Insight into an Ancient and Enigmatic Molecule Using Dictyostelium discoideum

Polyphosphate (polyP) is a linear polymer of orthophosphates linked by high energy phosphoanhydride bonds and is found in all cell types in nature. Despite this high conservation its role in cellular biology has remained enigmatic. This is due to a lack of reliable means of quantification and understanding of how polyP is synthesized in eukaryotes. We have identified polyP as a molecule that accumulates extracellularly in Dictyostelium discoideum. Proliferating Dictyostelium accumulate increasing amounts of polyP as a function of cell density. At high densities polyP acts an inhibitor of proliferation inducing a stationary phase, preventing high density populations from outgrowing their food source before the onset of starvation. The amount of polyP observed during stationary phase is sufficient to inhibit proliferation of mid-log actively dividing cells, and the addition of recombinant exopolyphosphatase to cultures results in reduced proliferation inhibition. We also found that inositol hexakisphosphate kinase (I6KA), a kinase responsible for inositol pyrophosphate synthesis, is needed for efficient polyP accumulation. I6KA has also been implicated in polyP metabolism in Yeast and Mice, marking the first case of a conserved mechanism for polyP synthesis in eukaryotic organisms. PolyP also has a role in regulating the transition from a vegetative state to a developmental state as Dictyostelium approach starvation. In low nutrients, cells treated with polyP form aggregates, the first step in development of Dictyostelium, while untreated cells remain vegetative. We found that polyP induces aggregation by inhibiting proteasome activity, and treatment with another proteasome inhibitor, Mg132, also induces aggregate formation. Ras and Akt are essential for polyP induced proteasome inhibition and aggregation. The effects of polyP were found to be mediated by the putative GPCR GrlD. Cells lacking GrlD do not bind biotinylated polyphosphate, suggesting it acts as an extracellular polyphosphate receptor. The effects of polyP on Dictyostelium were also found to be conserved to humans, as polyP inhibits proliferation and proteasome activity of human leukocytes and four human leukemia cell lines. Furthermore, as in Dictyostelium, polyphosphate induces differentiation of monocytes into fibrocytes. This work provides insight into the highly unusual and poorly understood signaling molecule polyP