PT  - JOURNAL ARTICLE
AU  - Alexander E. Epstein
AU  - Sofia Espinoza-Sanchez
AU  - Thomas D. Pollard
TI  - Phosphorylation of Arp2 is not essential for Arp2/3 complex activity in fission yeast
AID  - 10.1101/372706
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 372706
4099  - http://biorxiv.org/content/early/2018/07/19/372706.short
4100  - http://biorxiv.org/content/early/2018/07/19/372706.full
AB  - LeClaire et al. presented evidence that phosphorylation of three sites on the Arp2 subunit activates 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.Summary Previous research concluded that phosphorylation at three sites on Arp2 is necessary to activate Arp2/3 complex. Epstein et al. make genomic substitutions blocking or mimicking phosphorylation to demonstrate that phosphorylation of these three sites does not regulate Arp2/3 complex in fission yeast.NPFnucleation promoting factorWASpWiskott-Aldrich syndrome proteinMD simulationsmolecular dynamics simulationsNIKNck-interacting kinaseLC-MS/MSliquid chromatography-tandem mass spectrometry