Studies on Carp JAK1 Kinase

• Main Authors: Chang, Mau-Sun, 張茂山
• Other Authors: Huang, Fore-Lien
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/26496254488977163962

博士 === 國立臺灣大學 === 動物學研究所 === 85 === Using the PCR strategy, we cloned the carp JAK1 kinase that encoded 1,156 amino acid residues. The overall amino acid sequence identity between carp JAK1 and murine JAK1, JAK2, JAK3 and human TYK2 is 57 %, 35.5 %, 31.3% and 42.4 %, respectively. In addition, carp JAK1 shows higher sequence homology to mammalian JAK1 in both the kinase-like (JH2) and kinase (JHI) domains (approximately 70 % identity). Therefore, carp JAK1 is a homologue of mammalian JAK1. In order to investigate the possible function of JH2 domain, full-length and various truncated forms of carp JAK1 were produced in the baculovirus system. Our results demonstrate that c-JH1 and c-JH2 associate with each other and c-JH2 can be tyrosine-phosphorylated by c-JAK1 and by c-JH(1+2).The JAK1 gene was also isolated from a carp genomic library and characterized. This gene is divided into 24 exons spanning at least 31 kb of genomic DNA. Exon I contains the 5''-untranslated region, and exon 2 contains the putative translation initiation site. The 2.5 kb DNA region upstream of the transcription initiation site contains numerous potential binding sites for transcription factors including NF-IL6, HNF-5, AP1, GHF-5, and E2A. When this DNA fragment was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into a carp CF cell line, it could drive the synthesis of CAT enzyme 16 times more efficiently than the promoterless pCAT- Basic. Deletion analysis defined a positive regulatory region between -1023 and - 528. A smaller region (-181 to +59) without any typical TATA-box sequences, G+C-rich sequences, or other binding sequences for known transcription factors still had promoter activity. Constructs without this region did not have detectable promoter activity. This suggests that this region of DNA may play an important role in the expression of carp JAK1 gene. There are two AP1 binding motifs located in the promoter region of JAK1 gene, 1449 and 1221 bp upstream to the transcription start site. In order to investigate whether carp AP1 may regulate the expression of carp JAK1 gene, we cloned the carp c-fos and junB cDNA. The carp c-fos encoded 347 amino acid residues and carp junB encoded 308 amino acid residues. The amino acid sequence identity of carp and human c-fos is 55.2% , and that of carp and human junB is 59.6%. However, the basic region and leucine zipper region of carp/ human c-fos and carp/ human junB showed higher amino acid sequence identity, up to 80% amino acid identity. The carp c-fos and junB was constructed with transfection vector pREP4 and named as RSV-c-fos and RSV-junB, respectively. The pJP1-CAT was cotransfected with RSV-c-fos and RSV-junB into CF cells. Cell extracts were prepared and assayed for the CAT activity at various transfecting time intervals after transfection. The data showed that the CAT activity of cotransfection of pJPI-CAT with both RSV-c-fos and RSV-junB could increase 5-10 times greater than that of pJPI-CAT alone. In addition, cotransfection of both RSV-c-fos and RSV-junB with pJPI-CAT drives more CAT activity than that of either RSV-c-fos or RSV-junB with pJPI-CAT. However, the mechanism how carp API regulates the promoter activity of carp JAK1 gene is still not clear. Further investigations will be needed to shed light on this question.