Abstract: Background and purpose: As a tumor suppressor gene, ribosomal S6 kinase 4 (RSK4) plays important roles in inhibiting cell proliferation, migration and inducing cell apoptosis. However, the proteins interacting with RSK4 are still unknown. This study aimed to screen proteins interacting with RSK4 in breast cancer cell line MDA-MB-231 by flag-tag affinity purification and LC-MS/MS (liquid chromatography/mass spectrometry). Methods: The pcDNA3.1/EGFP-RSK4-Flag eukaryotic expression vector was constructed by inserting full length RSK4 gene into vector pcDNA3.1/EGFP-Flag. And then the recombinant plasmids were transferred into MDA-MB-231 cells. Real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) and Western blot were used to detect the expression of RSK4 in MDA-MB-231 cells. Affinity purification and LC-MS/MS were applied to screen proteins interacting with RSK4, and the related action mechanism of RSK4 with its interacted proteins was detected based on bioinformatics gene ontology (GO) and ingenuity pathway analysis (IPA). Results: Twenty-four proteins, such as serine/threonine-protein kinase 38 (STK38)/serine/threonine-protein kinase 38-like (STK38L), MOB kinase activator 2 (MOB2) and protein arginine N-methyltransferase 5 (PRMT5), were successfully identified by Flag-tag affinity purification followed by LCMS/ MS analysis, which probably interacted with RSK4. Bioinformatics analysis of the identified proteins suggested the proteins interacting with RSK4 were involved in diverse biological pathways, such as apoptosis and cell migration. Conclusion: According to bioinformatics results of proteins interacting with RSK4 identified by affinity purification and LC-MS/MS, biological networks of RSK4 are involved in apoptosis and migration in breast cancer cells.

Key words: Affinity purification, RSK4, Eukaryotic expression, Gene transfection