Cloning and expression analysis of a protein kinase C gene, PKCmu, and its regulation of the promoter region in zebrafish.
The cDNA and genomic DNA of zebrafish (Danio rerio) protein kinase Cmu (PKCmu), with its promoter region, were obtained. The 508-amino acid zebrafish PKCmu has 86.17% similarity to human PKCmu. 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 PKCmu 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 PKCmu 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, PKCmu may have important nuclear effects on cell growth and may involve nuclear localization. By transiently transfecting ZFL cells with various zebrafish PKCmu 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.
Chen
Jyh-Yih
JY
Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Jiaushi, Ilan, Taiwan. zoocjy@ate.sinica.edu.tw
Wei
Chuan-Chih
CC
Chiou
Ming-Jyun
MJ
Su
Hung-Yi
HY
Kuo
Ching-Ming
CM
D020121:5' Untranslated Regions; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006728:Hormones / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000235:genetics; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013217:Starvation / Q000201:enzymology / Q000235:genetics; D015027:Zebrafish / Q000235:genetics* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics*
D020121:5' Untranslated Regions; D017931:DNA Primers; D018076:DNA, Complementary; D006728:Hormones; D019913:Nuclear Localization Signals; D011994:Recombinant Proteins; D029961:Zebrafish Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C089212:protein kinase D; D011493:Protein Kinase C