Cloning and Characterization of Liver-specific Isoform(Cil) of Chk1 Gene from Rat: Implying a Role of Cil in Negative Regulation of Chk1 Kinase Activity

• Main Authors: Yih-Jyh Shann, 單亦智
• Other Authors: Ming-Ta Hsu
• Format: Others
• Language: en_US
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/31498198960601930430

博士 === 國立陽明大學 === 生物化學研究所 === 90 === In our search for novel kinases that are important for cells proliferation, we have isolated the Chk1 gene from rat embryo liver. Subsequent studies identified a variant mRNA molecule of Chk1 in rat adult liver by Northern blot analysis. This isoform of Chk1 gene from rat was named Cil (Cil for Chk1 isoform in liver). Cil was then cloned from rat liver cDNA library or from rat liver mRNA by 5’RACE method and characterized. Cil contains the C-terminal region of Chk1 gene but the 5’ end is derived from sequence in the intron of Chk1 preceding the C-terminal domain by differential RNA splicing; the kinase domain of Chk1 gene is absent in this isoform. Adult tissue RNA and protein blot analyses indicated that Cil is specifically expressed only in rat adult liver and its expression increases with liver development. Expression of Cil is greatly reduced in three rat hepatoma cell lines examined. Promoter-trap experiment suggests that a promoter is located in the intron 5 of Chk1 gene preceding the C-terminal domain of Chk1 and transcription from this novel promoter generated the new 5’ non-coding exon of Cil. Thus Cil is generated by both alternate promoter usage and differential RNA splicing. UV irradiation induced caffeine-sensitive phosphorylation of both Chk1 serine-345 and the corresponding site in Cil, implying that Atr kinase is involved in the phosphorylation of both proteins. We demonstrated the interaction between the kinase domain of Chk1 and Cil using yeast two-hybrid assay, pull-down analysis and in vitro kinase assay. Cil also was found to decrease the transactivating function of p53, in opposite to the effect of Chk1, and mutation at serine-63 abolished this effect. Further investigation of the effects of Chk1 or Cil overexpression on p53 phosphorylation revealed that Chk1 overexpression enhanced the UV-induced p53 serine-20 phosphorylation. On the other hand, overexpression of Cil abolished the UV-induced p53 serine-15 and -20 phosphorylation. Cdc25C was another Chk1 target protein and its serine-216 phosphorylation was increased in response to UV and bleomycin treatment. However, Chk1 serine-345 phosphorylation was found to be increased in response to UV but not bleomycin treatment, suggesting that Cdc25C serine-216 was not phosphorylated through ATR-Chk1-Cdc25C pathway in response to bleomycin treatment. We found that Cil overexpression also abolished the UV-induced phosphorylation of Cdc25C serine-216. Taking these data together, we suggest that Cil may play a role that negatively regulates Chk1 kinase activity.