The Identification And Function Analysis Of Rap2 Interaction Protein

Rap2 is a member of Rap subfamily among the Ras family, consisting of three isoforms, including Rap2 a, Rap2 b and Rap2 c. They are involved in regulation of a wide variety of cellular processes. Members of the Rap subfamily include Rap1 and Rap2. Rap1 are about 60% homologous with Rap2, therefore, the biological function of Rap2 is similar to Rap1. Rap2 can be associated with the common downstream effect factors of Ras family, and Rap2 also has its specific binding proteins, such as MINK, TINK and PARG1. Rap2 is involved in regulation of many cell signaling pathways through its specific binding proteins. This paper mainly studies the new Rap2 specific binding proteins.In this study, we investigated the unknown Rap2 binding proteins in the NC65 renal cancer cell line with the stable expression of Rap2 by Co-immunoprecipitation assay and protein mass spectrometry analysis, and found that Vimentin and H2 B could interact with Rap2 in NC65 cells, respectively. To further verify the interacting relationship between Vimentin and Rap2 or H2 B and Rap2,immunofluorescence assay, Co-immunoprecipitation assay and the yeast two-hybrid analysis were performed. The results showed that there are the trend of colocalization between Vimentin and Rap2 or H2 B and Rap2 in NC65 cells. And we confirmed that Vimentin was a Rap2 binding protein, the interacting relationship between Vimentin and Rap2 was indirect. However, the interacting relationship between Rap2 and H2 B in NC65 cells needed to be further clarified. We used the wound healing assay to investigate the effect of co-expression of Rap2 and Vimentin on cell movement, the results indicated that over-expression of Vimentin promoted NC65 cell migration and invasion, and the co-expression of Rap2 and Vimentin also enhanced NC65 cell migration ability. It provides the basis for the further study of Rap2 function and its mechanism.