- Email: [email protected]
[15] Phosphorylation assays for proteins of the two-component regulatory system controlling chemotaxis in Escherichia coli
188
PURIFICATION OF PROTEIN KINASES
[15]
[15] Phosphorylation Assays for Proteins of the Two-Component Regulatory System Controlling Chemotaxis in Escherichia coli By J. FRED HESS, ROBERT B. BOURRET, and MELVIN I. SIMON
Introduction
Chemotaxis and Two-Component Regulatory Systems In vitro phosphorylation assays using purified proteins have been critical to success in unraveling the signal transduction mechanism which controls bacterial chemotaxis. The known reactions of the excitation path way are summarized in Fig. I. The CheA protein autophosphorylates 1-3 and transfers the phosphoryl group to the CheY protein. 2,3 CheY-phosphate is believed to interact with the flagellar switch to regulate the swimming behavior of the cell. Activation of CheA-phosphate and CheY-phosphate formation is coupled to the ligand occupancy state of the transmembrane receptor Tar by the CheW protein. 4,4a CheY-phosphate spontaneously autodephosphorylates 5to liberate Pi ; this reaction is greatly accelerated by the CheZ protein. 2'5 The CheB protein, which is involved in adaptation, is also phosphorylated by CheA; however, dephosphorylation of CheB is not affected by the presence of CheZ. 2 From the perspective of this chapter CheB is little different than CheY and will not be discussed further. This chapter focuses on the central reactions involving CheA, CheY, and CheZ, whereas [16] in this volume describes the coupling of these reactions to Tar and CheW. Chemotaxis is only one example of a widespread paradigm in bacteria for sensing and responding appropriately to various environmental sig1 j. F. Hess, K. Oosawa, P. Matsumura, and M. I. Simon, Proc. Natl. Acad. Sci. U.S.A. 84, 7600 (1987). 2 j. F. Hess, K. Oosawa, N. Kaplan, and M. I. Simon, Cell (Cambridge, Mass.) 53, 79 (1988). 3 D. Wylie, A. M. Stock, C.-Y. Wong, and J. B. Stock, Biochem. Biophys. Res. Commun. 151, 891 (1988). 4 K. A. Borkovich, N. Kaplan, J. F. Hess, and M. I. Simon, Proc. Natl. Acad. Sci. U.S.A. 86, 1208 (1989). 4~ K. A. Borkovich and M. I. Simon, Cell 63, 1339 (1990), 5 j. F. Hess, R. B. Bourret, K. Oosawa, P. Matsumura, and M. I. Simon, Cold Spring Harbor Syrup. Quant. Biol. 53, 41 (1988).
METHODS IN ENZYMOLOGY,VOL. 200
Copyright© 1991by AcademicPress, Inc. All rights of reproductionin any form reserved.
[15]
PROTEINSCONTROLLINGE. coli CHEMOTAXIS
189
nals.6,6a Two-component regulatory systems consist of a CheA-like protein (termed the sensor) paired with a CheY-like protein (termed the regulator). There is now substantial experimental evidence Table I 1-3'5'7-24a)supporting a common mechanism of action, although each two-component regulatory system has unique properties. The proposed scheme involves autophosphorylation of a histidine residue in the sensor protein with the y-phosphoryl group of ATP, and subsequent transfer of the phosphoryl group to an aspartate residue in the regulator protein, which modifies regulator activity. Thus, formation of regulator-phosphate is believed to be controlled environmentally. The system is reset by a specific phosphatase activity which hydrolyzes regulator-phosphate. General Considerations
Chemotaxis is one of the best understood of the two-component regulatory systems. The phosphorylation assays we have employed to study the chemotaxis proteins are described below as a guide for investigating re6 j. B. Stock, A. J. Ninfa, and A. M. Stock, Microbiol. Rev. 53, 450 (1989). 6a R. B. Bourret, K. A. Borkovich, and M. I. Simon, Ann. Rev. Biochem. 60, 401 (1991). 7 j. F. Hess, R. B. Bourret, and M. I. Simon, Nature (London) 336, 139 (1988). s D. A. Sanders, B. L. Gillece-Castro, A. M. Stock, A. L. Burlingame, and D. E. Koshland, Jr., J. Biol. Chem. 264, 21770 (1989). 9 A. J. Ninfa and B. Magasanik, Proc. Natl. Acad. Sci. U.S.A. 83, 5909 (1986). l0 j. Keener and S. Kustu, Proc. Natl. Acad. Sci. U.S.A. 85, 4976 (1988). It V. Weiss and B. Magasanik, Proc. Natl. Acad. Sci. U.S.A. 85, 8919 (1988). 12 M. M. Igo and T. J. Silhavy, J. Bacteriol. 170, 5971 (1988). 13 M. M. Igo, A. J. Ninfa, and T. J. Silhavy, Genes Dev. 3, 598 (1989). 14 M. M. Igo, A. J. Ninfa, J. B. Stock, and T. J. Silhavy, Genes Dev. 3, 1725 (1989). ts H. Aiba, T. Mizuno, and S. Mizushima, J. Biol. Chem. 264, 8563 (1989). 16 H. Aiba, F. Nakasai, S. Mizushima, and T. Mizuno, J. Biol.Chem. 264, 14090 (1989). 17 S. Forst, J. Delgado, and M. Inouye, Proc. Natl. Acad. Sci. U.S.A. 86, 6052 (1989). t8 K. Makino, H. Shinagawa, M. Amemura, T. Kawamoto, M. Yamada, and A. Nakata, J. Mol. Biol. 210, 551 (1989). 19 M. Amemura, K. Makino, H. Shinagawa, and A. Nakata, J. Bacteriol. 172, 6300 (1990). 2o S. Jin, T. Roitsch, R. G. Ankenbauer, M. P. Gordon, and E. W. Nester, J. Bacteriol. 172, 525 (1990). 21 y. Huang, P. Morel, B. Powell, and C. I. Kado, J. Bacteriol. 172, 1142 (1990). 22 S. Jin, R. K. Prusti, T. Roitsch, R. G. Ankenbauer, and E. W. Nester, J. Bacteriol. 172, 4945 (1990). 23 M. Perego, S. P. Cole, D. Burbulys, K. Trach, and J. A. Hoch, J. Bacteriol. 171, 6187 (1989). 23a K. Mukai, M. Kawata, and T. Tanaka, J. Biol. Chem. 265, 20000 (1990). 23b W. R. McCleary and D. R. Zusman, J. Bacteriol. 172, 6661 (1990). 24 A. J. Ninfa, E. G. Ninfa, A. N. Lupas, A. Stock, B. Magasanik, and J. B. Stock, Proc. Natl. Acad. Sci. U.S.A. 85, 5492 (1988). 24~G. Olmedo, E. G. Ninfa, J. Stock, and P. Youngman, J. Mol. Biol. 215, 359 (1990).
190
PURIFICATION
OF PROTEIN
[15]
KINASES
go
m
~
~
eq
eq
o~ B
~
o,~
r~
0 ,,~ ,.I O [,,i.1
~:
a~
~.~
~
~
..
_~ ~ o~
Z ~.
Z
e-.
~8
8s >'"~
i
~
0l,u I-
%
Z 0
NO
M 0
::,, o ..=~
o ¢) ~ ~e
•
.~
~ .1:~ N
o .~ .,=
~'~ z~ --
~
..~ Z
=,.'< Z ~
[15]
PROTEINSCONTROLLINGE. coil CHEMOTAXIS
191
..........................................................................................................................
Tar
~
p
,f ) ~
ADP~"~ ( ~ / ~ " @ "
~'~Pi
FIG. 1. The excitation signaling pathway of chemotaxis in Escherichia coli. See text for details.
lated systems. Where available, relevant data from similar systems are included for comparison. The assays are especially useful for characterizing qualitative features of the various proteins, such as the nature and regulation of their biochemical activities with regard to phosphorylation, the functional relationships between proteins, and the altered properties of mutant proteins. There is great flexibility to customize the assay to emphasize the particular aspect of the reaction under consideration. Each assay incorporates different segments of the signal transduction pathway. Furthermore, the prominent radioactive species observed (ATP, sensor-phosphate, regulator-phosphate, or Pi) depends on the balance between the phosphorylation and dephosphorylation rates of the sensor and regulator proteins, and can thus be altered by adjusting the reaction time and the proportions of the various reaction components. One practical consequence of the apparently common reaction mechanism is the necessity of devising assay conditions under which the phosphoprotein linkages in question are stable. Boiling greatly accelerates hydrolysis of CheA-phosphate and especially CheY-phosphate; therefore samples are not heated at any point during analysis. In addition, phosphohistidine is acid labile, whereas phosphoaspartate is both acid and base labile. 25 Routine laboratory procedures which utilize acid [e.g., staining protein gels in acetic acid, or trichloroacetic acid (TCA) precipitation of proteins] result in significant loss of signal, but when limited in duration have nevertheless been usefully employed. The CheA, CheY, and CheZ proteins are purified and stored at - 2 0 ° z~J. M. Fujitaki and R. A. Smith, this series, Vol. 107 [2].
192
PURIFICATION OF PROTEIN KINASES
[15]
in TEDG buffer (see below); thus all phosphorylation reactions are performed in TEDG supplemented with the appropriate reagents. All reactions are performed at room temperature. Reaction components are thoroughly mixed and allowed to equilibrate at room temperature prior to addition of the component which initiates the reaction. Reaction volumes are governed by the concentration of purified proteins available and the method of analysis employed, but typically are -10-20/zl. Solutions
LB: 1% tryptone, 0.5% yeast extract, 1% NaC1 3fl-Indoleacrylic acid: 20 mg/ml stock solution in 100% ethanol SDS sample buffer: 0.125 M Tris/pH 6.8, 4% SDS, 20% sucrose, 10% (v/v) 2-mercaptoethanol, 0.02% Bromphenol Blue Stain: 45% (v/v) methanol, 10% (v/v) acetic acid, 0.15% Coomassie Blue Destain: 15% (v/v) methanol, 7.5% (v/v) acetic acid TEDG: 50 mM Tris/pH 7.5, 0.5 mM EDTA, 2 mM Dithiothreitol, 10% (v/v) glycerol TEDG20: TEDG made with 20% (v/v) glycerol [y-32p]ATP: A 5- to 10-fold concentrated stock solution is made by mixing the appropriate small amount of high specific activity [y32p]ATP (7000 Ci/mmol, ICN) with unlabeled ATP. The actual specific activity of the stock [y-32p]ATP thus prepared can be determined by diluting and counting an aliquot, which then permits expression of results in terms of picomoles of labeled protein Purification of Escherichia coli CheA, CheY, and CheZ Proteins A wide variety of protein phosphorylation reactions occurs in bacteria. 6'26'27'27a In most cases, the experimental advantages of working with purified proteins in a defined system, rather than with crude cell extracts, justify the additional effort necessary to purify the proteins in question. High-level expression of the chemotaxis proteins from plasmids made by Matsumura and co-workers was critical to development of simple purification procedures. CheA (and CheW) are expressed under the control of the Serratia marcescens trp promoter in pDV4, 2s whereas CheY 26 A. J. Cozzone, Annu. Rev. Microbiol. 42, 97 (1988). 27 A. J. Cozzone, ed., "Protein Phosphorylation in Prokaryotes," Special issue of Biochimie 71, 987 (1989). 27a J.-C. Cortay, D. Negre, and A. J. Cozzone, this volume [17]. 28 j. Stader, P. Matsumura, D. Vacante, G. E. Dean, and R. M. Macnab, J. Bacteriol. 166, 244 (1986).
[15]
PROTEINSCONTROLLINGE. coli CHEMOTAXIS
193
and CheZ are similarly expressed in pRL22. 29The chemotaxis proteins are produced at sufficiently high levels from these plasmids that purification progress can be monitored simply by following a prominent protein species that is (1) of the correct molecular weight and (2) observed only in the presence of the plasmid. Thus, at appropriate points in each purification, aliquots are subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12.5% polyacrylamide gel and Coomassie Blue staining to identify fractions containing the protein of interest. CheA is 71K, 3° CheZ is 24K, 31 and CheY is 14K. 29'31 To prepare cell lysates, plasmid-containing bacteria are grown in LB with ampicillin to OD600 = 1. The trp promoter is induced by the addition of 3fl-indoleacrylic acid to 100/zg/ml, and the culture is incubated until saturation. All subsequent steps are performed at 0-4 ° in TEDG, except as noted. The culture is harvested by centrifugation for 10 min at 6000 g. The pellet is resuspended in a smaller volume of TEDG (TEDG20 for CheA) to wash the cells, and the centrifugation repeated. The pellet is drained, and may be stored indefinitely at - 2 0 ° if desired. The pellet is resuspended in 5 to 15 ml of TEDG (TEDG20 for CheA) and lysed by sonication. Debris and membranes are removed by a 10-rain centrifugation at 8000 g, followed by a 1-hr centrifugation at 165,000 g. Purification of E. coli CheA, CheY, and CheZ from cell lysates is described below; alternate purification schemes for the closely related Salmonella typhimurium proteins have been reported. 32-34After the final gel-filtration step in each procedure, fractions are concentrated as desired by ultrafiltration and stored at - 2 0 °. Purified CheA, CheY, and CheZ retain activity during storage for a period of years.
Purification of CheA CheA is purified using ammonium sulfate precipitation, dye-ligand chromatography, and gel filtration. TEDG20 is used instead of TEDG in the early steps of the procedure to facilitate CheA solubility. CheA is precipitated from the cell lysate by the addition of ( N H 4 ) 2 S O 4 to 35% saturation. CheA is one of the first proteins to precipitate. The precipitate 29 p. Matsumura, J. J. Rydel, R. Linzmeier, and D. Vacante, J. Bacteriol. 160, 36 (1984). 30 E. C. Kofoid and J. S. Parkinson, J. Bacteriol. 173, in press (1991). 31 N. Mutoh and M. I. Simon, J. Bacteriol. 165, 161 (1986). 32 A. M. Stock, T. Chen, D. Welsh, and J. B. Stock, Proc. Natl. Acad. Sci. U.S.A. 85, 1403 (1988). 33 A. M. Stock, D. E. Koshland, Jr., and J. B. Stock, Proc. Natl. Acad. Sci. U.S.A. 82, 7989 (1985). 34 A. M. Stock and J. B. Stock, J. Bacteriol. 169, 3301 (1987),
194
PURIFICATION OF PROTEIN KINASES
[15]
is collected by a 15-min centrifugation at 19,000 g, resuspended in 5 ml TEDG20, and dialyzed against TEDG20 to remove salt. The material is loaded onto a 10-ml Affi-Gel Blue (100-200 mesh, BioRad, Richmond, CA) column equilibrated with TEDG and the column washed with two to three column volumes of TEDG. CheA is eluted using a 0.25 to 1 M NaCI/TEDG gradient developed over 10 column volumes. Column fractions containing CheA are pooled, concentrated by precipitation with (NH4)2SO 4 added to 45% saturation, and the precipitate resuspended in 200/zl TEDG. The blue column pool is loaded onto a Sepharose 12 fast protein liquid chromatography (FPLC) gel-filtration column (Pharmacia, Piscataway, N J) equilibrated with TEDG and chromatographed at room temperature. CheA elutes as a single peak of approximately tetrameric size (-300 kDa). This procedure yields - 1 to 2 mg CheA from a l-liter culture.
Purification of Che Y CheY is purified by a modification of the method of Matsumura et al.29 The procedure consists of dye-ligand chromatography, anion-exchange chromatography, and gel filtration. Cell lysate is loaded onto a 10-ml AffiGel Blue column equilibrated with TEDG and the column washed with two to three column volumes of TEDG. CheY is eluted using a gradient of 0 to 0.6 M NaC1 in TEDG, developed over eight column volumes. Fractions containing CheY are pooled and dialyzed against TEDG to remove salt. The blue column pool is loaded onto a 10-ml DEAE-Sepharose CL-6B (Pharmacia) column equilibrated with TEDG, the column washed with one column volume of TEDG, and CheY eluted with 0. I M NaC1 in TEDG. CheY binds weakly to this column, so it is important that salt is removed from the sample prior to loading the column. In fact, many mutant CheY proteins containing a 2 + charge change with respect to wild-type CheY do not bind to the DEAE column. Fractions containing CheY are pooled and concentrated by ultrafiltration. The DEAE po61 is loaded onto a Superose 12 FPLC column (Pharmacia) equilibrated with TEDG and chromatographed at room temperature. CheY elutes as a single peak of monomeric size (~14 kDa). This procedure yields - 5 mg CheY from a l-liter culture.
Purification of CheZ CheZ may be purified from the same cells as CheY, using dye-ligand chromatography, ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. CheZ does not bind to the blue column (see
[15]
PROTEINSCONTROLLINGE. coli CHEMOTAXIS
195
above) and is recovered in the wash effluent. Fractions containing CheZ are pooled and CheZ precipitated by the addition of (NH4)2SO4 to 43% saturation. This step results in a substantial purification. The precipitate is collected by a 15-min centrifugation at 19,000 g, resuspended in TEDG, and dialyzed against TEDG to remove salt. The material is loaded onto a Mono Q FPLC (Pharmacia) column equilibrated with TEDG and the column washed with two to three column volumes of 0.1 M NaCI/TEDG. CheZ is eluted with a linear gradient of 0.1 to 0.35 M NaC1 in TEDG, developed over - 1 0 column volumes. Fractions containing CheZ are pooled, concentrated by precipitation with (NH4)2SO4 added to 45% saturation, and the precipitate resuspended in TEDG. The Mono Q pool is loaded onto a Superose 12 FPLC column (Pharmacia) equilibrated with TEDG and chromatographed at room temperature. CheZ elutes as a peak of approximately tetrameric size ( - 110 kDa). Very large aggregates of Che Z are sometimes observed in the void volume. This procedure yields - 1 to 2 mg CheZ from a l-liter culture. Small-Scale Purification of Mutant Proteins Small-scale purification of mutant CheA or CheY proteins can be accomplished from 100-ml cultures using the basic strategy outlined above with minor modifications. The bed volumes are reduced to 3 ml for the Blue column and 1.5 ml for the DEAE column. Protein is eluted from the columns with steps of increasing salt concentration rather than a linear gradient. Buffer is applied in aliquots of the indicated size and the column drained by gravity flow, with manual collection of fractions. This allows one person to operate four columns simultaneously. The step gradient schemes are as follow: CheA, Blue column:
5 x 1 ml TEDG wash 5 x I ml 0.25 M NaC1/TEDG wash 10 x 1 ml 1 M NaC1/TEDG elution CheY, Blue column: 5 x 1 ml TEDG wash 9 x 1 ml 0.6 M NaC1/TEDG elution CheY, DEAE column: 4 x 1 ml TEDG wash 7 x 0.75 ml 0.1 M NaC1/TEDG elution The yield of pure protein per unit volume of culture is reduced approximately two- to three-fold with respect to the large-scale procedures; however, it is more than sufficient to perform the assays described in the remainder of this chapter.
196
Autophosphorylation
PURIFICATION OF PROTEIN KINASES
[15]
of CheA
Sensor autophosphorylation is the first step in the signal transduction pathway, and an obligate intermediate in regulator phosphorylation. To measure CheA autophosphorylation, CheA ( - 1 5 pmol/sample) is mixed with TEDG + 5 mM MgCl2 + 50 mM KC1. The reaction is initiated by the addition of [7-32P]ATP (specific activity typically -5000 cpm/pmol). With CheA the only protein species present, it is convenient to separate labeled CheA-phosphate from unincorporated [7-32p]ATP and [32P]Pi by trichloroacetic acid precipitation onto filters. Aliquots are spotted onto Whatman GF/C glass fiber filter disks and immediately dropped into icecold 10% trichloroacetic acid/l% sodium pyrophosphate. After 30 rain, the filters are washed three times for 30 rain each in ice-cold 5% trichloroacetic acid/l% sodium pyrophosphate, rinsed for 5 rain in 95% ethanol, air dried, and assayed by liquid scintillation counting. Because CheA-phosphate is somewhat acid labile, it is important that all samples are treated identically and that exposure to acid is minimized. This procedure results in hydrolysis of approximately 60-70% of the CheA-phosphate. A single large reaction can be sampled at various times to determine phosphorylation rate, or multiple parallel reactions can be incubated for a constant time to determine the effect of varying some other parameter (e.g., salt or ATP concentration, pH). CheA autophosphorylation is not observed in the absence of divalent cation. 1,3Among the divalent cations tested, Mg 2÷ best fulfills this requirement and Mn 2+ also works. 35 Potassium ion (but not Na ÷) further stimulates the reaction. 2 Similarly, EnvZ autophosphorylation is stimulated by both Mg 2+ (Refs. 15 and 17) and K ÷ . 13,15However, cation requirements do differ somewhat between various sensor proteins. Autophosphorylation of PhoM requires both K + and Mg 2+.19 In contrast, FrzE autophosphorylation is supported by Mn 2+ , but not by Ca 2+ , Cu 2+ , Fe 2+ , Mg 2÷ , or Zn 2+ .23b Increasing concentration of ATP results in an increased autophosphorylation rate, with a half-maximal value at - 0 . 2 mM ATP and saturation at 1 mM ATP. 3'35 An ATP concentration below saturation may be used to slow down the reaction for the sake of obtaining multiple time points when measuring the initial reaction rate, or to minimize the amount of radioactivity handled while maintaining high specific activity. Thus, reactions are typically performed at 0.1 to 0.5 mM ATP. The pH optimum for CheA autophosphorylation is 8.5 to 9.0. 2 The pH optima of the other chemotaxis phosphotransfer reactions have not been determined. For the sake of consistency and physiological relevance (the 35 K. A. Borkovich, personal communication (1990).
[15]
PROTEINSCONTROLLINGE. coli CHEMOTAXIS
197
internal pH of E. coli is 7.5-8.036), autophosphorylation and all other reactions are routinely done in TEDG (pH 7.5). The nucleotide specificity of the autophosphorylation reaction can be assessed by performing parallel reactions with [y-32p]ATP or [y-32p]GTP. Labeling with [y-32P]GTP has not been observed for the three sensor proteins tested so far (Table I). Similarly, confirming evidence that the labeled protein generated in a reaction containing [y-32p]ATP results from phosphorylation rather than adenylylation may be obtained by the failure to observe labeled protein in a parallel reaction containing [a-32P]ATP. This is the case for all sensor proteins tested to date (Table I). To demonstrate that the phosphorylation observed is an intramolecular event (i.e., autophosphorylation), the initial rate of phosphorylation is measured over a range of protein concentrations. The rate of an intramolecular reaction should be independent of protein concentration. 37 This prediction has been confirmed for CheA, 2 EnvZ, 13'15 and FrzE. 23b Purification of Phosphorylated CheA The protein product of the autophosphorylation reaction can be isolated and used to study the properties of CheA-phosphate directly, or as starting material for further phosphate transfer reactions (see below). CheA can be phosphorylated with approximately 1 mol of phosphate/mol of CheA monomer by performing a CheA autophosphorylation reaction in the presence of saturating ATP. 2 A typical reaction contains 1.4 nmol CheA, 0.8-1 mM [y-32p]ATP (specific activity of -6000 cpm/pmol), 5 mM MgC12, and 50 mM KC1 in TEDG. Following a 15-min incubation, the reaction is terminated by the addition of ammonium sulfate (45% saturation) to precipitate CheA-phosphate. The mixture is placed on ice for 20 min to facilitate precipitation and the precipitate collected by a 10-rain centrifugation in a microcentrifuge (15,000 g) at 4°. The supernatant, containing most of the radioactivity, is discarded. The precipitated CheA is then resuspended in TEDG, and the remaining free ATP removed by gel filtration on a Superose 12 FPLC column (Pharmacia). The column effluent is collected and the fractions assayed for radioactivity. Two peaks are detected; the first corresponds to CheA-phosphate and the second corresponds to unincorporated ATP. Generally, 70-100% of the CheA protein is phosphorylated. Phosphorylated CheA can be stored at - 2 0 ° for up to 1 month without significant (less than 10%) hydrolysis of the phosphoryl group. 36E. Padan and S. Schuldiner,this series, Vol. 125 [27]. 37j. A. Todhunterand D. L. Purich, Biochim. Biophys. Acta 4~, 87 (1977).
198
PURIFICATION OF PROTEIN KINASES
[15]
Identification of CheA Histidine Phosphorylation Site The first step in determining the site of phosphorylation is to test the stability of the phosphoprotein linkage to various chemical treatments. Phosphoramidates are sensitive to hydrolysis in the presence of acid, hydroxylamine, or pyridine, but are relatively stable to alkali. 25Results of such tests are consistent with the presence of phosphohistidine in CheA, 3 DegS,23a EnvZ,12,17 NtrB, i1 PhoM, 19 PhoR, 18 and VirA 2° (Table I). The site of phosphorylation in CheA, histidine-48, was identified by purifying 32p-labeled peptides generated by proteolytic digestion of purified CheA-phosphate. A seven-amino acid phosphorylated peptide with the sequence RAAHSIK was isolated after three rounds of proteolysis, purification of the labeled peptide by reversed-phase HPLC, and identification of the peptide by amino acid analysis. 7 This peptide was further digested to single amino acids by a cocktail of carboxypeptidases B and Y. The products of the carboxypeptidase digestion were analyzed by two-dimensional thin-layer chromatography on silica gel 60 K6 (Whatman, Hillsboro, OR) with chloroform : methanol : 17% (w/w) ammonia (2 : 2 : l, v/v) in the first dimension and phenol : water (3 : 1, w/w) in the second dimension. 3s The mobility of radiolabeled spots was compared with synthetic phosphohistidine, 39 phosphoarginine (Sigma, St. Louis, MO), phosphoserine (Sigma), phosphothreonine (Sigma), and phosphotyrosine (Sigma) visualized by ninhydrin staining. The mobility of the radiolabeled spot shifted with the sequential removal of amino acids from the carboxy terminus of the peptide as the digest proceeded; the end product of the digestion comigrated with phosphohistidine. NtrB,ll PhoM, 19and VirA 2° also contain phosphohistidine. When base hydrolysates of 32p-labeled proteins were subjected to two-dimensional thin-layer chromatography (NtrB, VirA) or ion exchange chromatography (PhoM), radioactivity comigrated with a phosphohistidine standard. Phosphorylation of CheY by CheA CheY may b~ phosphorylated starting either with CheA and ATP or with purified CheA-phosphate. These two reaction conditions yield different types of information, as will be described here and in the following section. The addition of CheY to a CheA autophosphorylation reaction permits preliminary examination of CheY phosphorylation. It is desirable to examine the radioactivity in the two proteins separately, so reactions are terminated by the addition of SDS sample buffer and the products 38 A. Niederwieser, this series, Vol. 25 [6]. 39 R. C. Sheridan, J. F. McCullough, and Z. T. Wakefield, Inorg. Synth. 13, 23 (1971).
[15]
PROTEINS CONTROLLINGE. coli CHEMOTAXIS
199
resolved by electrophoresis on a 12.5% polyacrylamide gel. The gel is treated with Stain for 15 min and Destain for 1 hr. After drying and autoradiography, the labeled bands can be cut out of the gel for direct quantitation by liquid scintillation counting. A disadvantage of this assay method is that there is no way to correct for the inevitable small variations in volumes between samples at each step of the experiment. Thus when comparing the amount of labeled protein observed in different samples, large differences are informative, whereas small differences cannot be determined with confidence. Results from different gels also cannot be compared, due to the variable loss of signal resulting from acid treatment. Under circumstances where CheY is a poor phosphoacceptor, allowing this reaction to proceed through many cycles can permit the accumulation of product, thus making it a sensitive assay for CheY phosphorylation. For example, in a 2.5-min reaction with a fourfold excess of CheY (15 pmol CheA:60 pmol CheY), phosphorylation of several mutant CheY proteins not labeled using other assay conditions was easily detected. 4° Furthermore, the reaction conditions were purposely adjusted so that no radioactivity was observed in CheA in the wild-type control lane, whereas CheY was strongly labeled. This was informative, because CheA-phosphate accumulated when many mutant CheY proteins were used as substrates. Such a change reflects an altered balance among the rates for the various reactions in the phosphotransfer pathway and suggests either a defect in phosphotransfer from CheA to CheY, or increased stability of the CheY-phosphate formed. A concurrent increase in CheY-phosphate levels would also be predicted in the latter case. CheZ can also be included in the reaction, typically in an amount equimolar to CheY. The overall reaction dynamics will be significantly altered by the resulting acceleration of CheY dephosphorylation, and the level of CheA-phosphate observed consequently declines. The addition of CheZ is a relatively easy way to screen CheY mutants for resistance to CheZ-stimulated dephosphorylation or, conversely, to assess the dephosphorylation-stimulating activity of CheZ mutants. Phosphotransfer from CheA-Phosphate to CheY One of the most informative assays of the chemotaxis phosphate transfer reactions is the transfer of the phosphoryl group from purified radiolabeled CheA-phosphate to CheY and finally to Pi. In the absence of ATP, CheA cannot cycle back to the phosphorylatcd form, and thus the flow of phosphate through the chemotaxis signal transduction pathway can be R. B. Bourret, J. F. Hess, and M. I. Simon, Proc. Natl. Acad. Sci. U.S.A. 87, 41 (1990).
200
PURIFICATION OF PROTEIN KINASES
[15]
analyzed. In a reaction containing a 5- to 10-fold molar excess of CheA-phosphate with respect to CheY, the dephosphorylation of CheY becomes the rate-limiting step in the production of Pi, allowing the detection of radiolabeled phosphate in CheY. The addition of CheZ to this reaction causes a substantial increase in the rate of hydrolysis of the phosphoryl group from CheA due to an increased rate of CheY dephosphorylation. 2 The products of the phosphotransfer assay are analyzed by both SDS-PAGE, to determine the amount of phosphate present in the individual proteins, and thin-layer chromatography (TLC), to quantitate the amount of Pi. A typical reaction contains 40 pmol CheA-phosphate (6000 cpm/pmol), 4 pmol CheY, and 5 mM MgCI2 in a volume of 30/zl. Note that in contrast to CheA autophosphorylation, KCI is neither required nor stimulatory for the phosphotransfer reaction. The reaction is initiated by the addition of CheY, and terminated at the desired times by the removal of 2.5-/zl aliquots to 7.5/zl SDS sample buffer containing prestained molecular weight standards (Bio-Rad, Bethesda Research Laboratories, Gaithersburg, MD). Time points are taken every 10 sec for the first minute, every 15 sec for the second minute, and finally an end point at 5 min. In a parallel control reaction, CheA-phosphate is incubated for 5 min with MgC12 in the absence of CheY to determine the stability of CheA-phosphate over the course of the reaction. A 1-/~1aliquot of each time point terminated in SDS sample buffer is removed, spotted onto a Polygram CEL 300 PEI (polyethyleneimine-cellulose)TLC plate (Brinkmann, Westbury, NY) and the plate developed with 1 M acetic acid/4 M LiCI, 8 : 2 (v/v). 41 The TLC plate is air dried and subjected to autoradiography. The remainder of each sample is loaded onto a 15% polyacrylamide minigel (Mighty Small II minigel apparatus, Hoefer Scientific Instruments, San Francisco, CA). Following SDS-PAGE, the gel is immediately dried and subjected to autoradiography. Three bands of radioactivity are detected on the gel: the CheA-phosphate band at -71 kDa, the CheY-phosphate band at -14 kDa, and Pi at the dye front. The amount of CheA-phosphate and CheY-phosphate is determined by excising the corresponding bands (using the prestained molecular weight standards and the autoradiograph as a guide) and counting the gel slices in scintillation fluid. It is impossible to accurately measure Pi since it migrates at the dye front and diffuses laterally out of the lanes; however, Pi can be quantitated by TLC. In the TLC system employed, CheA-phosphate and CheY-phosphate remain at the origin, whereas Pi migrates as a diffuse spot with an Rf of approximately 0.7. These regions are excised and counted. 4t K. R a n d e r a t h and E. Randerath, this series, Vol. 12A [40].
[15]
PROTEINSCONTROLLINGE. coli CHEMOTAXIS
201
Calculations Utilizing the counts obtained from the TLC plate in conjunction with the data from the gel, the amount of phosphate present in each species (CheA, CheY, and inorganic phosphate) is corrected for pipetting errors and for low-level spontaneous decay of CheA-phosphate. First, the percentage of Pi in the control sample containing CheA-phosphate with no CheY is used to correct for spontaneous hydrolysis of the phosphoryl group that occurred during storage. This value, typically 5-10%, is subtracted from the amount of Pi on the TLC plate for each point. (Note that this operation simultaneously corrects for background in the Pi samples.) Second, a piece of the plate is counted to determine background, and this value is subtracted from the counts at the origin of the TLC plate. The adjusted values for the origin and Pi are then summed for each time point, and the protein-bound phosphate and Pi are expressed as percentages of this total. Similarly, a piece of the gel is counted to determine background, and this value is subtracted from the counts for each gel slice. The adjusted values for each time point are summed and the fraction of protein-bound phosphate in CheA and in CheY is calculated. Since the percentage of the total counts in both CheA-phosphate and CheY-phosphate from the gel must be equal to the percentage protein bound counts obtained from the TLC, it is then possible to calculate the percentage of total counts which are in CheA-phosphate and CheY-phosphate, with the remaining counts being Pi. This normalization of CheA-phosphate, CheY-phosphate, and Pi permits valid comparison of the level of each species at the different time points in the reaction.
Assay Modifications The CheA to CheY phosphotransfer assay can be modified slightly to estimate the rate of hydrolysis of CheA-phosphate by CheY. z The conditions described above are optimized to detect the phosphorylated form of CheY, but the reaction occurs too rapidly to determine a rate accurately. Lowering the amount of CheY such that CheA-phosphate is present in a 100- to 200-fold molar excess slows the reaction, allowing for the collection of several data points in the linear phase of the reaction. CheY is diluted in 0.5 mg/ml BSA to enhance protein stability. In addition, only the TLC assay is utilized for this experiment, since the level of phosphorylated CheY is less than 1% of the total label in the reaction and thus cannot be accurately measured in the gel system. The addition of EDTA inhibits both the transfer of phosphate from CheA to CheY and the dephosphorylation of CheY to produce P~, indicat-
202
PURIFICATION OF PROTEIN KINASES
[15]
ing that divalent cations are required for both reactionsJ ,s'42 The cation requirement is not particularly specific. Phosphotransfer from CheA to CheY occurs rapidly in the presence of Co 2+ , Mg 2÷ , Mn 2+, or Zn 2+ , and slowly in the presence of Ca 2÷ or Cd2+. 42 Cadmium ion, Co 2+, Mg 2÷, Mn 2+, and Zn 2÷ each support CheY autodephosphorylation, whereas Ca 2+ does not. 42 CheY contains a single divalent metal ion binding site, with a KD for Mg 2÷ of about 0.5 mM at pH 7-8. 42 As mentioned previously, a divalent metal ion is also necessary for autophosphorylation of CheA. The amount of EDTA required to stop the phosphotransfer from CheA to CheY is less than the amount needed to stop dephosphorylation of the CheY protein. Therefore, high levels of EDTA are necessary to trap CheY in the phosphorylated form. The presence of Mg 2÷ similarly affects phosphate transfer reactions in other two-component regulatory systems; NtrB to NtrC phosphotransfer is accelerated by Mg 2÷ (Ref. I I) and EnvZ to OmpR phosphotransfer requires Mg 2+ (Ref. 17).
Purification and Use of CheY-Phosphate The purification of CheY-phosphate allows the questions of whether CheA is involved in either CheY dephosphorylation or in the acceleration of CheY dephosphorylation by CheZ to be addressed. Isolation of CheY-phosphate from a reaction containing 0.5 nmol purified CheA-phosphate and 1 nmol CheY is accomplished by allowing the reaction to proceed for only 15 sec and then adding 100 mM EDTA. 5 The reaction products are separated by gel filtration on a Superose 12 FPLC column (Pharmacia) in TEDG containing 70 mM EDTA. Three peaks of radioactivity are detected: one corresponding to CheA-phosphate (-300 kDa), one to CheY-phosphate ( - 1 4 kDa), and one to Pi eluting at the included volume of the column. The radioactivity associated with the CheY-phosphate peak indicates that only about 5% of the CheY molecules present are in the phosphorylated form; however, the CheY-phosphate isolated by this procedure is stable and can be stored for several weeks at - 2 0 °. Dephosphorylation of CheY is initiated by the addition of 100 mM MgCl2/100 mM Tris, pH 9.0 (the Tris is necessary to buffer against the drop in pH that occurs upon the addition of the Mg 2+ to the EDTA). Aliquots are removed at 5- to 10-sec intervals for 1 min, terminated in SDS sample buffer, and analyzed on TLC. The dephosphorylation of CheY is not affected by the addition of equimolar amounts of CheA. 5 The addition of equimolar amounts of CheZ accelerates the dephosphorylation of CheY 42 G. S. Lukat, A. M. Stock, and J. B. Stock,
Biochemistry 29, 5436 (1990).
[15]
PROTEINS COr~TROLLINGE. coli CHEMOTAXIS
203
with no requirement for CheA. 5 However, Mg 2÷ is required for maximal stimulation of CheY dephosphorylation by CheZ. 42 The known phosphatase activities of two-component regulatory systems differ in rate and molecular organization, presumably to meet the particular response time requirements of each system. NtrC has an autophosphatase activity that is somewhat slower than that of CheY, and NtrC-phosphate is also stabilized by EDTA. l°'H In contrast, FrzE-phosphate, 23b OmpR-phosphate, 14 and VirG-phosphate 22 are relatively stable (half-life - 1 hr) and lack the autophosphatase activity observed in CheY and NtrC entirely. In the case of Ntr and Omp (but not Che or Vir), stimulated regulator dephosphorylation is observed in the presence of the sensor. Dephosphorylation of NtrC is accelerated by the combined presence of the NtrB and Pti proteins and ATPg'I°; similarly, dephosphorylation of OmpR is accelerated by the combined presence of EnvZ and ATP. 14,16The rapid time scale of the chemotaxis reactions conceivably may prevent the detection of a similar role for CheA in CheY dephosphorylation by the experimental design employed, or additional proteins (e.g., CheW, Tar; see [16] in this volume) may be required. Alternatively, CheA may not be involved in CheY dephosphorylation at all. VirA apparently does not affect VirG dephosphorylation, in either the presence or absence of ATP. 22 Identification of CheY Aspartate Phosphorylation Site Acyl phosphates are sensitive to hydrolysis in the presence of acid, base, or hydroxylamine. 25 Results of such tests are consistent with the presence of phosphoaspartate in CheY, 43 DegU, 23a FrzE, 23b NtrC, H OmpR, 17 PhoB, 18 PhoMORF2,19 and VirG 22 (Table I). If stability to various chemical treatments suggests the presence of an acyl phosphate, confirmation depends on replacing the labile phosphate group with a stable modification. Reduction of an aspartylphosphate with NaBH4 generates homoserine. 44 Use of [3H]NaBH4 thus chemically and radioactively marks the site of phosphorylation. Several possibilities then exist for analyzing smaller pieces of the reduced, labeled protein. The site of CheY phosphorylation was identified by subjecting proteolytic fragments to mass spectrometry: The site of VirG phosphorylation was identified by sequencing radioactive proteolytic fragments by standard Edman degradation. 22Paper electrophoresis was used to identify homoserine lactone among the acid hydrolysis products of NtrC) 1 43A. M. Stock, D. C. Wylie, J. M. Mottonen, A. N. Lupas, E. G. Nifa, A. J. Ninfa, C. E. Schutt, and J. B. Stock, Cold Spring Harbor Syrup. Quant. Biol. 53, 49 (1988). 44C. Degani and P. D. Boyer, J. Biol. Chem. 248, 8222 (1973).
204
PURIFICATION OF PROTEIN KINASES
[15]
Establishing Biological Relevance of in Vitro Assays The in vitro phosphorylation assays described in this chapter provide a powerful method for characterizing a regulated protein phosphorylation cascade. It is absolutely critical, however, to establish a clear link between in vitro observations and the biological phenomenon under investigation. A detailed discussion is outside the scope of this chapter, but two general strategies are briefly mentioned. The first is to obtain mutants in the pathway by either random or site-specific mutagenesis, characterize the biochemical (phosphorylation) properties ofthe mutant proteins, and compare these properties to the phenotype of the mutant organism. This approach has been used successfully in the Che, T M Omp, 16,17,46Spo, 24a and Vir 2°'22'46~ systems. A second strategy is to establish that phosphorylation occurs in vivo, and directly monitor the effects of environmental stimuli or signaling pathway mutations on phosphorylation in the organism. The relative stability of the phosphoryl group made this feasible for OmpR. 47 Bacteria were grown in minimal medium containing [32P]Pi and lysed. OmpR was immunoprecipitated from soluble extracts and subjected to S D S - P A G E followed by autoradiography and immunoblotting. Another technique that may be more generally applicable has been described. 4a The characteristic. mobilities during two-dimensional gel electrophoresis of the phosphorylated and nonphosphorylated proteins are established using controls prepared in vitro. The charge difference due to the phosphoryl group provides a means for separation. Both forms of the protein are visualized by [35S]methionine labeling in the actual experiment. The signal can also be strengthened by overproducing the protein of interest. Acknowledgments We thank Kathy Borkovichand Kenji Oosawa for usefuldiscussionsduringthe development of these assays, and Kathy Borkovich, Juan Davagnino, and Hong Ma for critical reading of the manuscript. This work was supported by Damon Runyon-WalterWinchell Cancer Fund FellowshipDRG-915(to J.F.H.), NationalResearch ServiceAward Fellowship AI107798 (to R.B.B.), and Grant AI19296from the National Institutes of Health (to M.I.S.). 45K. Oosawa, J. F. Hess, and M. I. Simon, Cell (Cambridge, Mass.) 53, 89 (1988). 46K. Kanamaru, H. Aiba, S. Mizushima,and T. Mizuno,J. Biol. Chem. 264, 21633 (1990). T. Roitsch, H. Wang, S. Jin, and E. W. Nester, J. Bacteriol. 172, 6054 (1990). 47S. Forst, J. Delgado, A. Rampersaud, and M. Inouye,/. Bacteriol. 172, 3473 (1990). 4s O. Amster-Choderand A. Wright, Science 249, 540 (1990).
PURIFICATION OF PROTEIN KINASES
[15]
[15] Phosphorylation Assays for Proteins of the Two-Component Regulatory System Controlling Chemotaxis in Escherichia coli By J. FRED HESS, ROBERT B. BOURRET, and MELVIN I. SIMON
Introduction
Chemotaxis and Two-Component Regulatory Systems In vitro phosphorylation assays using purified proteins have been critical to success in unraveling the signal transduction mechanism which controls bacterial chemotaxis. The known reactions of the excitation path way are summarized in Fig. I. The CheA protein autophosphorylates 1-3 and transfers the phosphoryl group to the CheY protein. 2,3 CheY-phosphate is believed to interact with the flagellar switch to regulate the swimming behavior of the cell. Activation of CheA-phosphate and CheY-phosphate formation is coupled to the ligand occupancy state of the transmembrane receptor Tar by the CheW protein. 4,4a CheY-phosphate spontaneously autodephosphorylates 5to liberate Pi ; this reaction is greatly accelerated by the CheZ protein. 2'5 The CheB protein, which is involved in adaptation, is also phosphorylated by CheA; however, dephosphorylation of CheB is not affected by the presence of CheZ. 2 From the perspective of this chapter CheB is little different than CheY and will not be discussed further. This chapter focuses on the central reactions involving CheA, CheY, and CheZ, whereas [16] in this volume describes the coupling of these reactions to Tar and CheW. Chemotaxis is only one example of a widespread paradigm in bacteria for sensing and responding appropriately to various environmental sig1 j. F. Hess, K. Oosawa, P. Matsumura, and M. I. Simon, Proc. Natl. Acad. Sci. U.S.A. 84, 7600 (1987). 2 j. F. Hess, K. Oosawa, N. Kaplan, and M. I. Simon, Cell (Cambridge, Mass.) 53, 79 (1988). 3 D. Wylie, A. M. Stock, C.-Y. Wong, and J. B. Stock, Biochem. Biophys. Res. Commun. 151, 891 (1988). 4 K. A. Borkovich, N. Kaplan, J. F. Hess, and M. I. Simon, Proc. Natl. Acad. Sci. U.S.A. 86, 1208 (1989). 4~ K. A. Borkovich and M. I. Simon, Cell 63, 1339 (1990), 5 j. F. Hess, R. B. Bourret, K. Oosawa, P. Matsumura, and M. I. Simon, Cold Spring Harbor Syrup. Quant. Biol. 53, 41 (1988).
METHODS IN ENZYMOLOGY,VOL. 200
Copyright© 1991by AcademicPress, Inc. All rights of reproductionin any form reserved.
[15]
PROTEINSCONTROLLINGE. coli CHEMOTAXIS
189
nals.6,6a Two-component regulatory systems consist of a CheA-like protein (termed the sensor) paired with a CheY-like protein (termed the regulator). There is now substantial experimental evidence Table I 1-3'5'7-24a)supporting a common mechanism of action, although each two-component regulatory system has unique properties. The proposed scheme involves autophosphorylation of a histidine residue in the sensor protein with the y-phosphoryl group of ATP, and subsequent transfer of the phosphoryl group to an aspartate residue in the regulator protein, which modifies regulator activity. Thus, formation of regulator-phosphate is believed to be controlled environmentally. The system is reset by a specific phosphatase activity which hydrolyzes regulator-phosphate. General Considerations
Chemotaxis is one of the best understood of the two-component regulatory systems. The phosphorylation assays we have employed to study the chemotaxis proteins are described below as a guide for investigating re6 j. B. Stock, A. J. Ninfa, and A. M. Stock, Microbiol. Rev. 53, 450 (1989). 6a R. B. Bourret, K. A. Borkovich, and M. I. Simon, Ann. Rev. Biochem. 60, 401 (1991). 7 j. F. Hess, R. B. Bourret, and M. I. Simon, Nature (London) 336, 139 (1988). s D. A. Sanders, B. L. Gillece-Castro, A. M. Stock, A. L. Burlingame, and D. E. Koshland, Jr., J. Biol. Chem. 264, 21770 (1989). 9 A. J. Ninfa and B. Magasanik, Proc. Natl. Acad. Sci. U.S.A. 83, 5909 (1986). l0 j. Keener and S. Kustu, Proc. Natl. Acad. Sci. U.S.A. 85, 4976 (1988). It V. Weiss and B. Magasanik, Proc. Natl. Acad. Sci. U.S.A. 85, 8919 (1988). 12 M. M. Igo and T. J. Silhavy, J. Bacteriol. 170, 5971 (1988). 13 M. M. Igo, A. J. Ninfa, and T. J. Silhavy, Genes Dev. 3, 598 (1989). 14 M. M. Igo, A. J. Ninfa, J. B. Stock, and T. J. Silhavy, Genes Dev. 3, 1725 (1989). ts H. Aiba, T. Mizuno, and S. Mizushima, J. Biol. Chem. 264, 8563 (1989). 16 H. Aiba, F. Nakasai, S. Mizushima, and T. Mizuno, J. Biol.Chem. 264, 14090 (1989). 17 S. Forst, J. Delgado, and M. Inouye, Proc. Natl. Acad. Sci. U.S.A. 86, 6052 (1989). t8 K. Makino, H. Shinagawa, M. Amemura, T. Kawamoto, M. Yamada, and A. Nakata, J. Mol. Biol. 210, 551 (1989). 19 M. Amemura, K. Makino, H. Shinagawa, and A. Nakata, J. Bacteriol. 172, 6300 (1990). 2o S. Jin, T. Roitsch, R. G. Ankenbauer, M. P. Gordon, and E. W. Nester, J. Bacteriol. 172, 525 (1990). 21 y. Huang, P. Morel, B. Powell, and C. I. Kado, J. Bacteriol. 172, 1142 (1990). 22 S. Jin, R. K. Prusti, T. Roitsch, R. G. Ankenbauer, and E. W. Nester, J. Bacteriol. 172, 4945 (1990). 23 M. Perego, S. P. Cole, D. Burbulys, K. Trach, and J. A. Hoch, J. Bacteriol. 171, 6187 (1989). 23a K. Mukai, M. Kawata, and T. Tanaka, J. Biol. Chem. 265, 20000 (1990). 23b W. R. McCleary and D. R. Zusman, J. Bacteriol. 172, 6661 (1990). 24 A. J. Ninfa, E. G. Ninfa, A. N. Lupas, A. Stock, B. Magasanik, and J. B. Stock, Proc. Natl. Acad. Sci. U.S.A. 85, 5492 (1988). 24~G. Olmedo, E. G. Ninfa, J. Stock, and P. Youngman, J. Mol. Biol. 215, 359 (1990).
190
PURIFICATION
OF PROTEIN
[15]
KINASES
go
m
~
~
eq
eq
o~ B
~
o,~
r~
0 ,,~ ,.I O [,,i.1
~:
a~
~.~
~
~
..
_~ ~ o~
Z ~.
Z
e-.
~8
8s >'"~
i
~
0l,u I-
%
Z 0
NO
M 0
::,, o ..=~
o ¢) ~ ~e
•
.~
~ .1:~ N
o .~ .,=
~'~ z~ --
~
..~ Z
=,.'< Z ~
[15]
PROTEINSCONTROLLINGE. coil CHEMOTAXIS
191
..........................................................................................................................
Tar
~
p
,f ) ~
ADP~"~ ( ~ / ~ " @ "
~'~Pi
FIG. 1. The excitation signaling pathway of chemotaxis in Escherichia coli. See text for details.
lated systems. Where available, relevant data from similar systems are included for comparison. The assays are especially useful for characterizing qualitative features of the various proteins, such as the nature and regulation of their biochemical activities with regard to phosphorylation, the functional relationships between proteins, and the altered properties of mutant proteins. There is great flexibility to customize the assay to emphasize the particular aspect of the reaction under consideration. Each assay incorporates different segments of the signal transduction pathway. Furthermore, the prominent radioactive species observed (ATP, sensor-phosphate, regulator-phosphate, or Pi) depends on the balance between the phosphorylation and dephosphorylation rates of the sensor and regulator proteins, and can thus be altered by adjusting the reaction time and the proportions of the various reaction components. One practical consequence of the apparently common reaction mechanism is the necessity of devising assay conditions under which the phosphoprotein linkages in question are stable. Boiling greatly accelerates hydrolysis of CheA-phosphate and especially CheY-phosphate; therefore samples are not heated at any point during analysis. In addition, phosphohistidine is acid labile, whereas phosphoaspartate is both acid and base labile. 25 Routine laboratory procedures which utilize acid [e.g., staining protein gels in acetic acid, or trichloroacetic acid (TCA) precipitation of proteins] result in significant loss of signal, but when limited in duration have nevertheless been usefully employed. The CheA, CheY, and CheZ proteins are purified and stored at - 2 0 ° z~J. M. Fujitaki and R. A. Smith, this series, Vol. 107 [2].
192
PURIFICATION OF PROTEIN KINASES
[15]
in TEDG buffer (see below); thus all phosphorylation reactions are performed in TEDG supplemented with the appropriate reagents. All reactions are performed at room temperature. Reaction components are thoroughly mixed and allowed to equilibrate at room temperature prior to addition of the component which initiates the reaction. Reaction volumes are governed by the concentration of purified proteins available and the method of analysis employed, but typically are -10-20/zl. Solutions
LB: 1% tryptone, 0.5% yeast extract, 1% NaC1 3fl-Indoleacrylic acid: 20 mg/ml stock solution in 100% ethanol SDS sample buffer: 0.125 M Tris/pH 6.8, 4% SDS, 20% sucrose, 10% (v/v) 2-mercaptoethanol, 0.02% Bromphenol Blue Stain: 45% (v/v) methanol, 10% (v/v) acetic acid, 0.15% Coomassie Blue Destain: 15% (v/v) methanol, 7.5% (v/v) acetic acid TEDG: 50 mM Tris/pH 7.5, 0.5 mM EDTA, 2 mM Dithiothreitol, 10% (v/v) glycerol TEDG20: TEDG made with 20% (v/v) glycerol [y-32p]ATP: A 5- to 10-fold concentrated stock solution is made by mixing the appropriate small amount of high specific activity [y32p]ATP (7000 Ci/mmol, ICN) with unlabeled ATP. The actual specific activity of the stock [y-32p]ATP thus prepared can be determined by diluting and counting an aliquot, which then permits expression of results in terms of picomoles of labeled protein Purification of Escherichia coli CheA, CheY, and CheZ Proteins A wide variety of protein phosphorylation reactions occurs in bacteria. 6'26'27'27a In most cases, the experimental advantages of working with purified proteins in a defined system, rather than with crude cell extracts, justify the additional effort necessary to purify the proteins in question. High-level expression of the chemotaxis proteins from plasmids made by Matsumura and co-workers was critical to development of simple purification procedures. CheA (and CheW) are expressed under the control of the Serratia marcescens trp promoter in pDV4, 2s whereas CheY 26 A. J. Cozzone, Annu. Rev. Microbiol. 42, 97 (1988). 27 A. J. Cozzone, ed., "Protein Phosphorylation in Prokaryotes," Special issue of Biochimie 71, 987 (1989). 27a J.-C. Cortay, D. Negre, and A. J. Cozzone, this volume [17]. 28 j. Stader, P. Matsumura, D. Vacante, G. E. Dean, and R. M. Macnab, J. Bacteriol. 166, 244 (1986).
[15]
PROTEINSCONTROLLINGE. coli CHEMOTAXIS
193
and CheZ are similarly expressed in pRL22. 29The chemotaxis proteins are produced at sufficiently high levels from these plasmids that purification progress can be monitored simply by following a prominent protein species that is (1) of the correct molecular weight and (2) observed only in the presence of the plasmid. Thus, at appropriate points in each purification, aliquots are subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12.5% polyacrylamide gel and Coomassie Blue staining to identify fractions containing the protein of interest. CheA is 71K, 3° CheZ is 24K, 31 and CheY is 14K. 29'31 To prepare cell lysates, plasmid-containing bacteria are grown in LB with ampicillin to OD600 = 1. The trp promoter is induced by the addition of 3fl-indoleacrylic acid to 100/zg/ml, and the culture is incubated until saturation. All subsequent steps are performed at 0-4 ° in TEDG, except as noted. The culture is harvested by centrifugation for 10 min at 6000 g. The pellet is resuspended in a smaller volume of TEDG (TEDG20 for CheA) to wash the cells, and the centrifugation repeated. The pellet is drained, and may be stored indefinitely at - 2 0 ° if desired. The pellet is resuspended in 5 to 15 ml of TEDG (TEDG20 for CheA) and lysed by sonication. Debris and membranes are removed by a 10-rain centrifugation at 8000 g, followed by a 1-hr centrifugation at 165,000 g. Purification of E. coli CheA, CheY, and CheZ from cell lysates is described below; alternate purification schemes for the closely related Salmonella typhimurium proteins have been reported. 32-34After the final gel-filtration step in each procedure, fractions are concentrated as desired by ultrafiltration and stored at - 2 0 °. Purified CheA, CheY, and CheZ retain activity during storage for a period of years.
Purification of CheA CheA is purified using ammonium sulfate precipitation, dye-ligand chromatography, and gel filtration. TEDG20 is used instead of TEDG in the early steps of the procedure to facilitate CheA solubility. CheA is precipitated from the cell lysate by the addition of ( N H 4 ) 2 S O 4 to 35% saturation. CheA is one of the first proteins to precipitate. The precipitate 29 p. Matsumura, J. J. Rydel, R. Linzmeier, and D. Vacante, J. Bacteriol. 160, 36 (1984). 30 E. C. Kofoid and J. S. Parkinson, J. Bacteriol. 173, in press (1991). 31 N. Mutoh and M. I. Simon, J. Bacteriol. 165, 161 (1986). 32 A. M. Stock, T. Chen, D. Welsh, and J. B. Stock, Proc. Natl. Acad. Sci. U.S.A. 85, 1403 (1988). 33 A. M. Stock, D. E. Koshland, Jr., and J. B. Stock, Proc. Natl. Acad. Sci. U.S.A. 82, 7989 (1985). 34 A. M. Stock and J. B. Stock, J. Bacteriol. 169, 3301 (1987),
194
PURIFICATION OF PROTEIN KINASES
[15]
is collected by a 15-min centrifugation at 19,000 g, resuspended in 5 ml TEDG20, and dialyzed against TEDG20 to remove salt. The material is loaded onto a 10-ml Affi-Gel Blue (100-200 mesh, BioRad, Richmond, CA) column equilibrated with TEDG and the column washed with two to three column volumes of TEDG. CheA is eluted using a 0.25 to 1 M NaCI/TEDG gradient developed over 10 column volumes. Column fractions containing CheA are pooled, concentrated by precipitation with (NH4)2SO 4 added to 45% saturation, and the precipitate resuspended in 200/zl TEDG. The blue column pool is loaded onto a Sepharose 12 fast protein liquid chromatography (FPLC) gel-filtration column (Pharmacia, Piscataway, N J) equilibrated with TEDG and chromatographed at room temperature. CheA elutes as a single peak of approximately tetrameric size (-300 kDa). This procedure yields - 1 to 2 mg CheA from a l-liter culture.
Purification of Che Y CheY is purified by a modification of the method of Matsumura et al.29 The procedure consists of dye-ligand chromatography, anion-exchange chromatography, and gel filtration. Cell lysate is loaded onto a 10-ml AffiGel Blue column equilibrated with TEDG and the column washed with two to three column volumes of TEDG. CheY is eluted using a gradient of 0 to 0.6 M NaC1 in TEDG, developed over eight column volumes. Fractions containing CheY are pooled and dialyzed against TEDG to remove salt. The blue column pool is loaded onto a 10-ml DEAE-Sepharose CL-6B (Pharmacia) column equilibrated with TEDG, the column washed with one column volume of TEDG, and CheY eluted with 0. I M NaC1 in TEDG. CheY binds weakly to this column, so it is important that salt is removed from the sample prior to loading the column. In fact, many mutant CheY proteins containing a 2 + charge change with respect to wild-type CheY do not bind to the DEAE column. Fractions containing CheY are pooled and concentrated by ultrafiltration. The DEAE po61 is loaded onto a Superose 12 FPLC column (Pharmacia) equilibrated with TEDG and chromatographed at room temperature. CheY elutes as a single peak of monomeric size (~14 kDa). This procedure yields - 5 mg CheY from a l-liter culture.
Purification of CheZ CheZ may be purified from the same cells as CheY, using dye-ligand chromatography, ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. CheZ does not bind to the blue column (see
[15]
PROTEINSCONTROLLINGE. coli CHEMOTAXIS
195
above) and is recovered in the wash effluent. Fractions containing CheZ are pooled and CheZ precipitated by the addition of (NH4)2SO4 to 43% saturation. This step results in a substantial purification. The precipitate is collected by a 15-min centrifugation at 19,000 g, resuspended in TEDG, and dialyzed against TEDG to remove salt. The material is loaded onto a Mono Q FPLC (Pharmacia) column equilibrated with TEDG and the column washed with two to three column volumes of 0.1 M NaCI/TEDG. CheZ is eluted with a linear gradient of 0.1 to 0.35 M NaC1 in TEDG, developed over - 1 0 column volumes. Fractions containing CheZ are pooled, concentrated by precipitation with (NH4)2SO4 added to 45% saturation, and the precipitate resuspended in TEDG. The Mono Q pool is loaded onto a Superose 12 FPLC column (Pharmacia) equilibrated with TEDG and chromatographed at room temperature. CheZ elutes as a peak of approximately tetrameric size ( - 110 kDa). Very large aggregates of Che Z are sometimes observed in the void volume. This procedure yields - 1 to 2 mg CheZ from a l-liter culture. Small-Scale Purification of Mutant Proteins Small-scale purification of mutant CheA or CheY proteins can be accomplished from 100-ml cultures using the basic strategy outlined above with minor modifications. The bed volumes are reduced to 3 ml for the Blue column and 1.5 ml for the DEAE column. Protein is eluted from the columns with steps of increasing salt concentration rather than a linear gradient. Buffer is applied in aliquots of the indicated size and the column drained by gravity flow, with manual collection of fractions. This allows one person to operate four columns simultaneously. The step gradient schemes are as follow: CheA, Blue column:
5 x 1 ml TEDG wash 5 x I ml 0.25 M NaC1/TEDG wash 10 x 1 ml 1 M NaC1/TEDG elution CheY, Blue column: 5 x 1 ml TEDG wash 9 x 1 ml 0.6 M NaC1/TEDG elution CheY, DEAE column: 4 x 1 ml TEDG wash 7 x 0.75 ml 0.1 M NaC1/TEDG elution The yield of pure protein per unit volume of culture is reduced approximately two- to three-fold with respect to the large-scale procedures; however, it is more than sufficient to perform the assays described in the remainder of this chapter.
196
Autophosphorylation
PURIFICATION OF PROTEIN KINASES
[15]
of CheA
Sensor autophosphorylation is the first step in the signal transduction pathway, and an obligate intermediate in regulator phosphorylation. To measure CheA autophosphorylation, CheA ( - 1 5 pmol/sample) is mixed with TEDG + 5 mM MgCl2 + 50 mM KC1. The reaction is initiated by the addition of [7-32P]ATP (specific activity typically -5000 cpm/pmol). With CheA the only protein species present, it is convenient to separate labeled CheA-phosphate from unincorporated [7-32p]ATP and [32P]Pi by trichloroacetic acid precipitation onto filters. Aliquots are spotted onto Whatman GF/C glass fiber filter disks and immediately dropped into icecold 10% trichloroacetic acid/l% sodium pyrophosphate. After 30 rain, the filters are washed three times for 30 rain each in ice-cold 5% trichloroacetic acid/l% sodium pyrophosphate, rinsed for 5 rain in 95% ethanol, air dried, and assayed by liquid scintillation counting. Because CheA-phosphate is somewhat acid labile, it is important that all samples are treated identically and that exposure to acid is minimized. This procedure results in hydrolysis of approximately 60-70% of the CheA-phosphate. A single large reaction can be sampled at various times to determine phosphorylation rate, or multiple parallel reactions can be incubated for a constant time to determine the effect of varying some other parameter (e.g., salt or ATP concentration, pH). CheA autophosphorylation is not observed in the absence of divalent cation. 1,3Among the divalent cations tested, Mg 2÷ best fulfills this requirement and Mn 2+ also works. 35 Potassium ion (but not Na ÷) further stimulates the reaction. 2 Similarly, EnvZ autophosphorylation is stimulated by both Mg 2+ (Refs. 15 and 17) and K ÷ . 13,15However, cation requirements do differ somewhat between various sensor proteins. Autophosphorylation of PhoM requires both K + and Mg 2+.19 In contrast, FrzE autophosphorylation is supported by Mn 2+ , but not by Ca 2+ , Cu 2+ , Fe 2+ , Mg 2÷ , or Zn 2+ .23b Increasing concentration of ATP results in an increased autophosphorylation rate, with a half-maximal value at - 0 . 2 mM ATP and saturation at 1 mM ATP. 3'35 An ATP concentration below saturation may be used to slow down the reaction for the sake of obtaining multiple time points when measuring the initial reaction rate, or to minimize the amount of radioactivity handled while maintaining high specific activity. Thus, reactions are typically performed at 0.1 to 0.5 mM ATP. The pH optimum for CheA autophosphorylation is 8.5 to 9.0. 2 The pH optima of the other chemotaxis phosphotransfer reactions have not been determined. For the sake of consistency and physiological relevance (the 35 K. A. Borkovich, personal communication (1990).
[15]
PROTEINSCONTROLLINGE. coli CHEMOTAXIS
197
internal pH of E. coli is 7.5-8.036), autophosphorylation and all other reactions are routinely done in TEDG (pH 7.5). The nucleotide specificity of the autophosphorylation reaction can be assessed by performing parallel reactions with [y-32p]ATP or [y-32p]GTP. Labeling with [y-32P]GTP has not been observed for the three sensor proteins tested so far (Table I). Similarly, confirming evidence that the labeled protein generated in a reaction containing [y-32p]ATP results from phosphorylation rather than adenylylation may be obtained by the failure to observe labeled protein in a parallel reaction containing [a-32P]ATP. This is the case for all sensor proteins tested to date (Table I). To demonstrate that the phosphorylation observed is an intramolecular event (i.e., autophosphorylation), the initial rate of phosphorylation is measured over a range of protein concentrations. The rate of an intramolecular reaction should be independent of protein concentration. 37 This prediction has been confirmed for CheA, 2 EnvZ, 13'15 and FrzE. 23b Purification of Phosphorylated CheA The protein product of the autophosphorylation reaction can be isolated and used to study the properties of CheA-phosphate directly, or as starting material for further phosphate transfer reactions (see below). CheA can be phosphorylated with approximately 1 mol of phosphate/mol of CheA monomer by performing a CheA autophosphorylation reaction in the presence of saturating ATP. 2 A typical reaction contains 1.4 nmol CheA, 0.8-1 mM [y-32p]ATP (specific activity of -6000 cpm/pmol), 5 mM MgC12, and 50 mM KC1 in TEDG. Following a 15-min incubation, the reaction is terminated by the addition of ammonium sulfate (45% saturation) to precipitate CheA-phosphate. The mixture is placed on ice for 20 min to facilitate precipitation and the precipitate collected by a 10-rain centrifugation in a microcentrifuge (15,000 g) at 4°. The supernatant, containing most of the radioactivity, is discarded. The precipitated CheA is then resuspended in TEDG, and the remaining free ATP removed by gel filtration on a Superose 12 FPLC column (Pharmacia). The column effluent is collected and the fractions assayed for radioactivity. Two peaks are detected; the first corresponds to CheA-phosphate and the second corresponds to unincorporated ATP. Generally, 70-100% of the CheA protein is phosphorylated. Phosphorylated CheA can be stored at - 2 0 ° for up to 1 month without significant (less than 10%) hydrolysis of the phosphoryl group. 36E. Padan and S. Schuldiner,this series, Vol. 125 [27]. 37j. A. Todhunterand D. L. Purich, Biochim. Biophys. Acta 4~, 87 (1977).
198
PURIFICATION OF PROTEIN KINASES
[15]
Identification of CheA Histidine Phosphorylation Site The first step in determining the site of phosphorylation is to test the stability of the phosphoprotein linkage to various chemical treatments. Phosphoramidates are sensitive to hydrolysis in the presence of acid, hydroxylamine, or pyridine, but are relatively stable to alkali. 25Results of such tests are consistent with the presence of phosphohistidine in CheA, 3 DegS,23a EnvZ,12,17 NtrB, i1 PhoM, 19 PhoR, 18 and VirA 2° (Table I). The site of phosphorylation in CheA, histidine-48, was identified by purifying 32p-labeled peptides generated by proteolytic digestion of purified CheA-phosphate. A seven-amino acid phosphorylated peptide with the sequence RAAHSIK was isolated after three rounds of proteolysis, purification of the labeled peptide by reversed-phase HPLC, and identification of the peptide by amino acid analysis. 7 This peptide was further digested to single amino acids by a cocktail of carboxypeptidases B and Y. The products of the carboxypeptidase digestion were analyzed by two-dimensional thin-layer chromatography on silica gel 60 K6 (Whatman, Hillsboro, OR) with chloroform : methanol : 17% (w/w) ammonia (2 : 2 : l, v/v) in the first dimension and phenol : water (3 : 1, w/w) in the second dimension. 3s The mobility of radiolabeled spots was compared with synthetic phosphohistidine, 39 phosphoarginine (Sigma, St. Louis, MO), phosphoserine (Sigma), phosphothreonine (Sigma), and phosphotyrosine (Sigma) visualized by ninhydrin staining. The mobility of the radiolabeled spot shifted with the sequential removal of amino acids from the carboxy terminus of the peptide as the digest proceeded; the end product of the digestion comigrated with phosphohistidine. NtrB,ll PhoM, 19and VirA 2° also contain phosphohistidine. When base hydrolysates of 32p-labeled proteins were subjected to two-dimensional thin-layer chromatography (NtrB, VirA) or ion exchange chromatography (PhoM), radioactivity comigrated with a phosphohistidine standard. Phosphorylation of CheY by CheA CheY may b~ phosphorylated starting either with CheA and ATP or with purified CheA-phosphate. These two reaction conditions yield different types of information, as will be described here and in the following section. The addition of CheY to a CheA autophosphorylation reaction permits preliminary examination of CheY phosphorylation. It is desirable to examine the radioactivity in the two proteins separately, so reactions are terminated by the addition of SDS sample buffer and the products 38 A. Niederwieser, this series, Vol. 25 [6]. 39 R. C. Sheridan, J. F. McCullough, and Z. T. Wakefield, Inorg. Synth. 13, 23 (1971).
[15]
PROTEINS CONTROLLINGE. coli CHEMOTAXIS
199
resolved by electrophoresis on a 12.5% polyacrylamide gel. The gel is treated with Stain for 15 min and Destain for 1 hr. After drying and autoradiography, the labeled bands can be cut out of the gel for direct quantitation by liquid scintillation counting. A disadvantage of this assay method is that there is no way to correct for the inevitable small variations in volumes between samples at each step of the experiment. Thus when comparing the amount of labeled protein observed in different samples, large differences are informative, whereas small differences cannot be determined with confidence. Results from different gels also cannot be compared, due to the variable loss of signal resulting from acid treatment. Under circumstances where CheY is a poor phosphoacceptor, allowing this reaction to proceed through many cycles can permit the accumulation of product, thus making it a sensitive assay for CheY phosphorylation. For example, in a 2.5-min reaction with a fourfold excess of CheY (15 pmol CheA:60 pmol CheY), phosphorylation of several mutant CheY proteins not labeled using other assay conditions was easily detected. 4° Furthermore, the reaction conditions were purposely adjusted so that no radioactivity was observed in CheA in the wild-type control lane, whereas CheY was strongly labeled. This was informative, because CheA-phosphate accumulated when many mutant CheY proteins were used as substrates. Such a change reflects an altered balance among the rates for the various reactions in the phosphotransfer pathway and suggests either a defect in phosphotransfer from CheA to CheY, or increased stability of the CheY-phosphate formed. A concurrent increase in CheY-phosphate levels would also be predicted in the latter case. CheZ can also be included in the reaction, typically in an amount equimolar to CheY. The overall reaction dynamics will be significantly altered by the resulting acceleration of CheY dephosphorylation, and the level of CheA-phosphate observed consequently declines. The addition of CheZ is a relatively easy way to screen CheY mutants for resistance to CheZ-stimulated dephosphorylation or, conversely, to assess the dephosphorylation-stimulating activity of CheZ mutants. Phosphotransfer from CheA-Phosphate to CheY One of the most informative assays of the chemotaxis phosphate transfer reactions is the transfer of the phosphoryl group from purified radiolabeled CheA-phosphate to CheY and finally to Pi. In the absence of ATP, CheA cannot cycle back to the phosphorylatcd form, and thus the flow of phosphate through the chemotaxis signal transduction pathway can be R. B. Bourret, J. F. Hess, and M. I. Simon, Proc. Natl. Acad. Sci. U.S.A. 87, 41 (1990).
200
PURIFICATION OF PROTEIN KINASES
[15]
analyzed. In a reaction containing a 5- to 10-fold molar excess of CheA-phosphate with respect to CheY, the dephosphorylation of CheY becomes the rate-limiting step in the production of Pi, allowing the detection of radiolabeled phosphate in CheY. The addition of CheZ to this reaction causes a substantial increase in the rate of hydrolysis of the phosphoryl group from CheA due to an increased rate of CheY dephosphorylation. 2 The products of the phosphotransfer assay are analyzed by both SDS-PAGE, to determine the amount of phosphate present in the individual proteins, and thin-layer chromatography (TLC), to quantitate the amount of Pi. A typical reaction contains 40 pmol CheA-phosphate (6000 cpm/pmol), 4 pmol CheY, and 5 mM MgCI2 in a volume of 30/zl. Note that in contrast to CheA autophosphorylation, KCI is neither required nor stimulatory for the phosphotransfer reaction. The reaction is initiated by the addition of CheY, and terminated at the desired times by the removal of 2.5-/zl aliquots to 7.5/zl SDS sample buffer containing prestained molecular weight standards (Bio-Rad, Bethesda Research Laboratories, Gaithersburg, MD). Time points are taken every 10 sec for the first minute, every 15 sec for the second minute, and finally an end point at 5 min. In a parallel control reaction, CheA-phosphate is incubated for 5 min with MgC12 in the absence of CheY to determine the stability of CheA-phosphate over the course of the reaction. A 1-/~1aliquot of each time point terminated in SDS sample buffer is removed, spotted onto a Polygram CEL 300 PEI (polyethyleneimine-cellulose)TLC plate (Brinkmann, Westbury, NY) and the plate developed with 1 M acetic acid/4 M LiCI, 8 : 2 (v/v). 41 The TLC plate is air dried and subjected to autoradiography. The remainder of each sample is loaded onto a 15% polyacrylamide minigel (Mighty Small II minigel apparatus, Hoefer Scientific Instruments, San Francisco, CA). Following SDS-PAGE, the gel is immediately dried and subjected to autoradiography. Three bands of radioactivity are detected on the gel: the CheA-phosphate band at -71 kDa, the CheY-phosphate band at -14 kDa, and Pi at the dye front. The amount of CheA-phosphate and CheY-phosphate is determined by excising the corresponding bands (using the prestained molecular weight standards and the autoradiograph as a guide) and counting the gel slices in scintillation fluid. It is impossible to accurately measure Pi since it migrates at the dye front and diffuses laterally out of the lanes; however, Pi can be quantitated by TLC. In the TLC system employed, CheA-phosphate and CheY-phosphate remain at the origin, whereas Pi migrates as a diffuse spot with an Rf of approximately 0.7. These regions are excised and counted. 4t K. R a n d e r a t h and E. Randerath, this series, Vol. 12A [40].
[15]
PROTEINSCONTROLLINGE. coli CHEMOTAXIS
201
Calculations Utilizing the counts obtained from the TLC plate in conjunction with the data from the gel, the amount of phosphate present in each species (CheA, CheY, and inorganic phosphate) is corrected for pipetting errors and for low-level spontaneous decay of CheA-phosphate. First, the percentage of Pi in the control sample containing CheA-phosphate with no CheY is used to correct for spontaneous hydrolysis of the phosphoryl group that occurred during storage. This value, typically 5-10%, is subtracted from the amount of Pi on the TLC plate for each point. (Note that this operation simultaneously corrects for background in the Pi samples.) Second, a piece of the plate is counted to determine background, and this value is subtracted from the counts at the origin of the TLC plate. The adjusted values for the origin and Pi are then summed for each time point, and the protein-bound phosphate and Pi are expressed as percentages of this total. Similarly, a piece of the gel is counted to determine background, and this value is subtracted from the counts for each gel slice. The adjusted values for each time point are summed and the fraction of protein-bound phosphate in CheA and in CheY is calculated. Since the percentage of the total counts in both CheA-phosphate and CheY-phosphate from the gel must be equal to the percentage protein bound counts obtained from the TLC, it is then possible to calculate the percentage of total counts which are in CheA-phosphate and CheY-phosphate, with the remaining counts being Pi. This normalization of CheA-phosphate, CheY-phosphate, and Pi permits valid comparison of the level of each species at the different time points in the reaction.
Assay Modifications The CheA to CheY phosphotransfer assay can be modified slightly to estimate the rate of hydrolysis of CheA-phosphate by CheY. z The conditions described above are optimized to detect the phosphorylated form of CheY, but the reaction occurs too rapidly to determine a rate accurately. Lowering the amount of CheY such that CheA-phosphate is present in a 100- to 200-fold molar excess slows the reaction, allowing for the collection of several data points in the linear phase of the reaction. CheY is diluted in 0.5 mg/ml BSA to enhance protein stability. In addition, only the TLC assay is utilized for this experiment, since the level of phosphorylated CheY is less than 1% of the total label in the reaction and thus cannot be accurately measured in the gel system. The addition of EDTA inhibits both the transfer of phosphate from CheA to CheY and the dephosphorylation of CheY to produce P~, indicat-
202
PURIFICATION OF PROTEIN KINASES
[15]
ing that divalent cations are required for both reactionsJ ,s'42 The cation requirement is not particularly specific. Phosphotransfer from CheA to CheY occurs rapidly in the presence of Co 2+ , Mg 2÷ , Mn 2+, or Zn 2+ , and slowly in the presence of Ca 2÷ or Cd2+. 42 Cadmium ion, Co 2+, Mg 2÷, Mn 2+, and Zn 2÷ each support CheY autodephosphorylation, whereas Ca 2+ does not. 42 CheY contains a single divalent metal ion binding site, with a KD for Mg 2÷ of about 0.5 mM at pH 7-8. 42 As mentioned previously, a divalent metal ion is also necessary for autophosphorylation of CheA. The amount of EDTA required to stop the phosphotransfer from CheA to CheY is less than the amount needed to stop dephosphorylation of the CheY protein. Therefore, high levels of EDTA are necessary to trap CheY in the phosphorylated form. The presence of Mg 2÷ similarly affects phosphate transfer reactions in other two-component regulatory systems; NtrB to NtrC phosphotransfer is accelerated by Mg 2÷ (Ref. I I) and EnvZ to OmpR phosphotransfer requires Mg 2+ (Ref. 17).
Purification and Use of CheY-Phosphate The purification of CheY-phosphate allows the questions of whether CheA is involved in either CheY dephosphorylation or in the acceleration of CheY dephosphorylation by CheZ to be addressed. Isolation of CheY-phosphate from a reaction containing 0.5 nmol purified CheA-phosphate and 1 nmol CheY is accomplished by allowing the reaction to proceed for only 15 sec and then adding 100 mM EDTA. 5 The reaction products are separated by gel filtration on a Superose 12 FPLC column (Pharmacia) in TEDG containing 70 mM EDTA. Three peaks of radioactivity are detected: one corresponding to CheA-phosphate (-300 kDa), one to CheY-phosphate ( - 1 4 kDa), and one to Pi eluting at the included volume of the column. The radioactivity associated with the CheY-phosphate peak indicates that only about 5% of the CheY molecules present are in the phosphorylated form; however, the CheY-phosphate isolated by this procedure is stable and can be stored for several weeks at - 2 0 °. Dephosphorylation of CheY is initiated by the addition of 100 mM MgCl2/100 mM Tris, pH 9.0 (the Tris is necessary to buffer against the drop in pH that occurs upon the addition of the Mg 2+ to the EDTA). Aliquots are removed at 5- to 10-sec intervals for 1 min, terminated in SDS sample buffer, and analyzed on TLC. The dephosphorylation of CheY is not affected by the addition of equimolar amounts of CheA. 5 The addition of equimolar amounts of CheZ accelerates the dephosphorylation of CheY 42 G. S. Lukat, A. M. Stock, and J. B. Stock,
Biochemistry 29, 5436 (1990).
[15]
PROTEINS COr~TROLLINGE. coli CHEMOTAXIS
203
with no requirement for CheA. 5 However, Mg 2÷ is required for maximal stimulation of CheY dephosphorylation by CheZ. 42 The known phosphatase activities of two-component regulatory systems differ in rate and molecular organization, presumably to meet the particular response time requirements of each system. NtrC has an autophosphatase activity that is somewhat slower than that of CheY, and NtrC-phosphate is also stabilized by EDTA. l°'H In contrast, FrzE-phosphate, 23b OmpR-phosphate, 14 and VirG-phosphate 22 are relatively stable (half-life - 1 hr) and lack the autophosphatase activity observed in CheY and NtrC entirely. In the case of Ntr and Omp (but not Che or Vir), stimulated regulator dephosphorylation is observed in the presence of the sensor. Dephosphorylation of NtrC is accelerated by the combined presence of the NtrB and Pti proteins and ATPg'I°; similarly, dephosphorylation of OmpR is accelerated by the combined presence of EnvZ and ATP. 14,16The rapid time scale of the chemotaxis reactions conceivably may prevent the detection of a similar role for CheA in CheY dephosphorylation by the experimental design employed, or additional proteins (e.g., CheW, Tar; see [16] in this volume) may be required. Alternatively, CheA may not be involved in CheY dephosphorylation at all. VirA apparently does not affect VirG dephosphorylation, in either the presence or absence of ATP. 22 Identification of CheY Aspartate Phosphorylation Site Acyl phosphates are sensitive to hydrolysis in the presence of acid, base, or hydroxylamine. 25 Results of such tests are consistent with the presence of phosphoaspartate in CheY, 43 DegU, 23a FrzE, 23b NtrC, H OmpR, 17 PhoB, 18 PhoMORF2,19 and VirG 22 (Table I). If stability to various chemical treatments suggests the presence of an acyl phosphate, confirmation depends on replacing the labile phosphate group with a stable modification. Reduction of an aspartylphosphate with NaBH4 generates homoserine. 44 Use of [3H]NaBH4 thus chemically and radioactively marks the site of phosphorylation. Several possibilities then exist for analyzing smaller pieces of the reduced, labeled protein. The site of CheY phosphorylation was identified by subjecting proteolytic fragments to mass spectrometry: The site of VirG phosphorylation was identified by sequencing radioactive proteolytic fragments by standard Edman degradation. 22Paper electrophoresis was used to identify homoserine lactone among the acid hydrolysis products of NtrC) 1 43A. M. Stock, D. C. Wylie, J. M. Mottonen, A. N. Lupas, E. G. Nifa, A. J. Ninfa, C. E. Schutt, and J. B. Stock, Cold Spring Harbor Syrup. Quant. Biol. 53, 49 (1988). 44C. Degani and P. D. Boyer, J. Biol. Chem. 248, 8222 (1973).
204
PURIFICATION OF PROTEIN KINASES
[15]
Establishing Biological Relevance of in Vitro Assays The in vitro phosphorylation assays described in this chapter provide a powerful method for characterizing a regulated protein phosphorylation cascade. It is absolutely critical, however, to establish a clear link between in vitro observations and the biological phenomenon under investigation. A detailed discussion is outside the scope of this chapter, but two general strategies are briefly mentioned. The first is to obtain mutants in the pathway by either random or site-specific mutagenesis, characterize the biochemical (phosphorylation) properties ofthe mutant proteins, and compare these properties to the phenotype of the mutant organism. This approach has been used successfully in the Che, T M Omp, 16,17,46Spo, 24a and Vir 2°'22'46~ systems. A second strategy is to establish that phosphorylation occurs in vivo, and directly monitor the effects of environmental stimuli or signaling pathway mutations on phosphorylation in the organism. The relative stability of the phosphoryl group made this feasible for OmpR. 47 Bacteria were grown in minimal medium containing [32P]Pi and lysed. OmpR was immunoprecipitated from soluble extracts and subjected to S D S - P A G E followed by autoradiography and immunoblotting. Another technique that may be more generally applicable has been described. 4a The characteristic. mobilities during two-dimensional gel electrophoresis of the phosphorylated and nonphosphorylated proteins are established using controls prepared in vitro. The charge difference due to the phosphoryl group provides a means for separation. Both forms of the protein are visualized by [35S]methionine labeling in the actual experiment. The signal can also be strengthened by overproducing the protein of interest. Acknowledgments We thank Kathy Borkovichand Kenji Oosawa for usefuldiscussionsduringthe development of these assays, and Kathy Borkovich, Juan Davagnino, and Hong Ma for critical reading of the manuscript. This work was supported by Damon Runyon-WalterWinchell Cancer Fund FellowshipDRG-915(to J.F.H.), NationalResearch ServiceAward Fellowship AI107798 (to R.B.B.), and Grant AI19296from the National Institutes of Health (to M.I.S.). 45K. Oosawa, J. F. Hess, and M. I. Simon, Cell (Cambridge, Mass.) 53, 89 (1988). 46K. Kanamaru, H. Aiba, S. Mizushima,and T. Mizuno,J. Biol. Chem. 264, 21633 (1990). T. Roitsch, H. Wang, S. Jin, and E. W. Nester, J. Bacteriol. 172, 6054 (1990). 47S. Forst, J. Delgado, A. Rampersaud, and M. Inouye,/. Bacteriol. 172, 3473 (1990). 4s O. Amster-Choderand A. Wright, Science 249, 540 (1990).