Jak2-Stat5 interactions analyzed in yeast
Jak2-Stat5 interactions analyzed in yeast
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DOI：
10.1074/jbc.273.20.12567
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发表时间：
1998-05-15
影响因子：
4.8
通讯作者：
Decker, T
中科院分区：
文献类型：
--
作者：
Barahmand-Pour, F;Meinke, A;Decker, T
许多细胞因子受体采用Janus蛋白酪氨酸激酶（Jaks）和信号转导和转录激活因子（Stats）进行核信号传导。在此，我们建立了酵母菌株，其中自激活的Jak 2激酶诱导酪氨酸磷酸化、二聚化、核转位和伴随表达的Stat 5蛋白的DNA结合。Stat 5的转录活性稳定整合，Stat-dependent报告基因需要的C-末端融合的VP 16的反式激活结构域。在这样的酵母菌株中，分析了Jak 2和Stat 5之间的相互作用，而不受参与调节Jak-Stat信号传导的其他哺乳动物蛋白质的干扰，并分析了这两种蛋白质的突变形式的有效相互作用的能力。Jab 2和Stat 5之间的复合物被发现在严格的免疫共沉淀条件下是稳定的。Jak 2的Jak同源区2-7（JH 2-JH 7）的缺失，仅留下激酶结构域（JH 1）完整，降低了激酶磷酸化Stat 5的能力，而JH 2结构域的缺失导致酶活性增加。Stat 5 SH 2结构域中的定点R618 K突变消除了Jak 2的磷酸化，而C末端的缺失导致Stat 5过度磷酸化。单个磷酸酪氨酸-SH 2结构域相互作用足以使Stat 5二聚化，但这种二聚体与DNA的结合效率非常低。总之，我们的数据表明，酵母细胞是研究Jak-Stat或Stat-Stat相互作用的合适工具。我们的突变分析表明，Stat 5 SH 2结构域是必不可少的相互作用与Jak 2和激酶结构域的Jak 2是足够的Jak 2-Stat 5相互作用。因此，Jak激酶结构域可能是在受体对接被阻断的情况下引起Stat磷酸化所需的全部。
Many cytokine receptors employ Janus protein tyrosine kinases (Jaks) and signal transducers and activators of transcription (Stats) for nuclear signaling, Here, we have established yeast strains in which an autoactivated Jak2 kinase induces tyrosine phosphorylation, dimerization, nuclear translocation, and DNA binding of a concomitantly expressed Stat5 protein. Transcriptional activity of Stat5 on a stably integrated, Stat-dependent reporter gene required the C-terminal fusion of the VP16 transactivation domain. In such yeast strains, the interaction between Jak2 and Stat5 was analyzed without interference by other mammalian proteins involved in regulating Jak-Stat signaling, and mutant versions of both proteins were analyzed for their ability to productively interact. Complexes between Jab2 and Stat5 were found to be stable under stringent co-immunoprecipitation conditions. Deletion of the Jak homology regions 2-7 (JH2-JH7) of Jak2, leaving only the kinase domain (JH1) intact, reduced the ability of the kinase to phosphorylate Stat5, whereas deletion of the JH2 domain caused an increased enzymatic activity, A site-directed R618K mutation in the Stat5 SH2 domain abolished the phosphorylation by Jak2, while deletion of the C terminus led to Stat5 hyperphosphorylation. A single phosphotyrosine-SH2 domain interaction was sufficient for the dimerization of Stat5, but such dimers bound to DNA very inefficiently. Together, our data show that yeast cells are appropriate tools for studying Jak-Stat or Stat-Stat interactions. Our mutational analysis suggests that the Stat5 SH2 domain is essential for the interaction with Jak2 and that the kinase domain of Jak2 is sufficient for Jak2-Stat5 interaction. Therefore, the Jak kinase domain may be all that is needed to cause Stat phosphorylation in situations where receptor docking is dispensable.