Fascin is the most frequently upregulated actin regulatory protein in metastatic tumors. The elevated expression levels of fascin universally correlate with poor clinical course and shorter survival across different cancer types. It is believed that fascin promotes cancer cell migration and invasion by crosslinking actin filaments into bundles. However, the molecular mehcanisms underlying the regulation of fascin bundling activity are not completely understood. In this study we examine the regulation of Fascin activity by monoubiquitination. IP and LC-MS/MS was used to identify the posttranslational modification of Fascin A novel chemical monoubiquitination method was employed to synthesize monoubiquitinated Fascin. The monoubiquitinated fascin was purified, and the effect of monoubiquitination on fascin bundling activity was determined using low speed sedimentation assay, fluorescence microscopy and transmission electron microscopy. Here we identified monoubiquitination as a novel mechanism that regulates fascin bundling activity and dynamics. The monoubiquitination sites were identified to be K247 and K250, two residues located in a positive charge patch at the actin binding site 2 (ABS2) of fascin. Using a chemical ubiquitination method, we synthesized chemically monoubiquitinated fascin (mUb-fascin) and determined the effects of monoubiquitination on fascin bundling activity and dynamics. Our data demonstrated that monoubiquitination decreased the fascin bundling EC50, delayed the initiation of bundle assembly and accelerated the disassembly of existing bundles. By analyzing the electrostatic properties on the solvent accessible surface of fascin, we proposed that monoubiquitination introduced steric hindrance to interfere with the interaction between actin filaments and the positively charged patch at ABS2. Mutation of the monoubiquitination sites (K247 and K 250) to arginine inhibited fascin monoubiquitination and enhanced pro-migration ability of fascin, supporting the notion that monoubiquitination inhibit fascin activity. We also identified Smurf1 as an E3 ligase regulating the monoubiquitination of fascin. Our findings revealed a previously unidentified regulatory mechanism for fascin, which will have important implications for the understanding of actin bundle regulation under physiological and pathological conditions.

Citation Format: Shengchen Lin, Shuang Lu, Mentor Mulaj, Bin Fang, Tyler Keeley, Lixin Wan, Jihui Hao, Martin Muschol, Jianwei Sun, Shengyu Yang. Monoubiquitination inhibits the actin bundling activity of fascin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5333. doi:10.1158/1538-7445.AM2017-5333