Cytokine-dependent activation of the JAK-STAT pathway in Saccharomyces cerevisiae
Cytokine-dependent activation of the JAK-STAT pathway in Saccharomyces cerevisiae
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酿酒酵母中 JAK-STAT 通路的细胞因子依赖性激活
DOI：
10.1002/bit.25948
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发表时间：
2016
影响因子：
3.8
通讯作者：
S.
中科院分区：
文献类型：
--
作者：
Yoshimoto;N.;Ikeda;Y.;Tatematsu;K.;Iijima;M.;Nakai;T.;Okajima;T.;Tanizawa;K.;Kuroda;S.
蛋白质磷酸化是细胞内信号分子的重要翻译后修饰，主要存在于丝氨酸和苏氨酸残基中。酪氨酸磷酸化是非常少的事件（小于磷酸化丝氨酸/苏氨酸残基的0.1%），但能够控制涉及增殖、分化、凋亡和致癌转化的细胞命运决定。因此，测定细胞内磷酸酪氨酸水平对药物开发和系统生物学研究具有重要意义。尽管哺乳动物细胞已常规地用于此目的，但由于内源性信号传导分子之间的意想不到的串扰，磷酸酪氨酸水平的准确测定经常遭受高背景。这种情况使我们首先建立了配体诱导的同型受体酪氨酸激酶的激活（即，表皮生长因子受体），酿酒酵母是一种低等真核生物，具有与高等真核生物相似的细胞器，但没有显示出实质水平的酪氨酸激酶活性。在这项研究中，我们表达了异聚体受体酪氨酸激酶（即，白细胞介素-5受体（IL 5 R）α链、共同β链和JAK 2酪氨酸激酶的复合物）。当与细胞壁锚定形式的IL 5共表达时，酵母产生JAK 2的自磷酸化，随后是转录因子STAT 5a的磷酸化和随后的磷酸化STAT 5a的核积累。两者合计，酵母可能是一个理想的宿主，灵敏地检测由各种酪氨酸激酶产生的磷酸酪氨酸。Biotechnol. Bioeng. 2016;113：1796-1804.© 2016 Wiley Periodicals，Inc.
Protein phosphorylation is an important post‐translational modification for intracellular signaling molecules, mostly found in serine and threonine residues. Tyrosine phosphorylations are very few events (less than 0.1% to phosphorylated serine/threonine residues), but capable of governing cell fate decisions involved in proliferation, differentiation, apoptosis, and oncogenic transformation. Hence, it is important for drug discovery and system biology to measure the intracellular level of phosphotyrosine. Although mammalian cells have been conventionally utilized for this purpose, accurate determination of phosphotyrosine level often suffers from high background due to the unexpected crosstalk among endogenous signaling molecules. This situation led us firstly to establish the ligand‐induced activation of homomeric receptor tyrosine kinase (i.e., epidermal growth factor receptor) inSaccharomyces cerevisiae, a lower eukaryote possessing organelles similar to higher eukaryote but not showing substantial level of tyrosine kinase activity. In this study, we expressed heteromeric receptor tyrosine kinase (i.e., a complex of interleukin‐5 receptor (IL5R) α chain, common β chain, and JAK2 tyrosine kinase) in yeast. When coexpressed with a cell wall‐anchored form of IL5, the yeast exerted the autophosphorylation of JAK2, followed by the phosphorylation of transcription factor STAT5a and subsequent nuclear accumulation of phosphorylated STAT5a. Taken together, yeast could be an ideal host for sensitive detection of phosphotyrosine generated by a wide variety of tyrosine kinases. Biotechnol. Bioeng. 2016;113: 1796–1804. © 2016 Wiley Periodicals, Inc.