From the School of Life Sciences, University of Dundee, MSI/WTB complex, Dow Street, Dundee DD1 5EH, United Kingdom
Received for publication, September 19, 2002, and in revised form, January 21, 2003
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ABSTRACT |
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A cyclic nucleotide phosphodiesterase, PdeE, that
harbors two cyclic nucleotide binding motifs and a binuclear
Zn2+-binding domain was characterized in
Dictyostelium. In other eukaryotes, the
Dictyostelium domain shows greatest homology to the 73-kDa subunit of the pre-mRNA cleavage and polyadenylation specificity factor. The Dictyostelium PdeE gene is expressed at its
highest levels during aggregation, and its disruption causes the loss of a cAMP-phosphodiesterase activity. The pdeE null mutants
show a normal cAMP-induced cGMP response and a 1.5-fold increase of cAMP-induced cAMP relay. Overexpression of a PdeE-yellow fluorescent protein (YFP) fusion construct causes inhibition of aggregation and loss of the cAMP relay response, but the cells can aggregate in
synergy with wild-type cells. The PdeE-YFP fusion protein was partially
purified by immunoprecipitation and biochemically characterized. PdeE
and its Dictyostelium ortholog, PdeD, are both maximally active at pH 7.0. Both enzymes require bivalent cations for activity. The common cofactors Zn2+ and Mg2+ activated
PdeE and PdeD maximally at 10 mM, whereas Mn2+
activated the enzymes to 4-fold higher levels, with half-maximal activation between 10 and 100 µM. PdeE is an allosteric
enzyme, which is ~4-fold activated by cAMP, with half-maximal
activation occurring at about 10 µM and an apparent
Km of ~1 mM. cGMP is degraded at a
6-fold lower rate than cAMP. Neither cGMP nor 8-Br-cAMP are efficient
activators of PdeE activity.
In mammalian cells the inactivation of the ubiquitous second
messengers cAMP and cGMP is achieved by no less than 11 different families of the class I phosphodiesterases
(PDEs).1 These families
differ in substrate specificities, endogenous and exogenous regulators,
and targeting to different subcellular compartments. All families share
the same conserved catalytic domain at the carboxyl terminus, whereas
the domains required for targeting and regulation of enzyme activity
are mainly localized at the amino terminus (1-3).
In the social amoeba Dictyostelium discoideum, the class I
PDEs are represented by the cAMP-PDE RegA (4-6) and the cGMP-PDE PDE3
(7). Lower eukaryotes additionally use class II PDEs, which have an
entirely different catalytic center. These enzymes are exemplified by
PDE1 from yeast (8) and PdsA from Dictyostelium (9).
Recent screens of Dictyostelium genomic and cDNA data
banks for proteins with cyclic nucleotide monophosphate (cNMP) binding motifs identified two proteins that each contained two cNMP binding motifs and the binuclear Zn2+-binding domain that forms the
catalytic center of metallo- Here, we report gene ablation, overexpression, and biochemical
characterization of the other gene, PdeE/GbpB. This gene
encodes a cAMP-stimulated cAMP-PDE that is highly active during the
aggregation stage of development. A bioinformatics approach showed that
a subregion of the metallo- Cell Growth and Development--
D.discoideum strain
NC4 was grown in association with Klebsiella aerogenes on SM
agar plates, and all other strains were grown in HL5 medium, which was
supplemented with 200 µg/ml uracil for the ura Gene Identification--
The Dictyostelium genome and
cDNA databases were screened with consensus sequences for cyclic
nucleotide binding motifs. Two candidate genes, PdeD (10)
and PdeE, were identified. PdeE was assembled
after several cycles of data base screening, yielding the complete
coding sequence and 5'-untranslated region with at least 5-fold
coverage. The gene is part of the recently published sequence of
chromosome II (18) and was also identified by other workers (11).
Similar to PdeD, PdeE showed an ORF with two PFAM cNMP binding motifs
(19) when analyzed with SMART (20), and the sequence upstream of the
ORF was interrupted by two short AT-rich regions. To determine whether
these regions were introns, oligonucleotides were designed that flanked
the two regions (5'-ATGAATTCTAAATATGGGGATAACATTATAG-3' and
5'-CCAATTCTTGCTGAAATCACTGC-3' for the 5'-intron and
5'-TGCAGATCATGATAGTGGTATCCTTC-3' and 5'-GATGGTAACTCCAAAAGGTTTGCC-3' for
the 3'-intron) to perform reverse transcription PCR of the mRNA of
10-h starved NC4 cells. Bands of 192 and 410 bp, respectively, were
obtained and sequenced, showing the presence of a 202 and a 80 bp
intron. The 3288-kb ORF of PdeE could then be established, and its
sequence has been in GenBankTM under accession number
AY047364 since July 19, 2001.
Gene Inactivation--
The PdeE gene was inactivated in a
ura PdeE-YFP Fusion Constructs--
The complete PdeE ORF
was amplified by PCR using the AdvantageTM genomic PCR kit
(BD Biosciences) and the oligonucleotides
5'-CGGGGATCCAATTCTAAATATGGGGATAACATTATAGATTTTC-3' and
5'-GGGCTCGAGTTAAACCTTCAATTGGATAAACTTTTCTTGG-3', which generate 5'-BamHI and 3'-XhoI sites, respectively.
The BamHI/XhoI-digested PCR product was cloned
into the BamHI/XhoI-digested vector pB17SYFP, which is a derivative of pDXA-HC (24) that contains the coding sequence
for the enhanced YFP (25) downstream of the constitutive actin15
promoter. The construct yields a 1335-amino acid fusion protein with
YFP (239 amino acids) at the carboxyl terminus of PdeE (1096 amino
acids) in which the carboxyl terminal Ser and Ile of PdeE are replaced
by Thr and Arg and fused to Asp and the start Met of YFP. The integrity
of the PdeE sequence was verified by DNA sequencing. Two errors were
detected, i.e. a neutral AAT to AAC transition in
Asn540 and an ATT to GTT transition, which transforms
Ile1036 into Val1036. The Ile1036
is not conserved, and there is often a Val at this position in homologous proteins; thus, protein function is unlikely to be affected.
The actin15PdeE-YFP vector and pB17SYFP were transformed into parent
strain AX2. Cells were selected in HL5 medium with 200 µg/ml G418.
RNA Isolation and Analysis--
Total RNA was isolated from
2 × 107 cells, size fractionated on 1.5% agarose
gels containing 2.2 M formaldehyde (26), and transferred to
nylon membranes. Membranes were hybridized to
[32P]dATP-labeled DNA probes according to standard
procedures. 3 µl of 0.28-6.6-kb RNA markers (Promega) were run on
the same gel and stained with ethidium bromide to estimate the size of
the PdeE mRNA.
Cyclic Nucleotide Phosphodiesterase Assays--
To measure cAMP
or cGMP hydrolysis, cells were resuspended in 250 mM
sucrose in 20 mM Hepes, pH 7.0 and lysed through Nuclepore filters (pore size 3.0 µM). Lysates were cleared by
centrifugation for 20 min at 40,000 × g. Cleared cell
lysates or a suspension of matrix linked to cAMP Relay and cAMP-induced cGMP Response--
Cells were
starved for 8 h, resuspended in PB buffer to 108
cells/ml, and stimulated with 0.1 µM cAMP for the cGMP
response and 5 µM 2'-deoxy-cAMP in the presence of 5 mM DTT for cAMP-induced cAMP production (cAMP relay).
Aliquots of cell suspension were rapidly mixed with an equal volume of
3.5% perchloric acid (v/v) at various intervals after stimulation.
After neutralization of the lysates with KHCO3, cGMP levels
were measured by radioimmunoassay, and cAMP levels were measured by
competition with [3H]cAMP for binding to the bovine PKA
regulatory subunit (27).
Immunoprecipitation--
25 µg of mouse monoclonal GFP
antibody ( Structure and Developmental Regulation of PdeE--
Screening of
Dictyostelium cDNA and genome databases with consensus
sequences for cyclic nucleotide binding motifs yielded two novel genes
of similar structure, PdeD (10) and PdeE, which were also identified by others as gbpA and gbpB
(11, 13). PdeE is transcribed into a 3.4 kb mRNA and
shows low expression during growth and a very pronounced peak at 8-10
h of starvation, when cells are aggregating (Fig.
1A). This peak was not found for gbpB expression in earlier work (11), which may be due
to the fact that the 4-h interval used by these workers between
successive points in the developmental time course was too large.
The 1096-amino acid PdeE protein can be subdivided into an amino
terminal region with no homology to known proteins, a middle region,
containing the metallo-
cAMP and cGMP-binding domains are highly homologous and share a similar
structure that was first determined for the regulatory subunit of
bovine cAMP-dependent protein kinase (PKA RI) (29). The
major determinant for nucleotide specificity is Ala210
(Fig. 1C, site A, yellow)
and Ala334 (site B,
yellow) in PKA RI (30). The equivalent positions in human
cGMP-dependent protein kinase (huPKG2) are
Thr243 and Ser367 (in blue), which
allow hydrogen bond formation with the C2-NH2
group of the guanine base. Both sites A and B in PdeE harbor a serine
(Ser890 and Ser1030) at the positions
equivalent to Ala210 or Ala334, suggesting that
PdeE binds cGMP (Fig. 1C). The putative PdeE cGMP-binding
domains deviate rather strongly from the consensus motif. In site A, an
Ala889 replaces the essential Arg209 (in
red) of PKA RI that forms an ion pair with the equatorial phosphate oxygen of cNMP (29). In addition, insertions of two and seven
amino acids precede this key residue in sites A and B, respectively.
Additional residues at this position are absent in all characterized
cNMP-binding proteins.
Search for Putative Functional Homologues of PdeD and
PdeE--
The PFAM metallo- Disruption of the PdeE Gene--
The PdeE gene was
inactivated by homologous recombination in the ura
Similar to its homolog PdeD, PdeE is likely to
encode a cytosolic cyclic nucleotide hydrolyzing activity. We therefore
tested whether such an activity might be lacking in the KO cell lines during the course of their development. Assays were performed in
cleared lysates in the presence of DTT and IBMX to inhibit the PDE
activities of PdsA, RegA, and PDE3 (5, 7, 37). Fig.
3A shows that, in the control
cell line RI1, cytosolic [3H]cAMP-hydrolyzing activity
increased strongly to reach a peak at 8 h of starvation. The
activity persisted at low levels during the slug stage of development
and was completely absent in the pdeE null mutant KO2. There
was no significant difference in [3H]cGMP hydrolysis
between the KO and RI cell lines (Fig. 3B), which suggests
that PdeE encodes a cAMP phosphodiesterase. We next analyzed the dose
dependence of the cAMP-PDE activity that was detected at
t = 8 h in the control RI cells. Fig.
3C shows that [3H]cAMP hydrolysis in the
control cells is stimulated by micromolar concentrations of cAMP,
whereas competition only occurs at millimolar concentrations. This does
not resemble the dose-dependence of the known cAMP-PDEs, PdsA and RegA
in Dictyostelium, which have a Km of 10 µM and 5 µM, respectively (5, 6, 38).
Effects of PdeE Gene Disruption on Cyclic Nucleotide
Responses--
The developmental regulation of putative PdeE activity
agrees well with the appearance of PdeE transcripts, with
maximum levels at the aggregation stage (Fig. 1A). At this
stage, both cAMP and cGMP are transiently synthesized in response to
stimulation with cAMP receptor ligands. To test whether PdeE is
involved in regulating cAMP-induced cAMP production (cAMP relay) or the
cGMP response, we measured the two responses in the KO and RI cell
lines. Fig. 4A shows that the
cAMP-induced cGMP response and basal cGMP levels are not significantly
different between KO and RI cells. However, basal cAMP levels are
2-fold elevated in the KO cells. The initial rate of cAMP accumulation
is similar in KO and RI cells, but the KO cells continue to accumulate
cAMP for a longer period, resulting in about 1.5-fold higher cAMP
levels (Fig. 4B). These data suggest that PdeE has no
function in degrading cGMP but may play a role in regulating the cAMP
relay response.
Overexpression of a PdeE-YFP Gene Fusion--
To provide further
evidence that PdeE encodes the cAMP hydrolyzing activity
that is lacking in the pdeE null mutants, we fused the
full-length PdeE gene to the gene for the enhanced YFP (25) and expressed the fusion construct under control of the constitutive actin15 promoter (A15) in the wild-type strain AX2.
[3H]cAMP hydrolysis in growing PdeE-YFP cells was 100 times higher than that in control YFP cells (Fig.
5A). The YFP cell line
developed normally into fruiting bodies within 22 h, but the
PdeE-YFP line was blocked in early development (Fig.
5B).
PdeE activity in the overexpressers (2.2 nmol/min/mg protein) is about
30-fold higher than the maximum activity reached during development of
control cell lines (see Fig. 3A), and this could lead to
significant depletion of intracellular cAMP. Early development and
aggregation show a cell-autonomous requirement for PKA activation (39,
40) and a non-cell-autonomous requirement for oscillatory cAMP
signaling (41). Overexpressed PdeE could hydrolyze most of the
intracellular cAMP and block either or both of these processes. To test
whether defective development in the PdeE-YFP cells was cell-autonomous, we developed the cells in synergy with the wild-type AX2. Fig. 6A shows that the
PdeE-YFP cells (detected by YFP fluorescence) co-aggregated with the
AX2 cells and were incorporated into aggregates and slugs albeit that
development was somewhat delayed. This suggests that oscillatory cAMP
signaling was affected rather than the activation of PKA. To test this
directly, we compared the cAMP relay response in the PdeE-YFP and the
YFP overexpressers. Fig. 6B shows that the relay response in
the PdeE-YFP cells was very strongly reduced.
Metal and pH Dependence of PdeE--
To establish optimal assay
conditions for PdeE and its ortholog PdeD, we determined the pH and
metal dependence of the two enzymes. Fig.
7A shows that the pH
dependence of PdeD and PdeE is almost identical with optimal activity
at pH 7.0.
The catalytic activity of PdeD resides in its metallo- Cyclic Nucleotide Specificity of PdeE--
The experiments shown
in Fig. 3 indicate that PdeE is not a major contributor to
[3H]cGMP hydrolysis by cytosolic cell fractions. This
does not exclude cGMP as a putative PdeE substrate. We compared the
efficiency of hydrolysis of [3H]cAMP and
[3H]cGMP by PdeE-YFP at increasing substrate
concentrations, and we also determined whether 8-Br-cAMP is as good an
activator for PdeE as 8-Br-cGMP is for PdeD (10). Fig.
8 shows that [3H]cAMP
hydrolysis by PdeE-YFP was strongly stimulated by cAMP with
half-maximal activation occurring around 10 µM.
Competition by cAMP was half-maximal around 1 mM. The
absolute level of stimulation was somewhat higher than observed
for the endogenous enzyme (Fig. 3C). This could be due to
the fact that the endogenous enzyme, because of its low activity, must
be measured in concentrated lysates, which may contain some cAMP. cGMP
and 8-Br-cAMP activated [3H]cAMP hydrolysis less
efficiently than cAMP and required 10-fold higher concentrations.
When [3H]cGMP was used as a substrate, the rate of
hydrolysis was at least six times lower than that for cAMP over
the entire concentration range. These data suggest that PdeE most
likely functions as a cAMP-stimulated cAMP- phosphodiesterase.
The Role of PdeE in Dictyostelium Development--
We
characterized a cAMP-stimulated cAMP phosphodiesterase that is
expressed at high levels in aggregating cells. Ablation of the
PdeE gene has no noticeable effects on growth and
aggregation, but overexpression of PdeE blocks aggregation
completely. This effect is not cell-autonomous, because PdeE
overexpressers will aggregate when mixed with wild-type cells. The
cAMP-induced cAMP relay response, which is essential for chemotactic
signal propagation during aggregation, is strongly reduced in PdeE
overexpressers. This is the most likely cause for their failure to
aggregate. Mutants defective in adenylyl cyclase A (ACA) or CracA, the
adenylyl cyclase and its activating factor that are required for cAMP
production, can also only aggregate in synergy with wild-type cells
(41, 42). The pdeE null mutant shows a 1.5-fold increase in
cAMP-induced cAMP relay. This does not markedly affect the aggregation
process under standard conditions, but the very pronounced expression of the PdeE gene during aggregation (Fig. 1A)
suggests a role in cAMP signaling at this stage.
Oscillatory cAMP secretion requires regulation of the cAMP production
process by both positive and negative feedback loops (43, 44). The
positive loop is provided by cAMP receptor (cAR)-mediated activation of
ACA. The negative loop involves cAR-mediated adaptation of ACA, but the
exact nature of this process has proven hard to resolve (45). The
negative feedback loop can, in theory, be provided by a cAMP-stimulated
cAMP phosphodiesterase, and a model was proposed that was based on
indirect activation of the cAMP-PDE RegA by cAMP (46). However, cAMP
activation of RegA has not yet been documented. PdeE is strongly
activated by cAMP in a direct manner, and this could contribute to the
transient nature of the cAMP relay response. cAR-mediated adaptation of
ACA does not require cAMP production by ACA (47), which rules out the
possibility that the cessation of cAMP accumulation is solely due to a
cAMP-stimulated PDE. However, PdeE, RegA, and cAR-mediated ACA
adaptation may act together to terminate cAMP relay. This would make
the oscillatory signaling system very robust but difficult to
characterize by a single genetic lesion.
Metal Dependence of PdeE--
PdeE and its
Dictyostelium homolog, PdeD, both harbor a region with high
homology to the binuclear Zn2+ binding motifs that were
first characterized in the metallo- Cyclic Nucleotide Specificity of PdeE--
Similar to PdeD, PdeE
is an allosteric enzyme that is stimulated by its substrate. The
KA and Km for cAMP lie around 10 µM and 1 mM, respectively. cGMP is degraded
at a 6-fold lower rate than cAMP, and activation is also much less pronounced. Other workers recently reported that cGMP was degraded at a
9-fold lower rate and required three times higher concentrations for
activation than cAMP (54). Their KA and
Km for cAMP were at 0.7 µM and 0.2 mM, respectively, which are somewhat lower than the values
found by us and could be due to the different parental strains (AX3
versus AX2) that were used in the two studies. The PdeE
fusion with YFP may, in our case, also have slightly altered the
kinetics of the enzyme.
For the cGMP-stimulated cGMP-PDE/PdeD, allosteric activation can be
satisfactorily explained from the domain architecture of the protein.
The metallo-
For PdeE, the situation is less straightforward. The PdeE cNMP binding
motifs both show the characteristic serine residue that confers
specificity for cGMP rather than for cAMP (Fig. 1C). Nevertheless, cAMP is a more efficient activator of PdeE than cGMP. In
addition, 8-Br-cAMP is a poor activator of PdeE activity. Furthermore,
the PdeE cGMP binding motifs show severe deviations from the consensus
motif, and it is questionable whether they can bind cGMP or cAMP at
all. It is therefore possible that allosteric activation of PdeE is not
provided by these motifs.
Ablation of PdeE function causes the loss of a cytosolic cAMP
hydrolyzing activity during development and augmentation of the cAMP
relay response. There is no significant reduction of [3H]cGMP hydrolyzing activity, and the cGMP response is
unaffected. This means that, within the context of cellular physiology,
PdeE functions as a cAMP phosphodiesterase.
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ABSTRACT
INTRODUCTION
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-lactamases (10-12). The corresponding
genes were named PdeD and PdeE by us (10) and
GbpA and GbpB by others (11, 13). Gene disruption and overexpression studies of PdeD/GbpA showed that the gene
encodes the cGMP-stimulated cGMP phosphodiesterase, which is lacking in the chemotactic stmF mutants (10, 13, 14). Analysis of
truncated PdeD constructs revealed that the catalytic activity was
provided by the metallo-
-lactamase domain (10).
-lactamase domain of PdeE and PdeD shares additional homology with a 73-kDa factor that is part of the complex for pre-mRNA cleavage and polyadenylation (15). Because the endonuclease (phosphodiesterase) activity of this complex is still unknown (16), we speculate that it is provided by the highly conserved
metallo-
-lactamase domain of the 73-kDa factor.
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strain
DH1 (17) and 200 µg/ml G418 for the AX2 cells transformed with YFP or
PdeE-YFP constructs. For developmental time courses, cells were
harvested from bacterial plates or growth medium, washed with 10 mM PB buffer (NaH2PO4 and
K2HPO4, pH 6.5), plated at variable cell
densities on PB agar (1.5% agar in PB buffer), and incubated at
22 °C.
cell line by homologous recombination (21) with a
linear construct that contains ~1 kb each of the 5'- and 3'-regions
of the PdeE gene flanking the PJB1 vector, which contains
the URA+ selection cassette (22). Two PdeE
fragments comprising nucleotides 2961-4427 and 1689-2835 were
amplified with oligonucleotides that yielded a 5'-PstI and 3'-BamHI on the first fragment and a 5'-BamHI and
3'-XbaI site on the second fragment. The fragments were
cloned in tandem in the PstI-BamHI- and
XbaI-BamHI-digested URA vector PJB1 (gift of
Peter N. Devreotes). The two constructs were linearized with BamHI, which yielded the PJB1 plasmid flanked by ~1 kb of
5'- and 3'-DNA of the PdeE gene. Homologous recombination
with this construct causes insertion of the entire plasmid at position
2835 of PdeE and the deletion of about 100 nucleotides. The
knockout construct was introduced into the ura
strain DH1,
and transformed cells were selected by growth in FM medium (23) in the
absence of uracil. Selected clones were screened for homologous
recombination by two separate PCR reactions and analysis of Southern
blots of genomic digests (Fig. 2A). 11 of 90 clones derived
from two transformations carried a gene disruption (pdeE).
Three knockout (KO) and three random integrant (RI) clones were used
for further analysis.
GFP immunoprecipitate
were incubated for 30 min at 22 °C with 10 nM or 10 µM [3H]cGMP or [3H]cAMP, 5 mM dithiothreitol (DTT), 0.2 mM IBMX, and 1 mM MgCl2 in 20 mM Hepes, pH 7.0 and
assayed for 5'-[3H]AMP or 5'-[3H] GMP
levels as described previously (10). All PDE assays in this work were
performed in the presence of DTT and IBMX, which inhibit other
Dictyostelium PDEs such as PdsA, RegA, and PDE3, but not
PdeD or PdeE.
GFP; Roche Molecular Biochemicals) was incubated for
1 h at 4 °C with a mixture of 250 µl each of slurries of
protein G linked to Sepharose 4B (Sigma) and protein A linked to
Affiprep (Bio-Rad). The
GFP antibody also cross-reacts with and
immunoprecipitates YFP. The
GFP-linked matrix was washed with
phosphate-buffered saline (0.7% NaCl in PB buffer) and resuspended to
the original concentration. 1 ml of cleared lysate, prepared in
phosphate-buffered saline at 2 × 108 cells/ml, was
incubated for 4 h with 100 µl of
GFP matrix suspension. The
matrix was washed thoroughly with 0.2 M NaCl in PB buffer and resuspended in 20 mM Hepes, pH 7.0.
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ABSTRACT
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Fig. 1.
Developmental regulation and structure of
PdeE. A, developmental
regulation. D. discoideum NC4 cells were incubated on PB
agar until fruiting bodies had formed. RNA was isolated at 2-h
intervals and hybridized to a [32P]dATP-labeled
PdeE probe. RNA markers were run on the same gel to estimate
the size of the PdeE mRNA, which was ~3.4 kb.
B, schematic of PdeE structure, indicating the position of
the two introns, the metallo- -lactamase domain, and the two cyclic
nucleotide binding motifs. C, alignment of the cNMP binding
motifs of PdeE, bovine PKA-RI, Dictyostelium PKA-RI, human
PKG2, and Hydra PKG. Black, identical;
gray, functionally conserved; red, side chain
essential for hydrogen bond and ion pair formation with ribose 2'-OH
and exocyclic oxygens, respectively; blue, Ser/Thr that
distinguishes guanine from adenine (yellow) binding (29).
D, alignment of PdeD and PdeE with the 73-kDa CPSF subunits
from Homo sapiens, its homolog PF1 from Saccharomyces
cerevisiae, and the Bacillus halodurans protein BH3002.
Green/magenta, binding to Zn2+(I) and
Zn2+ (II), respectively, in Bacillus cereus
metallo-
-lactamase and human glyoxylase II (12, 32).
-lactamase domain that is common to a number
of hydrolases (28), and a carboxyl-terminal region that contains two
cyclic nucleotide binding cNMP motifs (19) (site
A and site B in Fig. 1B).
PdeD has a similar domain architecture and encodes a cGMP-stimulated
cGMP phosphodiesterase (10, 13).
-lactamase domain contains the
HXHXD sequence, two more conserved His
residues, and one Asp/Cys residue (19). These residues can bind one or
two Zn2+ ions that are essential for catalysis (12, 31).
The metallo-
-lactamase domain is also present in the well
characterized enzyme glyoxalase II (32) and in a variety of other
proteins that usually function as hydrolases (33). Class II cAMP-PDEs,
such as Dictyostelium PdsA, also contain the
HXHXD sequence but do not otherwise conform to
the PFAM metallo-
-lactamase domain (19). PdeE and PdeD do conform
but have little else in common with any enzymes of known substrate
specificity that carry this domain or with the class II PDEs. The
closest homolog of PdeE outside Dictyostelium is a
Bacillus halodurans protein (BH3002) of unknown function
(34). The homology between PdeE, PdeD, and BH3002 was greatest in a region preceding the conserved Asp669 in PdeE (Fig. 1,
magenta) and showed the motif
FXFFXT/SXHXXPXXXXXXEXXGXXXXYS/TXD (Fig. 1D). When GenBankTM was searched with this
motif, a class of highly homologous proteins was detected. These
proteins also carried the HXHXD motif of the metallo-
-lactamase domain upstream of the search motif and were either 73-kDa subunits of the pre-mRNA cleavage and polyadenylation specificity factor (CPSF) (15) or uncharacterized bacterial proteins
(Fig. 1D). CPSF73 is one of a complex of factors that binds
to the RNA sequence AAUAAA and causes 3'-endonucleolytic cleavage and
the addition of the poly(A) tail a few bases downstream from this
sequence. Another factor in the complex, CPSF100 (35), harbors a
degenerate metallo-
-lactamase motif, which does not display the
region of homology to PdeD and PdeE. CPSF100 also lacks an essential
residue for binding to Zn(I). Despite the fact that all components of
the polyadenylation complex have been cloned, the 3'-endonuclease has
not yet been identified (16, 36). Because 3'-endonucleases cleave
3'-nucleotide phosphodiester bonds, as is the case for PdeD and PdeE,
we consider it likely that CPSF73 provides the 3'-endonuclease
activity. The bacterial homologs may likewise be 3'-nucleotidases.
strain
DH1 to generate cell line pdeE. Construct integration was
verified by two separate PCR reactions and Southern analysis of genomic
digests (Fig. 2A). Three
randomly selected KO and RI clones were compared in an analysis of
phenotypes. Growth and developmental morphology on bacterial plates of
the KO and RI clones were identical (Fig. 2B). We also could
not distinguish differences in growth rate in axenic cultures or in the
morphology and rate of development of cells plated on PB agar at cell
densities of 104, 105, and 106
cells/cm2 (data not shown).
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Fig. 2.
Gene disruption of PdeE.
A, genomic digests of putative knockouts. The
ura strain DH1 was transformed with a linear construct of
the URA+ vector, PJB1, flanked by 1.1 and 1.4 kb of the 5'-
and 3'-PdeE sequences, respectively. Clones 2, 7, and 33 were
identified by two PCR reactions as putative PdeE gene KOs, and clones
1, 3, and 6 as RIs. Genomic DNA of the parent strain DH1 and the KO and
RI clones was digested with Bcl I. In DH1 and RI cells a 2.8-kb band
should be present, with additional bands of unknown size marking the
random vector integrations in RI cells. Insertion of vector PJB1 in the
PdeE gene should yield a band of 9.4 kb. B, the
morphologies of colonies of RI clones 1 and 6 and KO clones 2 and 33 growing on Klebsiella aerogenes lawns are virtually
identical.
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Fig. 3.
Developmental regulation of cNMP hydrolyzing
activity in PdeE mutants. A and
B, development. The pdeE null line KO2
(open symbol) and control line RI1 (closed
symbol) were plated on PB agar at 2 × 108
cells/plate and incubated at 22 °C for 20 h. Every 4 h,
cleared lysates of 108 cells/ml were prepared and tested at
1:0, 1:3, 1:10, and 1:30 dilutions for hydrolysis of 10 5
M [3H]cAMP (A) or
10
5 M [3H]cGMP (B)
in the presence of the PdsA inhibitor DTT and the RegA and PDE3
inhibitor IBMX. Activities were calculated from the dilution that gave
10-20% hydrolysis of the substrate and standardized on the protein
content of the cleared lysate. Means ± S.E. of two time courses
assayed in triplicate are presented. C, kinetics. The
activity detected at t = 8 h in RI1 was used to
perform a dose-response analysis of [3H]cAMP hydrolysis.
1:3 diluted cleared lysate was incubated for 30 min at 22 °C with
10
8 M [3H]cAMP and the
indicated concentrations of cAMP and assayed for [3H]cAMP
hydrolysis. Data are derived from two experiments and expressed as
percentage of hydrolysis achieved in the absence of added cAMP.
View larger version (16K):
[in a new window]
Fig. 4.
cAMP relay and cGMP response in the
PdeE mutants. PdeE KO and RI cells
were starved for 6 h and stimulated with 0.1 µM cAMP
for the cGMP response (A) and 5 µM
2'-deoxy-cAMP and 5 mM DTT for the cAMP relay response
(B) and assayed at the indicated time points for cGMP and
cAMP levels, respectively. The data represent means ± S.E. for
two sets of three time courses for the cGMP response and two
experiments performed in triplicate for cAMP relay. The
asterisks (*) indicate that cNMP levels at individual time
points are significantly different (p > 0.95) between
RI and KO cells as determined by analysis of variance (53).
View larger version (56K):
[in a new window]
Fig. 5.
Expression of a PdeE-YFP fusion
construct. A, PDE activity. AX2 cells expressing either
YFP or PdeE-YFP from the constitutive actin15
promoter were harvested in growth phase and lysed at 108
cells/ml. Hydrolysis of 10 5 M
[3H]cAMP was measured during 30 min in progressive
dilutions of cleared lysate. Enzyme activity was calculated from
dilutions (1:300 for PdeE-YFP and 1:0 for YFP) that did not degrade
more than 20% of the substrate and standardized on the protein content
of the lysate. B, phenotype. Vegetative AX2 cells expressing
either PdeE-YFP or YFP were distributed on PB
agar at 3 × 105 cells/cm2. Cells were
incubated for 22 h at 22 °C and photographed.
View larger version (51K):
[in a new window]
Fig. 6.
Synergistic development and cAMP
relay. A, synergistic development. AX2 cells and
PdeE-YFP cells were mixed at 1:0, 1:1, or 0:1 ratios and plated on PB
agar at 3 × 105 cells/cm2. Cells were
photographed after 16 h at 22 °C under both bright field and UV
illumination to monitor morphogenesis and YFP fluorescence
simultaneously. Bar length equals 1 mm. B, cAMP
relay. AX2 cells expressing either YFP or PdeE-YFP were starved for
6 h, stimulated with 5 µM 2'-deoxy-cAMP and 5 mM DTT, and assayed for cAMP at the indicated time points.
Means ± S.E. of two experiments performed in triplicate are
presented.
View larger version (17K):
[in a new window]
Fig. 7.
pH and metal dependence of PdeE and
PdeD. A, pH. Cells expressing PdeE-YFP or PdeD-YFP (10)
were lysed in 0.5 mM Hepes, pH 7.0. Lysates were diluted
1:300 in 50 mM potassium phosphate buffer at the indicated
pH and tested for hydrolysis of 10 5 M
[3H]cAMP or [3H]cGMP, respectively. Data
are expressed as percentage of hydrolysis obtained at pH 7.0. Means ± S.E. of two experiments performed in triplicate are
presented. B and C, metal. Immunoprecipitates of PdeE-YFP
(B) and PdeD-YFP (C) were incubated for 16 h
at 0 °C with 0.2 M EDTA in 20 mM Hepes, pH
7.0. After thorough washing with 20 mM Hepes, the
immunoprecipitates were tested for hydrolysis of 10
5
M [3H]cAMP or [3H]cGMP,
respectively, in the presence of the indicated concentrations of
ZnCl2, MgCl2, and MnCl2. Data are
expressed as percentage of hydrolysis obtained with 3×
10
3 M MnCl2. Means ± S.E. of two experiments performed in triplicate are
presented.
-lactamase
domain (10), which requires the binuclear Zn2+ binding
motif for hydrolysis (12, 32). PdeE has a similar domain, and the
activity of both PdeD and PdeE may therefore be expected to be
dependent on Zn2+ ions. However, pilot experiments showed
no stimulation of PdeD by Zn2+ ions. We assumed that the
metal was tightly bound to the enzyme. To relieve PdeE-YFP and PdeD-YFP
from their bound metals, we immunoprecipitated the enzymes with GFP
antibodies and incubated the immunoprecipitate overnight with 0.2 M EDTA. After removal of EDTA, the enzymes were no longer
active without added metals. Fig. 7, B and C show that EDTA-treated PdeE and PdeD were stimulated with equal
effectiveness by Zn2+ or Mg2+, showing maximal
stimulation at 10 mM. No synergistic effects of
Zn2+ and Mg2+ were observed (data not shown).
Unexpectedly, Mn2+ ions activated both enzymes to at least
4-fold higher levels than Zn2+ or Mg2+.
Increased activation was already evident at a concentration (~10
µM) where Mn2+ is present in the cell.
Co2+, Cu2+, and Cd2+ stimulated
PdeD and PdeE to similar levels as Zn2+ and
Mg2+ did (data not shown).
View larger version (24K):
[in a new window]
Fig. 8.
Analog specificity of PdeE. 1:300
diluted, cleared lysates of cells expressing PdeE-YFP were incubated
for 30 min at 22 °C with 2.10 8 M
[3H]cAMP or 2.10
8 M
[3H]cGMP in the presence of the indicated concentrations
of cAMP, cGMP, and 8-Br-cAMP and tested for cyclic nucleotide
hydrolysis. Data were standardized on the protein content of the
cleared lysate. Means ± S.E. of two or three experiments
performed in triplicate are presented.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactamases. The second
Zn2+ ion is not essential for all metallo-
-lactamases,
and Mg2+, Cd2+, and Co2+ can often
replace Zn2+ (48, 49). We found that Zn2+ and
Mg2+ were not very effective in supporting PdeE and PdeD
activity but that Mn2+ stimulated activity to much higher
levels. This was already evident at the micromolar concentrations that
equate Mn2+ levels in cells. It is therefore possible that
PdeD and PdeE use Mn2+ instead of Zn2+ as a
cofactor for hydrolysis.
-lactamase domain harbors the cGMP hydrolytic activity
(10), and the first of the two carboxyl-terminal cNMP binding motifs
shows the consensus cGMP binding motif that is found in the cGMP
dependent protein kinases (10, 11, 29). PKA and PKG bind
8-Br-substituted ligands with equal or greater affinity than cAMP or
cGMP itself, because the bulky bromine forces the molecule in the
syn conformation (purine above ribose) that is preferred by
the binding site (50, 51). In agreement with this, 8-Br-cGMP is a
better activator for PdeD then cGMP itself, whereas cAMP does not
activate at all (10, 52).
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ACKNOWLEDGEMENTS |
---|
Sequence data for D. discoideum were obtained from the Genome Sequencing Centre Jena, website at genome.imb-jena.de/dictyostelium/. The German part of the D. discoideum Genome Project is carried out by the Institute of Biochemistry I, Cologne, and the Department of Genome Analysis, Institut für Molekulare Biotechnologie, Jena, with support by the Deutsche Forschungsgemeinschaft (No 113/10-1 and 10-2). We thank Dr. Inke Näthke for advice on immunoprecipitation procedures.
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FOOTNOTES |
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* This research was funded by Wellcome Trust University Award Grant 057137 and Netherlands Organization for Scientific Research (NWO) Grant 805.17.048 (to K. E. W.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY047364.
To whom correspondence should be addressed. Tel.: 44-1382-348078;
Fax: 44-1382-345386, E-mail: p.schaap@dundee.ac.uk.
Published, JBC Papers in Press, February 6, 2003, DOI 10.1074/jbc.M209648200
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ABBREVIATIONS |
---|
The abbreviations used are: PDE, cyclic nucleotide phosphodiesterase; cNMP, cyclic nucleotide monophosphate; YFP, yellow fluorescent protein; GFP, green fluorescent protein; CPSF, cleavage and polyadenylation specificity factor; PKA, cAMP-dependent protein kinase; PKG, cGMP dependent protein kinase; cAR, cAMP receptor; ACA, adenylyl cyclase A; 8-Br-cAMP, 8-bromoadenosine 3':5'-monophosphate; 8-Br-cGMP, 8-bromoguanosine 3':5'-monophosphate; DTT, dithiothreitol; IBMX, isobutylmethylxanthine; ORF, open reading frame; PFAM, protein family database of alignments and hidden Markov models; SMART, simple modular architecture research tool; KO, knockout; RI, random integrant.
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