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(Received for publication, October 9, 1995) From the
EnvZ of Escherichia coli is a transmembrane histidine
kinase belonging to the family of two-component signal transducing
systems prevalent in prokaryotes and recently discovered in eukaryotes.
In response to changes in medium osmolarity EnvZ regulates the level of
phosphorylated OmpR, its conjugate response-regulating transcription
factor for ompF and ompC genes. EnvZ has dual
opposing enzymatic activities; OmpR-phosphorylase (kinase) and
phospho-OmpR-dephosphorylase (phosphatase). The osmotic signal is
proposed to regulate the ratio of the kinase to the phosphatase
activities of EnvZ to modulate the level of OmpR phosphorylation. In
this work we used a COOH-terminal fragment of a previously identified
kinase
Phosphorylation and dephosphorylation of cellular proteins plays
a critical role in signal transduction to regulate numerous cellular
functions in both prokaryotes and eukaryotes. Protein histidine kinases
and their response regulators, so-called ``two-component''
systems, constitute a large family (>50) of signal transducing
systems that enable bacteria to adapt to the changing environment
(reviewed by Parkinson and Kofoid(1992)). Classical eukaryotic protein
kinases are tyrosine, serine, and threonine kinases. Recently, however,
histidine kinases have been found not only in yeast (Maeda et
al., 1994; Ota and Varshavsky, 1993) but also in plants (Chang et al., 1993) (reviewed by Alex and Simon(1994) and Swanson et al.(1994)) and in mammalian cells (Crovello et
al., 1995). This represents a novel paradigm for eukaryotic cell
signaling. Prokaryotic histidine kinases exhibit considerable homology
in their COOH-terminal domains, and it is believed that they share a
common mechanism of action. Exposure of bacteria to high osmolarity
leads to dehydration, collapse of ion gradients over the cytoplasmic
membrane, and decrease in cell viability. Therefore, the first response
of bacteria to osmotic stress consists of changes in the activities of
enzymes and transport systems so that the turgor pressure is restored
and the cytoplasmic environment is optimized. Somewhat later, changes
in gene expression provide additional flexibility in adapting cells to
osmotic shock (for reviews see Csonka(1989), and Csonka and
Hanson(1991). One of the means that Escherichia coli adopts to
handle osmotic stress is to modulate the type of diffusion pores that
exist in the outer membrane (Csonka and Hanson, 1991). These pores are
formed by homotrimeric association of the porin proteins, OmpF and
OmpC. These proteins are highly expressed (approximately 10 EnvZ protein is a bifunctional histidine kinase/phosphatase. It is a
trans-inner membrane osmosensor consisting of 450 amino acids. It
contains two transmembrane domains (16-46 and 163-179), a
115-amino acid residue periplasmic domain(47-162), a 270-amino
acid resiue COOH-terminal cytoplasmic domain, and a
short(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) NH Taz1 is a hybrid receptor consisting of the
NH
Figure 1:
Autophosphorylation and
phosphotransferase activity of EnvZ
Figure 2:
Phosphorylation of EnvZ
Figure 3:
Reverse transfer of the phoshoryl group
from EnvZ
To examine whether OmpR(N) is a
better substrate than OmpR for the phosphotransfer reaction from
EnvZ
Figure 4:
Reverse transfer of the phoshoryl group
from EnvZ
Next, we examined the effect
of divalent cations on the phosphatase reaction. We carried out the
reaction in 25 mM Tris-HCl (pH 8.5) in the absence of ADP and
in the presence of 5 mM EDTA, 5 mM EGTA, 5 mM MgCl
Figure 5:
Effect of metal ions and their chelators
on the phosphatase activity of EnvZ
Figure 6:
Endoproteinase Lys-C digestion of
phospho-EnvZ(C) and phospho-EnvZ
Previous work from this laboratory demonstrated that
substitutions at the conserved asparagine residue blocks the kinase but
maintains the phosphatase activity (Yang and Inouye, 1991; Yang et
al., 1993). The present data show that although the N347D mutation
(corresponds to the N381D mutation in Taz) severely impaired the
ATP-dependent autophosphorylation, EnvZ The
mechanism of the phosphatase reaction has not been elucidated for EnvZ.
Also the role of the conserved asparagine is currently undefined. The
results obtained with the EnvZ EnvZ The present results suggest that the energy levels of
the phosphoryl group is similar between the sensor, EnvZ, and the
response regulator, OmpR, so that the phosphotransfer reaction is
readily reversible. We are currently investigating whether the wild
type EnvZ also dephosphorylates phospho-OmpR by the reverse reaction of
the OmpR-phosphorylation reaction.
Volume 271,
Number 3,
Issue of January 19, 1996 pp. 1424-1429
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
/Phosphatase
Mutant of EnvZ (EnvZ
N347D), a Bifunctional Signal
Transducer of Escherichia coli(*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/phosphatase
EnvZ mutant
(EnvZ
N347D) to demonstrate that the phosphoryl group on
phospho-OmpR is transferred back to EnvZ to the same histidine residue
(His) that is utilized for the autokinase reaction by the
wild type protein. Phospho-EnvZ
N347D thus formed could also
transfer its phosphoryl group back to OmpR. The phosphotransfer
reaction from phospho-OmpR to EnvZN347D was inhibited by ADP
while Mg ions stimulated the dephosphorylation
reaction, resulting in release of inorganic phosphate. These results
indicate that the energy levels of phosphoryl groups on OmpR and EnvZ
are very similar and that the phosphatase reaction in the
EnvZ
N347D mutant involves a reversal of the phosphotransfer
reaction from EnvZ to OmpR using the identical His residue.
molecules/cell), and the rate of diffusion through OmpF has been
measured to be 10 times faster than through OmpC (Nikaido and Vaara,
1985). The expression of these two porins is differentially regulated
through the members of the ompB operon, ompR and envZ (Hall and Silhavy, 1981; Forst and Inouye, 1988). OmpF is
preferentially produced at low osmolarity and OmpC at high osmolarity. 
-terminal cytoplasmic region. Conserved
amino acid residues that EnvZ shares with other histidine kinases
include: His, the autophosphorylation site, Asn
(function unknown), and two glycine-rich segments
DXGXG(373-377) and GXG(403-405)
(putative ATP-binding domains). EnvZ autophosphorylates His
using ATP and then transfers the phosphoryl group to a
transcriptional factor, OmpR (Igo and Silhavy, 1988; Forst et
al., 1989; Aiba et al., 1989). OmpR is a cytoplasmic
protein of 239 amino acid residues. It has an NH
-terminal
regulatory domain that bears significant homology to other response
regulators of the two-component system and a COOH-terminal DNA-binding
domain. EnvZ phosphorylates OmpR on Asp (Delgado et
al., 1993). EnvZ also dephosphorylates phospho-OmpR (Igo et
al., 1989; Aiba et al., 1989a). The concentration of
phosphorylated OmpR in the cell differentially regulates the
transcription of ompF and ompC genes for outer
membrane porins, OmpF and OmpC (Sarma and Reeves, 1977; Hall and
Silhavy, 1981; Mizuno and Mizushima, 1987; Slauch and Silhavy, 1989).
Of the kinase and phosphatase activities of EnvZ, the latter is
considered to be regulated by the osmotic signal, which thereby
controls the levels of phosphorylated OmpR in the cell (Yang and
Inouye, 1991; Russo and Silhavy, 1991; Yang et al., 1993; Jin
and Inouye, 1993).
-terminal (256-residue) ligand-binding domain of Tar (a
chemoreceptor for aspartate) and the COOH-terminal (228-residue)
signaling domain of EnvZ (Utsumi et al., 1989). This chimeric
receptor induces ompC-lacZ expression in response to
aspartate, thus enabling one to study EnvZ function in response to a
well defined ligand (the natural ligand for EnvZ is unknown). It has
been shown that all substitution mutants at the conserved Asn residue
(381 in Taz1) eliminated the kinase activity of Taz1, resulting in the
elimination of ompC-lacZ expression in either the absence or
presence of aspartate (Yang et al., 1993). However, all of
them retained the phosphatase activity at a similar level to that of
the wild type Taz1, regardless of the amino acid residues used for
substitution (D, E, Q, H, A, T, and C). The Asn substitution mutants could complement the H277V (the
autophosphorylation site) mutant of Taz1 in vivo to restore
wild type levels of osmoregulation. For a finer analysis of the role of
the conserved Asn in EnvZ, the 271-residue
(Arg
-Gly
) cytoplasmic domain of the
mutant EnvZ
N347D(C) was purified, and its enzymatic activity was
characterized. We now report that the phosphatase reaction of
EnvZN347D(C) involves the reverse phosphotransfer of the
phosphoryl group from OmpR to EnvZ to the identical His residue which is the site of autophosphorylation. The
dephosphorylation of phospho-OmpR by the mutant protein was found to be
inhibited by ADP but stimulated by Mg
ions. These
results indicate that the phosphate groups on EnvZ and OmpR are
energetically similar and that the phosphatase reaction of the mutant
EnvZ
N347D(C) involves the reverse phosphotransfer of the
phosphoryl group from OmpR to EnvZ using the identical
His, the autophosphorylation site.
Chemicals and Reagents
Nitrocellulose membranes were from Schleicher & Schuell.
Alkaline phosphatase-conjugated goat anti-rabbit antibody was from
Bio-Rad. Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl
phosphate p-toluidine salt was from Life Technologies, Inc.
[-
P]ATP was obtained from Amersham.
Endoproteinase Lys-C (sequencing grade) was purchased from Boehringer
Mannheim.
Cell Strain and Construction of Plasmids
E. coli B strain BL21-DE3 (FompT
r
m) was used for the expression of the envZ(C) and envZN347D(C) gene
fragments cloned into the pEt11a vector. The 1.5-kilobase pair EcoRII/EcoRI DNA fragment corresponding to the
271-amino acid residue (Arg
-Gly
)
cytoplasmic domain of EnvZ (EnvZ(C)) was recloned from the ompC:envZ
plasmid previously constructed in the laboratory (Forst et
al., 1989) into a pEt11a T7 expression system to construct
pEt11aE-Z(C). The EnvZ(C) protein thus expressed contains four extra
amino acids (Met, Ala, Gly, Ile) at the amino-terminal end. The
mutagenesis of envZ was performed by site-directed mutagenesis
using M13 as described previously (Forst et al., 1989). The envZ
N347D(C) mutant was then subcloned into pEt11aE in
the same manner as the wild type gene. (
)Purification of EnvZ(C) and
EnvZ
EnvZ(C) was purified from E. coli B strain BL21(DE3)
transformed with pEt11aE-Z(C) plasmid by a modification of the
procedure described previously (Forst et al., 1989). The
protein was precipitated using 30% ammonium sulfate saturation instead
of 40%. The Affi-Gel blue affinity column was substituted with a Green
A affinity column from which the protein was eluted with a linear
gradient 0-1.5 M KCl in buffer A (20 mM Tris-HCl (pH 7.8) containing 5% glycerol, 10 mM N347D(C) Protein Fragments
-mercaptoethanol, and 1 mM EDTA). Protein fractions
were pooled and centricon-concentrated.Preparation of Membrane Fractions
PDR 200 cells harboring wild type EnvZ was grown in LB
medium. Mid-logarithmic-phase cells were harvested by centrifugation
and washed once and sonicated in buffer B (0.1 M sodium
phosphate (pH 7.2) containing 10% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), ()5 mM EDTA, 5 mM 1,10-phenanthroline, 0.3 mMp-hydroxymercuribenzoate, and 1 µM leupeptin). Cell debris was removed by centrifugation at 10,000
g for 15 min. The supernatant was then
ultracentrifuged at 180,000 g for 90 min at 4 °C.
The pellet was washed with buffer C (20 mM Tris-HCl (pH 7.8),
5% glycerol, 1 mM EDTA, 10 mM -mercaptoethanol)
containing 2 M KCl and sonicated in the same buffer and then
centrifuged at 393,000 g for 14 min. This washing step
was repeated. The resulting membrane fraction (EnvZ(M)) was resuspended
in buffer C. The concentration of total protein in the membrane
preparation was estimated by the Bio-Rad assay.In Vitro Autophosphorylation and pH Stability Assays
EnvZ membrane preparations or soluble purified cytoplasmic
proteins were incubated in 0.1 M Tris-HCl buffer (pH 8.0)
containing 50 mM KCl, 5 mM CaCl, 1 mM PMSF, and 10% glycerol with 0.4
mM[-
P]ATP (1,100 cpm/pmol) at 25
°C for 10 min. The reaction was stopped by adding 5
SDS gel
loading buffer containing 10% (w/v) SDS, 3 mM -mercaptoethanol, and 40% glycerol. The reaction mixture was
then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
After electrophoresis protein bands were transferred to a BA85
nitrocellulose membrane (0.45 µm). The membrane was exposed for
autoradiography. Protein bands were visualized with anti-EnvZ
polyclonal antiserum and goat anti-rabbit IgG alkaline phosphatase
conjugate followed by a color reaction with nitro blue tetrazolium and
5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt. The pH
stability of of the phosphate linked to EnvZN347D(C) and to
EnvZ(C) was checked by the method described previously (Forst et
al., 1989).In Vitro Phosphorylation of OmpR Protein by EnvZ Membrane
or Cytoplasmic Proteins
EnvZ proteins were first autophosphorylated under the same
conditions as described above for 15 min. Purified OmpR (2 µl, 0.3
µg/µl, obtained from S. Harlocker (Delgado et al.,
1993)) was then added to the reaction mixture and incubated at 25
°C for 20 min. The reaction was stopped by adding 5 SDS gel
loading buffer, and the reaction mixture was analyzed by SDS-PAGE as
described above.Dephosphorylation of Phosphorylated OmpR by EnvZ
Cytoplasmic Proteins
The phosphatase activity of EnvZ-soluble cytoplasmic proteins
was measured as follows.Step 1: Autophosphorylation of EnvZ(M) for 20
Min
EnvZ(M) (50 µg) was autophosphorylated with
[-
P]ATP (100 µCi, specific activity:
3,000 Ci/mmol) in 200 µl of 0.1 M Tris-HCl buffer (pH 8.0)
(containing 50 mM KCl, 5 mM CaCl
, 1
mM PMSF, and 10% glycerol) at 25 °C for 20 min. The
reaction mixture was centrifuged at 393,000 g for 14
min at 4 °C using a Beckman TL100 ultracentrifuge. The membrane
pellet was washed three times with 0.1 M Tris-HCl buffer (pH
8.0) containing 50 mM KCl, 5 mM CaCl, 1
mM PMSF, and 10% glycerol, sonicated, and then resuspended in
the same buffer. The suspended membrane fraction was centrifuged and
washed six more times. The removal of
[-
P]ATP and P
in the membrane
fraction was monitored by thin-layer chromatography. The final membrane
fraction containing phosphorylated EnvZ(M) was resuspended in 200
µl of the same washing buffer.Step 2: Isolation of Phosphorylated OmpR
Protein
Purified OmpR protein (approximately 15 µg) was
incubated with the membrane fraction containing phosphorylated EnvZ
protein in a total volume of 140 µl for 20 min at 25 °C. After
incubation the reaction mixture was centrifuged at 393,000 g for 14 min to remove the EnvZ-containing membrane. The
120-µl supernatant containing phosphorylated OmpR was collected and
immediately used as substrate for the phosphatase assay.Step 3: OmpR-P Phosphatase Assay
Phosphorylated
OmpR protein (1.7 µM) was mixed with purified cytoplasmic
fragment of EnvZ (EnvZ(C) or EnvZN347D(C)) (8.7 µM)
and ADP (final concentration: 1 mM) in phosphatase buffer (0.1 M Tris-HCl buffer (pH 8.0) containing 50 mM KCl, 5
mM CaCl, 1 mM PMSF, and 10% glycerol) for
the required length of time at 25 °C. The reaction was stopped by
adding 5 SDS gel loading buffer and analyzed by SDS-PAGE,
transferred to nitrocellulose, and then subjected to autoradiography as
described above.Reverse Transfer of Phoshoryl Group from
EnvZ
Phosphorylated OmpR protein was used as substrate for the
phosphatase assay for EnvZN347D(C) to OmpR ProteinN347D(C) as described above. For the
study of the reverse transfer of the phosphoryl group from the mutant
protein, purified non-phosphorylated NH-terminal fragment
of OmpR (1 µg, obtained from S. Harlocker (Delgado et al.,
1993)) was added to the reaction mixture containing phospho-OmpR and
EnvZN347D(C) and incubated for an additional 10 min under the
same conditions. The results were analyzed in the same manner as
described above.Endoproteinase Lys-C Digestion of Phospho-EnvZ(C) and
Phospho-EnvZ
EnvZ(C) protein (8 µg) was autophosphorylated as
described above. EnvZN347D(C)N347D(C) protein was phosphorylated using
phosphorylated OmpR following the protocol described above except that
all the reactions were carried out in 25 mM Tris-HCl (pH 8.5).
After the 20-min incubation the reaction was stopped by the addition of
SDS loading buffer and an aliquot of the reaction mixture containing
approximately 8 µg of the mutant protein was subjected to
endoproteinase Lys-C digestion along with the wild type protein. The
phosphorylated proteins were incubated for 18 h at 37 °C in the
digestion buffer of 25 mM Tris-HCl (pH 8.5) containing 1
mM EDTA and 1 µg of endoproteinase Lys-C. After incubation
SDS loading buffer was added to the reaction mix and further incubated
for 30 min at 40 °C. The digestion products were separated on a
Tricine-SDS-PAGE gel (Schägger and von Jagow,
1987). The separating gel composition was 16.5% T and 6% C. the spacer
gel was of 10% T and 3% C while the stacking gel was 4% T and 3% C.
Insulin B chain was used as a marker. The protein bands were
transferred to nitrocellulose and exposed for autoradiography.
The COOH-terminal Fragment of EnvZ
The
purified EnvZN347D Is
Kinase/Phosphatase
N347D(C) was tested for its ability to
autophosphorylate in the presence of
[-
P]ATP and thence to transfer the
phosphoryl group to OmpR (kinase activity). Fig. 1A shows
that EnvZ(C) was autophosphorylated (lane 1) and could
concomitantly transfer the phosphoryl group to OmpR (lane 2).
EnvZ
N347D(C), however, was deficient in autophosphorylation (lane 3), and therefore subsequent transfer of the phosphoryl
group to OmpR was also not detected (lane 4). Equivalent
quantities of purified COOH-terminal proteins were used for the
reactions as determined by immunoblot (Fig. 1B).
N347D(C) and EnvZ(C). Two
micrograms of purified cytoplasmic proteins EnvZ(C) (lane 1) and EnvZN347D(C) (lane 3) were incubated in 0.1 M Tris-HCl buffer (pH 8.0) containing 50 mM KCl and 5
mM CaCl with 0.4
mM[-
P]ATP (1,100 cpm/pmol) at 25
°C for 10 min. The reaction was stopped by the addition of 5
SDS gel loading buffer. In a duplicate set of reactions (lanes 2 and 4) purified OmpR protein (0.6 µg)
was added to the reaction mixture at the end of 5 min and further
incubated for 15 min before stopping the reaction with 5 SDS
sample buffer. The reaction mixtures were then subjected to 17.5%
SDS-PAGE analysis. After electrophoresis protein bands were transferred
to a nitrocellulose membrane and exposed for autoradiography. Protein
bands were visualized with anti-EnvZ polyclonal antiserum as described
under ``Experimental Procedures.'' A, SDS-PAGE
autoradiogram. B, immunoblot of the same gel as in A.
Phosphorylation of EnvZ
We next examined whether EnvZN347D(C) by
Phospho-OmpRN347D(C) was
capable of dephosphorylating phospho-OmpR. The phosphatase assay was
performed as described under ``Experimental Procedures''; P-labeled EnvZ (Fig. 2, lane 1) was
incubated with purified OmpR, and the reaction mixture was incubated at
25 °C. [
P]phosphoryl group was transferred
to OmpR from phospho-EnvZ(M) as shown in Fig. 2, lane
2.
P-Labeled phospho-OmpR was then isolated by
ultracentrifugation of the reaction mixture to remove the EnvZ(M).
Phospho-OmpR, recovered in the supernatant, was then incubated with the
COOH-terminal fragments of EnvZ for 1 and 20 min. With wild type
EnvZ(C) approximately 90% of phospho-OmpR was dephosphorylated within 1
min (lane 7), and in 20 min phospho-OmpR was completely
hydrolyzed (lane 8). There was no significant
dephosphorylation of phospho-OmpR in the absence of EnvZ(C) during the
same time period (lanes 3 and 4). In contrast to the
wild type EnvZ(C), when EnvZ
N347D(C) was used for the reaction,
dephosphorylation of phospho-OmpR was accompanied with the concomitant
appearance of phospho-EnvZN347D(C), as shown in lanes 5 and 6. At 20 min there was an almost quantitative
transfer of phosphoryl group from OmpR to EnvZN347D(C) (lane
6).
N347D(C) by
phospho-OmpR. Phosphorylated OmpR was prepared as described under
``Experimental Procedures.'' Lane 1, phospho-EnvZ(M)
obtained by autophosphorylation with
[-
P]ATP. Lane 2, phospho-EnvZ(M)
incubated with OmpR. Lanes 3 and 4, phospho-OmpR (1
µg) incubated in phosphatase buffer for 1 and 20 min, respectively.
Phospho-OmpR (1 µg) was incubated in phosphatase buffer with
EnvZ(C) or EnvZ
N347D(C) (2 µg in each case) and ADP (final
concentration: 1 mM) for 1 and 20 min, at 25 °C. The
reaction was stopped by adding 5 SDS gel loading buffer and
analyzed by SDS-PAGE, transferred to nitrocellulose, and then subjected
to autoradiography as described in the legend to Fig. 1. Lanes 5 and 6, EnvZN347D(C), 1 and 20 min,
respectively; Lanes 7 and 8, EnvZ(C), 1 and 20 min,
respectively.
Reversibility of the Phosphoryl Group Transfer from
EnvZ
The kinase activity of EnvZ proceeds
in two steps; autophosphorylation at His243 with ATP, followed by
phosphoryl group transfer from phospho-EnvZ to OmpR. Although
EnvZN347D(C) to OmpRN347D(C) was deficient in autophosphorylation with ATP (see Fig. 1), the experiment described above clearly indicated that
it could become phosphorylated by phospho-OmpR. If the phosphorylated
EnvZN347D(C) thus obtained is identical to the autophosphorylated
wild type EnvZ(C), it is expected that the phosphoryl group on
EnvZN347D(C) should be transferred back to OmpR if an excess of
non-phospho-OmpR is added. To test this, purified
NH-terminal domain of OmpR (residues 1-134) was added
after 20 min incubation of EnvZN347D(C) with phospho-OmpR (Fig. 2, lane 6) and was further incubated for an
additional 10 min. OmpR(N) contains the Asp residue and
has been shown to be efficiently phosphorylated by EnvZ(C) (Delgado et al., 1993). As shown in Fig. 3, lane 2, the
phosphoryl group was almost quantitatively transferred from OmpR-P to
EnvZ
N347D(C) after a 20-min incubation. The molar ratio of OmpR
to EnvZN347D(C) in the reaction was 1:5. In a duplicate reaction,
after the initial 20-min incubation of EnvZN347D(C) with OmpR,
OmpR(N) was added to the reaction mixture and incubated for a further
10 min (molar ratio of OmpR to OmpR(N) was 1:5). On incubation with
OmpR(N) reverse transfer of the phosphoryl group was detected from
EnvZN347D(C) to OmpR(N) (Fig. 3, lane 1). This
result clearly indicated that the N347D mutation did not effect the
phosphotransferase activity of EnvZ(C). It was interesting to note that
no reverse transfer of phosphoryl group to OmpR was detected (Fig. 3, lane 1).
N347D(C) to OmpR and the inhibition of the phosphatase
reaction of EnvZN347D(C) by ADP. Lane 2, phosphorylated
OmpR protein was isolated and used as substrate for the phosphatase
assay for EnvZN347D(C) as described in the legend to Fig. 2, except that the reaction was performed in the absence of
ADP. Incubation time was 20 min. Lane 1, in a duplicate
reaction purified NH-terminal OmpR protein
(OmpR:OmpR(N)::1:5) was added to the reaction mixture after 20-min
incubation and further incubated for 10 min under the same conditions. Lanes 3, 4, and 5, the phosphatase assay was carried
out in the presence of 1, 5, and 10 mM ADP, respectively.
Incubation time was 20 min. The results were analyzed in the same
manner as described in the legend to Fig. 2.
N347D(C), the phosphatase assay was carried out with
phospho-OmpR and EnvZN347D(C) at a molar ratio of 1:2. Aliquots
were taken at 0.5, 1, 10, 30, and 45 min and analyzed by SDS gel
electrophoresis (Fig. 4, lanes 1-5). By 30 min (lane 4), the ratio of P in OmpR to that of
EnvZ
N347D(C) reaches a constant (by densitometry), indicating
that the phosphotransfer reaction between the two proteins have reached
an equilibrium. At 45 min, three aliquots were taken. To one,
additional non-phospho-OmpR was added (lane 6), to the second
non-phospho-OmpR(N) was added (lane 7), and to the third both
OmpR and OmpR(N) was added (lane 8). These reaction mixtures
were incubated for another 10 min. From lanes 6-8, it is
clear that on the addition of OmpR or OmpR(N) the phosphoryl group is
transferred to the added component. On the basis of the distribution of P among these proteins, it appears that all three compete
for the phosphoryl group. Also, OmpR(N) was not a better substrate than
OmpR for the phosphotransfer from EnvZ
N347D(C). It is to be noted
that when phospho-OmpR(N) was used instead of phospho-OmpR for the
phosphorylation of EnvZN347D(C), it was at least 95% less
efficient than that with phospho-OmpR (data not shown). This may
suggest that the COOH-terminal domain of OmpR has some role in the
reverse transfer of phosphoryl group from OmpR to EnvZN347D(C).
N347D(C) to OmpR and/or to OmpR(N). The phosphatase
assay was carried out as described in the legend to Fig. 2,
except that ADP was omitted from the reaction buffer. The time course
of the phosphotransfer from phospho-OmpR to EnvZN347D(C) was
examined. Aliquots from the reaction mixture were withdrawn at 0.5-,
1-, 10-, 30-, and 45-min intervals (lanes 1-5,
respectively). At the 45-min time point three aliquots were taken, to
one was added OmpR (lane 6), to the second was added OmpR(N) (lane 7), and to the third was added both in equimolar amounts (lane 8), and the reaction mixes were incubated for a further
10 min. The results were analyzed in the same manner as described in
the legend to Fig. 2.
Effects of ADP and Metal Ions on the Reverse
Phosphotransfer
Since ADP, ATP, and non-hydrolyzable analogues
of ATP are known co-factors that enhance the rate of the phosphatase
reaction (Aiba et al., 1989a; Igo et al., 1989), we
investigated the effect of ADP on the phosphatase reaction with
EnvZN347D(C). Interestingly, as the ADP concentrations increased
from 0 to 10 mM, a progressive inhibition in the phosphoryl
group transfer from phospho-OmpR to EnvZN347D(C) was observed (Fig. 3, lanes 2-5). At 10 mM ADP the
phosphotransfer was completely blocked., and 5 mM CaCl. As shown in Fig. 5, lane 3, 5 mM EDTA inhibits the
reaction. The EDTA inhibition can be reversed by addition of 5 mM MgCl (data not shown). In contrast, EGTA does not
inhibit the reaction (Fig. 5, lane 4). These results
indicate that Mg is a necessary co-factor of the
phosphatase reaction. Since very little phosphoprotein is detected in
the presence of Mg
, it clearly enhances the
phosphatase reaction. Ca
on the other hand stabilizes
phospho-OmpR and retards the phosphotransfer (lane 6).
N347D(C). The phosphatase
assay was carried out as described in the legend to Fig. 2except that 0.1 M Tris-HCl (pH 8.5) was used as
the buffer. EnvZN347D(C) (approximately 2.6 µg) was incubated
in the presence of 5 mM EDTA, EGTA, MgCl, and
CaCl for 20 min for lanes 3, 4, 5, and 6,
respectively. The results were analyzed in the same manner as in Fig. 2.
Involvement of the Conserved Autophosphorylation Site,
His
To identify the residue phosphorylated
in EnvZ, in the Phosphatase Reaction of
EnvZ
N347D(C)N347D(C) by phospho-OmpR, we first examined its pH
stability. Phospho-EnvZN347D(C) was alkali stable (pH 13) but
acid-labile (pH 1) (data not shown), which is characteristic of
histidyl phosphate. To determine which histidine residue(s), of the six
histidine residues in EnvZN347D(C) was phosphorylated, both
wild-type and mutant phosphoproteins were subjected to endoproteinase
Lys-C digestion. Non-phosphorylated EnvZ(C) was also digested. Note
that EnvZ(C) was autophosphorylated with
[-
P]ATP, while EnvZ
N347D(C) was
phosphorylated using [P]phospho-OmpR. The
products of digestion of non-phospho-EnvZ(C), autophosphorylated
EnvZ(C), and phospho-EnvZ
N347D(C) were separated on a
Tricine-SDS-PAGE gel (Fig. 6, lanes 2-4,
respectively). Lanes 1 and 2 of the gel were stained
with Serva blue dye. Lane 2 shows the seven predicted EnvZ(C)
Lys-C digestion bands. Lanes 3 and 4 of the gel were
subjected to autoradiography. For both phosphoproteins, a single band,
migrating at the identical position corresponding to the fourth band in
the stained gel, was phosphorylated. From its size the fragment was
judged to be the peptide Gln to Asn
,
containing His
, the only histidine residue present in
that fragment. This result clearly demonstrates that the same residue
(His
) which was autophosphorylated by ATP in the wild
type EnvZ(C) was phosphorylated in EnvZ
N347D(C) by phospho-OmpR.
N347D(C). EnvZ(C) protein (8
µg) was autophosphorylated with [-
P]ATP
as described in the text. EnvZ
N347D(C) protein (8 µg) was
phosphorylated using phospho-OmpR following the protocol described in
the legend to Fig. 2, except that all the reactions were carried
out in 25 mM Tris-HCl (pH 8.5). After the 20-min incubation
the reaction was stopped by the addition of 5 SDS loading
buffer. Phospho-EnvZ(C) and phospho-EnvZN347D(C) were then
subjected to endoproteinase Lys-C digestion as described under
``Experimental Procedures.'' Non-phosphorylated wild type
EnvZ(C) (8 µg) was also digested with endoproteinase Lys-C in the
same manner as the phosphoproteins. The digestion products were
separated on a Tricine-SDS-PAGE gel. Lane 1, insulin B chain,
a molecular size marker; lane 2, the digest of
non-phosphorylated EnvZ(C); lane 3, the digest of
phospho-EnvZ(C); lane 4, the digest of
phospho-EnvZN347D(C)). Part of the gel (lanes 1 and 2) was stained with Serva blue dye. The other part (lanes
3 and 4) was blotted on to a BA 85 nitrocellulose
membrane (0.45 µm) and exposed for
autoradiography.
N347D(C) can still receive
the phosphoryl group readily from phospho-OmpR. Moreover, once
phosphorylated it also retains its ability to transfer the phosphoryl
group back to OmpR or OmpR(N). The phosphatase assay yielded unexpected
results with the N347D mutant protein. The mutant could efficiently
dephosphorylate phospho-OmpR by removing the phosphoryl group from it
onto its own histidine (His) which is also the
autophosphorylation site. Similar reverse phosphotransfer from
phospho-OmpR to EnvZ was observed with purified EnvZ
N347H(C). In
the presence of 5 mM Mg and absence of ADP
very little phospho-protein is detected for both EnvZ
N347D and
OmpR. The phosphoryl group is released as inorganic phosphate under
these conditions (not shown). Therefore the ``phosphatase''
reaction by EnvZN347D(C) involves a phosphotransfer reaction from
phospho-OmpR to the mutant protein. It is important to point out that
the in vivo complementation experiments (Yang et al.,
1993) clearly demonstrated that the asparagine substituent mutants can
complement the H277V mutant (confers null phenotype) to restore
functional signal transduction, indicating that the phosphatase
activity of the conserved asparagine substituent mutants is
functionally equivalent to the wild type phosphatase activity.N347D(C) mutant protein offers some
insights into the roles of the conserved Asn and the
phosphatase function of EnvZ. Since EnvZ
N347D(C) is deficient in
autophosphorylation (Fig. 1), it is possible that the conserved
asparagine is involved in ATP binding and/or in the subsequent transfer
of the phosphoryl group to histidine. It has been postulated that EnvZ
may have a modulator binding site which may or may not overlap with the
putative ATP binding site. Contrary to what is observed with the wild
type protein ADP serves as a negative effector for the phosphatase
reaction of the N347D mutant protein (Fig. 3), suggesting that
the putative modulator binding site has been modified in the
EnvZN347D(C) mutant. Therefore the conserved asparagine in EnvZ
may be involved directly or indirectly in defining the putative ATP
and/or modulator binding sites in EnvZ.N347D(C) is
phosphorylated during the dephosphorylation of phospho-OmpR and in the
presence of Mg and the absence of ADP the phosphoryl
group is released very efficiently as inorganic phosphate. This raises
the possibility that the phosphatase reaction of the mutant
EnvZ
N347D(C) proceeds in two steps. In the first step phosphoryl
group transfer occurs from phospho-OmpR to His on EnvZ,
which is the autophosphorylation site of EnvZ. In the second step this
phosphorylated intermediate is hydrolyzed to inorganic phosphate which
is a Mg
-dependent reaction. Therefore the
phosphorylation of the mutant EnvZ during the dephosphorylation of
phospho-OmpR just represents the reverse phosphotransferase reaction of
the the phosphorylation of OmpR by EnvZ. If His
is
involved in the phosphatase reaction of wild type EnvZ (as follows from
the first possibility), an important implication would be that
non-phosphorylatable substitutions at His
should
inactivate both the kinase and phosphatase functions of EnvZ. Known
mutations at this position in EnvZ include H243V (Yang and Inouye,
1991; Yang et al., 1993) and H243R (Tokishita and Mizuno,
1994), all of which confer a null phenotype. This is in agreement with
the prediction. It is of interest to note here that in the nitrogen
regulatory system, mutating the conserved histidine at position 319 to
asparagine (H319N) does not eliminate the phosphatase activity of NtrB
(Atkinson and Ninfa, 1993; Kamberov et al., 1994). Although
the Ntr system closely parallels the osmoregulatory system, they differ
in many respects. EnvZ is a membrane-localized sensor kinase, while
NtrB is a soluble cytoplasmic protein. Unlike EnvZ, NtrB requires an
accessory protein, P
for its phosphatase activity (Ninfa
and Magasanik, 1986; Keener and Kustu, 1988). Thus it is possible that
the mechanism of the phosphatase reaction of NtrB may be different from
that of EnvZ.
))
We thank K. Takayama, K. Madura, and Linda Egger for
the critical reading of this manuscript; J. Delgado for initial
guidance; T. Jin for the critical discussions and S. Harlocker for
providing the purified OmpR proteins.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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