Genetic interactions of the E3 ubiquitin ligase component FbxA with cyclic AMP metabolism and a histidine kinase signaling pathway during Dictyostelium discoideum development

Abstract

Dictyostelium discoideum amoebae with an altered fbxA gene, which is thought to encode a component of an SCF E3 ubiquitin ligase, have defective regulation of cell type proportionality. In chimeras with wild-type cells, the mutant amoebae form mainly spores, leaving the construction of stalks to wild-type cells. To examine the role of fbxA and regulated proteolysis, we have recovered the promoter of fbxA and shown that it is expressed in a pattern resembling that of a prestalk-specific gene until late in development, when it is also expressed in developing spore cells. Because fbxA cells are developmentally deficient in pure culture, we were able to select suppressor mutations that promote sporulation of the original mutant. One suppressor mutation resides within the gene regA, which encodes a cyclic AMP (cAMP) phosphodiesterase linked to an activating response regulator domain. In another suppressor, there has been a disruption of dhkA, a gene encoding a two-component histidine kinase known to influence Dictyostelium development. RegA appears precociously and in greater amounts in the fbxA mutant than in the wild type, but in an fbxA/dhkA double mutant, RegA is restored to wild-type levels. Because the basis of regA suppression might involve alterations in cAMP levels during development, the concentrations of cAMP in all strains were determined. The levels of cAMP are relatively constant during multicellular development in all strains except the dhkA mutant, in which it is reduced at least sixfold. The level of cAMP in the double mutant dhkA/fbxA is relatively normal. The levels of cAMP in the various mutants do not correlate with spore formation, as would be expected on the basis of our present understanding of the signaling pathway leading to the induction of spores. Altered amounts of RegA and cAMP early in the development of the mutants suggest that both fbxA and dhkA genes act earlier than previously thought.

FIG. 1.

Map of the fbxA locus. The full sequence can be found under GenBank accession no. AF151111. CAP, transcription start.

FIG. 2.

Phenotypes of the wild type, DH1 (A), the fbxA mutant HR6 (B), and the mutant complemented by the myc-tagged structural gene expressed from its promoter-containing fragment, HR47 (C). Note the long strands of extracellular sheath material that are left by the migrating slugs of the fbxA mutant. Bar, 500 μm. (D) myc-stained Western blot with a time course of expression of the promoter.

FIG. 3.

Patterns of expression of GFP. The GFP gene coding sequence was fused to the fbxA promoter and transformed into cells for expression studies. Bars, 100 (A to H) and 50 μm (I). Fluorescence and transmitted-light images were superimposed and compiled from projected Z sections obtained with a Zeiss LSM 510 confocal microscope.

FIG. 4.

Morphology of suppressors of the fbxA mutant. Strains DH1 (wild type), 82 (fbxA), HR48 (fbxA/regA), HR45 (regA), HR49 (fbxA/dhkA), and HR52 (dhkA) were allowed to develop on lawns of bacteria on SM plates (36). Bar (first panel), 100 μm. fbxA cells (second panel) form elongated slugs; this phenotype is largely corrected by introduction of a second mutation in either the regA (third panel) or dhkA (fifth panel) gene. DH1 (first panel) and the other single-mutant strains (fourth and sixth panels) are also shown; development of the fbxA/dhkA strain is much improved over that of either single mutant.

FIG. 5.

RegA expression in the presence and absence of FbxA. Blots of extracts of the various strains developed on filters for the indicated times were prepared and probed with an anti-RegA antibody. The blots were stained with Ponceau S prior to antibody treatment to ensure that equal amounts of protein were applied to each lane. WT, wild type.

FIG. 6.

cAMP levels during development of various mutants. Intracellular cAMP concentrations were determined as described in Materials and Methods.