Cloning and characterization of liver-specific isoform of Chk1 gene from rat.
We have isolated and characterized an isoform of protein kinase Chk1 gene from rat liver and a rat liver cDNA library by 5'-rapid amplification of cDNA ends. The gene (Cil) contains the C-terminal region of the Chk1 gene, but the 5'-end is derived from a 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. Tissue RNA and protein blot analyses indicated that Cil was specifically expressed only in rat liver, and its expression increased with liver development. Expression of Cil was found to be reduced in three rat hepatoma cell lines examined. A promoter trap experiment suggested that a promoter was located in the intron preceding the C-terminal domain of Chk1, and transcription from this novel promoter generated the new 5' noncoding exon of Cil. Thus Cil was generated by both alternate promoter usage and differential RNA splicing. UV irradiation induced caffeine-sensitive phosphorylation of both Chk1 and Cil at Ser-345 in Chk1 and its equivalent site in Cil, implying a role for ATR kinase in the phosphorylation of both proteins. We demonstrated the interaction between the kinase domain of Chk1 and Cil using a yeast two-hybrid assay and pull-down technique. In contrast to the effect of Chk1, Cil was found to decrease the transactivating function of p53, and the S63A mutation of Cil abolished this effect. These results suggest that Cil may serve as a dominant negative competitor of Chk1 as suggested previously.
Shann
Y J
YJ
Institute of Biochemistry, National Yang-Ming University, 112, Taipei, Taiwan, Republic of China.
Hsu
M T
MT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002110:Caffeine / Q000494:pharmacology; D000071877:Checkpoint Kinase 1; D003001:Cloning, Molecular; D017930:Genes, Reporter; D006801:Humans; D007527:Isoenzymes; D008099:Liver / Q000201:enzymology* / Q000502:physiology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D016415:Sequence Alignment; D014018:Tissue Distribution; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D014466:Ultraviolet Rays
D007527:Isoenzymes; D016159:Tumor Suppressor Protein p53; D002110:Caffeine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605871:Chek1 protein, rat