E3Ligase SCF^β-TRCP Regulates The Mechanism Of MTSS1Protein In Tumor Cell Proliferation And Migration

MTSS1(metastasis suppressor-1) was first identified as a metastasis suppressorissing in metastatic bladder carcinoma cell lines. In the further study of MTSS1unction, MTSS1also acts as a scaffold protein that interacts with multiple partners toegulate actin dynamics. Recent research shows that MTSS1protein is closely relatedo the development of tumor, especially prostate cancer and breast cancer. The downegulation of MTSS1expression in prostate cancer contributes to the occurrence,evelopment and metastasis of tumor. MTSS1low expression is related to therognosis of breast cancer. However, the mechanism of down regulation of MTSS1inrostate and breast cancer cells is not clear.Ubiquitin proteasome system is the main protein degradation pathway in theukaryotic cells, through degradation of the cell cycle, DNA replication and repair,poptosis, and the degradation of some important signal pathways related proteins,own regulation intracellular protein expression level, indirect regulates a variety ofiological processes of cells. Ubiquitin proteasome system through E1biquitin-activating enzyme, E2ubiquitin-conjugating enzyme, and E3ubiquitinigase enzyme covalently attachs of multiple ubiquitin molecules to a substrate androteasome identifying and degradation of ubiquitin marker proteins for degradationf the substrate. The substrate specificity of ubiquitin proteasome system isetermined by the E3enzymes.β-TRCP is a kind of E3ligase, it can identify the specificity amino acid sequencef the protein, by combination with Cullin1, Skp1and Rbx1, SCFβ-TRCPcompoundsor degradation of the substrate. Before degradation, the substrate is first phosphorylated by intracellular kinase, then SCFβ-TRCPtransfer ubiquitin molecules tothe substrate and after the ubiquitin the substrate is degradated by proteasome.Dependent the observation of MTSS1protein structure, we found that theMTSS1protein has the amino acid sequence DSG(X)2+nS, which can be identified byE3ubiquitin ligase SCFβ-TRCP1, and its suggest MTSS1protein may be identificationand degradation by ubiquitin proteasome pathway.Based on the degradation characteristics of E3ubiquitin ligase SCFβ-TRCP1substrate, we designed this experiment. First, we detect the combination of theMTSS1and E3ubiquitin ligase SCFβ-TRCP1complexes，observe influence of substratelevel by inhibition of E3ubiquitin ligase complex and search phosphorylation MTSS1protein kinase and its phosphorylation site, to verify MTSS1protein degradation byE3ubiquitin ligase SCFβ-TRCP1complexes. Second, we respectively constructedoverexpression of MTSS1wild type and mutant kinase phosphorylation sites of celllines in prostate and breast cancer, to explored the affection of SCFβ-TRCP1on MTSS1cell function through cell proliferation and metastasis the experiment.Methods:（1） The combination between MTSS1protein and SCFβ-TRCP1E3ubiquitin ligasecompounds was detected by co-immunoprecipitation and immunoblotting.（2） We used the method of link shRNA fragments to slow virus vector, packagedvirus, infected cell to establish a stable knockout cell line, to observe theinhibition of the expression of genes within SCFβ-TRCP1E3ubiquitin ligasecompounds for the effect of MTSS1protein expression level.（3） We used the half-life experimental to detect the degradation rate of MTSS1protein in different cell lines.（4） The inhibiton of β-TRCP for the effect of MTSS1and β-TRCP mRNA level incell detected by RT-PCR. （5） We applied immunoblotting to detect exogenous MTSS1degradationmediated by CKIδ after transient transfection. We used the method of pointmutations to mutate the site phosphorylated by CKIδ, to observe the effect ofmutation site for the combination of β-TRCP and MTSS and the degradationof MTSS1.（6） We used retroviruses to establish the wide-type cell line of MTSS1highexpression and mutant cell line. And applied nick experimental to observe theeffect of wild-type or mutant MTSS1gene for cell migration velocity.（7） The change of cell proliferation was detected by bromodeoxyuridineexperimental.（8） The cell migration ability was detected by transwell experimental.Results:（1） The results of co-immunoprecipitation showed that MTSS1protein cancombine with Cullin1、β-TRCP1、Skp1and Rbx1Which are the compositionsof SCFβ-TRCP1E3ubiquitin ligase compounds.（2） The inhibition of Cullin1and β-TRCP by shRNA increased the expression ofMTSS1protein level. The half-life experimental showed that the inhibition ofβ-TRCP level obviously decreased the degradation rate of MTSS1protein.（3） Protein degradation experimental showed that kinase CKIδ involved in thedegradation of substrates protein by SCFβ-TRCP1E3ubiquitin ligase compounds.Mutate the serine of MTSS1322site inhibited the ubiquitylation of MTSS1protein mediated by β-TRCP.（4） Mutant-type MTSS1(S322A) inhibited the combination of β-TRCP and MTSSand the degradation of MTSS1mediated by CKIδ.（5） The wide-type cell line of MTSS1high expression and mutant cell line wereestablished, the results of cell counting and BrdU showed that cellproliferation rate of wide-type cell line of MTSS1high expression be notablydecreased, the cell number in S phase be obviously reduced, however the cell proliferation rate of MTSS1mutant-type cell line further be suppressed.（6） The results of nick experimental and transwell experimental showed that thehigh expression of MTSS1can obviously inhibite cell migration, and aftermutate the identification domain of β-TRCP, the cell migration ability furtherbe decreased.Conclusions:E3ubiquitin ligase SCFβ-TRCP1mediated the MTSS1ubiquitin proteindegradation process through phosphorylate322serine of MTSS1by CKIδ. Thedegradation of regulation MTSS1protein by SCFβ-TRCP1E3ubiquitin ligase involvedin cell growth and metastasis in prostate and breast cancer.