Cell–substrate adhesion drives Scar/WAVE activation and phosphorylation by a Ste20-family kinase, which controls pseudopod lifetime

Singh, S. P. , Thomason, P. A., Lilla, S., Schaks, M., Tang, Q., Goode, B. L., Machesky, L. M. , Rottner, K. and Insall, R. H. (2020) Cell–substrate adhesion drives Scar/WAVE activation and phosphorylation by a Ste20-family kinase, which controls pseudopod lifetime. PLoS Biology, 18(8), e3000774. (doi: 10.1371/journal.pbio.3000774) (PMID:32745097) (PMCID:PMC7425996)

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.

• Item Type: Articles
• Additional Information: This work was supported by Cancer Research UK core grant number A17196 and Multidisciplinary Award A20017 to RHI, by the Deutsche Forschungsgemeinschaft, grant GRK2223/1 to KR, and a grant from the NIH (GM063691) to BG.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Insall, Professor Robert and Lilla, Dr Sergio and Thomason, Dr Peter and Singh, Dr Shashi and Machesky, Professor Laura

Creator Roles

Thomason, P. A.: Conceptualization
Lilla, S.: Methodology
Machesky, L. M.: Conceptualization, Writing – review and editing
Insall, R. H.: Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review and editing
Singh, S. P.: Conceptualization, Data curation, Investigation, Methodology, Validation, Writing – original draft

• Authors: Singh, S. P., Thomason, P. A., Lilla, S., Schaks, M., Tang, Q., Goode, B. L., Machesky, L. M., Rottner, K., and Insall, R. H.
• College/School: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Journal Name: PLoS Biology
• Publisher: Public Library of Science
• ISSN: 1544-9173
• ISSN (Online): 1545-7885
• Copyright Holders: Copyright © 2020 Singh et al.
• First Published: First published in PLoS Biology 18(8): e3000774
• Publisher Policy: Reproduced under a Creative Commons License