Functional studies of mst3 kinase in the cell morphology and cell migration

• Main Authors: Wen-Yang Lai, 賴文揚
• Other Authors: Ming-Derg Lai
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/6jtf7k

碩士 === 國立成功大學 === 生物化學研究所 === 92 === 　　Mammalian Sterile 20-like kinase 3 (Mst3), the physiological functions of which is unknown, is a member of the germinal center kinase-III family. It contains a conserved kinase domain at its NH2 terminus, and a regulatory domain at its COOH terminus. There are another three members of MST family. The Mst1/Mst2 belongs to GCK-II subfamily and the Mst4 belongs to the GCK-III. The Mst1 and Mst2 are involved in inducing apoptosis. The Mst4, which shares 88% sequence similarity to Mst3, mediates cell growth, cell transformation, and cell motility instead of promote apoptosis. Nevertheless, the physiological functions of Mst3 are unknown. 　　The Mst3 is ubiquitously expressed in most tissues and cell lines. In order to study the biological function, we exploited the small interfering RNA (siRNA) technique to suppress Mst3 expression. We constructed Mst3 RNAi plasmid targeting six different locations of Mst3 mRNA. Transient transfection of RNAi and HA-Mst3 indicate that two of Mst3 RNAi could suppress exogenous Mst3 expression. One of the functional Mst3 RNAi plasmid was stably expressed in MCF-7 cell by transfection. Western blot analysis indicated that the Mst3 protein expression was significantly silenced by Mst3 RNAi plasmid comparing with the vector control. We investigated whether cell proliferation and cell motility was altered by down-regulating expression of Mst3. There was no difference of cell proliferation between Mst3 RNAi clone and control cells. In contrast, the cell motility was enhanced in Mst3 RNAi clone in wounding assay, boyden chamber assay, and cell Immunofluorescence. Further more, the cell migration was evidently reduced when we stably overexpressed Mst3 in MDCK cell. Therefore, we propose that Mst3 plays a role of inhibiting cell motility. Further study is required to understand the molecular mechanism how Mst3 regulates cell migration.