Interaction of DnaK with ATP: Binding, hydrolysis and Ca+2-stimulated autophosphorylation

Vol. 166, No. 3, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

February 14, 1990 Pages 1284-1292

INTERACTION OF DnaK WITH ATP: BINDING, HYDROLYSIS

AND ca+2-s TIMULATED AUTOPHOSPHORYLATION

Barbara L. Daliel, Diane A. Skaleris, Kathrin Kohle,

Herbert Weissbach and Nathan Brot

Roche Institute of Molecular Biology

Roche Research Center

Nutley, NJ 07110

Received December 13, 1989

The autophosphorylation of DnaK from Escherichia coli usin markedly stimulated by Ca+2 and to a lesser degree by Mn +2.

ATP as phosphate donor is Mg+ 5 and other divalent ions are

without effect in this reaction. Lanthanum, an agonist/antagonist of Ca+2, is also effective in stimulating the autophosphorylation. In contrast, Mg+2 but not Ca+2, markedly stimulates the hydrolysis of ATP catalyzed by DnaK. Also at O”, ATP forms a stable complex with DnaK without hydrolysis that is independent of cations. About 15% of the DnaK in E. coli is associated with membrane vesicles where it also can be phosphorylated in the presence of Ca+2. 0 1990 Academic Press, Inc.

The heat shock (HS) response in E. coli results in the increased synthesis of at least 17

proteins (1). It is now clear that this is due to the formation of a new species of RNA polymerase

containing a 32 Kda (T factor, in place of the normal 070 factor (2,3) that recognizes a consensus

sequence present in HS genes. Recent studies have shown that the extent of the HS response is

directly related to the increased level of 032 during HS (4-6).

Although the function of the HS proteins in protecting E. coli against heat and other

stresses is not yet known, several of the proteins have been purified. One of the major HS

proteins is the product of the dnaK gene. DnaK has been studied in some detail and it has been

shown to be required for bacteriophage and F-plasmid DNA replication (7- 12). In addition the

protein possesses a weak ATPase activity and can be autophosphorylated (7). The latter reaction is

unique since it occurs even after heating DnaK to 1OO’C. Previous in vitro studies using ATP as

the phosphate donor showed that threonine residues in the protein were phosphorylated, but the

cation specificity of the reaction was not investigaged in any detail (7,8). DnaK is also of special

interest since it has a high degree of homology with the eukaryotic Hsp70 HS protein family (13).

Recently we have shown (14) that DnaK has very similar characteristics to one of the eukaryotic

HSP 70 proteins, called P71 that is localized in mitochondria and is very likely identical to the

IPresent address: Schering-Plough, Department of Molecular Biology, Bloomfield, NJ 07003.

0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved. 1284


yeast SSCl product recently described by Craig er al. (15).. Not only does P71 strongly

crossreact with antibodies against DnaK, but both P71 and DnaK were shown to be

phosphorylated in a reaction markedly stimulated by Ca+2. In view of the Ca+2-dependent

autophosphorylation of DnaK (14), the present study was initiated to investigate in more detail the

cation specificity for the various reactions of DnaK with ATP.

MATERIALS AND METHODS

Materials. DnaK was prepared from E. coli RR1 that had been transformed with plasmid pCG203 which contains the DnaK gene (kindly supplied by C. Georgopolous, Univ. of Utah). The protein was purified as previously described (8) except that the glycerol gradient step was omitted and the protein was purified by ATP-agarose affinity chromatography (16). The purified DnaK preparations showed one major band when stained with Coomassie blue after polyacrylamide gel electrophoresis. The protein was stored at -70°C. Phosphoamino acids and nucleotides were purchased from Sigma Chemical Company and [Y-~~P]ATP, (3000 Ci/mmol) was obtained from Amersham Corp. Nitrocellulose filters were obtained from Schleicher and Schuell and Immobilon polyvinylidene difluoride (PVDF) membranes from Millipore Corp. Cellulose chromatography sheets were obtained from Eastman-Kodak. Protein concentrations were determined according to the method of Bradford (17). DnaK antiserum was prepared as described previously (18) and [1251]labeled donkey antirabbit IgG (0.37 M Bq/ug) was purchased from Amersham Corp.

DnaK autophosphorylation, DnaK autophosphorylation was performed as described previously (9) with some modifications. For analysis of the ionic requirements, the purified DnaK protein (2-5 pg) was incubated at 37’C in a 50 ~1 reaction mixture consistin buffer (pH 6.2), 5 mM 2-mercaptoethanol, 5 pM [Y-~~P]ATP (0.5-1.0 x 10 P

of 50 mM MES

various divalent cations as noted in the legends to the figures and tables. cpm /pmol) and

After 30 min, the protein was precipitated with 10% Cl3CCOOH and the precipitate suspended in 50 pl NaDodSO4 electrophoresis sample buffer (19), boiled for 5 min, and a 10 p.l aliquot was electrophoresed using a NaDodS04 10% polyacrylamide gel. The gels were fixed, stained with Coomassie blue, dried and exposed to Kodak XAR-5 film. The relative amounts of 32P-phosphoprotein were determined by scanning the autoradiograms using a soft laser densitometer (LKB-Pharmacia). For routine analyses, purified DnaK protein at the indicated concentrations was incubated as described above and at various times, aliquots (10 ~1) were applied to duplicate 25 mm nitrocellulose disks which were then washed twice, 10 min each, in 200 ml of 10% C13CCOOH containing 1 mM ATP. The disks were rinsed with 10% C13CCOOH, then dissolved in Bray’s scintillation fluid and the radioactivity determined in a Beckman LS-6800 liquid scintillation spectrometer.

ATP binding and hvdrolvsis. In experiments designed to detect ATP binding to DnaK, the purified protein was incubated at 0°C for 3 min under the conditions described above. Aliquots (10 ~1) were then applied to duplicate nitrocellulose disks which were subsequently washed twice with 10 ml of cold buffer containing 20 mM Tris-HCl pH 7.0. The filters were then dissolved in scintillation fluid and the radioactivity determined. The reaction mixture used to determine the ATPase activity of DnaK contained in a final volume of 25 pl; 50 mM Tris-HCl, pH 9.0,5 mM p- mercaptoethanol, 10% glycerol (v/v), 0.1 mM Y-~~P]ATP and 1 pg of DnaK. After 30 min at 37’ an aliquot was removed and the amount of [y-3 L PIATP of Conway and Lipmann (20).

hydrolyzed was determined by the method

Phosphoamino ac’d analysis To identify the phosphorylated amino acid, DnaK phosphorylated with [Y-~~P;ATP was precipitated with 10% Cl3CCOOH, the precipitate washed with 5% C13CCOOH and rinsed with acetone. The protein was hydrolyzed in 5.7 N HCl for 120 min at llO°C and the acid stable phosphoamino acids were separated by electrophoresis on cellulose plates in a buffer (pH 3.5) containing 5% acetic acid and 0.5% pyridine (21). The radioactive amino acids were detected by autoradiography and their migration compared to that of authentic 0-phospho-L-serine, 0-phospho-L-threonine, and 0-phospho-L-tyrosine.

Analvsis of DnaK in membrane vesicles. Membrane vesicles were prepared according to the procedure of Kaback (22) except that 0.05 mg/mL lysozyme was used to generate spheroplasts and the RNAase treatment and washes were omitted. The membrane vesicles were stored in 50

1285


mM MES buffer pH 6.2 at -2O’C. Phosphorylation reactions were carried out at 37’C for 30 min as described above except that membrane vesicles (25 pg protein) were used. Immunoblots of the membranes were performed as described previously (15) and the DnaK was quantitated by densitometer tracings of the autoradiograms. Determination of protein in the vesicle preparations was according to the method of Lowry ef al. (23).

RESULTS

Ionicu&phosuhorvlation of DnaK, Because of the recent finding (14)

that Ca+2 stimulated the autophosphorylation of both DnaK and the eukaryotic Hsp P71, we

investigated the effect of various cations on the autophosphorylation of DnaK, using gel analysis to

detect the [32P] phosphorylated protein (see Methods). Fig. 1 (lane 1) shows that there is a low

level of DnaK autophosphorylation in the absence of any added cations and that the presence of

Mg+2 does not appear to significantly affect the reaction (lane 2). However, the amount of DnaK

autophosphorylation is significantly higher in the presence of Mn+2 (lane 3) and as shown previously (14), is markedly increased when Ca+2 is present (lane 4). In addition, the stimulator-y

effect of Ca+2 can be reversed by the addition of 5 mM EGTA to the reaction mixture (lane 4 vs. lane 5). Heating the DnaK for 10 min at 1OOV prior to the addition of Ca+2 has little effect on the

autophosphorylation reaction (lane 4 vs. lane 6). Quantitation of these experiments using

densitometry (see Merho&) showed that Mn+2 and Ca+2 stimulated the reaction by about four-

fold and fourteen-fold, respectively. The Ca+2 stimulated autophosphorylation was found to be

maximal at pH 6.2 (data not shown) which is similar to previously reported results (8) and ATP saturating at 5 PM. A number of other metals including Na+, K+, Fe+21 Zn+2* Co+2, Cd+z, Be+2,

and SI+~ had no effect on the reaction. As shown below, La+3 can replace Ca+2 in this reaction.

The results in Fig. 1 were obtained using gel electrophoresis but for routine assays a filter

procedure based on acid precipitation of the [32P]-labelled protein was used which was faster and quantitative (see Methods). Fig. 2A shows the effect of Ca+2 concentration on the phosphorylation of DnaK. Unlike the ~10 pM Ca+2 concentrations required for other Ca+2

dependent kinases such as protein kinase C (24) and Ca+2/calmodulin kinase II (25) it was found

Dna K-

1 234 56

FiLla Effect of metal ions on DnaK autophosphorylation. DnaK (2 pg) was incubated at 37OC for 30 min with 5 FM [+P] ATP, various metals (5 mM) and EGTA (5mM) as indicated. The phosphorylated DnaK was separated by gel elech-ophoresis as described in Merhods. DnaK was present in all the experiments except that the DnaK was preheated at 1OOW for 10 min in experiment 6. Lane 1, no metal; lane 2, Mg +2; lane 3, Mn+2; lane 4, Ca+2; lane 5, Ca+2, EGTA; and lane 6. heated DnaK, Ca+2.

1286


2.5 12

2.0 10 -

s i= d 1.5

05 SE g 1.0

a w

0.5

o.o[ ’ ’ ’ ’ ’ ’ ’ 0 100 200 300 400 0 100 2ou 300

mM Ca+2 ,.

1C min

I

IziLL DnaK autophosphorylation in vitro. 32[P]-labeled DnaK was assayed by C13CCOOH precipitation as described in Methods. A) Effect of Ca+z concentration, B) Time course, and C) Effect of DnaK concentration. 5 pM [$2P]ATP wali used in these experiments and, unless otherwise noted, the reactions were carried out at 37OC for 30 min and contained 150 mM CaClp and 5 pg (70 pmol) DnaK.

that the optimum Ca+2 concentration for DnaK autophosphorylation was about 150 mM. The

reaction was linear for up to 240 min of incubation (Fig. 2B) and dependent on DnaK

concentration (Fig. 2C). Under optimal conditions, about 20% of the added DnaK was

phosphorylated in 4 h.

The high concentration of Ca+2 required for the reaction prompted further studies to

eliminate the possibility that a trace contaminant in the CaC12 used was responsible for the

stimulation of DnaK autophosphorylation. Similar results were obtained using other calcium salts

such as calcium acetate and calcium lactate, and CaC12 obtained from different manufacturers (data

not shown). In addition, it is known that the rare earth metal lanthanum (La+3) can act as an

agonistiantagonist of Ca+2 in other systems and is used to provide evidence for the involvement of

Ca+2 in biological reactions (26). When this metal was added in PM concentrations, it also

stimulated the autophosphorylation of DnaK at p.M concentrations (Fig. 3). The results with La+3

support the view that Ca+2 is responsible for stimulating the autophosphorylation of DnaK.

To investigate the nucleotide specificity of the autophosphorylation reaction, a series of

nucleotides were substituted for ATP in the reaction. CTP and UTP were inactive and GTP, at lo-

fold higher concentration (50 PM), gave only IO-20% of the Ca +2-dependent activity seen with

ATP (data not shown).

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pM Lat3

Eiez Effect of La+3 concentration on DnaK autophosphorylation. Purified DnaK protein (5 pg, 70 pmol) was incubated with 5 ~M[Y-~~P]ATP and the indicated concentrations of La+3 at 37’C for 30 min. Autophosphorylation was assayed by C13CCOOH precipitation, as described in Methods.

Identification of the DhosDhorvlated amino acid. Preliminary experiments showed that

when DnaK was autophosphorylated using y-labelled ATP as substrate in the presence of Ca+2 the

[32P]phosphate bond was stable at pH 2.5 at 45°C for up to 2 h. On the other hand, there was

complete hydrolysis of the phosphate bond when the protein was incubated in 2 N NaOH (4S’C, 2

h). This stability is characteristic of both 0-phosphoserine and 0-phosphothreonine bonds

(27,28). Using thin layer gel electrophoresis, 0-phosphothreonine was identified as the

phosphorylated amino acid in DnaK when either ATP or GTP was used as substrate. These

results are consistent with previous results which identified 0-phosphothreonine after

autophosphorylation of DnaK using ATP as substrate in the presence of Mg2+ (7).

ATP binding: and hvdrolvsis. Since DnaK can be autophosphorylated by ATP in a Ca+*-

stimulated reaction, experiments were carried out to investigate whether Ca+2 influenced either the

formation of a stable complex of ATP with DnaK or ATP hydrolysis catalyzed by DnaK.

Complex formation was assayed by determining the amount of radioactivity retained by a

nitrocellulose filter after incubation of DnaK at O’C with [y-32P]ATP (see Methods). At this

temperature, there is no autophosphorylation of DnaK or ATP hydrolysis. Table 1 shows that

DnaK forms a stable complex with ATP that is not dependent on either Ca+2 or Mg+2. Similar

results were obtained when [c&P]-labelled ATP was used. It is also seen in Table 1 that about

40% of the binding activity with ATP is retained after heating the protein for 10 min at 100°C.

Thus, the ability of DnaK to form a complex with ATP is also heat stable, but not to the same

degree as the autophosphorylation reaction.

Since it has previously been reported that DnaK has a weak ATPase activity (7), the cation

and nucleotide specificity of this reaction were also studied. Table 1 also shows that DnaK

possesses an ATPase activity that is markedly stimulated by Mg+2 and to a much lesser extent by

Ca+2. In addition, the protein loses almost all of its nucleoside triphosphatase activity after heating

at 100°.

Pren Qs. In eukaryotic cells the A

Hsp70 family of proteins is thought to be required for proper folding of proteins, as may be

1288


Table 1: Binding and Hydrolysis of ATP

Addition [+P]ATP

Bound Hydrolyzed

None 1.7

+Ca+2 1.5

+Mg+2 1.5

DnaK, heated 0.7a

pm01

1

10

432

14’

The nucleotide binding and hydrolysis assays were performed as described in Methods usin

1 5 PM [T-~~P]ATP , 1 pg (14 pmol) DnaK, and where indicated, 150 mM Ca+2 and 5 mM

Mg+ . The incubations were carried out at 0°C for 5 min for ATP binding and at 37’C for 30 min for ATP hydrol

3 sis. Where indicated, the DnaK was heated to 100°C for 10 min prior to the

reaction. a, Ca+ added, b, Mg+2 added.

required for the translocation of proteins through membranes (29-32). These findings coupled

with the known role of Ca+2 dependent protein kinases in the pathway of signal transduction in

eukaryotic membranes (33) prompted us to look for the presence of DnaK in washed E. coli

membrane vesicles (22). Fig. 4 (lane 1) shows an immunoblot of purified DnaK and lane 2

shows that a protein in the isolated membrane vesicles that corn&rates with purified DnaK also

reacts with the DnaK antibody. Extensive washing of the vesicles with H2O or with 0.5 M NaCl

removed less than 10% of this protein. The membrane bound protein, similar to purified DnaK,

could be phosphorylated with [y-s2P]ATP, in the presence of Ca+2 (lane 3), and was also heat

stable. Only a slight phosphorylation occurred in the absence of Ca+2 (not shown). On the basis

of these characteristics, the protein was identified as DnaK. It was estimated, based on a

comparison of densitometer analysis of immunoblots of different concentrations of DnaK with the

1 2 3

Fig. 4, Immunoblot analysis and autophosphorylation of DnaK in membrane vesicles. Membrane vesicles were prepared as described elsewhere (22; see Methods) . Lane 1, purified DnaK, 2.5 ng; lane 2, membrane vesicles, 0.5 pg protein. These samples were subjected to polyacrylamide gel electrophoresis followed by immunoblot analysis. Lane 3, membrane vesicles (25 pg protein). This sample was incubated with 5 pM [Y-P~~]ATP and 150 mM Ca+2 for 30 min at 37’ before electrophoresis and autoradiography.

1289


amounts found in the membrane vesicles, that -15% of the DnaK in cells grown at 30°C is present

in membranes and that this amount increases only slightly after HS.

DISCUSSION

The present study was initiated by the recent observation that E. coli DnaK and

mitochondrial Hsp P71 have similar characteristics including a Ca+2-stimulated

authophosphorylation (14). Although it is well established that E. coli maintains a physiological

level of Ca+2 through the action of specific extrusion mechanisms (34), the role of intracellular

Ca+2 in prokaryotes is less well defined. Ca+2, as well as Mg+2, have been reported to be

involved in stimulating an ATPase activity in a variety of organisms (35). In addition, calcium

ions have been found to regulate the chemotactic behavior of Bacillus subtilis (36). Since many of

the effects of calcium in eukaryotic cells are mediated by calcium-binding regulatory proteins such

as calmodulin, it is of interest that a heat stable factor with properties similar to those of calmodulin

has been identified in E. coli (37), B. subtilis (38), and Streptomyces erythraceus (39). Amino

acid sequence analysis of the purified protein from the latter organism showed a sequence similar

to the calcium binding site domain in calmodulin (40).

To our knowledge, the autophosphorylation of DnaK is the first Ca+2-stimulated

phosphorylation activity reported in E. coli. Because of the high levels of Ca+2 required for the

reaction, the possibility that a contaminant in the calcium preparations is responsible for the

stimulation cannot be completely ruled out. However, different sources of CaC12, as well as

different calcium salts gave the same results. In addition, when La+3 (10 FM), which has been

shown to act specifically as either an antagonist or agonist of Ca+2 reactions (26), was used, it

could substitute for Ca+2 in the reaction. Taken together, these results suggest that Ca+2 can

stimulate the autophosphorylation reaction, but the physiological significance of these results

should be viewed with caution. A variety of phospholipids were tested to see if they had any effect

on the autophosphorylation reaction but the results were negative. Also the addition of crude E.

coli extracts (S-100 fractions) inhibited the autophosphorylation of DnaK, and there was no

evidence that DnaK was involved in the phosphorylation of other proteins in these extracts.

However, previous in vivo experiments with DnaK mutants showed that the phosphorylated

species of both glutaminyl and threonyl-tRNA synthetases were absent in these mutant cells (41).

It should be noted that DnaK has been shown to be phosphorylated in vivo (7) indicating

that the in vitro phosphorylation may not be an artifact. However, as yet there is no known effect

of phosphorylation on DnaK activity. DnaK is required for the shutting off of the HS response in

E. coli (42) which could be related to the present finding that large amounts of DnaK are associated

with membranes where the protein could act as a sensor. Directly or indirectly, DnaK may be

involved in the release of & from the HS polymerase Eo32 resulting in decreased transcription of

HS genes.

The present studies also demonstrate that DnaK forms a stable complex with ATP and that

complex formation can occur at O” in the absence of Ca+2, conditions in which phosphorylation of

the protein or ATP hydrolysis do not occur. ATP can be hydrolyzed by DnaK, but this reaction is

Mg+2 dependent and heat sensitive. Whether the binding of ATP to DnaK is an intermediate step

1290


in either autophosphorylation or hydrolysis has not been determined. Based on studies by

Georgopoulos and colleagues (8,9) concerning the role of DnaK in h-phage DNA replication, it

will be of interest to re-examine many of the reactions of DnaK in the presence of DnaJ and GrpE

proteins, both of which are known to interact with DnaK (9,43).

The studies with DnaK might provide some insight into the role of the Hsp P7 1, a

mitochondrial protein, that has properties similar to DnaK (14). As an example, P71 could be

involved in mitochondrial DNA replication, similar to the known role of DnaK in the bacteriophage

system (7-12). This protein (P71) also appears to be identical to the product of the SSCl gene

(15) an essential gene of yeast (44) and may be similar to a recently described mitochondrial

protein (16,45). Studies in progress are aimed at understanding the role of phosphorylation in the

function of both DnaK and mitochondrial W 1.

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4.

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7.

10. 11.

12. 13. 14.

15.

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Documents

Interaction of DnaK with ATP: Binding, hydrolysis and Ca+2-stimulated autophosphorylation