https://www.cell.com/cell/pdf/S0092-8674(00)80276-7.pdf

SH2 Signaling in a Lower Eukaryote: A STAT Protein That Regulates Stalk Cell Differentiation in Dictyostelium

▪ Abstract In Dictyostelium amoebae, cell-type differentiation, spatial patterning, and morphogenesis are controlled by a combination of cell-autonomous mechanisms and intercellular signaling. A chemotactic aggregation of ∼105 cells leads to the formation of a multicellular organism. Cell-type differentiation and cell sorting result in a small number of defined cell types organized along an anteroposterior axis. Finally, a mature fruiting body is created by the terminal differentiation of stalk and spore cells. Analysis of the regulatory program demonstrates a role for several molecules, including GSK-3, signal transducers and activators of transcription (STAT) factors, and cAMP-dependent protein kinase (PKA), that control spatial patterning in metazoans. Unexpectedly, two component systems containing histidine kinases and response regulators also play essential roles in controlling Dictyostelium development. This review focuses on the role of cAMP, which functions intracellularly to mediate the activity ...

Integration of signaling networks that regulate Dictyostelium differentiation.

We have found conditions for saturation mutagenesis by restriction
 enzyme mediated integration that result in plasmid tagging of disrupted
 genes. Using this method we selected for mutations in genes that act at
 checkpoints downstream of the intercellular signaling system that
 controls encapsulation in Dictyostelium
 discoideum . One of these genes, mkcA , is a
 member of the mitogen-activating protein kinase cascade family while
 the other, regA , is a novel bipartite gene homologous to
 response regulators in one part and to cyclic nucleotide
 phosphodiesterases in the other part. Disruption of either of these
 genes results in partial suppression of the block to spore formation
 resulting from the loss of the prestalk genes, tagB and
 tagC . The products of the tag genes have
 conserved domains of serine proteases attached to ATP-driven
 transporters, suggesting that they process and export peptide signals.
 Together, these genes outline an intercellular communication system
 that coordinates organismal shape with cellular differentiation during
 development.

Developmental signal transduction pathways uncovered by genetic suppressors.

The novel tyrosine kinase ZAK1 activates GSK3 to direct cell fate specification.

Terminal differentiation of both stalk and spore cells in Dictyostelium can be triggered by activation of cAMP-dependent protein kinase (PKA). A screen for mutants where stalk and spore cells mature in isolation produced three genes which may act as negative regulators of PKA: rdeC (encoding the PKA regulatory subunit), regA and rdeA. The biochemical properties of RegA were studied in detail. One domain is a cAMP phosphodiesterase (Km approximately 5 microM); the other is homologous to response regulators (RRs) of two-component signal transduction systems. It can accept phosphate from acetyl phosphate in a reaction typical of RRs, with transfer dependent on Asp212, the predicted phosphoacceptor. RegA phosphodiesterase activity is stimulated up to 8-fold by the phosphodonor phosphoramidate, with stimulation again dependent on Asp212. This indicates that phosphorylation of the RR domain activates the phosphodiesterase domain. Overexpression of the RR domain in wild-type cells phenocopies a regA null. We interpret this dominant-negative effect as due to a diversion of the normal flow of phosphates from RegA, thus preventing its activation. Mutation of rdeA is known to produce elevated cAMP levels. We propose that cAMP breakdown is controlled by a phosphorelay system which activates RegA, and may include RdeA. Cell maturation should be triggered when this system is inhibited.

An intersection of the cAMP/PKA and two- component signal transduction systems in Dictyostelium

spiA, a marker for sporulation, is expressed during the culmination stage of Dictyostelium development, when the mass of prespore cells has moved partly up the newly formed stalk. Strains containing a full-length spiA promoter/lacZ fusion were stained for beta-galactosidase activity at intervals during development. The results indicate that expression of spiA initiates in prespore cells at the prestalk/prespore boundary (near the apex) and extends downward into the prespore mass as culmination continues. A spatial gradient of staining expands from the top of the prespore mass and intensifies until the front of activation reaches the bottom, whereupon the entire region stains darkly. The spiA promoter can be deleted to within 301 bp of the transcriptional start site with no effect on the relative strength, timing or spatial localization of expression. Further 5′ deletions from −301 to −175 reduce promoter strength incrementally, although timing and spatial expression are not affected. Deletions to −159 and beyond result in inactive promoters. Treatment of early developmental structures with 8-Br-cAMP in situ activates the intracellular cAMP-dependent protein kinase (PKA) and precociously induces spiA expression and sporulation. The absence of an apparent gradient of staining in these structures suggest that PKA is equivalently activatable throughout the prespore region and that all prespore cells are competent to express spiA. Thus, we postulate that the pattern of expression of spiA reveals the progression of an inductive signal for sporulation and suggest that this signal may originate from the prestalk cells at the apex.

D.L. Richardson

Papers

Progression of an inductive signal activates sporulation in Dictyostelium discoideum