214
P U R I F I C A T I O N OF PROTEIN KINASES
[17]
that the ligand-binding domain of Tar can regulate both the phosphatetransfer activity of a eukaryotic receptor kinase domain, and also cause it to behave as an activator of other kinases in a heterologous system. Acknowledgments We thank Elliot Altman, Bob Bourret, Juan Davagnino, Fred Hess, Chris O'Day, and Peggy Saks for critical reading of the manuscript. This work was supported by National Research Service Award Fellowship GM11223 (to K.A.B.) and by Grant AI19296 from the National Institutes of Health (to M.I.S.).
[17] A n a l y z i n g P r o t e i n P h o s p h o r y l a t i o n in P r o k a r y o t e s
By JEAN-CLAUDE CORTAY, DIDIER NI~GRE, and ALAIN-JEAN COZZONE In prokaryotes, the interest in protein phosphorylation has taken longer to gather momentum than in eukaryotes. In fact, even the occurrence of this chemical modification in microorganisms has long been a matter of controversy. The first attempt to characterize a protein kinase activity in bacteria was made in 1969 by Kuo and Greengard) The authors reported the presence in Escherichia coli extracts of a cyclic AMP-dependent enzyme that could catalyze the phosphorylation by ATP of histones, which are exogenous basic proteins. Soon thereafter, two different protein kinases, regulated in a reciprocal fashion by cyclic AMP, were described in oral streptococci, also phosphorylating histones and protamines, 2 and a few more reports were published on this topic, r-5 But still no definite conclusion could be drawn on the existence of a protein kinase activity in prokaryotes, namely because of the irreproducibility of certain results, as in the case of ribosomal protein phosphorylation, 3'6 or because of the incomplete chemical characterization of the phosphorylated moiety of the proteins. The latter point was critical, since bacteria are known to contain some kinds of kinases (e.g., polyphosphate kinase) that are quite different I j. F. Kuo and P. Greengard, J. Biol. Chem. 244, 3417 (1969). 2 R. L. Khandelwal, T. N. Spearman, and I. R. Hamilton, FEBS Lett. 31, 246 (1973). 3 j. Gordon, Biochem. Biophys. Res, Commun. 44, 579 (1971). 4 E. Kurek, N. Grankowski, and E. Gasio/', Acta Microbiol. Pol. 4, 171 (1972). 5 D. M. Powers and A. Ginsburg, in "Metabolic Interconversions of Enzymes" (E. H. Fischer, E. G. Krebs, and E. R. Stadtman, eds.), p. 131. Springer-Verlag, New York, 1973. 6 E. Kurek, N. Grankowski, and E. Gasior, Acta Microbiol. Pol. 4, 177 (1972).
METHODS IN ENZYMOLOGY, VOL. 200
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
[17]
PROTEIN PHOSPHORYLATION IN PROKARYOTES
215
from protein kinases and whose in vitro activity is stimulated especially by basic proteins, 7,a thus leading to possible errors in the investigations of protein phosphorylation. 7'9 The first clear demonstration of a protein kinase in a prokaryotic system came from an analysis of virus-infected bacteria. Rahmsdorf et a/. 1°'1~ showed that a cyclic nucleotide-independent protein kinase appears in E. coli following infection with bacteriophage T7. The induced enzyme can phosphorylate both endogenous and exogenous proteins, and the products of the reaction have the chemical characteristics of phosphoserine and phosphothreonine. However, the authors simultaneously showed H that this protein kinase is, in fact, encoded by a specific viral gene, since its appearance is prevented by ultraviolet irradiation of the phage genome but not by that of the host genome. It was therefore generally assumed for several years that uninfected bacteria did not carry any protein kinase activity and, consequently, that protein phosphorylation was restricted to eukaryotic cells. 9,11 In the past decade, however, this concept has been reversed by conclusive evidence that bacteria do contain specific protein kinases) 2 At this time, the occurrence of protein phosphorylation has been demonstrated in over 30 different species that belong to the 2 bacterial kingdoms, the eubacteria and archaebacteria, as well as to the line of cyanobacteria, which strongly suggests that this chemical modification is a universal phenomenon in microorganisms.12 The methods used in the corresponding experiments share a number of common features that will be presented in this chapter.
Labeling of Phosphoproteins The detection ofphosphorylated proteins necessitates their initial labeling either in vivo in a culture medium containing ortho-[32p]phosphate or in vitro at the expense of [y-32p]ATP.
7 H. C. Li and G. G. Brown, Biochem. Biophys. Res. Commun. 53, 875 (1973). s N. Agabian, O. M. Rosen, and L. Shapiro, Biochem. Biophys. Res. Commun. 49, 1690 (1972). 9 I. Pastan and S. Adhya, Bacteriol. Rev. 40, 527 (1976). ~0H. J. Rahmsdorf, P. Herrlich, S. H. Pai, M. Schweiger, and H. G. Wittmann, Mol. Gen. Genet. 127, 259 (1973). tl H. J. Rahmsdorf, S. H. Pai, H. Ponta, P. Herrlich, and R. Roskoski, Proc. Natl. Acad. Sci. U.S.A. 71, 586 (1974). t2 A. J. Cozzone, Annu. Rev. Microbiol. 42, 97 (1988).
216
PURIFICATION OF PROTEIN KINASES
[17]
Labeling in Vivo Cells are grown at 30 or 37° in a low-phosphate medium 13 containing 100 mM Tris-HC1, pH 7.5, 37 mM NH4CI, 27 mM KCI, 2.5 mM MgC12, 140/zM Na2SO4, 176/xM Na2HPO4, 7.5/zM FeC13, and 150/.~M CaCI2. This medium is supplemented with either 20 mM glucose, 40 mM glycerol, 50 mM sodium acetate, 50 mM sodium citrate, or 50 mM sodium succinate as the carbon source. Other low-phosphate media with similar composition can be used as well. 14-17To activate the growth of cells, casein hydrolysate (I mg/ml) or yeast extract (0.5 mg/ml) can be added. However, it is wise to purify these compounds before use by eliminating the phosphate 18that is frequently contained in commercial preparations. The same treatment should be applied to broth preparations whenever used for growing bacteria, although this type of rich medium usually does not favor the detection of phosphorylated proteins. The labeling of cells is achieved in the presence of carrier-free ortho-[32p]phosphate (20-100/zCi/ml). Double-labeling experiments are performed by adding simultaneously either [35S]sulfate (100-200/xCi/ml; specific activity 25-40/zCi/mg) or [35S]methionine (5-10 /zCi/ml; specific activity 900-1 I00 Ci/mmol). The labeling time varies from 15 min to a few hours.
Labeling in Vitro A typical incubation mixture 19'z° contains 0.8-I mg/ml of protein, 25 mM Tris-HCl at pH 7.5, 5 mM MgCI2, 5 mM 2-mercaptoethanol, and 100 ~M (100/zCi/ml) [y-32p]ATP (specific activity 1000-3000 Ci/mmol). Incubation is carried out for 15-60 min at 30-37 °. When required, 20-100 /~M cyclic AMP or cyclic GMP is added. Some assays are carried out in the presence of 2-10 mM sodium fluoride, an inhibitor of certain phosphatases, or in the presence of molybdate or vanadate in the micromolar range. 2L22
13 M. Mana'i and A. J. Cozzone, Biochem. Biophys. Res. Commun. 91, 819 (1979). 14 M. Garnak and H. C. Reeves, Science 203, 1111 (1979). 15 G. Ferro-Luzzi Ames and K. Nikaido, Eur. J. Biochem. 115, 525 (1981). 16 A. C. Borthwick, W. H. Holms, and H. G. Nimmo, Biochem. J. 222, 797 (1984). 17j. Babul and D. G. Fraenkel, Biochem. Biophys. Res. Commun. 151, 1033 (1988). t8 H. Inouye, S. Michaelis, A. Wright, and J. Beckwith, J. Bacteriol. 146, 668 (1981). 19 M. Dadssi and A. J. Cozzone, FEBS Lett. 186, 187 (1985). 2o j. y. Wang and D. E. Koshland, J. Biol. Chem. 256, 4640 (1981). 21 T. M. Chiang, J. Reizer, and E. H. Beachey, J. Biol. Chem. 264, 2957 (1989). 22 D. B. Karr and D. W. Emerich, J. Bacteriol. 171, 3420 (1989).
[17]
PROTEIN PHOSPHORYLATION IN PROKARYOTES
217
Preparation of Phosphoprotein Samples
Total Cellular Phosphoproteins After labeling in vivo, cells are collected by low-speed centrifugation, suspended in a buffer made of 10 mM Tris-HCl, pH 7.4, 5 mM MgC12, and 50/zg/ml pancreatic ribonuclease, then opened by alumina grinding or repeated ultrasonic disruption. The cellular extract is incubated for 15 min at 4° in the presence of 50/xg/ml pancreatic deoxyribonuclease and treated with 0.12 vol of 3% (w/v) sodium dodecyl sulfate (SDS)-10% (v/v) 2mercaptoethanol. After centrifugation for 20-30 min at 30,000 g at 4°C, the supernatant fraction ($30) is collected, proteins are precipitated for 8-12 hr with 5 vol of 95% (v/v) acetone at - 2 0 °, then centrifuged and dried under vacuum. 23 Alternatively, proteins are precipitated by 5-10% (w/v) trichloroacetic acid (TCA) or 25 to 80% (w/v) ammonium sulfate. 22,z4 This preparation contains total cellular phosphoproteins devoid of contaminating nucleic acids. It can also be obtained, through the same procedure, from a cellular extract incubated in vitro with radioactive ATP. Comparative experiments performed in the presence of protease inhibitors throughout the extraction and purification steps indicate that no degradation of phosphoprotein takes place in these experimental conditions.23'25 Similarly no loss of phosphoryl groups by phosphatase action seems to occur. 22
Phosphoproteins of Subcellular Fractions In most cases the analysis of phosphoproteins concerns total cellular preparations. However, experiments have been performed to study phosphoproteins from individual subcellular fractions. 23'26 For this purpose, the conventional techniques of purification can be used. Thus, to prepare cytoplasmic proteins, fraction $30 is collected and further centrifuged for 180 min at 225,000 g, the resulting supernatant is removed, and proteins are precipitated as above. The pellet contains the total ribosomes which are suspended in 1 vol of 10 mM Tris-HC1, pH 7.7, 100 mM magnesium acetate, and treated for 60 min at 4° with 2 vol of glacial acetic acid. Ribosomal proteins thus solubilized are dialyzed against 1 M acetic acid and lyophilized, z7 On the other hand, nucleoids can be prepared by the 23 j. C. Cortay, C. Rieul, B. Duclos, and A. J. Cozzone, Eur. J. Biochem. 159, 227 (1986). 24 M. Zylicz, J. H. LeBowitz, R. McMacken, and C. Georgopoulos, Proc. Natl. Acad. Sci. U.S.A. 811, 6431 (1983). A. M. Turner and N. H. Mann, F E M S Microbiol. Lett. 57, 301 (1989). 26 C. S. Mimura, F. Poy, and G. R. Jacobson, J. Cell. Biochern. 33, 161 (1987). 27 j. S. Hardy, C. G. Kurland, P. Voynow, and G. Mora, Biochemistry 8, 2897 (1969).
218
PURIFICATION OF PROTEIN KINASES
[17]
low-salt spermidine technique2a after conversion of bacterial cells to spheroplasts, proteins are then precipitated with 15% (w/v) trichloroacetic acid, washed with a mixture of acetone-ether (1 : 1), and dried under vacuum. Proteins can also be prepared from membranes purified by detergent treatment of cellular extracts and centrifugation through EDTA-sucrose step gradient. 29 Separation and Detection of Phosphoproteins The techniques more frequently utilized to separate phosphoproteins are polyacrylamide gel electrophoresis and, to a lesser extent, chromatography.
One-Dimensional Electrophoresis Migration can be performed in nondenaturing conditions by using 7.5% (w/v) polyacrylamide gels at pH 7.4. 3o-32However, phosphoproteins are more often analyzed under denaturing conditions in the presence of 0.5-1% (w/v) sodium dodecyl sulfate by using continuous 7.5-12% gels, 33 or various linear or exponential gradient gels which involve acrylamide concentrations ranging from 4 to 30% (w/v).34'35But, except for prepurified phosphoprotein preparations containing a limited number of molecules,36'37 the resolving power of the one-dimensional electrophoresis technique is generally insufficient to separate all the constituents of a complex protein mixture, e.g., total cellular extract. Two-dimensional analytical systems are then required.
Two-Dimensional Electrophoresis Protein samples (20-200/xg) are analyzed mostly by the two-dimensional technique described by O'Farrell. 38Separation in the first dimension is achieved by either isoelectric focusing to equilibrium in pH 5 to 7 ~s T. Kornberg, A. Lockwood, and A. Worcel, Proc. Natl. Acad. Sci. U.S.A. 71, 3189 (1974). 29 K. Ito, T. Sato, and T. Yura, Cell (Cambridge, Mass) 11, 551 (1977). 3o A. C. Peacock andlC. W. Dingman, Biochemistry 7, 668 (1968). 3l G. Antranikian, C. Herzberg, and G. Gottschalk, Eur. J. Biochem. 153, 413 (1985). 32 D. B. Karr and D. W. Emerich, J. Bacteriol. 171, 3420 (1989). 33 U. K. Laemmli, Nature (London) 227, 680 (1970). 34 j. Londesborough, J. Bacteriol. 165, 595 (1986). 35 G. M. F. Watson and N. H. Mann, J. Gen. Microbiol. 134, 2559 (1988). 36 j. F. Hess, K. Osawa, P. Matsumura, ~nd M. I. Simon, Proc. Natl. Acad. Sci. U.S.A. 84, 7609 (1987). 37 j. Reizer, M. J. Novotny, W. Hengstenberg, and M. H. Saier, J. Bacteriol. 160, 333 (1984). 38 p. H. O'Farrell, J. Biol. Chem. 250, 4007 (1975).
[17]
PROTEIN PHOSPHORYLATION IN PROKARYOTES
219
ampholine or nonequilibrium electrophoresis in pH 3 to I0 ampholine. 23 In either case a 4% (w/v) polyacrylamide gel in 9.5 M urea is used. Electrophoresis in the second dimension is usually performed in SDS-polyacrylamide gels, 39-41 under conditions similar to those described above for the one-dimensional system. In some instances, migration in the first dimension is done using a nondenaturing gel 42 and separation in the second dimension is done in a 7.5 to 20% (w/v) polyacrylamide gradient gel. 4°
Detection of Phosphoproteins Aside from 32p autoradiography or radioactive counting, no specific and sensitive procedure for easily detecting phosphoproteins is currently available. The immunoreactivity with anti-phosphoamino acid antibodies basically affords an efficient means of detection, but its application is often complicated, especially with complex protein mixtures. After migration of 32P-labeled proteins, polyacrylamide gels are soaked in 10-16% (w/v) trichloroacetic acid for 30-45 min at 90°. 23,42 This treatment is essential for removing some contaminating phosphorylated molecules, such as polyphosphates or nucleic acids, which may render erroneous the analysis of phosphoproteins. But this also releases the phosphoryl groups from phosphoramidates and acyl phosphates in proteins, and therefore allows only the detection of proteins carrying acid-stable phosphohydroxyamino acids. 43 After hot trichloroacetic acid (TCA) treatment, gels are washed overnight in 5% (w/v) TCA-0.5% (w/v) NaEHPO4, then incubated for 1-2 hr in 7.5% (v/v) acetic acid-30% (v/v) methanol 23 or in 1% (v/v) glycerol-5% (w/v) TCA-0.5% (w/v) Na2HP04-45% (v/v) methanol, 42 and finally dried under vacuum and autoradiographed for 1-4 days. When required, a more sensitive detection of labeled proteins is obtained by fluorography. In this case, after treatment with hot TCA, gels are soaked in glacial acetic acid for 1 hr, then in a 10% (w/v) solution of 2,5-diphenyloxazole in glacial acetic acid. After several washes with water, gels are shrunk to suitable size in 50-70% (v/v) methanol and dried. In double-labeling experiments where total proteins incorporate laSS]sulfur or [35S]methionine and phosphoproteins specifically incorporate [32p]phosphate, the differential detection of the two radioisotopes can be made. 44 Two films are exposed for each gel: one records directly the/3 39 B. Averhoff, G. Antranikian, and G. Gottschalk, FEMS Microbiol. Lett. 33, 299 (1986). 4o M. Wada, K. Sekine, and H. Itikawa, J. Bacteriol. 168, 213 (1986). 41 M. Enami and A. Ishihama, J. Biol. Chem. 259, 526 (1984). 42 A. M. Turner and N. H. Mann, J. Gen. Microbiol. 132, 3433 (1986). 43 B. Duclos, S. Marcandier, and A. J. Cozzone, this series, Volume 201 [2]. 44 p. C. Cooper and A. W. Burgess, Anal. Biochem. 126, 301 (1982).
220
PURIFICATION OF PROTEIN KINASES
[17]
emissions from 35S and the other, shielded by aluminum foil, records scintillation photons from an intensifying screen excited by the 32p emissions. Autoradiography is performed at - 80° so that there is no detection of 35S on the 32p film, and only a few percent of the/3 emissions detected on the 32p film are found on the 35S film, which allows virtually complete discrimination of the two isotopes. Figure 1 shows a typical pattern23 of the total 35S-labeled proteins of E. coli, and Fig. 2 indicates the location of the 130 32p-labeled phosphoproteins present in this bacterium. On the other hand, both nonphosphorylated and phosphorylated proteins can be visualized, in any case, by the standard staining procedures, including Coomassie Blue, PAGE-Blue, and silver nitrate.
Chromatography For both analytical and preparative purposes, some bacterial phosphoproteins have been purified by chromatographic procedures including ionexchange and affinity columns, gel filtration and, recently, fast protein liquid chromatography (FPLC). Thus, isocitrate dehydrogenase purification requires DEAE-cellulose, Porcion Red-Sepharose, and Sephadex G150 columns. 14,16The phosphoprotein DnaK of E. coli is purified by phosphocellulose and DEAE-Sephacel chromatography,24 and the 61K phosphoprotein of Streptococcus is obtained by Sephadex G-100 filtration. 26 The purification of the phosphorylated nitrogen regulator protein is achieved on heparin-Sepharose45 and that of the chemotaxis phosphoproteins on a Pharmacia Superose 12 FPLC column.46 A combination of ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-75 is needed to purify the phosphorylated form of protein HPr from gram-positive bacteria. 37 Characterization of Phosphorylated Moiety of Proteins As in eukaryotes, one important criterion in demonstrating protein phosphorylation in prokaryotes is showing that phosphoryl groups are covalently bound to certain amino acids of protein substrates and characterizing the nature of the linkage involved. The four major classes of phosphoamino acids (O-phosphomonoesters, phosphoramidates, acyl phosphates, and thiophosphates) are all present in bacterial proteins. A number of techniques have been developed to identify these various residues. They are described elsewhere in this vol45 V. Weiss and B. Magasanik, Proc. Natl. Acad. Sci. U.S.A. 85, 8919 (1988). 46 j. F. Heiss, K. Osawa, N. Kaplan, and M. I. Simon, Cell (Cambridge, Mass,) 53, 79 (1988).
(earl)
SSVW
VVVvVvY v ! v Y
v
"10~ !
~ = ~ ~=~ v
!
=l ¢) ' ~ t% l= 0~ >= ~ ~~ - . ,-.5 ~ ,,,1:: ~ ~ ',~
o
~-
E E = ~ N
0
~"
¢)
x=Z=.= ea
=a ,~ a= _ "~
o
•. .
c
o ~ . ~
= . . g ~ aca. -
"O
e,
o
=
P U R I F I C A T I O N OF PROTEIN KINASES
[17]
that the ligand-binding domain of Tar can regulate both the phosphatetransfer activity of a eukaryotic receptor kinase domain, and also cause it to behave as an activator of other kinases in a heterologous system. Acknowledgments We thank Elliot Altman, Bob Bourret, Juan Davagnino, Fred Hess, Chris O'Day, and Peggy Saks for critical reading of the manuscript. This work was supported by National Research Service Award Fellowship GM11223 (to K.A.B.) and by Grant AI19296 from the National Institutes of Health (to M.I.S.).
[17] A n a l y z i n g P r o t e i n P h o s p h o r y l a t i o n in P r o k a r y o t e s
By JEAN-CLAUDE CORTAY, DIDIER NI~GRE, and ALAIN-JEAN COZZONE In prokaryotes, the interest in protein phosphorylation has taken longer to gather momentum than in eukaryotes. In fact, even the occurrence of this chemical modification in microorganisms has long been a matter of controversy. The first attempt to characterize a protein kinase activity in bacteria was made in 1969 by Kuo and Greengard) The authors reported the presence in Escherichia coli extracts of a cyclic AMP-dependent enzyme that could catalyze the phosphorylation by ATP of histones, which are exogenous basic proteins. Soon thereafter, two different protein kinases, regulated in a reciprocal fashion by cyclic AMP, were described in oral streptococci, also phosphorylating histones and protamines, 2 and a few more reports were published on this topic, r-5 But still no definite conclusion could be drawn on the existence of a protein kinase activity in prokaryotes, namely because of the irreproducibility of certain results, as in the case of ribosomal protein phosphorylation, 3'6 or because of the incomplete chemical characterization of the phosphorylated moiety of the proteins. The latter point was critical, since bacteria are known to contain some kinds of kinases (e.g., polyphosphate kinase) that are quite different I j. F. Kuo and P. Greengard, J. Biol. Chem. 244, 3417 (1969). 2 R. L. Khandelwal, T. N. Spearman, and I. R. Hamilton, FEBS Lett. 31, 246 (1973). 3 j. Gordon, Biochem. Biophys. Res, Commun. 44, 579 (1971). 4 E. Kurek, N. Grankowski, and E. Gasio/', Acta Microbiol. Pol. 4, 171 (1972). 5 D. M. Powers and A. Ginsburg, in "Metabolic Interconversions of Enzymes" (E. H. Fischer, E. G. Krebs, and E. R. Stadtman, eds.), p. 131. Springer-Verlag, New York, 1973. 6 E. Kurek, N. Grankowski, and E. Gasior, Acta Microbiol. Pol. 4, 177 (1972).
METHODS IN ENZYMOLOGY, VOL. 200
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
[17]
PROTEIN PHOSPHORYLATION IN PROKARYOTES
215
from protein kinases and whose in vitro activity is stimulated especially by basic proteins, 7,a thus leading to possible errors in the investigations of protein phosphorylation. 7'9 The first clear demonstration of a protein kinase in a prokaryotic system came from an analysis of virus-infected bacteria. Rahmsdorf et a/. 1°'1~ showed that a cyclic nucleotide-independent protein kinase appears in E. coli following infection with bacteriophage T7. The induced enzyme can phosphorylate both endogenous and exogenous proteins, and the products of the reaction have the chemical characteristics of phosphoserine and phosphothreonine. However, the authors simultaneously showed H that this protein kinase is, in fact, encoded by a specific viral gene, since its appearance is prevented by ultraviolet irradiation of the phage genome but not by that of the host genome. It was therefore generally assumed for several years that uninfected bacteria did not carry any protein kinase activity and, consequently, that protein phosphorylation was restricted to eukaryotic cells. 9,11 In the past decade, however, this concept has been reversed by conclusive evidence that bacteria do contain specific protein kinases) 2 At this time, the occurrence of protein phosphorylation has been demonstrated in over 30 different species that belong to the 2 bacterial kingdoms, the eubacteria and archaebacteria, as well as to the line of cyanobacteria, which strongly suggests that this chemical modification is a universal phenomenon in microorganisms.12 The methods used in the corresponding experiments share a number of common features that will be presented in this chapter.
Labeling of Phosphoproteins The detection ofphosphorylated proteins necessitates their initial labeling either in vivo in a culture medium containing ortho-[32p]phosphate or in vitro at the expense of [y-32p]ATP.
7 H. C. Li and G. G. Brown, Biochem. Biophys. Res. Commun. 53, 875 (1973). s N. Agabian, O. M. Rosen, and L. Shapiro, Biochem. Biophys. Res. Commun. 49, 1690 (1972). 9 I. Pastan and S. Adhya, Bacteriol. Rev. 40, 527 (1976). ~0H. J. Rahmsdorf, P. Herrlich, S. H. Pai, M. Schweiger, and H. G. Wittmann, Mol. Gen. Genet. 127, 259 (1973). tl H. J. Rahmsdorf, S. H. Pai, H. Ponta, P. Herrlich, and R. Roskoski, Proc. Natl. Acad. Sci. U.S.A. 71, 586 (1974). t2 A. J. Cozzone, Annu. Rev. Microbiol. 42, 97 (1988).
216
PURIFICATION OF PROTEIN KINASES
[17]
Labeling in Vivo Cells are grown at 30 or 37° in a low-phosphate medium 13 containing 100 mM Tris-HC1, pH 7.5, 37 mM NH4CI, 27 mM KCI, 2.5 mM MgC12, 140/zM Na2SO4, 176/xM Na2HPO4, 7.5/zM FeC13, and 150/.~M CaCI2. This medium is supplemented with either 20 mM glucose, 40 mM glycerol, 50 mM sodium acetate, 50 mM sodium citrate, or 50 mM sodium succinate as the carbon source. Other low-phosphate media with similar composition can be used as well. 14-17To activate the growth of cells, casein hydrolysate (I mg/ml) or yeast extract (0.5 mg/ml) can be added. However, it is wise to purify these compounds before use by eliminating the phosphate 18that is frequently contained in commercial preparations. The same treatment should be applied to broth preparations whenever used for growing bacteria, although this type of rich medium usually does not favor the detection of phosphorylated proteins. The labeling of cells is achieved in the presence of carrier-free ortho-[32p]phosphate (20-100/zCi/ml). Double-labeling experiments are performed by adding simultaneously either [35S]sulfate (100-200/xCi/ml; specific activity 25-40/zCi/mg) or [35S]methionine (5-10 /zCi/ml; specific activity 900-1 I00 Ci/mmol). The labeling time varies from 15 min to a few hours.
Labeling in Vitro A typical incubation mixture 19'z° contains 0.8-I mg/ml of protein, 25 mM Tris-HCl at pH 7.5, 5 mM MgCI2, 5 mM 2-mercaptoethanol, and 100 ~M (100/zCi/ml) [y-32p]ATP (specific activity 1000-3000 Ci/mmol). Incubation is carried out for 15-60 min at 30-37 °. When required, 20-100 /~M cyclic AMP or cyclic GMP is added. Some assays are carried out in the presence of 2-10 mM sodium fluoride, an inhibitor of certain phosphatases, or in the presence of molybdate or vanadate in the micromolar range. 2L22
13 M. Mana'i and A. J. Cozzone, Biochem. Biophys. Res. Commun. 91, 819 (1979). 14 M. Garnak and H. C. Reeves, Science 203, 1111 (1979). 15 G. Ferro-Luzzi Ames and K. Nikaido, Eur. J. Biochem. 115, 525 (1981). 16 A. C. Borthwick, W. H. Holms, and H. G. Nimmo, Biochem. J. 222, 797 (1984). 17j. Babul and D. G. Fraenkel, Biochem. Biophys. Res. Commun. 151, 1033 (1988). t8 H. Inouye, S. Michaelis, A. Wright, and J. Beckwith, J. Bacteriol. 146, 668 (1981). 19 M. Dadssi and A. J. Cozzone, FEBS Lett. 186, 187 (1985). 2o j. y. Wang and D. E. Koshland, J. Biol. Chem. 256, 4640 (1981). 21 T. M. Chiang, J. Reizer, and E. H. Beachey, J. Biol. Chem. 264, 2957 (1989). 22 D. B. Karr and D. W. Emerich, J. Bacteriol. 171, 3420 (1989).
[17]
PROTEIN PHOSPHORYLATION IN PROKARYOTES
217
Preparation of Phosphoprotein Samples
Total Cellular Phosphoproteins After labeling in vivo, cells are collected by low-speed centrifugation, suspended in a buffer made of 10 mM Tris-HCl, pH 7.4, 5 mM MgC12, and 50/zg/ml pancreatic ribonuclease, then opened by alumina grinding or repeated ultrasonic disruption. The cellular extract is incubated for 15 min at 4° in the presence of 50/xg/ml pancreatic deoxyribonuclease and treated with 0.12 vol of 3% (w/v) sodium dodecyl sulfate (SDS)-10% (v/v) 2mercaptoethanol. After centrifugation for 20-30 min at 30,000 g at 4°C, the supernatant fraction ($30) is collected, proteins are precipitated for 8-12 hr with 5 vol of 95% (v/v) acetone at - 2 0 °, then centrifuged and dried under vacuum. 23 Alternatively, proteins are precipitated by 5-10% (w/v) trichloroacetic acid (TCA) or 25 to 80% (w/v) ammonium sulfate. 22,z4 This preparation contains total cellular phosphoproteins devoid of contaminating nucleic acids. It can also be obtained, through the same procedure, from a cellular extract incubated in vitro with radioactive ATP. Comparative experiments performed in the presence of protease inhibitors throughout the extraction and purification steps indicate that no degradation of phosphoprotein takes place in these experimental conditions.23'25 Similarly no loss of phosphoryl groups by phosphatase action seems to occur. 22
Phosphoproteins of Subcellular Fractions In most cases the analysis of phosphoproteins concerns total cellular preparations. However, experiments have been performed to study phosphoproteins from individual subcellular fractions. 23'26 For this purpose, the conventional techniques of purification can be used. Thus, to prepare cytoplasmic proteins, fraction $30 is collected and further centrifuged for 180 min at 225,000 g, the resulting supernatant is removed, and proteins are precipitated as above. The pellet contains the total ribosomes which are suspended in 1 vol of 10 mM Tris-HC1, pH 7.7, 100 mM magnesium acetate, and treated for 60 min at 4° with 2 vol of glacial acetic acid. Ribosomal proteins thus solubilized are dialyzed against 1 M acetic acid and lyophilized, z7 On the other hand, nucleoids can be prepared by the 23 j. C. Cortay, C. Rieul, B. Duclos, and A. J. Cozzone, Eur. J. Biochem. 159, 227 (1986). 24 M. Zylicz, J. H. LeBowitz, R. McMacken, and C. Georgopoulos, Proc. Natl. Acad. Sci. U.S.A. 811, 6431 (1983). A. M. Turner and N. H. Mann, F E M S Microbiol. Lett. 57, 301 (1989). 26 C. S. Mimura, F. Poy, and G. R. Jacobson, J. Cell. Biochern. 33, 161 (1987). 27 j. S. Hardy, C. G. Kurland, P. Voynow, and G. Mora, Biochemistry 8, 2897 (1969).
218
PURIFICATION OF PROTEIN KINASES
[17]
low-salt spermidine technique2a after conversion of bacterial cells to spheroplasts, proteins are then precipitated with 15% (w/v) trichloroacetic acid, washed with a mixture of acetone-ether (1 : 1), and dried under vacuum. Proteins can also be prepared from membranes purified by detergent treatment of cellular extracts and centrifugation through EDTA-sucrose step gradient. 29 Separation and Detection of Phosphoproteins The techniques more frequently utilized to separate phosphoproteins are polyacrylamide gel electrophoresis and, to a lesser extent, chromatography.
One-Dimensional Electrophoresis Migration can be performed in nondenaturing conditions by using 7.5% (w/v) polyacrylamide gels at pH 7.4. 3o-32However, phosphoproteins are more often analyzed under denaturing conditions in the presence of 0.5-1% (w/v) sodium dodecyl sulfate by using continuous 7.5-12% gels, 33 or various linear or exponential gradient gels which involve acrylamide concentrations ranging from 4 to 30% (w/v).34'35But, except for prepurified phosphoprotein preparations containing a limited number of molecules,36'37 the resolving power of the one-dimensional electrophoresis technique is generally insufficient to separate all the constituents of a complex protein mixture, e.g., total cellular extract. Two-dimensional analytical systems are then required.
Two-Dimensional Electrophoresis Protein samples (20-200/xg) are analyzed mostly by the two-dimensional technique described by O'Farrell. 38Separation in the first dimension is achieved by either isoelectric focusing to equilibrium in pH 5 to 7 ~s T. Kornberg, A. Lockwood, and A. Worcel, Proc. Natl. Acad. Sci. U.S.A. 71, 3189 (1974). 29 K. Ito, T. Sato, and T. Yura, Cell (Cambridge, Mass) 11, 551 (1977). 3o A. C. Peacock andlC. W. Dingman, Biochemistry 7, 668 (1968). 3l G. Antranikian, C. Herzberg, and G. Gottschalk, Eur. J. Biochem. 153, 413 (1985). 32 D. B. Karr and D. W. Emerich, J. Bacteriol. 171, 3420 (1989). 33 U. K. Laemmli, Nature (London) 227, 680 (1970). 34 j. Londesborough, J. Bacteriol. 165, 595 (1986). 35 G. M. F. Watson and N. H. Mann, J. Gen. Microbiol. 134, 2559 (1988). 36 j. F. Hess, K. Osawa, P. Matsumura, ~nd M. I. Simon, Proc. Natl. Acad. Sci. U.S.A. 84, 7609 (1987). 37 j. Reizer, M. J. Novotny, W. Hengstenberg, and M. H. Saier, J. Bacteriol. 160, 333 (1984). 38 p. H. O'Farrell, J. Biol. Chem. 250, 4007 (1975).
[17]
PROTEIN PHOSPHORYLATION IN PROKARYOTES
219
ampholine or nonequilibrium electrophoresis in pH 3 to I0 ampholine. 23 In either case a 4% (w/v) polyacrylamide gel in 9.5 M urea is used. Electrophoresis in the second dimension is usually performed in SDS-polyacrylamide gels, 39-41 under conditions similar to those described above for the one-dimensional system. In some instances, migration in the first dimension is done using a nondenaturing gel 42 and separation in the second dimension is done in a 7.5 to 20% (w/v) polyacrylamide gradient gel. 4°
Detection of Phosphoproteins Aside from 32p autoradiography or radioactive counting, no specific and sensitive procedure for easily detecting phosphoproteins is currently available. The immunoreactivity with anti-phosphoamino acid antibodies basically affords an efficient means of detection, but its application is often complicated, especially with complex protein mixtures. After migration of 32P-labeled proteins, polyacrylamide gels are soaked in 10-16% (w/v) trichloroacetic acid for 30-45 min at 90°. 23,42 This treatment is essential for removing some contaminating phosphorylated molecules, such as polyphosphates or nucleic acids, which may render erroneous the analysis of phosphoproteins. But this also releases the phosphoryl groups from phosphoramidates and acyl phosphates in proteins, and therefore allows only the detection of proteins carrying acid-stable phosphohydroxyamino acids. 43 After hot trichloroacetic acid (TCA) treatment, gels are washed overnight in 5% (w/v) TCA-0.5% (w/v) NaEHPO4, then incubated for 1-2 hr in 7.5% (v/v) acetic acid-30% (v/v) methanol 23 or in 1% (v/v) glycerol-5% (w/v) TCA-0.5% (w/v) Na2HP04-45% (v/v) methanol, 42 and finally dried under vacuum and autoradiographed for 1-4 days. When required, a more sensitive detection of labeled proteins is obtained by fluorography. In this case, after treatment with hot TCA, gels are soaked in glacial acetic acid for 1 hr, then in a 10% (w/v) solution of 2,5-diphenyloxazole in glacial acetic acid. After several washes with water, gels are shrunk to suitable size in 50-70% (v/v) methanol and dried. In double-labeling experiments where total proteins incorporate laSS]sulfur or [35S]methionine and phosphoproteins specifically incorporate [32p]phosphate, the differential detection of the two radioisotopes can be made. 44 Two films are exposed for each gel: one records directly the/3 39 B. Averhoff, G. Antranikian, and G. Gottschalk, FEMS Microbiol. Lett. 33, 299 (1986). 4o M. Wada, K. Sekine, and H. Itikawa, J. Bacteriol. 168, 213 (1986). 41 M. Enami and A. Ishihama, J. Biol. Chem. 259, 526 (1984). 42 A. M. Turner and N. H. Mann, J. Gen. Microbiol. 132, 3433 (1986). 43 B. Duclos, S. Marcandier, and A. J. Cozzone, this series, Volume 201 [2]. 44 p. C. Cooper and A. W. Burgess, Anal. Biochem. 126, 301 (1982).
220
PURIFICATION OF PROTEIN KINASES
[17]
emissions from 35S and the other, shielded by aluminum foil, records scintillation photons from an intensifying screen excited by the 32p emissions. Autoradiography is performed at - 80° so that there is no detection of 35S on the 32p film, and only a few percent of the/3 emissions detected on the 32p film are found on the 35S film, which allows virtually complete discrimination of the two isotopes. Figure 1 shows a typical pattern23 of the total 35S-labeled proteins of E. coli, and Fig. 2 indicates the location of the 130 32p-labeled phosphoproteins present in this bacterium. On the other hand, both nonphosphorylated and phosphorylated proteins can be visualized, in any case, by the standard staining procedures, including Coomassie Blue, PAGE-Blue, and silver nitrate.
Chromatography For both analytical and preparative purposes, some bacterial phosphoproteins have been purified by chromatographic procedures including ionexchange and affinity columns, gel filtration and, recently, fast protein liquid chromatography (FPLC). Thus, isocitrate dehydrogenase purification requires DEAE-cellulose, Porcion Red-Sepharose, and Sephadex G150 columns. 14,16The phosphoprotein DnaK of E. coli is purified by phosphocellulose and DEAE-Sephacel chromatography,24 and the 61K phosphoprotein of Streptococcus is obtained by Sephadex G-100 filtration. 26 The purification of the phosphorylated nitrogen regulator protein is achieved on heparin-Sepharose45 and that of the chemotaxis phosphoproteins on a Pharmacia Superose 12 FPLC column.46 A combination of ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-75 is needed to purify the phosphorylated form of protein HPr from gram-positive bacteria. 37 Characterization of Phosphorylated Moiety of Proteins As in eukaryotes, one important criterion in demonstrating protein phosphorylation in prokaryotes is showing that phosphoryl groups are covalently bound to certain amino acids of protein substrates and characterizing the nature of the linkage involved. The four major classes of phosphoamino acids (O-phosphomonoesters, phosphoramidates, acyl phosphates, and thiophosphates) are all present in bacterial proteins. A number of techniques have been developed to identify these various residues. They are described elsewhere in this vol45 V. Weiss and B. Magasanik, Proc. Natl. Acad. Sci. U.S.A. 85, 8919 (1988). 46 j. F. Heiss, K. Osawa, N. Kaplan, and M. I. Simon, Cell (Cambridge, Mass,) 53, 79 (1988).
(earl)
SSVW
VVVvVvY v ! v Y
v
"10~ !
~ = ~ ~=~ v
!
=l ¢) ' ~ t% l= 0~ >= ~ ~~ - . ,-.5 ~ ,,,1:: ~ ~ ',~
o
~-
E E = ~ N
0
~"
¢)
x=Z=.= ea
=a ,~ a= _ "~
o
•. .
c
o ~ . ~
= . . g ~ aca. -
"O
e,
o
=
