SCAR Phosphorylation in the Regulation of Cell Movement - an Analysis Using Dictyostelium

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SCAR/WAVE proteins are among the most important regulators of actin polymerization, and thus of how most eukaryotic cells move. Despite this, the way in which SCAR/WAVE activity is controlled remains unclear. Working in Dictyostelium, one of the best experimental organisms for studying the genetics and cell biology of cell movement, we have discovered a novel and complex pattern of phosphorylation which is essential for SCAR/WAVE function. This includes at least two phosphorylation sites, in particular a conserved tyrosine near the middle of the protein. It seems likely that each site is responsible for a specific and regulated biological function. This grant proposes a thorough investigation of the biological meaning of these phosphorylations ? what stimuli cause each one, the effects on SCAR/WAVE function and thus on cell movement in general, and the biochemical pathways they use.
Specific aims include:
(a) Precisely defining the sites that can be phosphorylated, using FT-ICR mass spectrometry to determine the precise location of each site. We will then develop assays which specifically identify the level of phosphorylation at each site, and create mutants which have lost a single site or can only be phosphorylated in one site.
(b) Analysing how phosphorylation at each site alters the function of SCAR/WAVE, and the physiological consequences to the cell. We will find out whether each phosphorylation acts to change the stoichiometry of SCAR/WAVE s regulatory complex, its interactions with actin through the Arp2/3 complex, its breakdown, or allows the binding of novel binding partners. We will also test the effects of mutants at each site on a wide range of actin-based processes, in particular chemotaxis and cytokinesis.
(c) Uncovering the pathways upstream and downstream of each phosphorylation event. We will assess how different signalling pathways affect SCAR/WAVE phosphorylation, with particular attention to Rac, G-protein signalling and adhesion. We will also screen for novel downstream binding partners that are specific for phosphorylation at each site.
Together these results will constitute a major improvement in our understanding of how actin polymerization is initiated, and thus how movement in healthy cells is controlled.