Osmotic stress response in Dictyostelium is mediated by cAMP

Abstract

DokA, a homolog of bacterial hybrid histidine kinases, is essential for hyperosmotic stress resistance in Dictyostelium: We show that a transient intracellular cAMP signal, dependent on the presence of DokA, is generated in response to an osmotic shock. This variation of cAMP levels contributes to survival under hypertonic conditions. In contrast to the low cAMP levels observed in dokA(-) strains, overexpression of the receiver domain of DokA causes an increase in cAMP levels, resulting in a rapidly developing phenotype. We present biochemical and cell biological data indicating that the DokA receiver domain is a dominant-negative regulator of a phosphorelay, which controls the intracellular cAMP phosphodiesterase RegA. The activity of the DokA receiver domain depends on a conserved aspartate, mutation of which reverses the developmental phenotype, as well as the deregulation of cAMP metabolism.

Fig. 1. (A) Concentration of intracellular cAMP in D.discoideum cells in response to osmotic stress. Cells were shaken in SPB and shocked osmotically with 400 mM sorbitol. Ax2 cells (diamonds) accumulate cAMP transiently when exposed to high osmolarity, whereas dokA^– cells (triangles) show a smaller increase without a maximum after the shock. Means and standard deviations of three independent experiments are shown. (B) Viability assay of osmotically shocked Ax2 and dokA^– cells. Cells were incubated for 10 min in 400 mM sorbitol buffer containing 5 mM 8-Br-cAMP as indicated and for 110 min without 8-Br-cAMP. Cells incubated constantly in SPB correspond to 100%. Means and standard deviations of three (Ax2) and four (dokA^–) independent experiments are shown. The simulation of a cAMP peak at the onset of the shock increases the viability rate of dokA^– cells ∼4-fold.

Fig. 2. Construction of cell lines overexpressing DokA fragments. (A) Domain structure of DokA as predicted from sequence analysis. (B) Three truncated forms of DokA (PHKR, HK and RR) were overexpressed in D.discoideum Ax2 cells under the control of the actin 15 promoter using the plasmid pDEX-RH. ‘H’ represents the conserved histidine 1053, ‘D’ represents the conserved aspartate 1567. Mutant forms of DokA fragments were expressed as indicated.

Fig. 3. Developmental phenotype of cells overexpressing DokA fragments. (A) Cells were developed on non-nutrient agar at a density of ∼1.5 × 10⁶ cells/cm². Photographs were taken after 36 h. Wild-type and HK cells develop normally; in contrast, RR-expressing cells form small aggregates and aberrant fruiting bodies. PHKR cells show only a weak developmental phenotype. The bar equals 3 mm. (B) Cells overexpressing the dominant-negative RR and its mutant form RR D1567A were developed as in (A) at 1 × 10⁶ cells/cm². Photographs were taken after 36 h. Mutation of D1567 reverses the developmental phenotype observed. The bar equals 1.5 mm. (C) Cells were grown on low-nutrient agar together with E.coli B/2. Photographs were taken after 3 days. Ax2 cells form streams during aggregation, PHKR- and RR-overexpressing cell lines do not. The bar equals 0.5 mm. (D) Wild-type cells and cells overexpressing the receiver domains of DokA, DhkA and Ssg478 were developed as in (A). RR cells (dokA-RR⁺) remain in a mound-like stage, while dhkA-RR⁺ and ssg478-RR⁺ cells form normal fruiting bodies slightly smaller than Ax2 cells. (E) Axenically growing wild-type cells and cells expressing the receiver domains of DokA, DhkA and Ssg478 were probed by RT–PCR to demonstrate overexpression in all three strains. M, marker; lanes 1 and 2, dokA-RR⁺ (RR) and Ax2 cells probed with dokA-RR primer; lanes 3 and 4, dhkA-RR⁺ and Ax2 cells probed with dhkA-RR primer; lanes 5 and 6, ssg478-RR⁺ and Ax2 cells probed with ssg478-RR primer.

Fig. 4. Development of D.discoideum strains on cAMP-S agar. Cells overexpressing DokA fragments (RR, RR D1567A), null mutants (dokA^–, regA^–) and Ax2 cells were grown on low-nutrient agar with E.coli B/2. Agar plates were used with or without 2 µM cAMP-S. RR and regA^– cells aggregate in the presence of cAMP-S, whereas aggregation of the other strains is completely blocked under these conditions. Photographs were taken after 3 days. The bar equals 2.6 mm.

Fig. 5. (A) Concentration of intracellular cAMP in D.discoideum cells. Cells were synchronized by shaking for 1 h in SPB. (B) Phosphodiesterase activity in lysates of Ax2, RR and regA^– cells. Cells overproducing RR show a lower activity compared with wild-type cells. The addition of 0.4 mM IBMX, an inhibitor of RegA, reduces cAMP degradation in all strains to a level similar to the decay measured in regA^– lysates. cAMP degradation in Ax2 cell lysates was set as 100%. Means and standard deviations of at least three independent experiments are shown.

Fig. 6. (A) Concentration of total cAMP in D.discoideum cells in response to hyperosmotic stress. Ax2 cells show a markedly stronger increase in cAMP when exposed to high tonicity (filled squares) compared with cells in SPB (open squares), whereas the addition of sorbitol has no influence on total cAMP levels in dokA^– cells (filled triangles in sorbitol buffer, open triangles in SPB). (B) Total cAMP levels of Ax2 and dokA^– cells in SPB (open symbols) and sorbitol buffer (closed symbols) and the fraction of intracellular cAMP (–, in SPB; +, in sorbitol buffer), respectively. The amounts of cAMP in the buffer and in the cell pellets were determined in parallel and normalized on the total cAMP concentration. (C) Comparison of total cAMP concentrations in rdeA^– cells and their parent strain DH1 in response to hyperosmotic stress. DH1 cells show an additional increase in 400 mM sorbitol (filled squares) compared with cells in SPB (open squares), while the elevated cAMP levels in rdeA^– cells do not increase further (filled diamonds in sorbitol buffer, open diamonds in SPB). In all panels, the means of at least three independent experiments are shown; the lines represent best linear fits. In (B), standard deviations of the total cAMP levels are depicted. Measurements were performed with DTT as inhibitor of the extracellular PDE. Therefore, values cannot be compared with those in Figures 1A and 5A.

Fig. 7. Dephosphorylation of GST–RdeA by DokA RR. The HPt protein RdeA was phosphorylated using the catalytic domain of E.coli CheA and [γ-³²P]ATP. Subsequently, either GST–RR or GST–RRDA was added. Proteins were separated by SDS–PAGE, electroblotted, exposed to X-ray film and stained with Coomassie Blue. The addition of RR reduces the phosphorylation level of RdeA, while addition of the D1567A mutant has no influence on RdeA labeling.

Fig. 8. Model. Nomenclature of gene products: ACA, adenylyl cyclase A; CAR, cAMP receptor; PKA, protein kinase A; XX, unidentified histidine kinase.