Identification Of Interacting Protein Of RSK4M By Tandem Affinity Purification Coupled To Nano LC-MS/MS

Background:Breast cancer, noun a dreaded, According to the latest statistics, in China’s annual newly diagnosed breast cancer cases and deaths accounted for global 12.2% and 9.6% respectively, the continued increase in the numerical, accelerating the deepening of our research on breast cancer.Berns Kand SUN Y, who has proved thatRSK4 is a crucial factor in suppressing the invasion and metastasis of breast cancer, it can inhibit the proliferation and induce the aging of breast cancer cells, but on the rsk4 mRNA alternative splicing downward into the variant (rsk4 variants, RSK4m) we are poorly understood.Deletion mutations (-E21) of 15 bases deletion mutations (-15nt) and exon twenty-first () occurred in exon nineteenth of RSK4.Caused by the free combination formed three variants (1) lack the penultimate exon, namely +15nt-E21 (variant 1); (2) the lack of the 19 exons of 15 bases, namely-15nt+E21 (variant 2); third, the lack the 19th exon 15 bases and the penultimate exons that-15nt-E21 (Variant 3).This article is aimed at the newly discovered RSK4 variants, their research is still in its infancy, especially its functional differences with the wild type RSK4, is the same or opposite, we do not understand,if you lose the inhibition function, is that as a new target to treat marker, if the same ability to inhibit, that is have lead to the same mechanism still has a different mechanism of action, which is easier to control the invasion and metastasis of tumor cells. The purpose of this study is to search for the interaction protein of RSK4m, so as to facilitate the further study of its molecular mechanism and function in breast cancer.Methods:We separately established overexpressing cell models of RSK4m2 and RSK4m3 containing two tandem affinity tags (StrepII and Flag).Using tandem affinity purification, we obtained protein complexes which interacted with RSK4m2 or RSK4m3. Mass spectrum analysis was performed to authenticate the obtained protein complexes, and bioinformatics analyses, includingIngenuity Pathway andGene Ontology Analysis, were performed for their functional annotations and depicted protein-protein interaction networks. To confirm bioinformatic analytic results, we also utilizedCO-IP(Co Immunoprecipitation) on some proteins interacting with RSK4m for validation.To verify the interaction between selected from the results of heat shock protein HSP90aal and RSK4m2. Finally, the growth and proliferation of breast cancer cell MDA-MB-231 by CCK-8 kit after transfection.Results:In this study, we isolated and identified 452RSK4m2-associated proteins and 713 RSK4m3-associated proteins using two STREPII and a single Flag tag-based tandem affinity purification (SF-TAP) coupled with nano LC-MS/MS in MDA-MB-231 cells. Functional annotations and protein-protein interaction networks of the protein interactome of RSK4m2 and RSK4m3 were provided separately, The application of biological information analysis method go (gene ontology) and IPA (ingenuity pathway analysis) analysis, respectively RSK4m2 RSK4m3 related protein for functional annotation and protein-protein interaction network graph drawing. Go analysis results:RSK4m2 RSK4m3 interaction proteins are involved in diverse cellular events, IPA analysis results: get RSK4m2 RSK4m3 enrichment the highest protein interaction network, which RSK4m2 HSP90aal exist direct interaction between. Comprehensive analysis of GO and IPA analysis results show that RSK4m2 and RSK4m3 are involved in various cellular activities, have a variety of biological functions. By Core Analyze dateset: ①RSK4m2and RSK4m3 mainly involved ribosomal biosynthesis. Some set of proteins in the protein-protein interaction networks,were closely concerned with ribosomal biosynthesis. ②Interactome of both were related with pre-RNA processes.③RSK4wt and RSK4m also join chromosome recombination and metabolic. Take GO annotation and IPA analyses together, they manifested that both of RSK4m2 and RSK4m3 were related with multiple cellular activities and had various biological functions. At the same time, we conformed there exited interacting relationship between HSP90aal and RSK4 by CO-IP.CCK-8 showed the expression RSK4m2 group and over expression RSK4m3 group at 24,48,72,96 cell inhibition rate (%) reached 3.9+0.01,5.6+0.01,31.1+0.01,23.6+0.02; 1.5+0,4.4+0.03,22.1 +0.05,19.7+0.02, differences in 48,72 and 96 hours of statistically significant (P<0.05).Conclusion:RSK4m2 RSK4m3 can inhibit the proliferation of breast cancer cell line MDA-MB-231. Results show that RSK4m may play and wild-type rsk4 as inhibition of the proliferation of breast cancer cell function, but may exist different regulatory mechanisms and rsk4 wild type (RSK4w), and RSK4m may have biological function of more than RSK4w, but the exact function remains to be further research. RSKm2 may is through inhibition of HSP90aal to hinder the growth of cancer cells.