# Phosphoproteomic Profiling Reveals Overlapping and Distinct Signaling Pathways in Dictyostelium discoideum in Response to Two Different Chemorepellents

## TL;DR

This study explores how Dictyostelium discoideum cells respond to two chemorepellents, AprA and polyP, revealing shared and distinct signaling pathways involving RNA metabolism and motility proteins.

## Contribution

The study identifies partially overlapping chemorepulsion mechanisms and two new components (RipA and GxcT) involved in response to both chemorepellents.

## Key findings

- AprA and polyP regulate over 200 proteins, with shared and distinct effects on RNA metabolism and ribosome-associated proteins.
- Both chemorepellents affect phosphorylation of signal transduction and RNA-associated proteins, including RipA and GxcT.
- Mutants lacking RipA or GxcT are unresponsive to AprA and polyP chemorepulsion.

## Abstract

Chemorepulsion 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). AprA and polyP affected levels of more than 200 proteins, with an overlap of both upregulating 25 proteins and downregulating two proteins. Two proteins were upregulated by AprA but downregulated by polyP, while two others showed the opposite trend. Surprisingly, many of the AprA- and polyP-regulated proteins are associated with RNA metabolism and ribosomes. AprA increased phosphorylation of 15 proteins and decreased phosphorylation of 36 proteins. PolyP increased phosphorylation of 12 proteins and decreased phosphorylation of 12 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 five 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.