ARID1A interacts with nonmuscle myosin IIA to regulate cancer cell motility.

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e17036 Background: ARID1A (BAF250A), a member of the SWI/SNF chromatin remodeling complex, is one of the most frequently mutated genes in human cancer. Here we report the discovery of a novel protein-protein interaction between ARID1A and the actin-binding motor protein, non-muscle myosin IIA (NM IIA) encoded by the myosin heavy chain 9 ( MYH9). Methods: The ARID1A immunoprecipitated protein complex was separated by gel electrophoresis followed by analysis of the peptide digested gel bands by C18-Reversed Phase chromatography using an Ultimate 3000 RSLCnano System (Thermo Scientific) equipped with an Acclaim PepMap C18 column (Thermo Scientific) and connected to a TriVersa NanoMate nanoelectrospray source (Advion) and a linear ion trap LTQ-XL mass spectrometer (Thermo Scientific). Protein identification was performed by Mascot search engine v. 2.5.1 (Matrix Science) against NCBI Homo sapiens database. Scaffold software v. 4.5.1 (Proteome Software Inc.) was used to validate the MS/MS peptide and protein identification based on 99% protein and 95% peptide probabilities. Immunoprecipitation and immunoblotting were done to evaluate the protein-protein interaction in ARID1A-wild type cell lines. Isogenic engineered cell lines, ES2 shRNA-control or shRNA- ARID1A stable transfection , and HCT116 control or ARID1A knockout by CRISPR-Cas9 (Horizon Discovery) were used to evaluate the effect of ARID1A loss on NM IIA expression and phosphorylation, and on cell migration by in vitro scratch assay with time lapse imaging. Results: Scaffold analysis of peptide spectra identified NM IIA with > 99% probability in the ARID1A immunopurified protein complex. In the ARID1A wildtype cell lines ES2 and KLE, endogenous NM IIA co-immunoprecipitated with ARID1A and vice versa. ES2 sh ARID1A cells had decreased total and phosphorylated NM IIA expression, and impaired cell migration compared to control cells. Similarly, HCT116 ARID1A homozygous knockout cells had impaired cell migration compared with HCT116 control cells. Conclusions: We report for the first time that ARID1A interacts with NM IIA to regulate cancer cell motility. Further investigation is ongoing to elucidate the significance of this newly identified function of ARID1A.