Peptide signaling during terminal differentiation of Dictyostelium

Abstract

A wide variety of mechanisms have evolved for intercellular communication in metazoans, but some of the signaling molecules were already used in their predecessors. The social amoeba, Dictyostelium discoideum, is known to use peptides to trigger sporulation within fruiting bodies, but their sequences have not been defined. We found that the peptide signal spore differentiation factor 2 (SDF-2) is processed from acyl-CoA binding protein, AcbA. The mammalian homolog of AcbA is processed to diazepam binding inhibitor that binds to the GABA(A) receptor in the brain and to peripheral 1,4 benzodiazepine receptors. Although Dictyostelium has neither GABA(A) nor peripheral-type benzodiazepine receptors, we find that both a diazepam binding inhibitor peptide and diazepam (Valium) can mimic SDF-2 in a Dictyostelium bioassay. Mutants lacking AcbA sporulate well only when developed in chimeras with WT cells. Using a yeast system we show that ligand binding to the SDF-2 receptor histidine kinase, DhkA, inhibits phosphorelay, which can account for its ability to induce rapid sporulation.

Fig. 1.

AcbA from Dictyostelium (Dicty AcbA) aligned to human ACBP. The neuropeptides TTN and ODN generated by proteolysis at trypsin sites of ACBP are indicated as well as the three synthetic peptides derived from Dictyostelium AcbA. Identities are given in bold.

Fig. 2.

Responses to peptides and diazepam. Rapid spore induction was determined in low-density populations of the sporogenous strain (KP) incubated in the presence of cAMP for 18 h (3). Shown are peptide 1 (▴), trypsinized AcbA (▿), peptides 2 and 3 together (▪), peptide 2 (○), peptide 3 (□), diazepam (•), and human ODN (▵). Values are the averages of three to five independent determinations with error bars showing 1 SD.

Fig. 3.

Growth of sln1^– yeast cells. Strain TM182 (sln1^–) transformed with empty vector (open bars) or the dhkA expression vector (stippled bars) were inoculated into synthetic complete-glucose medium in the presence or absence of 10 nM SDF-2 as indicated. After incubation for 4 days at 30°C, growth was measured (OD₆₀₀) (average of at least three independent experiments). Only cells expressing dhkA grew significantly under these conditions, and their growth was inhibited by SDF-2. Results are representative of three independent experiments.

Fig. 4.

AcbA during synchronous development. (A) Axenically growing Dictyostelium cells of strain AX4 were washed and deposited on buffer-saturated filters at time 0. The cells aggregated by 12 h and formed fruiting bodies by 24 h. At the times shown, 5 × 10⁷ cells were collected and prepared for Western analyses (see Materials and Methods). Each sample had 10 μgof protein. After transfer of electrophoretically separated proteins to poly(vinylidene difluoride) membranes, blots were stained with antiserum to recombinant AcbA diluted 1:500. The band at the expected size of AcbA (10 kDa) is indicated by an arrow. (B) Western blot of purified prespore and prestalk extracts. No staining was observed on Western blots of extracts prepared from acbA^– cells (data not shown).

Fig. 5.

Priming and processing. KP cells were incubated in the presence of cAMP for 20 h and either left untreated (open bars) or primed with 0.1 pM of the synthesized SDF-2 peptide (filled bars). (A) The supernatants were collected and assayed for SDF-2 activity. Addition of 0.5% Triton X-100 to unprimed cells did not release SDF-2 into the supernatant. Primed KP cells carrying a null mutation in acbA did not release measurable SDF-2. (B) Five minutes after being primed KP cells were washed to remove SDF-2 and then incubated with or without 1 pmol of recombinant AcbA. The supernatants was assayed for SDF-2 activity after 1 h. The serine protease inhibitor tosyl phenylalanyl chloromethylketone (TPCK) was added to 10 μM just before addition of AcbA. Primed KP cells carrying a null mutation in acbA (diagonal stripes) generated SDF-2 from AcbA, whereas KP cells carrying a null mutation in tagC (vertical stripes) did not generate measurable SDF-2 from AcbA even after being primed with 10 pM SDF-2. Results are representative of three independent experiments.

Fig. 6.

Proposed signaling pathway leading to sporulation. AcbA is released from prespore cells and proteolytically cleaved by TagC on prestalk cells to generate the SDF-2 peptide. SDF-2 binds to the histidine kinase receptor DhkA present on the surface of both prespore and prestalk cells and inhibits phosphorelay via RdeA to the cAMP phosphodiesterase RegA. The activity of RegA is reduced when its response regulator region is not phosphorylated, leading to an increased accumulation of cAMP generated by the late adenylyl cyclase ACR. Encapsulation is triggered by the resulting increase in PKA activity (3, 5). A similar pathway in prestalk cells results in extracellular TagC activity (3, 30).