Characterization of a cAMP-stimulated cAMP Phosphodiesterase in Dictyostelium discoideum*

Marcel E. Meima, Karin E. Weening, and Pauline SchaapDagger

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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-beta -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-beta -lactamase domain (10).

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-beta -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-beta -lactamase domain of the 73-kDa factor.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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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- 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.

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- 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.

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 alpha 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.

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 (alpha 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 alpha GFP antibody also cross-reacts with and immunoprecipitates YFP. The alpha 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 alpha 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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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.


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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-beta -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-beta -lactamase and human glyoxylase II (12, 32).

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-beta -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).

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-beta -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-beta -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-beta -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-beta -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-beta -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.

Disruption of the PdeE Gene-- The PdeE gene was inactivated by homologous recombination in the ura- 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.

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).


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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.

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.


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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).

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).


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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.

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.


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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.

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. 


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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.

The catalytic activity of PdeD resides in its metallo-beta -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).

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.


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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.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -lactamases. The second Zn2+ ion is not essential for all metallo-beta -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.

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-beta -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).

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.

    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.

    FOOTNOTES

* 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.

Dagger 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

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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