of autoradiography is very easy, since no gel drying is required; (iii) after phosphorylation, immobilized proteins may be used for additional labeling procedures, such as immunolabeling. Certain possible limitations of the solidphase phosphorylation assay should be mentioned. Adsorbance of autophosphorylated protein kinases to the nitrocellulose filters might cause a high background. This problem was largely circumvented by occupying unbound sites of the filter by preincubation with
SOLID-PHASE
PHOSPHORYLATION
a blocking solution prior to the phosphorylation assay (see Methods). Preincubating the enzyme with unlabeled ATP further lowered the background deriving from adherence of the autophosphorylated protein kinase (data not shown). Limitations to the applicability of the technique might also arise from other factors affecting the overall efficiency of the blotting procedure. For example, it is well known that blots give a biased representation of gel patterns, as the efficiency of the elution of proteins from polyacrylamide gels is inversely related to the peptide molecular weight (9,12,13). In addition, under certain conditions, low molecular weight-proteins are not very well retained by nitrdcellulose filters (12,13). In principle, these problems could be circumvented by using large pore polyacrylamide gels and long transfer times for high molecular weight proteins, whereas the use of small pore size nitrocellulose membranes or of other matrices could be considered for low molecular weight proteins (13- 15). REFERENCES 1. Nestler, E. J., and Greengard, P. (1984) Protein Phosphorylation in the Nervous System, Wiley, New York. 2. Rosen, 0. M., and Krebs, E. G. (eds.) (1981) Protein Phosphotylation, Cold Spring Harbor Conf. Cell Prolif., Vol. 8, Cold Spring Harbor, New York. 3. Ueda, T., and Greengard, P. (1977) Adenosine 3’:5’monophosphate-regulated phosphoprotein system of neuronal membranes. I. Solubilization, purification, and some properties of an endogenous phosphoprotein. J. Biol. Chem. 252,5 155-5 163. 4. Huttner, W. B., and Greengard, P. (1979) Multiple phosphorylation sites in Protein I and their differ-
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ential regulation by cyclic AMP and calcium. Proc. Natl. Acad. Sci. USA 76, 5402-5406. 5. Schiebler, W., Jahn, R., Doucet, J. P., Rothlein, J., and Greengard, P. ( 1986) Characterization of synapsin I binding to small synaptic vesicles. J. Biol. Chem. 261,8383-8390. 6. Laemmli, U. K. ( 1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. 7. Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76,4350-4354.
Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72,248-254. 9. Gershoni, J. M., and Palade, G. E. (1983) Protein blotting: Principle and applications. Anal. Biochem. 131, 1-15. 10. Gershoni, J. M. ( 1985) Protein blotting: Development and perspectives. Trends Biochem. Sci. (Vol. 10) 8.
3, 103-106.
Il. Towbin, H., and Gordon, J. (1984) Immunoblotting and dot immunobinding: Current status and outlook. J. Immunol. Methods 72, 3 13-340. 12. Bumette, W. N. (198 1) Western blotting: Electrophoretie transfer of proteins from sodium dodecylsulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated Protein A. Anal. Biochem. 112, 195203.
13. Lin, W., and Kasamatsu, H. (1983) On the electrotransfer of polypeptides from gels to nitrocellulose membranes. Anal. Biochem. 128, 303-3 11. 14. Bittner, M., Kupferer, P., and Morris, C. F. (1980) Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzyloxymethyl celhtlose or nitrocellulose sheets.Anal. Biochem. 102,459471. 15.
Gershoni, J. M., and Palade, G. E. (1982) Electrophoretic transfer of proteins from sodium dodecylsulfate-polyacrylamide gels to a positively charged membrane filter. Anal. Biochem. 124,396405.