Explore The Relationship Between Human Serine / Threonine Kinase Aik And Cell Cycle Regulation

Liver cancer is a kind of malignant tumor, with high prevalence among Chinese. Seeking liver cancer-related genes and characterizing their biological functions will help to reveal the mechanism of liver tumorigenesis. Aik2 is a human serine/threonine protein kinase gene, cloned by our laboratory in 1998. Recent studies show that it may be involved in the process of cell mitosis and liver tumorigenesis. Then we tried to do the research of the functions of Aik2 and the other members of the family, Aik1 and Aik3. Because the cell cycle is out of control during the malignant proliferation of tumor cells, to understand the relationship between Aiks and liver tumorigenesis, we first used the the Semi RT－PCR to study the expression abundance of HsAiks family in 40 cases of liver tumor tissues. We found that the expression of Aik3 does not change evidently, while the expression of Aik2 and Aik1 obviously increase in most of the tumor tissues. This result is similar to those found in other tumor tissues previously. From these findings we have more reasons to believe that Aik1 and Aik2 are oncogene。To learn the functions of increased expression of Aiks kinase in liver tumor, first we should know their positions in the cell signal transduction network and their cascade orders. So we used Aik2 as a bait protein to isolate proteins, interacting with it by the yeast two-hybrid LexA assay. Then we found RACK1, CDC20, Rcl, HC8, and CIB can interact with Aik2. Among them, CDC20 is a known protein, which has interactions with Aik1. Their interactions can affect the G2/M phase during the cell mitosis. By the methods of GST-pull down and co-immunoprecipitation, we confirmed that RACK1, Rcl, and HC8 are new found proteins, which can interact with Aik2. It is interesting that RACK1 has the similar structure of CDC20. RACK1 is a very important binding protein, which has many significant functions and participates in many protein interactions in the cells. The result of co-immunoprecipitation indicated the binding domains are the catalytic domain of Aik2 and WD5-WD6 domain of RACK1. RACK1 is known to have interactions with PKCβII and to play an important role in the cell sub-localization of PKCβII. We infer that PKCβII may regulate the activation of Aik2 and then regulate the process of cell mitosis through this pathway. To reveal the unique significance of this interaction in the mitosis process, we tested the binding abilities of Aik2 mutations to RACK1 by co-immunoprecipitation. We learned that this interactionis not related to the activation of Aik2. And the in vitro kinase assay confirmed the phosphorylation of PKCβII to Aik2 and its function of activating Aik2. Furthermore, we studied the effect of Aik2 and RACK1's interaction to the growth of NIH3T3 cell lines. We carried out the NIH3T3 cell's transgenetic experiments of RACK1, Aik2, RACK1W＋Aik2, RACK1m1＋Aik2, and RACK1m2＋Aik2 with the cell selection marker of pEGFP. The results of flowcytometry show that the over expression of RACK1 can inhibit the cell mitosis, while the over expression of Aik2 can promote the mitosis. The cell mitosis is promoted again, When Aik2 and RACK1 both over expressing in the cells. Thus, we speculate that RACK1 may bind to the Aiks and competitively regulate the binding of CDC20 and Aiks. Through this way, it can affect the cell cycle indirectly. When a cell divides into two daughter cells, the cytoskeleton proteins need to be assembled and de-assembled. Aiks can interact with CDC20, regulate the formation of spindle and the segregation of chromosome. Then we want to know whether Aiks are involved in the process of cytoskeleton assembly and de-assembly. However, there are no reports about it by now. But during the process of cell mitosis, some scientists found that Aiks and Septin both appear in the region of fission furrow. These two proteins' distribution and colocalization in the cell region show that they have some relationship on their functions. We used the methods co-immunoprecipitation and yeast two-hybrid LexA assa...