[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.
424
[34]
RENATURATION OF PROTEIN KINASES
a
b
c
d
e
205 --
f
--SNF1 8gal
116-97--
SNF1
66--
45_
:i!!!iii!!!!~;iii~i~ii!ii!iiii!!!iiii!ii~;!!~:?~!iii!i-i!?:ii i i ? i~¸::
.......
FIG. 1. Assay of the SNF1 protein kinase of S. cerevisiae on a protein blot. Proteins were prepared from cultures (glucose repressed, except for lane c) of yeast strains with the following genotypes: (a) wild type (SNFI +) carrying the SNF1 gene on a multicopy plasmid,. (b) wild type, (c) wild type, glucose derepressed, (d) snfl-A3 (deletion mutation), (e) snfl::HIS3 (insertion mutation), (f) snfl::HIS3 carrying a bifunctional SNFI-lacZ gene fusion on a plasmid. Proteins (50 p,g) were separated by electrophoresis in 7.5% SDS-polyacrylamide and transferred to nitrocellulose. Protein kinase activity was assayed on the protein blot as described here, with only minor differences. An automdiograph is shown. The positions of the SNFI and SNFl-fl-galactosidase (SNFI-/3gal) fusion proteins are marked. The identification of these proteins was confirmed by immunoblot analysis with anti-SNF1 antibody (not shown). Faint bands above the SNF 1 band were detected frequently, but not reproducibly (see lane b). Protein size standards are indicated in kilodaltons. [Adapted from Ref. 1 (copyright 1986 by the AAAS).]
[34]
PROTEIN KINASE ACTIVITY ON PROTEIN BLOTS
425
method is generally applicable for assaying protein kinase activity and does not require antibody. It is particularly useful for demonstrating that a cloned putative protein kinase gene or cDNA encodes a protein kinase. Other applications are also discussed. In this method, a protein blot is prepared by transferring electrophoretically fractionated proteins to a nitrocellulose membrane. The proteins bound to the filter are exposed to the denaturant guanidine hydrochloride and then are allowed to renature in buffer. An autophosphorylation reaction is carried out by incubating the filter-bound proteins in buffer containlng [y-32p]ATP. The labeled products are detected by autoradiography. Genetic or immunological methods can be applied to identify the protein kinase activity of interest. Method Preparing Protein Blot
Separate proteins by SDS-polyacrylamide gel electrophoresis 2 and electrophoretically transfer to a nitrocellulose membrane 3 in cold 25 mM Tris, 192 mM glycine (pH 8.3), without methanol. Carry out the transfer in the cold room and submerge the electroblotter in an ice bath. Apply a voltage gradient of 12-14 V/cm for 90 min. Blocking Filter
Block the filter by incubation in 30 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonate), pH 7.5, containing 5% (w/v) nonfat dry milk (Carnation), for 30 min at 25°. This and subsequent incubations and washes are carried out on a rotating platform. Comments. Nonfat dry milk is commonly used as a blocking agent. We have substituted 3% (w/v) gelatin or 100 /~g/ml phenol-extracted salmon sperm DNA as the blocking agent and then omitted nonfat dry milk from renaturation and denaturation buffers. These changes did not affect the array of phosphorylated proteins detected. Omitting any blocking agent reduced labeling severalfold and increased the background. Roussou et al. 4 substituted 1% (w/v) bovine serum albumin or 1% (w/v) total histones. Varying the blocking agent provides a control to determine whether proteins in the blocking agent are serving as a substrate for the kinase of 2 U. K. Laemmli, Nature (London) 227, 680 (1970). 3 H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979). 4 I. Roussou, G. Thireos, and B. M. Hauge, Mol. Cell. Biol. 8, 2132 (1988).
426
RENATURATION OF PROTEIN KINASES
[34]
interest or in some way affecting kinase activity on the blot. The blocking agent can also be chosen deliberately to provide a substrate; for example, the yeast GCN2 protein kinase is not autophosphorylated in this assay but is able to phosphorylate histones coating the filter. 4 Denaturation and Renaturation o f Filter-Bound Proteins
Incubate the filter in 100 ml of denaturation buffer for 1 hr at 25°. Wash the filter several times in cold renaturation buffer and incubate in 500 ml of renaturation buffer for 16 hr at 4°. (Suggested volumes are suitable for filters up to 12 x 14 cm.) Denaturation buffer: 7 M guanidine hydrochloride, 50 mM Tris-HCl, pH 8.3, 50 m M dithiothreitol (DTT), 2 mM EDTA, 0.25% (w/v) nonfat dry milk Renaturation buffer: 50 mM Tris-HC1, pH 7.5, I00 mM NaCI, 2 mM DTT, 2 mM EDTA, 0.1% (v/v) Nonidet P-40 (NP-40), 0.25% (w/v) nonfat dry milk Comments. This denaturation and renaturation procedure is adapted 5
from the method of Hager and B u r g e s s . 6 Omission of the denaturation step reduces phosphorylation of SNFI and other proteins; however, this might not be the case for all protein kinases. This and similar denaturation and renaturation procedures have been effective for renaturing a variety of activities on protein blots: phosphorylation of histones by the yeast GCN2 protein kinase4; DNA binding by retroviral gene products required for integration, 7 by a human enhancer-binding protein, 8 and by yeast transcription factor IIIAg; and erythrocyte binding by a Giardia lectin. 10Vinson et ai. 11 described a related method involving graded removal of the denaturant that allowed in situ detection of DNA binding activity for a nitrocellulose-bound protein expressed from a recombinant bacteriophage.
5 M. J. Roth, personal communication (1986). 6 D. A. Hager and R. R. Burgess, Anal. Biochem. 109, 76 (1980). 7 M. J. Roth, N. Tanese, and S. P. Goff, J. Mol. Biol. 203, 131 (1988). s H. Singh, J. H. LeBowitz, A. S. Baldwin, Jr., and P. A. Sharp, Cell (Cambridge, Mass.) 52, 415 (1988). 9 C. K. Wang and P. A. Weil, J. Biol. Chem. 264, 1092 (1989). 10 H. D. Ward, B. I. Lev, A. V. Kane, G. T. Keusch, and M. E. A. Pereira, Biochemistry 26, 8669 (1987). 11 C. R. Vinson, K. L. LaMarco, P. F. Johnson, W. H. Landschulz, and S. L. McKnight, Genes Dev. 2, 801 (1988).
[34]
PROTEIN KINASE ACTIVITY ON PROTEIN BLOTS
427
Autophosphorylation Reaction Rinse the filter several times in 30 mM HEPES, pH 7.5, at 25°. Place the filter in a sealable plastic bag with 5 ml of kinase buffer containing [T-32p]ATP. Incubate 20-30 rain at 25°. Kinase buffer: 30 mM HEPES, pH 7.5, l0 mM MgC12, 2 mM MnC12, 0.1 ftM ATP, 0.03 /zM [T-32p]ATP (3000 Ci/mmol, New England Nuclear, Boston, MA)
Comments. The kinase buffer should be adjusted according to the requirements of particular protein kinases. The reaction can also be optimized with respect to ATP concentration. The reported Km values for most protein kinases are in the range of 10 to 100/,tM. 12 To assay the SNF1 kinase, we originally used only 0.03/.tM [T-aEp]ATP. Addition of 0.1/xM cold ATP did not change the incorporation of label into the SNF1 protein and reduced the background. Addition of 1 btM cold ATP reduced the labeling of SNFI about twofold, and addition of I0/.tM cold ATP reduced the signal substantially. Washing Filter and Autoradiography Wash the filter in 30 mM HEPES, pH 7.5, for 2 hr at 25° several times, until the radioactivity bound to the filter no longer decreases with continued washing. Dry the filter in air until slightly damp; a damp filter can be successfully washed more extensively, if necessary. Expose to film for 1 to 10 hr at - 7 0 ° with an intensifying screen for autoradiography. Longer exposures may result in unacceptable background. Comments. If the washing procedure described above does not reduce the background to an acceptable level, the filter can be washed again in 1 N HC1 for 1 hr at 25°. Washing the filter in 1 N HCI for 1 hr at 70° greatly reduces background, but also reduces the signal; we found that the amount of SNF1 protein detectable by subsequent immunoblot assay was also reduced. Roussou et al. 4 included 1 mM ATP in the washing buffer.
Identifying Activity of Interest Genetic Methods. In genetic systems, the protein kinase activity of interest can be identified by examining mutants lacking the protein, overexpressing the protein, or expressing a derivative with different electrophoretic mobility (Fig. 1). Similar methods apply for proteins expressed 12 p. j. Roach, in "Methods in Enzymology" (F. Wold and K. Moldave, eds.), Vol. 107, p. 81. Academic Press, Orlando, Florida, 1984.
428
RENATURATION OF PROTEIN KINASES
[34]
from cloned sequences in bacteria; in this case identifying the activity of interest should be trivial. Immunological Methods. If antibody specific to the protein kinase of interest is available, the filter can be used for an immunoblot assay after a suitable autoradiograph is obtained. Analysis o f Phosphoprotein
To demonstrate that the labeling of a protein results from phosphorylation of an amino acid residue, the phosphoprotein can be recovered from the filter by the method of Parekh et al. ~3and subjected to phosphoamino acid analysis. ~4 Estimate the amount of protein required to yield sufficient labeled material and, if necessary, run a preparative gel. After carrying out the assay, excise the region of the filter containing the phosphoprotein, taking care to minimize the excised region. Incubate the filter strip in 0.5 ml of 40% (v/v) acetonitrile, 0.1 M ammonium acetate, pH 8.9, for 3 hr at 37°. About 30-60% of the radioactive label should be recovered. Lyophilize the eluate. Subject the recovered material to partial acid hydrolysis and separate phosphoamino acids by two-dimensional thin-layer electrophoresis as described by Cooper et al. 14 As a control, carry out the same procedure using an equal-sized strip of filter that does not have labeled protein bound to it. Applications This is a rapid method for demonstrating biochemically that a gene that is homologous to protein kinase genes does in fact encode a functional protein kinase. This method does not require either biochemical characterization of the protein or specific antibody. Cloned genes or cDNAs can be expressed in Escherichia coli, and the protein produced can be assayed directly. In genetic systems, mutants can be employed to show that a gene encodes a protein kinase. In systems where altered genes can.be transformed into the organism or into cultured cells, the gene can be manipulated in vitro to change the size and level of expression of the gene product, thereby facilitating proof that the phosphorylated protein is encoded by the gene in question. If antibody specific to the protein is available, this assay can be used 13 B. S. Parekh, H. B. Mehta, M. D. West, and R. C. Montelaro, Anal. Biochem. 148, 87 (1985). 14j. A. Cooper, B. M. Sefton, and T. Hunter, in "Methods in Enzymology" (J. D. Corbin and J. D. Hardman, eds.), Vol. 99, p. 387. Academic Press, New York, 1983.
[341
PROTEIN KINASE ACTIVITY ON PROTEIN BLOTS
429
as an alternate to, or in conjunction with, immune complex assays. This assay offers the advantage that the kinase activity is associated with a particular polypeptide. Thus, it can be used to confirm that the activity detected in an immune complex assay is due to the protein against which the antibody is directed, rather than to a minor protein kinase contaminating the immune complex. This assay may also be useful during the purification of a protein kinase to assign the catalytic activity to a particular polypeptide. The activity of a protein kinase is in many cases regulated or modulated in some way. This assay can be useful in distinguishing between possible regulatory mechanisms. For example, if the kinase activity is altered by a mechanism involving covalent modification, differences in activity may be apparent in this assay, presuming that the modification is stable. In contrast, if the kinase activity is modulated by physical association with another protein, then the electrophoretic separation of the two proteins in this procedure should preclude detection of regulatory effects. Limitations and Possible Problems The method is not suitable for detection of any protein kinase that requires two different subunits for activity because the different polypeptides would be separated electrophoretically. A possible remedy would be electrophoresis on a nondenaturing gel so that the polypeptides remain associated. Some proteins may not regain activity in response to a denaturation and renaturation regimen. It is possible that some proteins may retain. activity or renature more efficiently following electrophoresis under nondenaturing conditions. The activity of a protein kinase may be too low, relative to background, for detection by this assay. If the cloned gene is available, overexpression of the protein would remedy this problem. Another possible problem is that the protein kinase may comigrate with another kinase so that the activity of interest is obscured. For example, assays of yeast proteins revealed many highly phosphorylated proteins of 45 to 63 kDa (Fig. 1), and it would therefore be difficult to identify a minor activity in this size range. For proteins expressed in E. coil, comigration of two activities is less likely to pose a problem as there are few phosphorylated species detected by this assay. If the cloned gene or eDNA encoding the protein kinase is available, the gene can usually be manipulated in vitro to shift the activity to a more convenient region of the protein blot. It is often possible to construct a fusion to another protein, such as fl-galactosidase, that is larger than the native protein and yet
430
RENATURATION OF PROTEIN KINASES
[35]
retains protein kinase activity (Fig. 1). Construction of fusion proteins, or other derivatives, also provides a valuable control. Demonstration that two different-sized derivatives both exhibit kinase activity confirms that the protein of interest is not merely serving as a substrate for another, comigrating kinase. Related methods are described in [33] and [35] in this volume. Acknowledgments W e thank Monica Roth for suggesting the method. This work was supported by Pubfic Health Service Grant GM34095 from the N.I.H. and an American Cancer Society Faculty Research Award to M.C.
[35] A s s e s s i n g A c t i v i t i e s o f B l o t t e d P r o t e i n K i n a s e s
By JAMES E. FERRELL, JR., and G. STEVEN MARTIN Celenza and C a r l s o n 1,2 have described a method for assessing the activities of protein kinases bound to nitrocellulose-blotting membranes. The method exploits the ability of SDS-denatured enzymes to regain activity after treatment with guanidine and nonionic detergent. The resulting kinase activities might arise from disinhibition of partially denatured enzymes or from renaturation of denatured enzymes. Here we present a modification of the Celenza and Carlson assay. In this assay, poly(vinylidene difluoride) (PVDF) membranes are used in place of nitrocellulose. The main advantage of PVDF here is that it can be washed with strong bases, which significantly lowers the background of unincorporated radiolabel without diminishing the kinase signal. In addition, milk is not used in the protocol. At least some batches of nonfat dry milk possess detectable levels of kinase activity (G. Schieven and J. E. FerreU, unpublished results), which means that blotted proteins that appear to possess kinase activity could actually represent preferred substrates for the milk kinase. This precaution may be of a more theoretical than practical significance; we have not found any kinase bands that are detectable when milk is used as a blocking agent that are not when albumin or no blocking agent is used. This protocol is the most sensitive and reliable of many variations we I j. L. Celenza and M. Carlson, this volume [34]. 2 j. L. Celenza and M. Carlson, Science 233, 1175 (1986).
METHODS IN ENZYMOLOGY, VOL. 200
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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.
424
[34]
RENATURATION OF PROTEIN KINASES
a
b
c
d
e
205 --
f
--SNF1 8gal
116-97--
SNF1
66--
45_
:i!!!iii!!!!~;iii~i~ii!ii!iiii!!!iiii!ii~;!!~:?~!iii!i-i!?:ii i i ? i~¸::
.......
FIG. 1. Assay of the SNF1 protein kinase of S. cerevisiae on a protein blot. Proteins were prepared from cultures (glucose repressed, except for lane c) of yeast strains with the following genotypes: (a) wild type (SNFI +) carrying the SNF1 gene on a multicopy plasmid,. (b) wild type, (c) wild type, glucose derepressed, (d) snfl-A3 (deletion mutation), (e) snfl::HIS3 (insertion mutation), (f) snfl::HIS3 carrying a bifunctional SNFI-lacZ gene fusion on a plasmid. Proteins (50 p,g) were separated by electrophoresis in 7.5% SDS-polyacrylamide and transferred to nitrocellulose. Protein kinase activity was assayed on the protein blot as described here, with only minor differences. An automdiograph is shown. The positions of the SNFI and SNFl-fl-galactosidase (SNFI-/3gal) fusion proteins are marked. The identification of these proteins was confirmed by immunoblot analysis with anti-SNF1 antibody (not shown). Faint bands above the SNF 1 band were detected frequently, but not reproducibly (see lane b). Protein size standards are indicated in kilodaltons. [Adapted from Ref. 1 (copyright 1986 by the AAAS).]
[34]
PROTEIN KINASE ACTIVITY ON PROTEIN BLOTS
425
method is generally applicable for assaying protein kinase activity and does not require antibody. It is particularly useful for demonstrating that a cloned putative protein kinase gene or cDNA encodes a protein kinase. Other applications are also discussed. In this method, a protein blot is prepared by transferring electrophoretically fractionated proteins to a nitrocellulose membrane. The proteins bound to the filter are exposed to the denaturant guanidine hydrochloride and then are allowed to renature in buffer. An autophosphorylation reaction is carried out by incubating the filter-bound proteins in buffer containlng [y-32p]ATP. The labeled products are detected by autoradiography. Genetic or immunological methods can be applied to identify the protein kinase activity of interest. Method Preparing Protein Blot
Separate proteins by SDS-polyacrylamide gel electrophoresis 2 and electrophoretically transfer to a nitrocellulose membrane 3 in cold 25 mM Tris, 192 mM glycine (pH 8.3), without methanol. Carry out the transfer in the cold room and submerge the electroblotter in an ice bath. Apply a voltage gradient of 12-14 V/cm for 90 min. Blocking Filter
Block the filter by incubation in 30 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonate), pH 7.5, containing 5% (w/v) nonfat dry milk (Carnation), for 30 min at 25°. This and subsequent incubations and washes are carried out on a rotating platform. Comments. Nonfat dry milk is commonly used as a blocking agent. We have substituted 3% (w/v) gelatin or 100 /~g/ml phenol-extracted salmon sperm DNA as the blocking agent and then omitted nonfat dry milk from renaturation and denaturation buffers. These changes did not affect the array of phosphorylated proteins detected. Omitting any blocking agent reduced labeling severalfold and increased the background. Roussou et al. 4 substituted 1% (w/v) bovine serum albumin or 1% (w/v) total histones. Varying the blocking agent provides a control to determine whether proteins in the blocking agent are serving as a substrate for the kinase of 2 U. K. Laemmli, Nature (London) 227, 680 (1970). 3 H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979). 4 I. Roussou, G. Thireos, and B. M. Hauge, Mol. Cell. Biol. 8, 2132 (1988).
426
RENATURATION OF PROTEIN KINASES
[34]
interest or in some way affecting kinase activity on the blot. The blocking agent can also be chosen deliberately to provide a substrate; for example, the yeast GCN2 protein kinase is not autophosphorylated in this assay but is able to phosphorylate histones coating the filter. 4 Denaturation and Renaturation o f Filter-Bound Proteins
Incubate the filter in 100 ml of denaturation buffer for 1 hr at 25°. Wash the filter several times in cold renaturation buffer and incubate in 500 ml of renaturation buffer for 16 hr at 4°. (Suggested volumes are suitable for filters up to 12 x 14 cm.) Denaturation buffer: 7 M guanidine hydrochloride, 50 mM Tris-HCl, pH 8.3, 50 m M dithiothreitol (DTT), 2 mM EDTA, 0.25% (w/v) nonfat dry milk Renaturation buffer: 50 mM Tris-HC1, pH 7.5, I00 mM NaCI, 2 mM DTT, 2 mM EDTA, 0.1% (v/v) Nonidet P-40 (NP-40), 0.25% (w/v) nonfat dry milk Comments. This denaturation and renaturation procedure is adapted 5
from the method of Hager and B u r g e s s . 6 Omission of the denaturation step reduces phosphorylation of SNFI and other proteins; however, this might not be the case for all protein kinases. This and similar denaturation and renaturation procedures have been effective for renaturing a variety of activities on protein blots: phosphorylation of histones by the yeast GCN2 protein kinase4; DNA binding by retroviral gene products required for integration, 7 by a human enhancer-binding protein, 8 and by yeast transcription factor IIIAg; and erythrocyte binding by a Giardia lectin. 10Vinson et ai. 11 described a related method involving graded removal of the denaturant that allowed in situ detection of DNA binding activity for a nitrocellulose-bound protein expressed from a recombinant bacteriophage.
5 M. J. Roth, personal communication (1986). 6 D. A. Hager and R. R. Burgess, Anal. Biochem. 109, 76 (1980). 7 M. J. Roth, N. Tanese, and S. P. Goff, J. Mol. Biol. 203, 131 (1988). s H. Singh, J. H. LeBowitz, A. S. Baldwin, Jr., and P. A. Sharp, Cell (Cambridge, Mass.) 52, 415 (1988). 9 C. K. Wang and P. A. Weil, J. Biol. Chem. 264, 1092 (1989). 10 H. D. Ward, B. I. Lev, A. V. Kane, G. T. Keusch, and M. E. A. Pereira, Biochemistry 26, 8669 (1987). 11 C. R. Vinson, K. L. LaMarco, P. F. Johnson, W. H. Landschulz, and S. L. McKnight, Genes Dev. 2, 801 (1988).
[34]
PROTEIN KINASE ACTIVITY ON PROTEIN BLOTS
427
Autophosphorylation Reaction Rinse the filter several times in 30 mM HEPES, pH 7.5, at 25°. Place the filter in a sealable plastic bag with 5 ml of kinase buffer containing [T-32p]ATP. Incubate 20-30 rain at 25°. Kinase buffer: 30 mM HEPES, pH 7.5, l0 mM MgC12, 2 mM MnC12, 0.1 ftM ATP, 0.03 /zM [T-32p]ATP (3000 Ci/mmol, New England Nuclear, Boston, MA)
Comments. The kinase buffer should be adjusted according to the requirements of particular protein kinases. The reaction can also be optimized with respect to ATP concentration. The reported Km values for most protein kinases are in the range of 10 to 100/,tM. 12 To assay the SNF1 kinase, we originally used only 0.03/.tM [T-aEp]ATP. Addition of 0.1/xM cold ATP did not change the incorporation of label into the SNF1 protein and reduced the background. Addition of 1 btM cold ATP reduced the labeling of SNFI about twofold, and addition of I0/.tM cold ATP reduced the signal substantially. Washing Filter and Autoradiography Wash the filter in 30 mM HEPES, pH 7.5, for 2 hr at 25° several times, until the radioactivity bound to the filter no longer decreases with continued washing. Dry the filter in air until slightly damp; a damp filter can be successfully washed more extensively, if necessary. Expose to film for 1 to 10 hr at - 7 0 ° with an intensifying screen for autoradiography. Longer exposures may result in unacceptable background. Comments. If the washing procedure described above does not reduce the background to an acceptable level, the filter can be washed again in 1 N HC1 for 1 hr at 25°. Washing the filter in 1 N HCI for 1 hr at 70° greatly reduces background, but also reduces the signal; we found that the amount of SNF1 protein detectable by subsequent immunoblot assay was also reduced. Roussou et al. 4 included 1 mM ATP in the washing buffer.
Identifying Activity of Interest Genetic Methods. In genetic systems, the protein kinase activity of interest can be identified by examining mutants lacking the protein, overexpressing the protein, or expressing a derivative with different electrophoretic mobility (Fig. 1). Similar methods apply for proteins expressed 12 p. j. Roach, in "Methods in Enzymology" (F. Wold and K. Moldave, eds.), Vol. 107, p. 81. Academic Press, Orlando, Florida, 1984.
428
RENATURATION OF PROTEIN KINASES
[34]
from cloned sequences in bacteria; in this case identifying the activity of interest should be trivial. Immunological Methods. If antibody specific to the protein kinase of interest is available, the filter can be used for an immunoblot assay after a suitable autoradiograph is obtained. Analysis o f Phosphoprotein
To demonstrate that the labeling of a protein results from phosphorylation of an amino acid residue, the phosphoprotein can be recovered from the filter by the method of Parekh et al. ~3and subjected to phosphoamino acid analysis. ~4 Estimate the amount of protein required to yield sufficient labeled material and, if necessary, run a preparative gel. After carrying out the assay, excise the region of the filter containing the phosphoprotein, taking care to minimize the excised region. Incubate the filter strip in 0.5 ml of 40% (v/v) acetonitrile, 0.1 M ammonium acetate, pH 8.9, for 3 hr at 37°. About 30-60% of the radioactive label should be recovered. Lyophilize the eluate. Subject the recovered material to partial acid hydrolysis and separate phosphoamino acids by two-dimensional thin-layer electrophoresis as described by Cooper et al. 14 As a control, carry out the same procedure using an equal-sized strip of filter that does not have labeled protein bound to it. Applications This is a rapid method for demonstrating biochemically that a gene that is homologous to protein kinase genes does in fact encode a functional protein kinase. This method does not require either biochemical characterization of the protein or specific antibody. Cloned genes or cDNAs can be expressed in Escherichia coli, and the protein produced can be assayed directly. In genetic systems, mutants can be employed to show that a gene encodes a protein kinase. In systems where altered genes can.be transformed into the organism or into cultured cells, the gene can be manipulated in vitro to change the size and level of expression of the gene product, thereby facilitating proof that the phosphorylated protein is encoded by the gene in question. If antibody specific to the protein is available, this assay can be used 13 B. S. Parekh, H. B. Mehta, M. D. West, and R. C. Montelaro, Anal. Biochem. 148, 87 (1985). 14j. A. Cooper, B. M. Sefton, and T. Hunter, in "Methods in Enzymology" (J. D. Corbin and J. D. Hardman, eds.), Vol. 99, p. 387. Academic Press, New York, 1983.
[341
PROTEIN KINASE ACTIVITY ON PROTEIN BLOTS
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as an alternate to, or in conjunction with, immune complex assays. This assay offers the advantage that the kinase activity is associated with a particular polypeptide. Thus, it can be used to confirm that the activity detected in an immune complex assay is due to the protein against which the antibody is directed, rather than to a minor protein kinase contaminating the immune complex. This assay may also be useful during the purification of a protein kinase to assign the catalytic activity to a particular polypeptide. The activity of a protein kinase is in many cases regulated or modulated in some way. This assay can be useful in distinguishing between possible regulatory mechanisms. For example, if the kinase activity is altered by a mechanism involving covalent modification, differences in activity may be apparent in this assay, presuming that the modification is stable. In contrast, if the kinase activity is modulated by physical association with another protein, then the electrophoretic separation of the two proteins in this procedure should preclude detection of regulatory effects. Limitations and Possible Problems The method is not suitable for detection of any protein kinase that requires two different subunits for activity because the different polypeptides would be separated electrophoretically. A possible remedy would be electrophoresis on a nondenaturing gel so that the polypeptides remain associated. Some proteins may not regain activity in response to a denaturation and renaturation regimen. It is possible that some proteins may retain. activity or renature more efficiently following electrophoresis under nondenaturing conditions. The activity of a protein kinase may be too low, relative to background, for detection by this assay. If the cloned gene is available, overexpression of the protein would remedy this problem. Another possible problem is that the protein kinase may comigrate with another kinase so that the activity of interest is obscured. For example, assays of yeast proteins revealed many highly phosphorylated proteins of 45 to 63 kDa (Fig. 1), and it would therefore be difficult to identify a minor activity in this size range. For proteins expressed in E. coil, comigration of two activities is less likely to pose a problem as there are few phosphorylated species detected by this assay. If the cloned gene or eDNA encoding the protein kinase is available, the gene can usually be manipulated in vitro to shift the activity to a more convenient region of the protein blot. It is often possible to construct a fusion to another protein, such as fl-galactosidase, that is larger than the native protein and yet
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[35]
retains protein kinase activity (Fig. 1). Construction of fusion proteins, or other derivatives, also provides a valuable control. Demonstration that two different-sized derivatives both exhibit kinase activity confirms that the protein of interest is not merely serving as a substrate for another, comigrating kinase. Related methods are described in [33] and [35] in this volume. Acknowledgments W e thank Monica Roth for suggesting the method. This work was supported by Pubfic Health Service Grant GM34095 from the N.I.H. and an American Cancer Society Faculty Research Award to M.C.
[35] A s s e s s i n g A c t i v i t i e s o f B l o t t e d P r o t e i n K i n a s e s
By JAMES E. FERRELL, JR., and G. STEVEN MARTIN Celenza and C a r l s o n 1,2 have described a method for assessing the activities of protein kinases bound to nitrocellulose-blotting membranes. The method exploits the ability of SDS-denatured enzymes to regain activity after treatment with guanidine and nonionic detergent. The resulting kinase activities might arise from disinhibition of partially denatured enzymes or from renaturation of denatured enzymes. Here we present a modification of the Celenza and Carlson assay. In this assay, poly(vinylidene difluoride) (PVDF) membranes are used in place of nitrocellulose. The main advantage of PVDF here is that it can be washed with strong bases, which significantly lowers the background of unincorporated radiolabel without diminishing the kinase signal. In addition, milk is not used in the protocol. At least some batches of nonfat dry milk possess detectable levels of kinase activity (G. Schieven and J. E. FerreU, unpublished results), which means that blotted proteins that appear to possess kinase activity could actually represent preferred substrates for the milk kinase. This precaution may be of a more theoretical than practical significance; we have not found any kinase bands that are detectable when milk is used as a blocking agent that are not when albumin or no blocking agent is used. This protocol is the most sensitive and reliable of many variations we I j. L. Celenza and M. Carlson, this volume [34]. 2 j. L. Celenza and M. Carlson, Science 233, 1175 (1986).
METHODS IN ENZYMOLOGY, VOL. 200
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
