[33]
R E N A T U R A T I O N O F P R O T E I N KINASES IN S D S GELS
417
[33] R e n a t u r a t i o n a n d Assay of P r o t e i n Kinases after E l e c t r o p h o r e s i s in S o d i u m D o d e c y l S u l f a t e - P o l y a c r y l a m i d e Gels By JILL n . HUTCHCROFT, MICHAEL ANOSTARIO, JR.,
MARIETTA L. HARRISON, and ROBERT L. GEAHLEN
An increasing appreciation of the importance of protein kinases in metabolic regulation has generated considerable interest in methods for their separation, detection, and characterization.1 A powerful method for the resolution of complex mixtures of proteins containing kinases is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although SDS-enzyme complexes are typically devoid of enzyme activity, it has proved possible in many cases to recover activity after removal of the detergent. Thus, procedures can be developed that couple the high resolving power of SDS-polyacrylamide gel electrophoresis with "on the spot" detection of specific enzyme activities. An extensive review of techniques used to localize functionally active enzymes following gel electrophoresis has been previously published in this series. 2We have adapted and extended such procedures to develop a simple and convenient method for the in situ (in the gel) renaturation and assay of protein kinases. 3 Our gel-renaturation technique is summarized in Fig. 1. Briefly, proteins are separated by electrophoresis through SDS-polyacrylamide gels, renatured by removal of the SDS, and incubated with buffer containing [~/-32p]ATP. The phosphorylation of substrates polymerized into the gel or the autophosphorylation of individual kinases is detected by autoradiography. Individual protein bands may be excised from the gel for quantification of radioactivity, analysis of phosphoamino acid content, or use in subsequent electrophoretic or chromatographic steps. The procedure can be applied both to the study of kinases present in crude protein mixtures and to the characterization of purified or partially purified enzymes.
1 This work was supported by Grant CA37372 awarded by the National Cancer Institute, National Institutes of Health. 2 M. J. Heeb and O. Gabriel, this series, Vol. 104, p. 416. 3 R. L. Geahlen, M. Anostario, Jr., P. S. Low, and M. L. Harrison, Anal. Biochem. 153, 151 (1986).
METHODS IN ENZYMOLOGY, VOL 200
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
418
RENATURATION OF PROTEIN KINASES
[33]
Sample ~-Substrate
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
°
.
.
SDS-PAGE NI/ -SDS
.~ [a2P]ATP I I I I I
AUTORADIOGRAM FIG. l. Diagram of the major steps in the method for renaturation and assay of protein kinase activity following electrophoresis in SDS-polyacrylamide gels.
General Procedure 1. Protein samples are solubilized in an equal volume of SDS-sample buffer [2.5% (w/v) SDS, 25% (w/v) sucrose, 25 mM Tris-HC1, pH 8.0, 2.5 mM EDTA, 15 mM 2-mercaptoethanol, 2.5 mg% pyronin Y). The sample mixture is then heated in a boiling water bath for 5 min prior to loading on the gel. The boiling step does not inhibit the subsequent renaturation of protein kinase activity. Proteins are separated by electrophoresis on an SDS-PAGE slab gel consisting of a 9% (w/v) acrylamide running gel and a 4% (w/v) stacking gel using the buffer system described by Laemmli. 4 If desired, 1 mg/ml of a protein kinase substrate may be added to the running gel solution prior to polymerization. Electrophoresis is performed overnight at 7 mA/gel until the tracking dye reaches the bottom of the gel. 2. The stacking gel is cut away from the separating gel and discarded. 4 U. K. Laemmli, Nature (London) 277, 680 (1970).
[33]
RENATURATION OF PROTEIN KINASES IN S D S GELS
419
The remaining polyacrylamide gel is carefully placed into a clean plastic dish with a tight lid for the remaining steps. All of the incubations are at room temperature on an orbital shaker. 3. To remove the SDS, the polyacrylamide gel is washed six times with 200 ml of 40 mM HEPES, pH 7.4, over a period of 4-6 hr. 4. The gel is then incubated with 50 to 100 ml ofphosphorylation buffer [25 mM HEPES, pH 7.4, I0 mM MnC12,250-500/zCi [y-32p]ATP (>7000 Ci/mmol; New England Nuclear, Boston, MA] for 3 hr. From this step on, care should be taken to use the appropriate P!exiglas shielding around the radioactive gel. 5. To remove excess [y-32p]ATP, the gel is rinsed briefly with distilled water and incubated for 4-12 hr in 600 ml of 40 mM HEPES, pH 7.4. To the buffer is added 20 g of Dowex 2X8-50 anion-exchange resin packed into dialysis tubing, which acts to bind the free [y-32p]ATP. 6. Substrate-containing gels are washed for an additional 3 hr in 40 mM HEPES, pH 7.4, containing 1% (w/v) sodium pyrophosphate. 7. The proteins in the gel are fixed by a 1-hr incubation in 250 ml of 10% 2-propanol, 5% acetic acid, 1% sodium pyrophosphate; stained for 2 hr in 250 ml 0.1% Coomassie Brilliant Blue R in 25% 2-propanol, 10% acetic acid; and then destained in 250 ml of 10% 2-propanol, 5% acetic acid. The destaining time can be shortened by adding pieces of packaging foam to the buffer. 8. The gel is vacuum dried and exposed to Kodak (Rochester, NY) X-Omat AR film to detect labeled bands. Technical Comments and Applications The in situ renaturation procedure can be used to identify and characterize protein kinase activity in a number of different systems. Some of the parameters that can or have been modified for specific applications are discussed below. Strategies for SDS-PAGE The in situ renaturation technique is generally applied to the detection of protein kinases either by autophosphorylation or by the phosphorylation of specific substrates included in the gel matrix. The experimental design determines the amount of protein that needs to be loaded onto the gel. Small quantities of purified proteins (0.01 to 2/~g/gel lane) are sufficient for renaturation of activity in gels containing protein substrates. When detecting protein kinase activities in crude cell extracts by autophosphorylation, the best results are obtained when large amounts of protein are
420
RENATURATION OF PROTEIN K1NASES
1
[33]
2
FIG. 2. Detection of protein kinase activity in particulate fractions from mouse spleen T lymphocytes. Postnuclear particulate fractions were prepared as described previously [M. L. Harrison, P. S. Low, and R. L. Geahlen, J. Biol. Chem. 259, 9348 (1984)] from untreated T lymphocytes (lane 1) or from cells treated for 10 min with 100 ng/ml TPA (lane 2). Proteins were separated by electrophoresis using a Hoeffer minigel apparatus. Protein kinases were detected by autophosphorylation following in situ renaturation. Radiolabeled proteins were detected by autoradiography. The arrow indicates the migration position of an 80K protein. Molecular weight (× 10 -3) given on left-hand side.
loaded (0.25-1.0 mg/gel lane for 12 × 14 × 0.15 cm separating gels). For this purpose, it is not necessary to also include a substrate protein in the separating gel. By using a minigel apparatus, up to 10-fold less protein can be loaded and incubation times can be decreased considerably. An example renaturation experiment using the minigel format is shown in Fig. 2. Since many protein kinases catalyze autophosphorylation reactions, it is possible to identify several different enzymes in relatively crude protein mixtures. This is illustrated in Fig. 2 for particulate fraction proteins prepared from murine spleen T lymphocytes. The distribution of specific kinases can then be examined by comparing protein fractions prepared from different cells or different subcellular compartments. Changes in the distribution of kinases in response to various stimuli can also be examined
[33]
RENATURATION OF PROTEIN KINASES IN S D S GELS
421
using this approach. For example, pretreatment of T lymphocytes with 100 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA) results in the appearance of an 80-kDa kinase in the membrane preparation (Fig. 2). Such studies can also lead to the identification of new enzymes. 5 Different protein substrates can be incorporated into the gel matrix. From a technical point of view, the substrate needs only to be soluble under the SDS-PAGE conditions and not so small that it diffuses from the gel. When the substrate is included in the gel solution prior to initiation of polymerization, much of the protein becomes trapped in the gel matrix and does not electrophorese from the gel. Recovery of the protein from the polymerized gel is difficult, suggesting that some of it may even be covalently attached to the acrylamide polymers. Substrate specificity studies may be performed to characterize purified kinases. For example, the active subunits of multisubunit protein kinases such as the a subunit of casein kinase 113 or the 7 subunit of phosphorylase kinase 6 have been identified in this fashion. Also, the enzymes capable of phosphorylating specific substrates can be identified in crude protein mixtures, as illustrated by Paudel and C a r l s o n 6 for the phosphorylation of phosphorylase b by extracts of rabbit skeletal muscle. The technique should also be useful for the correlation of protein kinase activity with specific protein bands on polyacrylamide gels to verify the identity of purified or partially purified enzymes. The concentration of acrylamide in the SDS-polyacrylamide gels can be varied to optimize the resolution of kinases of interest. We have performed renaturation assays using concentrations of acrylamide ranging from 9 to 15% in the separating gel mixtures. The renaturation of enzymes separated on two-dimensional gels has been reported. 7 The addition of large amounts (1 mg/ml) of protein substrates to the polyacrylamide separating gel precludes the use of Coomassie Blue or other protein stains because of the high level of background staining. The use of prestained molecular weight markers provides a convenient way of determining the relative sizes of specific protein kinases in such gels. They may also be used to avoid the fixing, staining, and destaining steps. Renaturation Procedure
In some cases the inclusion of 25% (v/v) 2-propanol in the renaturation washes helps to remove the SDS and renature the enzymes. It has been suggested that 2-propanol may help remove renaturation-inhibiting con5 R. L. Geahlen and M. L. Harrison, Biochem. Biophys. Res. Commun. 134, 963 (1986). 6 H. K. Paudel and G. M. Carlson, Arch. Biochem. Biophys. 264, 641 (1988). 7 j. H. Keen, M. H. Chestnut, and K. A. Beck, J. Biol. Chem. 262, 3864 (1987).
422
RENATURATION OF PROTEIN KINASES
[33]
taminants present in certain batches of SDS. 8,9 We have had consistently good results using electrophoresis-grade SDS purchased from Bio-Rad (Richmond, CA), without the inclusion of 2-propanol in our buffers. After removal of SDS, denaturants such as 6 M guanidine hydrochloride or 8 M urea can be used to completely denature the proteins. Removal of the denaturants by subsequent washes allows the gradual refolding of the kinases into their enzymatically active conformations. 1° This has been reported to increase the sensitivity for the detection of Ca 2÷/calmodulindependent protein kinase II. H Renaturation times reported in the literature vary widely. The incubations should be long enough to ensure the complete removal of SDS, yet not so long as to cause a significant diffusion of proteins. Assay Conditions
Specific effectors of kinase activity such as Ca z+ or Ca 2+ and calmodulin can be added during the assay to detect kinases that are stimulated by these agents. In fact, the procedure can be utilized to decipher the cofactor requirements of certain enzymes. Harmon et al.12 used the renaturation method to demonstrate that a Ca 2+-dependent protein kinase from soybean was regulated by Ca 2÷, but did not require calmodulin. Similar results were obtained by Gunderson and Nelson 13 for a Ca2+-dependent kinase from Paramecium. 14 Ca2+/calmodulin dependent kinases from a variety of sources can be detected by including the activators in the assay mixture.~ ~,14,15 The phosphorylation buffer may be modified depending on the characteristics of a given protein kinase. For example, the catalytic subunit of cAMP-dependent protein kinase shows a clear preference for Mg 2+ over Mn 2+ in the presence of high concentrations of ATP, both in solution and when renatured in gels. T M However, at the low ATP concentrations described in our procedure above, Mn 2+ is much more effective than Mg 2+ at supporting the kinase reaction) It is important to verify that the presumptive kinase activity detected under a given set of experimental conditions is due to covalent binding of 8 S. A. Lacks, S. S. Springhorn, and A. L. Rosenthal, Anal. Biochem. 100, 357 (1979). 9 A. Blank, R. H. Sugiyama, and C. A. Dekker, Anal. Biochem. 120, 267 (1982). 10 K. Weber and D. J. Kuter, J. Biol. Chem. 2,46, 4504 (1971). H I. Kameshita and H. Fujisawa, Anal. Biochem. 183, 139 (1989). Iz A. C. Harmon, C. Putnam-Evans, and M. J. Cormier, Plant Physiol. 83, 830 (1987). 13 R. E. Gundersen and D. L. Nelson, J. Biol. Chem. 262, 4602 (1987). t4 D. P. Blowers and A. J. Trewavas, Biochem. Biophys. Res. Commun. 143, 691 (1987). 15 T. Miyakawa, Y. Oka, E. Tsuchiya, and S. Fukui, J. Bacteriol. 171, 1417 (1989). 16 E. M. Reimann, D. A. Walsh, and E. G. Krebs, J. Biol. Chem. 246, 1986 (1971).
[34]
PROTEIN KINASE ACTIVITY ON PROTEIN BLOTS
423
the radiolabeled phosphate rather than nonspecific binding or entrapment of [y32p]ATP. This can be done by performing a number of control experiments including the following: (1) the addition of excess unlabeled ATP to the phosphorylation buffer to compete with the [y-32p]ATP, (2) the performance of labeling studies with [a-32p]ATP,H (3) the reelectrophoresis of an excised 32p-labeled protein band on a second gel, or (4) analysis of the phosphoamino acid content of excised bands. In gels containing immobilized substrate, it is possible to differentiate between autophosphorylation of a protein kinase and substrate phosphorylation. This is easily done by reelectrophoresis of the 32p-labeled protein band in a second SDS-polyacrylamide gel and analysis of the molecular weight of the labeled protein. Recovery of the radiolabeled protein is improved if the proteins are not fixed prior to drying the initial gel. To be successful, the above techniques depend on the ability of a denatured protein kinase to refold to regain enzymatic activity. It is likely that some enzymes will not renature under these conditions. While many protein-serine/threonine kinases have been renatured from SDS-PAGE gels, relatively few protein-tyrosine kinases have been successfully detected. For example, we have not been successful at renaturing the proteintyrosine kinase p56 tckin gels containing proteins obtained from membranes of LSTRA, a cell line that overexpresses the enzyme.~7.~8Thus, the ability to detect a particular enzyme of interest must be empirically determined. However, for those enzymes that can be detected, in situ renaturation can be a simple and useful method for their further characterization. 17 A. F. Voronova and B. M. Sefton, Nature (London) 319, 682 (1986). 18 j. D. Marth, R. Peet, E. G. Krebs, and R. M. Pedmutter, Cell (Cambridge, Mass.) 43, 393 (1985).
[34] R e n a t u r a t i o n o f P r o t e i n K i n a s e A c t i v i t y o n P r o t e i n Blots By JOHN L. CELENZA and MARIAN CARLSON
We describe here a method for renaturing proteins bound to a nitrocellulose membrane and then assaying for protein kinase activity in situ on the protein blot. We first developed this assay to prove that a gene with sequence homology to protein kinases (the SNF1 gene of Saccharomyces cerevisiae) in fact encodes a protein with kinase activity (Fig. 1). ~ The 1 j. L. Celenza and M. Carlson, Science 233, 1175 (1986).
METHODS IN ENZYMOLOGY,VOL. 200
Copyright © 1991by AcademicPress, Inc. All rights of reproductionin any form reserved.
R E N A T U R A T I O N O F P R O T E I N KINASES IN S D S GELS
417
[33] R e n a t u r a t i o n a n d Assay of P r o t e i n Kinases after E l e c t r o p h o r e s i s in S o d i u m D o d e c y l S u l f a t e - P o l y a c r y l a m i d e Gels By JILL n . HUTCHCROFT, MICHAEL ANOSTARIO, JR.,
MARIETTA L. HARRISON, and ROBERT L. GEAHLEN
An increasing appreciation of the importance of protein kinases in metabolic regulation has generated considerable interest in methods for their separation, detection, and characterization.1 A powerful method for the resolution of complex mixtures of proteins containing kinases is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although SDS-enzyme complexes are typically devoid of enzyme activity, it has proved possible in many cases to recover activity after removal of the detergent. Thus, procedures can be developed that couple the high resolving power of SDS-polyacrylamide gel electrophoresis with "on the spot" detection of specific enzyme activities. An extensive review of techniques used to localize functionally active enzymes following gel electrophoresis has been previously published in this series. 2We have adapted and extended such procedures to develop a simple and convenient method for the in situ (in the gel) renaturation and assay of protein kinases. 3 Our gel-renaturation technique is summarized in Fig. 1. Briefly, proteins are separated by electrophoresis through SDS-polyacrylamide gels, renatured by removal of the SDS, and incubated with buffer containing [~/-32p]ATP. The phosphorylation of substrates polymerized into the gel or the autophosphorylation of individual kinases is detected by autoradiography. Individual protein bands may be excised from the gel for quantification of radioactivity, analysis of phosphoamino acid content, or use in subsequent electrophoretic or chromatographic steps. The procedure can be applied both to the study of kinases present in crude protein mixtures and to the characterization of purified or partially purified enzymes.
1 This work was supported by Grant CA37372 awarded by the National Cancer Institute, National Institutes of Health. 2 M. J. Heeb and O. Gabriel, this series, Vol. 104, p. 416. 3 R. L. Geahlen, M. Anostario, Jr., P. S. Low, and M. L. Harrison, Anal. Biochem. 153, 151 (1986).
METHODS IN ENZYMOLOGY, VOL 200
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
418
RENATURATION OF PROTEIN KINASES
[33]
Sample ~-Substrate
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
°
.
.
SDS-PAGE NI/ -SDS
.~ [a2P]ATP I I I I I
AUTORADIOGRAM FIG. l. Diagram of the major steps in the method for renaturation and assay of protein kinase activity following electrophoresis in SDS-polyacrylamide gels.
General Procedure 1. Protein samples are solubilized in an equal volume of SDS-sample buffer [2.5% (w/v) SDS, 25% (w/v) sucrose, 25 mM Tris-HC1, pH 8.0, 2.5 mM EDTA, 15 mM 2-mercaptoethanol, 2.5 mg% pyronin Y). The sample mixture is then heated in a boiling water bath for 5 min prior to loading on the gel. The boiling step does not inhibit the subsequent renaturation of protein kinase activity. Proteins are separated by electrophoresis on an SDS-PAGE slab gel consisting of a 9% (w/v) acrylamide running gel and a 4% (w/v) stacking gel using the buffer system described by Laemmli. 4 If desired, 1 mg/ml of a protein kinase substrate may be added to the running gel solution prior to polymerization. Electrophoresis is performed overnight at 7 mA/gel until the tracking dye reaches the bottom of the gel. 2. The stacking gel is cut away from the separating gel and discarded. 4 U. K. Laemmli, Nature (London) 277, 680 (1970).
[33]
RENATURATION OF PROTEIN KINASES IN S D S GELS
419
The remaining polyacrylamide gel is carefully placed into a clean plastic dish with a tight lid for the remaining steps. All of the incubations are at room temperature on an orbital shaker. 3. To remove the SDS, the polyacrylamide gel is washed six times with 200 ml of 40 mM HEPES, pH 7.4, over a period of 4-6 hr. 4. The gel is then incubated with 50 to 100 ml ofphosphorylation buffer [25 mM HEPES, pH 7.4, I0 mM MnC12,250-500/zCi [y-32p]ATP (>7000 Ci/mmol; New England Nuclear, Boston, MA] for 3 hr. From this step on, care should be taken to use the appropriate P!exiglas shielding around the radioactive gel. 5. To remove excess [y-32p]ATP, the gel is rinsed briefly with distilled water and incubated for 4-12 hr in 600 ml of 40 mM HEPES, pH 7.4. To the buffer is added 20 g of Dowex 2X8-50 anion-exchange resin packed into dialysis tubing, which acts to bind the free [y-32p]ATP. 6. Substrate-containing gels are washed for an additional 3 hr in 40 mM HEPES, pH 7.4, containing 1% (w/v) sodium pyrophosphate. 7. The proteins in the gel are fixed by a 1-hr incubation in 250 ml of 10% 2-propanol, 5% acetic acid, 1% sodium pyrophosphate; stained for 2 hr in 250 ml 0.1% Coomassie Brilliant Blue R in 25% 2-propanol, 10% acetic acid; and then destained in 250 ml of 10% 2-propanol, 5% acetic acid. The destaining time can be shortened by adding pieces of packaging foam to the buffer. 8. The gel is vacuum dried and exposed to Kodak (Rochester, NY) X-Omat AR film to detect labeled bands. Technical Comments and Applications The in situ renaturation procedure can be used to identify and characterize protein kinase activity in a number of different systems. Some of the parameters that can or have been modified for specific applications are discussed below. Strategies for SDS-PAGE The in situ renaturation technique is generally applied to the detection of protein kinases either by autophosphorylation or by the phosphorylation of specific substrates included in the gel matrix. The experimental design determines the amount of protein that needs to be loaded onto the gel. Small quantities of purified proteins (0.01 to 2/~g/gel lane) are sufficient for renaturation of activity in gels containing protein substrates. When detecting protein kinase activities in crude cell extracts by autophosphorylation, the best results are obtained when large amounts of protein are
420
RENATURATION OF PROTEIN K1NASES
1
[33]
2
FIG. 2. Detection of protein kinase activity in particulate fractions from mouse spleen T lymphocytes. Postnuclear particulate fractions were prepared as described previously [M. L. Harrison, P. S. Low, and R. L. Geahlen, J. Biol. Chem. 259, 9348 (1984)] from untreated T lymphocytes (lane 1) or from cells treated for 10 min with 100 ng/ml TPA (lane 2). Proteins were separated by electrophoresis using a Hoeffer minigel apparatus. Protein kinases were detected by autophosphorylation following in situ renaturation. Radiolabeled proteins were detected by autoradiography. The arrow indicates the migration position of an 80K protein. Molecular weight (× 10 -3) given on left-hand side.
loaded (0.25-1.0 mg/gel lane for 12 × 14 × 0.15 cm separating gels). For this purpose, it is not necessary to also include a substrate protein in the separating gel. By using a minigel apparatus, up to 10-fold less protein can be loaded and incubation times can be decreased considerably. An example renaturation experiment using the minigel format is shown in Fig. 2. Since many protein kinases catalyze autophosphorylation reactions, it is possible to identify several different enzymes in relatively crude protein mixtures. This is illustrated in Fig. 2 for particulate fraction proteins prepared from murine spleen T lymphocytes. The distribution of specific kinases can then be examined by comparing protein fractions prepared from different cells or different subcellular compartments. Changes in the distribution of kinases in response to various stimuli can also be examined
[33]
RENATURATION OF PROTEIN KINASES IN S D S GELS
421
using this approach. For example, pretreatment of T lymphocytes with 100 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA) results in the appearance of an 80-kDa kinase in the membrane preparation (Fig. 2). Such studies can also lead to the identification of new enzymes. 5 Different protein substrates can be incorporated into the gel matrix. From a technical point of view, the substrate needs only to be soluble under the SDS-PAGE conditions and not so small that it diffuses from the gel. When the substrate is included in the gel solution prior to initiation of polymerization, much of the protein becomes trapped in the gel matrix and does not electrophorese from the gel. Recovery of the protein from the polymerized gel is difficult, suggesting that some of it may even be covalently attached to the acrylamide polymers. Substrate specificity studies may be performed to characterize purified kinases. For example, the active subunits of multisubunit protein kinases such as the a subunit of casein kinase 113 or the 7 subunit of phosphorylase kinase 6 have been identified in this fashion. Also, the enzymes capable of phosphorylating specific substrates can be identified in crude protein mixtures, as illustrated by Paudel and C a r l s o n 6 for the phosphorylation of phosphorylase b by extracts of rabbit skeletal muscle. The technique should also be useful for the correlation of protein kinase activity with specific protein bands on polyacrylamide gels to verify the identity of purified or partially purified enzymes. The concentration of acrylamide in the SDS-polyacrylamide gels can be varied to optimize the resolution of kinases of interest. We have performed renaturation assays using concentrations of acrylamide ranging from 9 to 15% in the separating gel mixtures. The renaturation of enzymes separated on two-dimensional gels has been reported. 7 The addition of large amounts (1 mg/ml) of protein substrates to the polyacrylamide separating gel precludes the use of Coomassie Blue or other protein stains because of the high level of background staining. The use of prestained molecular weight markers provides a convenient way of determining the relative sizes of specific protein kinases in such gels. They may also be used to avoid the fixing, staining, and destaining steps. Renaturation Procedure
In some cases the inclusion of 25% (v/v) 2-propanol in the renaturation washes helps to remove the SDS and renature the enzymes. It has been suggested that 2-propanol may help remove renaturation-inhibiting con5 R. L. Geahlen and M. L. Harrison, Biochem. Biophys. Res. Commun. 134, 963 (1986). 6 H. K. Paudel and G. M. Carlson, Arch. Biochem. Biophys. 264, 641 (1988). 7 j. H. Keen, M. H. Chestnut, and K. A. Beck, J. Biol. Chem. 262, 3864 (1987).
422
RENATURATION OF PROTEIN KINASES
[33]
taminants present in certain batches of SDS. 8,9 We have had consistently good results using electrophoresis-grade SDS purchased from Bio-Rad (Richmond, CA), without the inclusion of 2-propanol in our buffers. After removal of SDS, denaturants such as 6 M guanidine hydrochloride or 8 M urea can be used to completely denature the proteins. Removal of the denaturants by subsequent washes allows the gradual refolding of the kinases into their enzymatically active conformations. 1° This has been reported to increase the sensitivity for the detection of Ca 2÷/calmodulindependent protein kinase II. H Renaturation times reported in the literature vary widely. The incubations should be long enough to ensure the complete removal of SDS, yet not so long as to cause a significant diffusion of proteins. Assay Conditions
Specific effectors of kinase activity such as Ca z+ or Ca 2+ and calmodulin can be added during the assay to detect kinases that are stimulated by these agents. In fact, the procedure can be utilized to decipher the cofactor requirements of certain enzymes. Harmon et al.12 used the renaturation method to demonstrate that a Ca 2+-dependent protein kinase from soybean was regulated by Ca 2÷, but did not require calmodulin. Similar results were obtained by Gunderson and Nelson 13 for a Ca2+-dependent kinase from Paramecium. 14 Ca2+/calmodulin dependent kinases from a variety of sources can be detected by including the activators in the assay mixture.~ ~,14,15 The phosphorylation buffer may be modified depending on the characteristics of a given protein kinase. For example, the catalytic subunit of cAMP-dependent protein kinase shows a clear preference for Mg 2+ over Mn 2+ in the presence of high concentrations of ATP, both in solution and when renatured in gels. T M However, at the low ATP concentrations described in our procedure above, Mn 2+ is much more effective than Mg 2+ at supporting the kinase reaction) It is important to verify that the presumptive kinase activity detected under a given set of experimental conditions is due to covalent binding of 8 S. A. Lacks, S. S. Springhorn, and A. L. Rosenthal, Anal. Biochem. 100, 357 (1979). 9 A. Blank, R. H. Sugiyama, and C. A. Dekker, Anal. Biochem. 120, 267 (1982). 10 K. Weber and D. J. Kuter, J. Biol. Chem. 2,46, 4504 (1971). H I. Kameshita and H. Fujisawa, Anal. Biochem. 183, 139 (1989). Iz A. C. Harmon, C. Putnam-Evans, and M. J. Cormier, Plant Physiol. 83, 830 (1987). 13 R. E. Gundersen and D. L. Nelson, J. Biol. Chem. 262, 4602 (1987). t4 D. P. Blowers and A. J. Trewavas, Biochem. Biophys. Res. Commun. 143, 691 (1987). 15 T. Miyakawa, Y. Oka, E. Tsuchiya, and S. Fukui, J. Bacteriol. 171, 1417 (1989). 16 E. M. Reimann, D. A. Walsh, and E. G. Krebs, J. Biol. Chem. 246, 1986 (1971).
[34]
PROTEIN KINASE ACTIVITY ON PROTEIN BLOTS
423
the radiolabeled phosphate rather than nonspecific binding or entrapment of [y32p]ATP. This can be done by performing a number of control experiments including the following: (1) the addition of excess unlabeled ATP to the phosphorylation buffer to compete with the [y-32p]ATP, (2) the performance of labeling studies with [a-32p]ATP,H (3) the reelectrophoresis of an excised 32p-labeled protein band on a second gel, or (4) analysis of the phosphoamino acid content of excised bands. In gels containing immobilized substrate, it is possible to differentiate between autophosphorylation of a protein kinase and substrate phosphorylation. This is easily done by reelectrophoresis of the 32p-labeled protein band in a second SDS-polyacrylamide gel and analysis of the molecular weight of the labeled protein. Recovery of the radiolabeled protein is improved if the proteins are not fixed prior to drying the initial gel. To be successful, the above techniques depend on the ability of a denatured protein kinase to refold to regain enzymatic activity. It is likely that some enzymes will not renature under these conditions. While many protein-serine/threonine kinases have been renatured from SDS-PAGE gels, relatively few protein-tyrosine kinases have been successfully detected. For example, we have not been successful at renaturing the proteintyrosine kinase p56 tckin gels containing proteins obtained from membranes of LSTRA, a cell line that overexpresses the enzyme.~7.~8Thus, the ability to detect a particular enzyme of interest must be empirically determined. However, for those enzymes that can be detected, in situ renaturation can be a simple and useful method for their further characterization. 17 A. F. Voronova and B. M. Sefton, Nature (London) 319, 682 (1986). 18 j. D. Marth, R. Peet, E. G. Krebs, and R. M. Pedmutter, Cell (Cambridge, Mass.) 43, 393 (1985).
[34] R e n a t u r a t i o n o f P r o t e i n K i n a s e A c t i v i t y o n P r o t e i n Blots By JOHN L. CELENZA and MARIAN CARLSON
We describe here a method for renaturing proteins bound to a nitrocellulose membrane and then assaying for protein kinase activity in situ on the protein blot. We first developed this assay to prove that a gene with sequence homology to protein kinases (the SNF1 gene of Saccharomyces cerevisiae) in fact encodes a protein with kinase activity (Fig. 1). ~ The 1 j. L. Celenza and M. Carlson, Science 233, 1175 (1986).
METHODS IN ENZYMOLOGY,VOL. 200
Copyright © 1991by AcademicPress, Inc. All rights of reproductionin any form reserved.
