From the Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro, Tokyo 153, Japan
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ABSTRACT |
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The CyaC protein, a cyanobacterial adenylate
cyclase, has a unique primary structure composed of the catalytic
domain of adenylate cyclase and the conserved domains of bacterial
two-component regulatory systems, one transmitter domain and two
receiver domains. In the present work, CyaC was produced in
Escherichia coli as a histidine-tagged recombinant protein
and purified to homogeneity. CyaC showed ability to autophosphorylate
in vitro with the Cyclic AMP (cAMP) is widely distributed from prokaryotes to
eukaryotes as an important signaling molecule. In cyanobacteria, Gram-negative bacteria that are able to perform higher plant-type oxygen-evolving photosynthesis, it has been shown that cellular cAMP
levels change in response to changes in environmental conditions such
as light-dark, low pH-high pH, oxic-anoxic (1, 2), and nitrogen
replete-deplete (3). In a gliding cyanobacterium, Spirulina
platensis, extracellular cAMP stimulates the activity of
respiration and gliding movement (4). These results suggest that cAMP
functions as a signaling molecule in cyanobacteria, although the
regulatory mechanism of cellular cAMP is yet to be elucidated.
cAMP is synthesized from ATP by an adenylate cyclase, and the activity
of adenylate cyclase is regulated by various mechanisms. Mammalian
transmembrane adenylate cyclases are regulated by the stimulatory and
inhibitory heterotrimeric G proteins in response to the binding of
chemical ligands to appropriate signal receptors (5). Ca2+
and Ca2+/calmodulin also regulate mammalian transmembrane
adenylate cyclases (5). The mammalian type V adenylate cyclase is
phosphorylated by protein kinase C and protein kinase A, and
phosphorylation regulates the activity of the enzyme (6, 7). In budding yeast Saccharomyces cerevisiae, adenylate cyclase is
activated by Ras, a small GTP-binding protein (8, 9). In
Escherichia coli, a phosphoenolpyruvate:carbohydrate
phosphotransferase system regulates the adenylate cyclase activity (10,
11).
Recently, a soluble adenylate cyclase has been isolated from mammalian
testis (12). The catalytic domains of this soluble adenylate cyclase
are more similar to those of cyanobacterial adenylate cyclases than
they are to those of mammalian transmembrane adenylate cyclases
(12).
Ten adenylate cyclase genes have been isolated from cyanobacteria
(13-17). They have a fairly conserved catalytic domain near the
C-terminal region and have characteristically different regulatory domains upstream of the catalytic domains. Among these genes, cyaC encodes a novel protein containing domains homologous
to members of the bacterial two-component regulatory system, one transmitter domain and two receiver domains, in addition to the catalytic domain of adenylate cyclase (16). The CyaC protein seems to
bind to the membrane of S. platensis cells, although it has
no sufficient hydrophobic regions to pierce the membrane (16).
Bacterial two-component regulatory systems, which provide the dominant
modes of signal transduction in bacteria for adaptation to
environmental changes, consist of two families of signal transduction proteins, the sensory kinase family and the response regulator family
(18, 19). The sensory kinases undergo autophosphorylation at a
histidine residue in their transmitter domain in response to
environmental stimuli, and the phosphoryl group once bound to the
histidine residue is transferred to an aspartate residue in the
receiver domain of the cognate response regulator. In most cases,
response regulators act as transcriptional factors and control the
expression of target genes. CheB in E. coli and RegA in
Dictyostelium discoideum exceptionally consist of a receiver domain and a catalytic domain of the enzyme, from methylesterase and
phosphodiesterase, respectively. The enzymes are activated by
phosphorylation of their receiver domains (20, 21). Several two-component regulatory proteins, so-called "hybrid sensory
kinases," contain both transmitter and receiver domains (22-25). It
can be said that CyaC has a novel structure consisting of both "a
hybrid sensory kinase" and a catalytic domain in one molecule.
Studies on the function of CyaC are important not only to understand
regulation of adenylate cyclase in cyanobacteria, but also to address
general issues about the regulation of enzyme activity by two-component regulatory systems.
In this study, we used recombinant proteins to investigate how CyaC is
phosphorylated and whether the phosphorylation state controls the
adenylate cyclase activity of CyaC.
Bacterial Strains and Growth Media--
The E. coli
strains used as hosts were JM109 (recA1, endA1,
gyrA96, thi, hsdR17 (rK Construction of CyaC Site-directed Mutants--
pETA (16)
contains the entire cyaC gene fused to a His tag sequence
from pET28a vector (Novagen), which is placed under the control of the
phage T7 promoter.
The CyaC D60A mutant was constructed with mismatch oligonucleotides and
PCR1 using ExTaq DNA
polymerase (Takara). pETA was used for the template DNA. PCR was
performed with the primers pD11 (5' position 159, GATCTGCTATACCGGAC, 3'
position 175) and pD13 (5' position 281, CATCCGCTGTGCACTGATAATT, 3' position
260) or pD12 (5' position 260, AATTATCAGTGCACAGCGGATG, 3' position 281) and pD14 (5' position 467, ACCTTGCTGAACGATCG, 3' position 451),
with mutated bases in boldface and the D60A substitution underlined. A
new restriction site (ApaLI) was created at position 268-273 without changing the amino acids. The ApaLI
restriction enzyme was used in the screening for the mutagenized
plasmid. The resulting PCR products were mixed, and PCR was repeated
with primers pD11 and pD14. The PCR product was ligated into pCRII vector (Invitrogen) to make pCRD1. The DraI fragment of
pCRD1 (220 base pairs) was ligated into pETA digested with
DraI. The resulting construct, pETD1, contained the
cyaC gene with the CyaC D60A mutation.
The CyaC H572Q mutant was constructed using methods similar to those
for the D60A mutation. PCR was performed with primers pH1 (5' position
1149, ACGATTACCAATGCTATTC, 3' position 1167) and pH3 (5' position 1819, GTGCGGAATTCTTGGGAGAC, 3'
position 1800) or pH2 (5' position 1800, GTCTCCCAAGAATTCCGCAC, 3'
position 1819) and pH4 (5' position 2010, ACTCCACCAGGTCACAG, 3'
position 1994), with mutated bases in boldface and the H572Q substitution underlined. A new restriction site (EcoRI) was
created at position 1809-1814 without changing the amino acids. The
EcoRI restriction enzyme was used in the screening for the
mutagenized plasmid. The resulting PCR products were mixed, and PCR was
repeated with primers pH1 and pH4. The PCR product was ligated into
pCRII vector to make pCRH. The MfeI-BsmI fragment
of pCRH (770 base pairs) was ligated into pETA digested with
MfeI and BsmI (partial digestion). The resulting
construct, pETH, contained the cyaC gene with the CyaC H572Q mutation.
The CyaC D895A mutant was constructed using methods similar to those
for the D60A mutation. PCR was performed with primers pD21 (5' position
2504, CGTAGAATTGGCTGATG, 3' position 2520) and pD23 (5' position 2788, GGCATCATTAATGCAGTAACA, 3' position
2768), or pD22 (5' position 2768, TGTTACTGCATTAATGATGCC, 3' position
2788) and pD24 (5' position 3268, ACAAACTTGTCAACAGTC, 3' position
3251), with mutated bases in boldface and the D895A substitution
underlined. A new restriction site (AseI) was created at
position 2777-2782 without changing the amino acids. The
AseI restriction enzyme was used in the screening for the
mutagenized plasmid. The resulting PCR products were mixed, and PCR was
repeated with primers pD21 and pD24. The PCR product was ligated into
pCRII vector to make pCRD2. The BsrGI-AvaI
fragment of pCRD2 (670 base pairs) was ligated into pETA digested with
BsrGI and AvaI (partial digestion). The resulting
construct, pETD2, contained the cyaC gene with the CyaC
D895A mutation.
The portions of pCRD1, pCRH, and pCRD2 derived from the PCR fragment
were sequenced to ensure the existence of no other mutations.
The CyaC D60A,D895A double mutant was constructed using pETD1 and
pETD2. Both pETD1 and pETD2 were digested with BamHI and BsrG I. The 2.5-kb pETD1 fragment was ligated into the
6.5-kb pETD2 fragment. The resulting construct, pETD1D2, contains the cyaC gene with the CyaC D60A,D895A double mutation.
The CyaC D60A,H572Q double mutant was constructed using the
MfeI-BsrGI fragments of pETD1 (7.7 kb) and pETH
(1.3 kb). The CyaC H572Q,D895A double mutant was constructed using the
MfeI-BsrGI fragments of pETH (1.3 kb) and pETD2
(7.7 kb). The CyaC D60A,H572Q,D895A triple mutant was constructed using
the MfeI-BsrGI fragments of pETD1D2 (7.7 kb) and
pETH (1.3 kb). These constructs were produced by methods similar to
those used for pETD1D2 construction.
Purification of Wild-type and Mutant His-CyaC Proteins--
The
purification procedure was essentially the same as that described
previously (16). The transformants, BL21(DE3)pLysS cells harboring one
of the expression constructs of the CyaC derivatives, were grown at
25 °C in Luria-Bertani medium (1.5 liters) supplemented with
kanamycin (25 µg ml Protein Phosphorylation Assay--
Unless otherwise stated, the
assay mixture (final volume 15 µl) contained, besides the enzyme, 50 mM Tris-HCl (pH 8.0), 200 mM KCl, 0.1 mM [
The phosphorylation assay using acetyl [32P]phosphate was
essentially the same as that described by Quon et al. (28).
The synthesized acetyl [32P]phosphate (7.5 µl) was
combined with each purified His-CyaC derivative in 7.5 µl of 50 mM Tris-HCl (pH 8.0) buffer containing 2 mM DTT
and 1 mM PMSF. The reactions were run at 30 °C for 15 min and terminated by the addition of 5 µl of 4 × SDS-PAGE
loading buffer. The samples were heated immediately at 50 °C for 5 min and applied to an 8% SDS-PAGE gel. The proteins were subsequently blotted onto an Immobilon-P membrane (Millipore). The phosphorylated proteins were analyzed using a BAS1000 Imaging Analyzer (Fuji film).
Adenylate Cyclase Activity Assay--
Unless otherwise stated,
the assay mixture (final volume 0.2 ml) contained, besides the enzyme,
50 mM Tris-HCl (pH 8.0), 200 mM KCl, 0.1 mM ATP, 1 mM MgCl2, 2 mM DTT, and 1 mM PMSF. The reaction was run for
15 min at 30 °C, terminated by the addition of 0.3 ml of 10%
perchloric acid, and neutralized with 0.5 ml of 1.01 M KOH.
The mixture was centrifuged at 200 × g for 10 min, and the cAMP content in the supernatants was measured by an enzyme immunoassay system according to the manufacture's protocol (EIA system, Amersham Pharmacia Biotech).
Stability of Phosphate Links in Wild-type and Mutant His-CyaC
Proteins--
Wild-type and mutant His-CyaC proteins were
phosphorylated and blotted onto an Immobilon-P membrane (Millipore) as
described above. The blots were incubated in 1 M HCl, 3 M NaOH, or 50 mM Tris-HCl (pH 7.2) at 25 °C.
After incubation for 1 h, the membranes were washed briefly with
distilled water, dried in air, and then exposed to an x-ray film.
Purification of His-CyaC--
The CyaC protein has a multidomain
structure consisting of the catalytic domain of adenylate cyclase and
domains that are homologous to those of bacterial two-component
regulatory systems. These domains exist in line from the N terminus: a
receiver domain (R1), a transmitter domain, a receiver domain (R2), and
a catalytic domain. The region between the receiver (R1) domain and the
transmitter domain shows similarity to the ETR1 protein that has been
identified as an ethylene receptor in plants (29, 30).
Eight kinds of CyaC derivatives were constructed and each protein was
fused with a histidine tag (Fig.
1A). The proteins were purified as described under "Experimental Procedures," and the purities were determined by SDS-PAGE (Fig. 1B). One band
with a molecular mass of 140 kDa was observed in each lane.
In the previous work, we showed that the catalytic activity of the CyaC
protein is stimulated by Mn2+ and less extensively by
Mg2+ (16). The specific activities (Mg2+- or
Mn2+-dependent adenylate cyclase activity) of
the purified wild-type His-CyaC protein used in this study were 7.4 and
260 nmol of cAMP formed mg
The molecular mass of His-CyaC was estimated to be 590 kDa by Superose
6 column chromatography, a kind of gel permeation column chromatography, suggesting that His-CyaC forms a homotetramer in
E. coli.
Autophosphorylation of His-CyaC--
Autophosphorylation of
His-CyaC was assayed by incubating the proteins with
[ Autophosphorylation of Mutant His-CyaC Proteins--
The
His572 residue of CyaC corresponds to the highly conserved
histidine residue found in sensory kinases of bacterial two-component regulatory systems. The Asp60 and Asp895
residues of CyaC correspond to a highly conserved aspartate residue among the response regulators of bacterial two-component regulatory systems (16, 18, 19).
To determine the role of these residues in the phosphorylation of CyaC,
autophosphorylation activities were assayed using His-CyaC(wild-type),
His-CyaC(D60A), His-CyaC(H572Q), His-CyaC(D895A), and
His-CyaC(D60A,D895A) (Fig. 1). The results show that His-CyaC(H572Q) is
unable to autophosphorylate in vitro (Fig.
3). Thus, His572 is thought
to be the initial phosphorylation site in CyaC.
The levels of phosphorylation observed in His-CyaC(wild-type) and
His-CyaC(D60A) were lower than those observed in His-CyaC(D895A) and
His-CyaC(D60A,D895A).
Stability of the Phosphoryl Groups of the Phosphorylated Wild-type
and Mutant His-CyaC Proteins--
Chemical stability assays have been
performed to determine the class of phosphorylated amino acid.
N-Phosphate bonds (phosphohistidine or phospholysine) are
base-stable and acid-labile. Acyl phosphate bonds (phosphoaspartate or
phosphoglutamate) are labile to both acid and base, and
O-phosphate bonds (phosphoserine, phosphothreonine, or
phosphotyrosine) are stable to acid (31, 32). To compare the stability
of phosphate links in the phosphorylated wild-type and mutant His-CyaC
proteins, each phosphorylated product was subjected to SDS-PAGE and
transferred to an Immobilon-P membrane. The membrane strips were
treated with acid, base, or neutral buffer.
Compared with the control treated with neutral buffer,
His-CyaC(wild-type) and His-CyaC(D60A) retained 64 and 69% of their label after base treatment, whereas His-CyaC(D895A) and
His-CyaC(D60A,D895A) retained 94 and 89% of their label, respectively
(Fig. 4). When treated with acid,
His-CyaC(wild-type) and His-CyaC(D60A) retained 19 and 20%,
respectively, of their label, whereas His-CyaC(D895A) and
His-CyaC(D60A,D895A) retained no label (Fig. 4). The phosphoryl groups
in phosphorylated His-CyaC(wild-type) and His-CyaC(D60A) were partially
labile under base conditions and labile under acid conditions, whereas
those in phosphorylated His-CyaC(D895A) and His-CyaC(D60A,D895A) were
stable under base conditions but labile under acid conditions. Portions
of the phosphate links in phosphorylated His-CyaC(wild-type) and
His-CyaC(D60A) seem to be phosphoacyl linkages, which are labile under
both acid and base conditions. The phosphate links in
phosphorylated His-CyaC(D895A) and His-CyaC(D60A,D895A) seem
to be N-phosphate linkages, which are stable under base
conditions but labile under acid conditions (32).
Adenylate Cyclase Activities of Wild-type and Mutant His-CyaC
Proteins--
The adenylate cyclase activities of His-CyaC derivatives
were compared with that of His-CyaC(wild-type) in vitro
(Fig. 5). The components in the reaction
mixtures and the assay conditions were the same as those used for the
autophosphorylation reaction except that the final volume of the
reaction mixtures differed. The adenylate cyclase activities of
His-CyaC(D60A), His-CyaC(H572Q), His-CyaC(D895A), and
His-CyaC(D60A,D895A) were 75, 23, 21, and 11% that of
His-CyaC(wild-type), respectively. The adenylate cyclase activity of
His-CyaC was decreased considerably by replacement of the
His572 or Asp895 residues with other amino
acids.
Phosphorylation of His-CyaC Proteins by Acetyl
[32P]Phosphate--
Several response regulator proteins
have been shown to be phosphorylated at a conserved aspartate
residue in the receiver domain by small molecular weight
phospho-donors such as acetyl phosphate, carbamoyl phosphate, and
phosphoramidate (33, 34). It was determined whether Asp60
and Asp895, the highly conserved phosphorylation
sites in the receiver domains of CyaC, are phosphorylated
by acetyl [32P]phosphate. When His-CyaC(H572Q),
His-CyaC(H572Q,D60A), His-CyaC(H572Q,D895A), and
His-CyaC(H572Q,D60A,D895A) (Fig. 1) were incubated with acetyl [32P]phosphate, the His-CyaC(H572Q) and
His-CyaC (H572Q,D60A) proteins were phosphorylated but the
His-CyaC(H572Q,D895A) and His-CyaC(H572Q,D60A,D895A) proteins were not
(Fig. 6). These results show that
Asp895 is phosphorylated but Asp60 is not
phosphorylated by acetyl phosphate and that these two amino acid
residues have somewhat different physiological functions.
Effect of Acetyl Phosphate on the Adenylate Cyclase Activities of
His-CyaC Proteins--
The adenylate cyclase activities of
His-CyaC(H572Q), His-CyaC(H572Q,D60A), His-CyaC(H572Q, D895A), and
His-CyaC(H572Q,D60A,D895A) were measured in the presence or absence of
acetyl phosphate. Acetyl phosphate greatly activated the adenylate
cyclase activities of His-CyaC(H572Q) and His-CyaC(H572Q,D60A)
(Fig. 7), while
His-CyaC(H572Q,D895A) and His-CyaC(H572Q,D60A,D895A) were
largely unaffected. Thus, the presence of Asp895 is
essential for the activation of adenylate cyclase activity. Acetyl
phosphate might activate the catalytic activity of the adenylate
cyclase by phosphorylating Asp895 in His-CyaC.
A cyanobacterial adenylate cyclase, CyaC, has been shown to have a
unique structure consisting of one transmitter domain and two receiver
domains of bacterial two-component regulatory systems in addition to
the catalytic domain of adenylate cyclase (15, 16). The possibility has
been considered that the autophosphorylation of the transmitter domain
and subsequent phosphotransfer to the receiver domains within the
molecule regulates the catalytic activity.
In the present study, CyaC was shown to have the ability to
autophosphorylate with [ The adenylate cyclase activity of the CyaC H572Q mutant, which does not
autophosphorylate, is about 4-fold lower than that of wild-type CyaC.
Furthermore, the adenylate cyclase activities of the CyaC D895A mutant
and CyaC D60A,D895A double mutant, which are able to autophosphorylate
but unable to transfer the phosphoryl group from His572 to
Asp895, are at least 5-fold lower than that of wild-type
CyaC. Thus the phosphorylation of Asp895 is required for
the full activity of CyaC, consistent with the results that CyaC is
phosphorylated at Asp895 by acetyl phosphate (Fig. 6) and
that the adenylate cyclase activity is stimulated in the presence of
acetyl phosphate (Fig. 7). These results indicate that CyaC
autophosphorylates first at His572 in the transmitter
domain, after which the phosphoryl group is transferred to
Asp895 in the receiver (R2) domain, and this stimulates the
adenylate cyclase activity (Fig. 8).
-phosphate of
[
-32P]ATP. CyaC derivatives were constructed by
site-directed mutagenesis in which the highly conserved phosphorylation
sites in the transmitter domain (His572) and receiver
domains (Asp60 or Asp895) were replaced by
glutamine and alanine residues, respectively. After autophosphorylation
of the CyaC derivatives, the chemical stabilities of the phosphoryl
groups bound to the derivatives were determined. It was found that
His572 is the initial phosphorylation site and that the
phosphoryl group once bound to His572 is transferred to
Asp895. The enzyme activities of the CyaC derivatives
defective in His572 or Asp895 were considerably
reduced. Asp895 is phosphorylated by acetyl
[32P]phosphate, a small phosphoryl molecule, but
Asp60 is not. Acetyl phosphate stimulates adenylate cyclase
activity only when Asp895 is intact. These results suggest
that the phosphorylation of Asp895 is essential for the
activation of adenylate cyclase and that Asp60 functions
differently from Asp895 in regulating the enzyme activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
mK+), supE44, relA1,
(lac-proAB)/F' (traD36, proAB,
lacIq,
(lacZ)M15)) for cloning and
BL21(DE3)pLysS (F
, ompT, hsdS
(rB
, mB
), dcm, gal,
(DE3), pLysS) for expression of recombinant proteins. Bacteria were
grown in Luria-Bertani medium (26). When required, kanamycin or
chloramphenicol was added at 25 or 30 µg ml
1, respectively.
1) and chloramphenicol (30 µg
ml
1). Each recombinant cyaC gene was expressed
in exponentially growing cells by adding 1 mM
isopropyl-
-D-thiogalactopyranoside. After 6 h, the
cells were harvested by centrifugation, washed with 50 mM
Tris-HCl (pH 7.5) buffer containing 150 mM NaCl, and
resuspended in 50 ml of extraction buffer consisting of 50 mM Tris-HCl (pH 8.0), 10% (w/v) glycerol, 0.5 M NaCl, 0.05% (w/v) Tween 80, 5 mM imidazole,
1 mM PMSF, and 1 mg ml
1 lysozyme. The cell
suspension was incubated on ice for 30 min and then sonicated at
4 °C for 9 min (3 min × 3) using a Kubota model
200M sonicator. The cell extract was centrifuged at
16,000 × g for 10 min, and the supernatant was further
centrifuged at 150,000 × g for 40 min. The
150,000 × g supernatant was loaded onto a HiTrap
Chelating column (Amersham Pharmacia Biotech; 1.6 × 2.5 cm)
connected to a fast protein liquid chromatography system (Amersham
Pharmacia Biotech) and eluted using step gradients of 5, 30, 60, and
200 mM imidazole in Buffer A (50 mM Tris-HCl
(pH 8.0), 10% (w/v) glycerol, 0.5 M NaCl, 0.05% (w/v)
Tween 80). The volume of eluent used in each step gradient was 32, 16, 13, and 8.5 ml, respectively. The 200 mM imidazole fraction
was concentrated to 0.5 ml using Ultrafree-15 (Millipore). The
concentrated eluate was loaded onto a Superose 6 column (Amersham
Pharmacia Biotech; 1 × 30 cm) and eluted with Buffer A containing
5 mM imidazole at a flow rate of 0.3 ml
min
1.
-32P]ATP (~6.7 µCi
nmol
1), 1 mM MgCl2, 2 mM DTT, and 1 mM PMSF. The reaction, started by
adding the enzyme, was run for 15 min at 30 °C and then terminated by the addition of 5 µl of 4 × SDS-PAGE loading buffer (200 mM Tris-HCl (pH 6.8), 400 mM DTT, 8% SDS, 40%
glycerol, and 0.04% bromphenol blue). To avoid the release of
phosphoryl groups, samples were heated immediately at 50 °C for 5 min (27) and applied to an 8% SDS-PAGE gel. The proteins were
subsequently blotted onto an Immobilon-P membrane (Millipore) and
subjected to autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, domain organizations of wild-type and
mutant CyaC proteins expressed as His-tag fusion proteins. The
full-length CyaC protein consists of 1202 amino acid residues. The
black boxes, white boxes, and ovals
show the catalytic domain of adenylate cyclase, the transmitter domain,
and the receiver domains, respectively. In wild-type, H
indicates histidine 572, and D indicates aspartate 60 and
aspartate 895. These amino acid residues are identified as highly
conserved phosphorylation sites. For site-directed mutations, the
altered amino acid residues are shown as outline characters.
A and Q indicate alanine and glutamine residues,
respectively. B, purification of wild-type and mutant CyaC
proteins expressed as His-tag fusion proteins. Each protein (2 µg)
was loaded onto an 8% SDS-PAGE gel for electrophoresis. The gel was
stained with Coomassie Brilliant Blue R-250. Lane 1,
His-CyaC(wild-type); lane 2, His-CyaC(D60A); lane
3, His-CyaC(H572Q); lane 4, His-CyaC(D895A); lane
5, His-CyaC(D60A,D895A); lane 6, His-CyaC(H572Q,D60A);
lane 7, His-CyaC(H572Q,D895A); lane 8,
His-CyaC(H572Q,D60A,D895A). The arrowheads indicate the
positions of molecular size standards.
1·min
1,
respectively. The specific activities are higher than those used in the
previous work (16). This is due to improvements in the purification
procedure as described under "Experimental Procedures."
-32P]ATP or [
-32P]ATP and
Mg2+ or Mn2+. Radiolabeling was obtained when
His-CyaC was incubated with [
-32P]ATP but not with
[
-32P]ATP (Fig. 2),
indicating that the phosphorylation of His-CyaC is
-phosphate
specific. Mg2+ and Mn2+ had similar effects on
the autophosphorylation activity of His-CyaC (Fig. 2, lanes
2 and 4), although the adenylate cyclase activity of
His-CyaC was stimulated by Mn2+ (16). Mn2+
might activate the catalytic activity of CyaC independently of the
autophosphorylation activity.
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Fig. 2.
Autophosphorylation of His-CyaC.
Purified His-CyaC(wild-type) protein (0.5 µg) was assayed as
described under "Experimental Procedures."
[ -32P]ATP or [
-32P]ATP was used as
the substrate. 1 mM MgC2 or 1 mM
MnC2 was added as the divalent cation.
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Fig. 3.
Autophosphorylation of wild-type and mutant
His-CyaC proteins. The purified proteins (0.37 µg) were assayed
as described under "Experimental Procedures." Lane 1,
His-CyaC(wild-type); lane 2, His-CyaC(D60A); lane
3, His-CyaC(H572Q); lane 4, His-CyaC(D895A); lane
5, His-CyaC(D60A,D895A). The phosphorylated proteins were
visualized by autoradiography.
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Fig. 4.
Chemical stability of phosphorylated
wild-type and mutant His-CyaC proteins. Aliquots (0.37 µg) of
purified wild-type or mutant His-CyaC proteins were phosphorylated,
loaded onto an 8% SDS-PAGE gel for electrophoresis, and then
transferred to an Immobilon-P membrane as described under
"Experimental Procedures." The membrane was cut into three strips,
and each strip was incubated in one of three solutions (50 mM Tris-HCl (pH 7.2) (black bars), 3 M NaOH (gray bars), or 1 M HCl
(hatched bars)) for 1 h at 25 °C. After treatment,
all strips were washed briefly with distilled water, dried in air, and
exposed to x-ray film. The remaining radioactivity was quantified by
densitometry, and the results are presented as relative
values. Wild, D60A, D895A, and
D60A,D895A represent His-CyaC(wild-type), His-CyaC(D60A),
His-CyaC(D895A), and His-CyaC(D60A,D895A), respectively.
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Fig. 5.
Adenylate cyclase activities of wild-type and
mutant His-CyaC proteins. Adenylate cyclase activity was measured
as described under "Experimental Procedures." Purified wild-type
and mutant His-CyaC proteins (0.17 µg) were used for the assays.
Wild, D60A, D895A, and
D60A,D895A represent His-CyaC(wild-type), His-CyaC (D60A),
His-CyaC(D895A), and His-CyaC(D60A,D895A), respectively.
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Fig. 6.
Phosphorylation of mutant His-CyaC proteins
by acetyl phosphate. Purified mutant His-CyaC proteins (0.5 µg)
were incubated with acetyl [32P]phosphate as described
under "Experimental Procedures." Phosphorylated proteins were
analyzed using a BAS1000 Imaging Analyzer (Fuji film). The
arrowhead indicates the position of the His-CyaC proteins.
Lane 1, His-CyaC(H572Q); lane 2,
His-CyaC(H572Q,D60A); lane 3, His-CyaC(H572Q,D895A);
lane 4, His-CyaC(H572Q,D60A,D895A).
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Fig. 7.
Effect of acetyl phosphate on the adenylate
cyclase activities of His-CyaC proteins. Adenylate cyclase
activities of His-CyaC derivatives were measured in the presence (+) or
absence ( ) of acetyl phosphate. Each His-CyaC protein fraction (0.6 µg) was preincubated in 0.1 ml of phosphorylation mixture (50 mM Tris-HCl (pH 8.0), 0.2 M KCl, 5 mM MgCl2, 2 mM DTT, and 1 mM PMSF with (+) or without (
) 10 mM acetyl
phosphate) for 30 min at 30 °C. After preincubation, each sample was
immediately cooled on ice for 5 min and then 0.1 ml of phosphorylation
mixture containing 0.2 mM ATP was added. After incubating
for 15 min at 30 °C, the reaction was terminated by adding 0.3 ml of
10% perchloric acid. The cAMP content was measured as described under
"Experimental Procedures." His-CyaC(H572Q), His-CyaC(H572Q,D60A),
His-CyaC(H572Q,D895A), and His-CyaC(H572Q, D60A,D895A) were used as
protein samples.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP in vitro.
On the other hand, the CyaC H572Q mutant was shown to be incapable of
autophosphorylation. Thus, His572 in the transmitter domain
is thought to be the initial phosphorylation site in CyaC. Studies on
the stability of phosphate links in phosphorylated CyaC derivatives
showed that the wild-type and D60A mutant proteins contain both an acyl
phosphate linkage and N-phosphate linkage, whereas the D895A
mutant and the D60A,D895A double mutant CyaC contain an
N-phosphate linkage alone. The fact that the acyl phosphate linkage in phosphorylated CyaC is lost when Asp895 is
replaced by an alanine residue indicates that Asp895 is
actually phosphorylated. Thus, after the autophosphorylation of
His572 in the transmitter domain, the phosphoryl group
bound to His572 is transferred to Asp895 in the
receiver (R2) domain. Asp60, the predicted phosphorylation
site in the receiver (R1) domain, is not likely to be phosphorylated by
the phosphotransfer reaction from the transmitter domain of CyaC. AsgA
of Myxococcus xanthus, which is required for the formation
of fruiting bodies, consists of a receiver domain on the N-terminal
side and a transmitter domain on the C-terminal side. The primary
structure of the two domains of AsgA is similar to the receiver (R1)
domain and the transmitter domain of CyaC. It has been shown that AsgA
autophosphorylates in the transmitter domain but that the phosphoryl
group once bound to the transmitter domain is not transferred to the
receiver domain. Plamann et al. (22) proposed that the AsgA
receiver domain functions as an input domain for the transmitter.
Interestingly, when a homology search was performed using the
GenBankTM BLAST server, the receiver (R1) domain of CyaC
showed especially high similarity to the receiver domain of AsgA in
comparison with other receiver domains in response regulator proteins.
The receiver (R1) domain of CyaC may have a function similar to that of
the receiver domain of AsgA.
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Fig. 8.
Proposed mechanism for the stimulation of the
adenylate cyclase activity of CyaC. CyaC autophosphorylates at
His572 (H) in the transmitter domain
(white box) using ATP, and the phosphoryl group
(circled P) is transferred to Asp895
(D) in the receiver domain (dotted oval).
Adenylate cyclase activity is stimulated by the phosphorylation of
Asp895 (shown by the bent arrow). The
black box indicates the catalytic domain.
Small phosphorylated molecules, such as acetyl phosphate, phosphoramidate, and carbamoyl phosphate, can act as phosphodonors for the phosphorylation of the receiver domains of response regulators in place of phosphorylated transmitter domains. Each response regulator protein shows different reactivities toward these small compounds (33). Spo0F, a response regulator protein in Bacillus subtilis, is not phosphorylated by acetyl phosphate in vitro while it is phosphorylated by a sensory kinase, KinA (35). In CyaC, the two receiver domains show different responses to acetyl phosphate; Asp895 is phosphorylated by acetyl phosphate, while Asp60 is not (Fig. 6). This probably reflects that the two aspartate residues have different physiological functions. It is probable that phosphoryl groups may be transferred to Asp60 residue of the receiver (R1) domain from the transmitter domains of other sensory kinases that have not yet been identified. The autophosphorylation activity, the phosphotransfer from His572 to Asp895, or the adenylate cyclase activity of CyaC may be regulated by the phosphorylation of Asp60.
We note that the phosphorylation levels observed in His-CyaC(wild-type) and His-CyaC(D60A), which are phosphorylated at both His572 and Asp895, are lower than those observed in His-CyaC(D895A) and His-CyaC(D60A,D895A). In the receiver domain (R2) derivatives, phosphoryl groups accumulate at His572, because phosphoryl groups bound to His572 are not transferred (Fig. 3). The low levels of phosphorylation observed in His-CyaC(wild-type) and His-CyaC(D60A) may be caused by dephosphorylation from phosphorylated Asp895. The phosphoryl groups of several phosphorylated response regulators are known to hydrolyze very rapidly. The half-life of hydrolysis of the phosphoaspartate group in phospho-CheY, a response regulator protein required for chemotaxis, is a few seconds (36). This instability is called autophosphatase activity. In addition, some sensory kinases are known to function to facilitate the rate of dephosphorylation of their cognate response regulators (37). CyaC also must have a mechanism to dephosphorylate its phosphorylated form. The rapid dephosphorylation from phosphorylated Asp895 may be required to respond to subsequent signals. However, there is a possibility that the rate of autophosphorylation decreases when Asp895 is phosphorylated.
It is noted that CyaC shows significant adenylate cyclase activity even if Asp895 is not phosphorylated. This is similar to CheB and RegA, which are alternative proteins with the catalytic domain of an enzyme beside the receiver domain of the bacterial two-component regulatory systems (20, 21).
It is likely that the autophosphorylation activity of CyaC is regulated by sensing a specific signal that is transferred from a primary signal sensor of the cell. In sensory kinases, the N-terminal side of the transmitter domain is thought to be a signal input domain (18, 19). The presumed signal input domain of CyaC, which was called the ETR1-like domain in the previous work (16), is similar to the ethylene sensor of Arabidopsis thaliana (ETR1) and to RcaE, the sensor for the complementary chromatic adaptation of Fremyella diplosiphon. This domain also exists in several other sensory kinases of Synechocystis PCC 6803 (17). Although the function of the ETR1-like domain remains unknown, it may participate in the recognition of a specific signal.
A CyaC homologue is found in the cyanobacterium Anabaena sp.
strain PCC 7120 (15), in which cellular cAMP levels are reduced in
response to a light-on signal. On the other hand, cAMP levels in the
disruptant of the cyaC homologue are not affected by the light-on signal (15). It has been suggested that CyaC responds to a
light signal; however, CyaC itself would not be a photoreceptor because
it has no typical chromophore binding motifs in its amino acid
sequence. Recently, a phytochrome that acts as a sensory photoreceptor
in plants has been found in the cyanobacterium Synechocystis PCC 6803 (17, 38). The cyanobacterial phytochrome consists of two
functional domains, an N-terminal domain homologous to the chromophore
attachment domain of the phytochrome and a C-terminal domain homologous
to the transmitter domain of the sensory kinase. Such a photoreceptor
having phosphorylation capability might control the activity of CyaC
through the mechanism of a phosphotransfer reaction.
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ACKNOWLEDGEMENTS |
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We thank Munehiko Asayama (Ibaraki University) and Kazuhito Inoue (Kanagawa University) for helpful discussions.
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FOOTNOTES |
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* This work was supported by a Grant-in-aid for General Scientific Research (07404045) from the Ministry of Education, Science, Sports and Culture of Japan (to M. O.) and also by a grant from Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists (to M. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Life
Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro, Tokyo 153, Japan. Tel.: 81-3-5454-6631; Fax:
81-3-5454-4333; E-mail: cohmori{at}komaba.ecc.u-tokyo.ac.jp.
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; kb, kilobase pair(s); PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis.
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