Cloning and Expression Analysis of a Protein Kinase C Gene,PKCμ,and Its Regulation of the Promoter Region in Zebrafish

The cDNA and genomic DNA of zebrafish (Danio rerio) protein kinase Cμ (PKCμ), with its promoter region, were obtained. The 508–amino acid zebrafish PKCμ has 86.17% similarity to human PKCμ. Real-time reverse-transcription polymerase chain reaction analysis with starvation and hormonal treatment found significant differences between the control group and the experimental group after 14 days of starvation. After injecting insulin-like growth factor II (IGF-II), growth hormone (GH), insulin, or human chorionic gonadotropin, significant differences were observed between the control and experimental groups 24 h after treatment. After injecting the gonadotropin-releasing hormone or luteotropin-releasing hormone, significant differences were seen between the control and experimental groups 15 h after treatment. These results suggest that in vivo PKCμ expression is regulated by the insulin family or by the GH, but other sex hormones produced a significant expression level more quickly than the insulin family and GH. The zebrafish PKCμ gene is located on zebrafish chromosome 17 and consists of 16 exons. A 2.6 kilobase pair on the 5′ flanking region displayed maximal promoter activity in the zebrafish liver (ZFL) cell line after treatment with IGF-I, IGF-II, and GH. However, a 1.6 kilobase pair on the 5′ flanking region displayed maximal promoter activity in the HeLa cell line after treatment with IGF-I, IGF-II, and GH. Finally, PKCμ may have important nuclear effects on cell growth and may involve nuclear localization. By transiently transfecting ZFL cells with various zebrafish PKCμ segments, we identified a nuclear localization signal: the amino acid sequence between amino acids 206 and 209 was able to predominantly direct enhanced green fluorescence protein (EGFP) into the nucleus, whereas a deletion of this motif abrogated the nuclear localization property.