Phosphorylation of Arp2 is not essential for Arp2/3 complex activity in fission yeast.

Alexander E Epstein,Thomas D Pollard,Sofia Espinoza-Sanchez

doi:10.26508/lsa.201800202

Abstract

LeClaire et al presented evidence that phosphorylation of three sites on the Arp2 subunit activates the Arp2/3 complex to nucleate actin filaments. We mutated the homologous residues of Arp2 (Y198, T233, and T234) in the fission yeast genome to amino acids that preclude or mimic phosphorylation. Arp2/3 complex is essential for the viability of fission yeast, yet strains unable to phosphorylate these sites grew normally. Y198F/T233A/T234A Arp2 was only nonfunctional if GFP-tagged, as observed by LeClaire et al in Drosophila cells. Replacing both T233 and T234 with aspartic acid was lethal, suggesting that phosphorylation might be inhibitory. Nevertheless, blocking phosphorylation at these sites had the same effect as mimicking it: slowing assembly of endocytic actin patches. Mass spectrometry revealed phosphorylation at a fourth conserved Arp2 residue, Y218, but both blocking and mimicking phosphorylation of Y218 only slowed actin patch assembly slightly. Therefore, phosphorylation of Y198, T233, T234, and Y218 is not required for the activity of fission yeast Arp2/3 complex.

Highlights

Of branched actin filament networks drives cellular processes including cell motility and clathrin-mediated endocytosis (Weinberg & Drubin, 2012; Blanchoin et al, 2014)
Mass spectrometry of the Arp2/3 complex purified in the presence of phosphatase inhibitors revealed phosphorylation of Arp2 at Y198 and Y218, previously unexplored but widely conserved sites in eukaryotes (Fig S1C and D)
We did not detect phosphorylation of T233 or T234. This does not rule out their phosphorylation in vivo, since phosphorylation of Arp2 at Y198 and Y218 was lost in the S. pombe Arp2/3 complex purified without phosphatase inhibitors

Summary

Introduction

Of branched actin filament networks drives cellular processes including cell motility and clathrin-mediated endocytosis (Weinberg & Drubin, 2012; Blanchoin et al, 2014). We explore the effect of Arp2 phosphorylation on Arp2/3 complex activity in the fission yeast Schizosaccharomyces pombe, where efficient homologous recombination facilitates making mutations in the genome (Grimm et al, 1988; Bahler et al, 1998), and quantitative fluorescence microscopy assays are available to characterize actin assembly during endocytosis (Berro & Pollard, 2014a).

Results

Conclusion