Phosphorylation of the WH2 domain in yeast Las17/WASP regulates G-actin binding and protein function during endocytosis

J J Tyler,L Abugharsa,K R Ayscough,I I Smaczynska-De Rooij,J S Palmer,L P Hancock,E G Allwood

doi:10.1038/s41598-021-88826-z

Abstract

Actin nucleation is the key rate limiting step in the process of actin polymerization, and tight regulation of this process is critical to ensure actin filaments form only at specific times and at defined regions of the cell. WH2 domains are short sequence motifs found in many different actin binding proteins including WASP family proteins which regulate the actin nucleating complex Arp2/3. In this study we reveal a phosphorylation site, Serine 554, within the WH2 domain of the yeast WASP homologue Las17. Both phosphorylation and a phospho-mimetic mutation reduce actin monomer binding affinity while an alanine mutation, generated to mimic the non-phosphorylated state, increases actin binding affinity. The effect of these mutations on the Las17-dependent process of endocytosis in vivo was analysed and leads us to propose that switching of Las17 phosphorylation states may allow progression through distinct phases of endocytosis from site assembly through to the final scission stage. While the study is focused on Las17, the sole WASP family protein in yeast, our results have broad implications for our understanding of how a key residue in this conserved motif can underpin the many different actin regulatory roles with which WH2 domains have been associated.

Highlights

The WH2 motif is an abundant and versatile G-actin binding sequence found in a wide array of actin binding proteins[1,2]
Two approaches were employed during the analysis, multidimensional protein identification technology (MudPIT) and neutral loss multi stage activation (MSA)
A number of these sites lie in the C-terminal region of the protein with T543 just upstream of the WH2, while S554 lies within the predicted N-terminal helix forming region of the WH2 domain

Summary

Introduction

The WH2 motif is an abundant and versatile G-actin binding sequence found in a wide array of actin binding proteins[1,2]. This, as well as the dynamic nature of the interaction between the WH2 motif and the actin monomer, helps to explain the broad array of divergent actin related processes in which WH2 motifs are implicated These include: nucleation, filament severing, monomer sequestration, monomer delivery and regulation of barbed-end dynamics[2,4,5,6,7,8]. In terms of a role in actin filament nucleation, in some proteins, for example those of the WASP family, WH2 domains are located adjacent to sites that bind Arp2/3. Arrow indicates position equivalent to S554 in Las[17] (D) Thermophoresis trace to measure G-actin binding affinity of wild type, phosphorylated and mutant WH2 peptides. Phosphorylation within the C-terminal intrinsically disordered region e.g. on WASP/N-WASP S483,S484/S484,S485 has been detected[15], though to date phosphorylation has mostly been identified and considered in terms of its potential to promote nucleation by disruption of the auto-inhibitory interface between the C-terminal CA region and the GTPase binding CRIB d omain[3,16,17]

Methods

Results

Conclusion