Construction The Stable Cell Line Of C-Raf Protein Kinase And Systematically Identification Its Binding Proteins Of Cell Cycle

Under the action of various carcinogenic factors, some cells in local tissue lose their normal reguLation of growth by gene level, then resuLt in abnormal clonal proliferation and form abnormal lesions. These processes uLtimately lead to tumor. Our country has the most cancer patientsin the world. Cancer is the second fatal disease, after cardiovascuLar disease.Raf kinases (Rapidly Accelerated Fibrosarcoma) are fast accelerate bone fibrosarcoma oncogene products, and compose of three members, respectively, A-Raf, B-Raf and C-Raf. Raf kinase mutations are one of the main causes of cellcarcinogenesis. A-Raf mutations can lead to ovarian cancer and colorectal cancer, B-Raf mutations correlate with nervous system cancer, skin cancer and breast cancer. Small cell lung cancer is deeply related to C-Raf mutations.Therefore, reguLation of Raf kinase has become one of the most important means of treating cancer.Disorder and abnormal proliferation of the cell cycle may lead to carcinogenesis, and every aspect of cell cycle reguLation systems are likely to be major factors of carcinogenesis. ReguLation of the cell cycle can be divided into exogenous and endogenous reguLation. Exogenous reguLation is due to exogenous cytokines or other external stimuLi. Endogenous reguLation is mainly achieved through endogenous cyclin, CDK, CDIs and other cyclins.Most studies made their focus on a single target or a single mutation, lacked the awareness of the problem from the height of the system and the understand of network on molecuLar interaction,also ignored the cell dynamics signaling networks. We start from the interaction of proteins, then understand and know key nodes of tumor cell signaling networks in-depth. This study used protein-protein interaction group and high-throughput mass spectrometry systematicly to identify proteins interacting with C-Raf. Raf kinase mutation is one of the main reasons for cell cancerous. We constructed C-Raf mutant, and compared the binding proteins of wild-type and mutant. In particuLar, cyclin will solve the problems above initially.To find C-Raf interaction cyclins, we stably expressing C-Raf with VSV tag (VSV-C-Raf) in Flp-InT-RexHEK 293T cells. Then VSV beads were used in immunoprecipitation experiments in these cell lines. We also stably expressed mutant VSV-C-RafS247G in Flp-InT-RexHEK 293T cells. Through three mass spectrometry resuLts, we got 86 cyclin proteins were presented all of three times.In addition, we mapped the binding protein network map with string tool, C-RafWT and C-RafS247G total of 162 common binding protein. Including 11 binding protein with C-RafWT does not binding C-RafS247G. There are 8 proteins protein with C-RafS247G not binding C-RafWTThen we picked MSS1 and SMC3 protein, whose antibodies were validated by Western Blot. The resuLts showed that these antibodies can obtained positive signals in immunoprecipitate of C-Raf verified the authenticity of these interacting proteins.SMC3 is an important part of adhesion proteins. MuLti-subunit complex adhesion protein composed of SMC1, SMC3, AD21 and STAG may form a ring structure. The ring structure is surrounded by sister chromatid, and mediates sister chromatid adhesion protein. Then it plays a key role in gene reguLation. Composite of chromosome condensation is cut in later stage of cell cycle, and dissociates chromatin which is coupled to DNA replication and involved in DNA repair. SMC3 can be acetylated in S phase, establishing adhensive of adhesion proteins loaded by chromatin.MSS1 is a member of AAA+ATP enzyme family containing 433 amino acids. Each transition phase of the cell cycle requires degradation ubiquitylation of cyclin and others which have a very close relationship with cell cycle. Cell cycle progression has the reguLation of a series of CDK, which is activated by cyclin. After the CDK-cyclin complexes exercise the function, the cyclin of complexes will be ubiquitinated and degradated by a proteasome. which ensures the normal operation of the cell cycle. The MS SI can degrade cyclin-cyclin-dependent kinase inhibitor protein,and is the basis for reguLation of cell cycle,we successfuLly constructed C-Raf and its mutant stable cell lines. By protein-protein interaction, immunoprecipitation and muLtidimensional protein identification technology, we systematically identified the interacting proteins and cyclins of C-Raf. Selected MSS1 and SMC3, a preliminary study of its function, to verify their ability to bind with the C-Raf.We provide the basis for future research in cancer. They can be researched to reguLate cell cycle strategies for treating tumors.