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.

Scar/WAVE复合物是伪足和片足形成的主要催化剂。在这里，我们展示了 Scar/WAVE 的富含脯氨酸的结构域在复合物被激活后被多磷酸化。阻断 Scar/WAVE 激活会阻止盘基网柄菌和哺乳动物细胞中的磷酸化，这意味着磷酸化在伪足形成后对其进行调节，而不是控制它们是否启动。出乎意料的是，磷酸化不是由趋化信号传导促进的，而是受到细胞：底物粘附的极大刺激，并且当细胞死亡时磷酸化减弱。磷酸化缺陷或拟磷化 Scar/WAVE 突变体均具有正常功能并挽救敲除细胞的表型，这表明磷酸化对于激活和肌动蛋白调节来说是可有可无的。然而，伪足和磷酸化缺陷的 Scar/WAVE 斑块在突变体中持续时间要长得多，改变了伪足和片足类的动力学和大小，从而改变了迁移速度。在盘基网柄菌或哺乳动物细胞中，Scar/WAVE 磷酸化不需要 ERK2。然而，MAPKKK 同源物 SepA 的贡献很大——sep A 突变体的稳态磷酸化程度较低，不会因粘附而增加。这些突变体的行为也与表达磷酸化缺陷的 Scar 的细胞相似，具有更长寿命的伪足和 Scar 募集斑块。我们得出的结论是，在调节 Scar/WAVE 活性方面​​，伪足与基质的接触比细胞外信号更重要，并且磷酸化通过促进 Scar/WAVE 周转来充当伪足计时器。