A Preliminary Study On The Interaction And Function Of Human Protein (SIAH1 And PHC2; RIOK3 And PAK2)

This thesis is composed of two separate parts:the degradation of PHC2by SIAH; the study on the function of the interaction between RIOK3and PAK2.In part I, the human liver cDNA library was screened with pDBLeu-SIAH1as bait by yeast two-hybrid system and PHC2was identified as a SIAH interactive protein. The interaction was then confirmed by GST Pull-down and co-immunoprecipitation assays. Immunofluorescent localization experiments revealed that SIAH1localized in the cytoplasm and nucleus and co-localized with14-3-3η in the nucleus. The Cys-rich domain of SIAH and the PxVxAxP motif of PHC2were demonstrated to be essential for their interaction. SIAH facilitated the ubiquitination and degradation of PHC2via ubiquitin-proteasome pathway in HEK293T cells. Mutations in the PcG genes result in developmental defects and have been implicated in human cancer. PHC2, as a functional component of class II PcG complexes, plays a role in embryogenesis and developing brain. The novel discovery of the degradation of PHC2by SIAH might help to investigate the road of PHC2in the early development.In part II, we identified the interaction between RIOK3and PAK2by screening the human fetal brain cDNA library of yeast two-hybrid system and confirmed the interaction by co-immunoprecipitation assays in vitro.Immunofluorescent localization experiments revealed that RIOK3co-localized with PAK2in the cytoplasm. When overexpressing RIOK3in cells, the expression of PAK2would be decreased. The result suggested that RIOK3would possibly regulate the expression of ITPKC. Few reports are concerned with the function of RIOK3, and PAK2is important in cytoskeletal reorganization, cell migration and invasion, thus the interaction between RIOK3and PAK2may provide new clues for the study on the function of RIOK3.