Novel Proteins Identified By Tandem Affinity Purification Coupled To Nano LC-MS/MS Interact With Ribosomal S6 Protein Kinase 4 (RSK4) And Its Variant Protein (RSK4M)

Background:Ribosomal S6 protein kinase 4 (RSK4) is an important novel tumor suppressor that inhibits breast cancer cell growth and induces senescence. However, RSK4m, which is a variant of RSK4 resulting from alternative splicing, may play distinct roles in some aspects. Protein is the main substance of the activities of life. To achieve its function, protein can not be separated from the interaction with other proteins. Alternative splicing is an important mechanism for regulating gene expression and generating protein diversity. Our pervious study suggested that the p90 ribosomal protein S6 kinase 4 (RSK4) may be a key molecule to suppress breast cancer invasion and metastasis. RSK4 pre-mRNAs undergo alternative splicing at different sites and these splices generate different mRNA variants. Each of the RSK4 variants has its own function. We proposed that different protein complexes formed by RSK4 and its variants with other proteins interaction might play an important role in breast cancer occurrence and development. Our study will establish the technique of tandem affinity purification in mammals. Afterwards, the study will identify the RSK4-interacting and RSK4 variants-interacting proteins by mass spectral analysis, and identify the key protein by bioinformatics analysis. Through the above studies, we expect to establish new ideas and ways for further clarifying mechanism of occurrence and development of breast cancer. Analysis of the RSK4 and RSK4m interactome could provide insight into their specific functions and integrative mechanisms, finding new therapeutic targets of beast caner.Methods:We separately established overexpressing cell models of RSK4 and RSK4m containing two tandem affinity tags (STREPII and Flag). Using tandem affinity purification, we obtained protein complexes that interacted with RSK4 or RSK4m. Mass spectrum analysis was performed to identify the obtained protein complexes, and bioinformatics analyses, including Gene Ontology and Ingenuity Pathway Analysis, were performed for their functional annotations and draw protein-protein interaction networks. To confirm bioinformatic analytic results, we also applied co-immunoprecipitation (Co Immunoprecipitation, Co-IP) on some proteins interacting with RSK4 for validation.Results:In this study, we isolated and identified 82 RSK4-associated proteins and 137 RSK4m-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 RSK4 and RSK4m were provided separately, by bioinformatics analyses (Gene Ontology and Ingenuity Pathway Analysis). The results of GO analysis showed RSK4wt or RSK4m jion various biological activities, such as tRNA metabolic process, amino acid activation, protein folding and so on, and most of them, which are the same as RSK4, are present in cytoplasm and play roles in functions in cytoplasm. IPA demonstrated that we obtained three signal pathway and protein-protein interaction networks with the highest enrichment which belong to interactome of RSK4wt or RSK4m, respectively, by Core Analyze dateset:(DRSK4wt and RSK4m mainly involved ribosomal biosynthesis. Some set of proteins in the protein-protein interaction networks such as PRMT5, STAT3, HSP90AB1, ATP5B, RPLs, were closely concerned with ribosomal biosynthesis. â‘¡ Interactome of both were related with pre-RNA processes. There are some interactome belonging to hnRNPs family (heterogeneous nuclear ribonucleproteins):HNRNPH1, HNRNPK, HNRNPM and they were RNA-binding proteins, interacted with pre-RNA, affected pre-RNA processing, mRNA metabolic and transport. â‘¢RSK4wt and RSK4m also join chromosome recombination and metabolic, those interactome like STK38, STK38L, MOB2, DDX3X, joined replication of cell centrosome and chromatin recombinant in matotic phase. Take GO annotation and IPA analyses together, they manifested that both of RSK4wt and RSK4m were related with multiple cellular activities and had various biological functions. Otherwise, RSK4m had more interactome and involved more biological pathway than RSK4. At the same time, we conformed there exited interacting relationship between PRMT5 and RSK4 by CO-IP, which suggested PRMT5 maybe inhibited RSK4 negative regulate to cell growth by methylating RSK4.Conclusions:There exist interactome with RSK4 or RSK4m. These interactome mainly involve in ribosomal biosynthesis, pre-RNA processing, chromatin remodeling and DNA metabolic, and mediate multiple biological processes, especially cell senescence. Then the different interactome are present in interacting proteins of RSK4 and RSK4m, which suggest that regulatory role and mechanism between them maybe differ and RSK4m has more molecular functions than RSK4, although their special functions need to be further studied. PRMT5, one of proteins interacting with RSK4, interacts with RSK4 directly.