SHARPIN serine 146 phosphorylation mediates ARP2/3 interaction, cancer cell invasion, and metastasis

Abstract

SHARPIN is involved in several cellular processes and promotes cancer progression. However, how the choice between different functions of SHARPIN is post-translationally regulated is unclear. Here we have characterized SHARPIN phosphorylation by mass spectrometry and in vitro kinase assay. Focusing on serine 131 and 146, we demonstrate their role in SHARPIN-ARP2/3 complex interaction, whereas they play no role in integrin inhibition or LUBAC activation. Consistent with its novel role in ARP2/3 regulation, serine 146 (S146) phosphorylation of SHARPIN promoted lamellipodia formation. We also demonstrate that SHARPIN S146 phosphorylation-mediated ARP2/3 interaction is sensitive to inhibition of ERK1/2 kinase, or reactivation of protein phosphatase 2A (PP2A). Notably, CRISPR-Cas9 mediated knockout of SHARPIN abrogated three-dimensional (3D) invasion of several cancer cell lines. The 3D invasion of cancer cells was rescued by overexpression of the wild-type SHARPIN, but not by SHARPIN S146A mutant,. Finally, we demonstrate that inhibition of phosphorylation at S146 significantly reduces the in vivo metastasis in the zebrafish model. Collectively, these results map SHARPIN phosphorylation sites and identify S146 as a novel phosphorylation switch defining ARP2/3 interaction and cancer cell invasion.