Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan, ROC.
Fish Shellfish Immunol. 2010 May-Jun;28(5-6):774-82. doi: 10.1016/j.fsi.2010.01.022. Epub 2010 Feb 13.
In this study, we isolated and characterized both JAK and STAT genes from Artemia, Artemia franciscana. Although AfJAK showed only 19% identity (33% similarity) to the Drosophila Hop protein, AfJAK contained the characteristic JAK homology domain (JH domain) from JH1 to JH7. On the other hand, AfSTAT showed higher identity (30%) to Drosophila STAT (STAT92E). The low identities of AfJAK and AfSTAT to Drosophila Hop and STAT92E suggest that JAK and STAT proteins are unique in each different species of invertebrate. RT-PCR analysis showed that both AfJAK and AfSTAT transcripts were ubiquitously expressed in the embryo, which is similar to the expression patterns of Drosophila Hop and STAT92E mRNAs during development. In addition, we generated a constitutively active form of AfSTAT by fusing the JH1 domain of AfJAK to the C-terminal end of AfSTAT. This fusion protein, AfSTAT-HA-JH1, autophosphorylated on its tyrosine residue and was able to bind to specific DNA motifs including the STAT-binding motifs in the Drosophila Raf promoter. Both AfJAK and AfSTAT proteins elicited the transactivation potential toward the fly Raf promoter in Sf9 cells. However, tyrosine phosphorylation of AfSTAT was not detected, which is consistent with the cellular localization analysis that most AfSTAT proteins were in the cytoplasm. Our results demonstrate that both JAK and STAT are present in the genome of Artemia, which can serve as the basis for further investigations to explore the role of the JAK/STAT signal pathway in the development and immune response of brine shrimp.
在这项研究中，我们从卤虫（Artemia franciscana）中分离并鉴定了 JAK 和 STAT 基因。虽然 AfJAK 与果蝇 Hop 蛋白的同源性仅为 19%（33%相似性），但它包含了 JH1 到 JH7 的特征 JAK 同源结构域（JH 结构域）。另一方面，AfSTAT 与果蝇 STAT（STAT92E）具有更高的同源性（30%）。AfJAK 和 AfSTAT 与果蝇 Hop 和 STAT92E 的低同源性表明，JAK 和 STAT 蛋白在不同无脊椎动物物种中是独特的。RT-PCR 分析显示，AfJAK 和 AfSTAT 的转录本在胚胎中广泛表达，这与果蝇 Hop 和 STAT92E mRNA 在发育过程中的表达模式相似。此外，我们通过将 AfJAK 的 JH1 结构域融合到 AfSTAT 的 C 末端，生成了一种组成型激活形式的 AfSTAT。这种融合蛋白 AfSTAT-HA-JH1 在其酪氨酸残基上发生自身磷酸化，并能够与包括果蝇 Raf 启动子中的 STAT 结合基序在内的特定 DNA 基序结合。AfJAK 和 AfSTAT 蛋白都能在 Sf9 细胞中激活果蝇 Raf 启动子的转录活性。然而，未检测到 AfSTAT 的酪氨酸磷酸化，这与细胞质中大多数 AfSTAT 蛋白的细胞定位分析结果一致。我们的结果表明，JAK 和 STAT 都存在于卤虫的基因组中，这可以为进一步研究探索 JAK/STAT 信号通路在卤虫发育和免疫反应中的作用提供基础。
