(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/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 EnvZ
N347D 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.
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 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.
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-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).
Taz1 is a hybrid receptor consisting of the
NH-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
EnvZ
N347D(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.
Figure 1:
Autophosphorylation and
phosphotransferase activity of EnvZN347D(C) and EnvZ(C). Two
micrograms of purified cytoplasmic proteins EnvZ(C) (lane 1) and EnvZ
N347D(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.
Figure 2:
Phosphorylation of EnvZN347D(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, EnvZ
N347D(C), 1 and 20 min,
respectively; Lanes 7 and 8, EnvZ(C), 1 and 20 min,
respectively.
Figure 3:
Reverse transfer of the phoshoryl group
from EnvZN347D(C) to OmpR and the inhibition of the phosphatase
reaction of EnvZ
N347D(C) by ADP. Lane 2, phosphorylated
OmpR protein was isolated and used as substrate for the phosphatase
assay for EnvZ
N347D(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.
To examine whether OmpR(N) is a
better substrate than OmpR for the phosphotransfer reaction from
EnvZN347D(C), the phosphatase assay was carried out with
phospho-OmpR and EnvZ
N347D(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 EnvZ
N347D(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 EnvZ
N347D(C).
Figure 4:
Reverse transfer of the phoshoryl group
from EnvZN347D(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 EnvZ
N347D(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.
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, 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).
Figure 5:
Effect of metal ions and their chelators
on the phosphatase activity of EnvZN347D(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. EnvZ
N347D(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.
Figure 6:
Endoproteinase Lys-C digestion of
phospho-EnvZ(C) and phospho-EnvZN347D(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-EnvZ
N347D(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-EnvZ
N347D(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.
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, EnvZN347D(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 EnvZ
N347D(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.
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 EnvZN347D(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
EnvZ
N347D(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.
EnvZN347D(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.
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.