Abstract
The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE's proline-rich domain is polyphosphorylated after the complex is activated. Blocking Scar/WAVE activation stops phosphorylation in both Dictyostelium and mammalian cells, implying that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether they are initiated. Unexpectedly, phosphorylation is not promoted by chemotactic signaling but is greatly stimulated by cell:substrate adhesion and diminished when cells deadhere. Phosphorylation-deficient or phosphomimetic Scar/WAVE mutants are both normally functional and rescue the phenotype of knockout cells, demonstrating that phosphorylation is dispensable for activation and actin regulation. However, pseudopods and patches of phosphorylation-deficient Scar/WAVE last substantially longer in mutants, altering the dynamics and size of pseudopods and lamellipods and thus changing migration speed. Scar/WAVE phosphorylation does not require ERK2 in Dictyostelium or mammalian cells. However, the MAPKKK homologue SepA contributes substantially-sepA mutants have less steady-state phosphorylation, which does not increase in response to adhesion. The mutants also behave similarly to cells expressing phosphorylation-deficient Scar, with longer-lived pseudopods and patches of Scar recruitment. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE's activity and that phosphorylation acts as a pseudopod timer by promoting Scar/WAVE turnover.
Highlights
Scar/WAVE is the dominant source of actin protrusions at the edge of migrating cells
Lamellipods and pseudopods are driven by Scar/WAVE recruiting the actin-related proteins (Arp2/3) complex, which in turn promotes an increase in the number of polymerizing actin filaments and growth of actin structures [1]
Lambda phosphatase resolved the multiple bands to a single one, shifting the multiple intensity peaks to single peak, confirming that the multiple bands are due to phosphorylation, and Scar is typically hyperphosphorylated to varying degrees
Summary
Scar/WAVE is the dominant source of actin protrusions at the edge of migrating cells. Lamellipods (in mammalian cells cultured in 2-D) and pseudopods (in cells in 3-D environments, or cells such as amoebas) are driven by Scar/WAVE recruiting the actin-related proteins (Arp2/3) complex, which in turn promotes an increase in the number of polymerizing actin filaments and growth of actin structures [1]. It works as part of a large, 5-membered complex, whose members have multiple names [2]; in this paper, they will be referred to as Nap, PIR121, Scar, Abi, and Brk in Dictyostelium, and Nap, PIR121, WAVE2, Abi, and HSPC300 in mammals. It will be vital to enumerate different modes of Scar/ WAVE regulation
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