Phosphoproteomic Profiling Reveals Overlapping and Distinct Signaling Pathways in Dictyostelium discoideum in Response to two different chemorepellents

Chemorepulsion causes cells to move away from the source of a signal, but the underlying mechanisms for eukaryotic cells are poorly understood. We performed proteomics and phosphoproteomics to elucidate how Dictyostelium discoideum responds to its two endogenous chemorepellent signals, the protein AprA and inorganic polyphosphate (polyP). At 60 minutes, AprA increased levels of 211 proteins and reduced levels of 57 proteins while polyP increased levels of 152 proteins and reduced levels of 168 proteins. Surprisingly, many of the AprA- and polyP-regulated proteins are associated with RNA metabolism and ribosomes. AprA and polyP both upregulated 19 proteins, one protein was downregulated by both, and one was upregulated by AprA and downregulated by polyP. AprA increased phosphorylation of 12 proteins and decreased phosphorylation of 60 proteins. PolyP increased phosphorylation of 7 proteins and decreased phosphorylation of 18 proteins. As expected, the two chemorepellents affected phosphorylation of signal transduction/ motility proteins, but unexpectedly affected phosphorylation of RNA-associated proteins. Both AprA and polyP decreased phosphorylation of six proteins including the Ras-interacting protein RipA and guanine nucleotide exchange factors (GEFs) such as the RacGEF GxcT. Mutants lacking RipA or GxcT were unresponsive to both AprA and polyP chemorepulsion. Together, this work supports the idea that rather than activating the same chemorepulsion mechanism, AprA and polyP activate only partially overlapping chemorepulsion mechanisms, and identifies two new components that are used by both chemorepellents.