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

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.

Contrasting activities of the aggregative and late PDSA promoters in Dictyostelium development.

Expression of the Dictyostelium PdsA gene from the aggregative (PdA) and late (PdL) promoter is essential for aggregation and slug morphogenesis, respectively. We studied the regulation of the PdA and PdL promoters in slugs using labile beta-galactosidase (gal) reporter enzymes. PdL was active in prestalk cells as was also found with stable gal. PdA activity decreased strongly in slugs from all cells, except those at the rear. This is almost opposite to PdA activity traced with stable gal, where slugs showed sustained activity with highest levels at the front. PdA was down-regulated after aggregation irrespective of stimulation with any of the factors known to control gene expression. PdL activity was induced in cell suspension by cAMP and DIF acting in synergy. However, a DIF-less mutant showed normal PdL activity during development, suggesting that DIF does not control PdL in vivo. Dissection of the PdL promoter showed that all sequences essential for correct spatiotemporal control of promoter activity are downstream of the transcription start site in a region between -383 and -19 nucleotides relative to the start codon. Removal of nucleotides to position -364 eliminated responsiveness to DIF and cAMP, but normal PdL activity in prestalk cells in slugs was retained. Further 5' deletions abolished all promoter activity. This result also indicates that the induction by DIF and cAMP as seen in cell suspensions is not essential for PdL activity in normal development.