Pseudopod Growth and Evolution during Cell Movement Is Controlled through SCAR/WAVE Dephosphorylation

Background: SCAR/WAVE is a principal regulator of pseudopod growth in crawling cells. It exists in a stable pentameric complex, which is regulated at multiple levels that are only beginning to be understood. SCAR/WAVE is phosphorylated at multiple sites, but how this affects its biological activity is unclear. Here we show that dephosphorylation of Dictyostelium SCAR controls normal pseudopod dynamics. Results: We demonstrate that the C-terminal acidic domain of most Dictyostelium SCAR is basally phosphorylated at four serine residues. A small amount of singly phosphorylated SCAR is also found. SCAR phosphorylation site mutants cannot replace SCAR's role in the pseudopod cycle, though they rescue cell size and growth. Unphosphorylatable SCAR is hyperactive-excessive recruitment to the front results in large pseudopods that fail to bifurcate because they continually grow forward. Conversely, phosphomimetic SCAR is weakly active, causing frequent small, disorganized pseudopods. Even in its regulatory complex, SCAR is normally held inactive by an interaction between the phosphorylated acidic and basic domains. Loss of basic residues complementary to the acidic phosphosites yields a hyperactive protein similar to unphosphorylatable SCAR. Conclusions: Regulated dephosphorylation of a fraction of the cellular SCAR pool is a key step in SCAR activation during pseudopod growth. Phosphorylation increases autoinhibition of the intact complex. Dephosphorylation weakens this interaction and facilitates SCAR activation but also destabilizes the protein. We show that SCAR is specifically dephosphorylated in pseudopods, increasing activation by Rac and lipids and supporting positive feedback of pseudopod growth.