Identification of a Novel Type of cGMP Phosphodiesterase That Is Defective in the ChemotacticstmFMutants

Abstract

StmF mutants are chemotactic mutants that are defective in a cGMP phosphodiesterase (PDE) activity. We identified a novel gene, PdeD, that harbors two cyclic nucleotide-binding domains and a metallo-␤-lactamase homology domain. Similar to stmF mutants, pdeD-null mutants displayed extensively streaming aggregates, prolonged elevation of cGMP levels after chemotactic stimulation, and reduced cGMP-PDE activity. PdeD transcripts were lacking in stmF mutant NP377, indicating that this mutant carries a PdeD lesion. Expression of a PdeD-YFP fusion protein in pdeD-null cells restored the normal cGMP response and showed that PdeD resides in the cytosol. When purified by immunoprecipitation, the PdeD-YFP fusion protein displayed cGMP-PDE activity, which was retained in a truncated construct that contained only the metallo-␤-lactamase domain. Newell, 1979, 1981;Kuwayama et al., 1993). Mutants KI8 and KI10 show no ligand-induced cGMP response and cannot chemotax (Kuwayama et al., 1993). Streamer F (stmF) mutants are defective in cGMP-PDE activity (Van Haastert et al., 1982). These mutants show an elevated and prolonged cGMP response and prolonged association of myosin with the cytoskeleton (Ross and Newell, 1981;Liu and Newell, 1988, 1994. When grown as colonies on dense bacterial lawns, stmF mutants form aggregates with very pronounced aggregation streams, a phenotype that under these conditions is not displayed by wild-type cells. INTRODUCTION Mutants in cGMP metabolism have implicated cGMP as intermediate for ligand-induced chemotaxis in DictyosteliumThree PDE genes have been identified in Dictyostelium; two of those, RegA (Shaulsky et al., 1996;Thomason et al., 1998) and PDE3 (Kuwayama et al., 2001), belong to the large class of HD-domain PDEs that is commonly found in vertebrates (Mehats et al., 2002). The third enzyme, PdsA (Lacombe et al., 1986), is a class II PDE that is also found in yeast (Nikawa et al., 1987). None of these genes are associated with the stmF locus.In our search for targets of cyclic nucleotides, we identified a gene, PdeD, with two putative cyclic nucleotide (cNMP)-binding domains and a binuclear Zn 2ϩ -binding domain. The latter domain forms the catalytic center of bacterial metallo-␤-lactamases, which hydrolyze an amide bond in the ␤-lactam ring of carbapenem antibiotics (Carfi et al., 1995) and are a major cause for widespread bacterial antibiotic resistance (Payne, 1993). We show that pdeD-null mutants phenocopy stmF mutants. One of the cNMP-binding domains of PdeD most likely functions as an allosteric activator of the enzyme, whereas the metallo-␤-lactamase homology domain catalyzes the hydrolysis of cGMP. MATERIALS AND METHODS Cell Growth and DevelopmentD. discoideum strains NC4, XP55, and NP377 were grown in association with Klebsiella aerogenes on SM agar plates, and all other strains were grown in HL5 medium, supplemented with 5 g/ml blasticidin or 20 -200 g/ml G418 for strains transformed with pBsr⌬Bam or neomycin selection markers, respectively. For developmental time courses, cells ...

Release of Ca 2+ from the Endoplasmic Reticulum Contributes to Ca 2+ Signaling in Dictyostelium discoideum

“…A pdeD null mutant lacking the major cGMP-specific phosphodiesterase exhibits enhanced and prolonged cGMP responses like those of the stmF mutant (27). Yet in the early stages of differentiation (3 h) its Ca 2ϩ responses are reduced rather than enhanced, consistent with the early suggestion that cGMP acts to inhibit Ca 2ϩ influx (24).…”

Characterization of a cAMP-stimulated cAMP Phosphodiesterase inDictyostelium discoideum

“…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 5Ј-untranslated region with at least 5-fold coverage.…”

“…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).…”

DictyosteliumStress-activated Protein Kinase α, a Novel Stress-activated Mitogen-activated Protein Kinase Kinase Kinase-like Kinase, Is Important for the Proper Regulation of the Cytoskeleton

“…This increased sensitivity was complemented by expression of SAPK␣ in these cells. Because cGMP can activate SAPK␣ kinase activity, we reinvestigated chemoattractant activation of SAPK␣ in cells lacking the cGMP phosphodiesterase (pdeD null cells) primarily responsible for degrading cGMP in response to cAMP stimulation (Meima et al, 2002). In this phosphodiesterase null mutation, cAMP-stimulated CGMP levels are highly elevated.…”