Reconstitution of the mammalian PI3K/PTEN/Akt pathway in yeast
Reconstitution of the mammalian PI3K/PTEN/Akt pathway in yeast
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DOI：
10.1042/bj20050574
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发表时间：
2005-09-01
影响因子：
4.1
通讯作者：
Cid, VJ
中科院分区：
文献类型：
--
作者：
Rodríguez-Escudero, I;Roelants, FM;Cid, VJ
哺乳动物信号转导通路涉及磷脂酰肌醇3-磷酸酶、磷脂酰肌醇3-磷酸酶和蛋白激酶B/c-Akt，参与细胞增殖和凋亡等多个过程。为了促进这些蛋白质的遗传、分子和药理学分析的新方法，我们通过在单细胞真核生物酿酒酵母(酵母)中异源表达来重建这一信号通路。哺乳动物PI3K的p110催化亚基的高水平表达显著抑制酵母细胞的生长。这种作用依赖于PI3K激酶的活性，并被PI3K抑制剂(LY294002)部分逆转，并被催化活性的PTEN(但不是其所谓的酵母同源基因Tepl)的共表达完全逆转。PI3K的生长停滞与PIP2(磷脂酰肌醇4，5-二磷酸)的丧失及其转化为PIP(磷脂酰肌醇3，4，5-三磷酸)有关。PIP2的缺失会导致肌动蛋白和隔膜蛋白结构的严重重排，分泌和内吞作用的缺陷，以及丝裂原激活的蛋白激酶SLT2的激活。在产生PIP3的酵母中，PKB/c-Akt定位于质膜并增强其磷酸化。磷酸化特异性抗体表明，活性的和死亡的PKB/c-Akt都在Thr(308)和Ser(473)Thr(301)被磷酸化，但不是Ser(473)磷酸化，需要哺乳动物PDK1(3-磷酸肌醇依赖的蛋白激酶-1)的酵母同源物：Pkh1和Pkh2。消除酵母Tor1和Tor2的功能，或相关的激酶(TEL1、MEC1和TRA1)，并不阻止Ser(473)的磷酸化，暗示另一种激酶(S)。在酵母中重建PI3K/PTEN/Akt通路可以在简化的环境中深入研究这些酶并分析它们的功能相互作用，建立一种新的工具来筛选新的激动剂和拮抗剂，并提供一种在酵母细胞中唯一耗尽PIP2的方法。
The mammalian signalling pathway involving class IPI3K (phosphoinositide 3-kinase), PTEN (phosphatidylinositol 3-phosphatase) and PKB (protein kinase B)/c-Akt has roles in multiple processes, including cell proliferation and apoptosis. To facilitate novel approaches for genetic, molecular and pharmacological analyses of these proteins, we have reconstituted this signalling pathway by heterologous expression in the unicellular eukaryote, Saccharomyces cerevisiae (yeast). High-level expression of the p110 catalytic subunit of mammalian PI3K dramatically inhibits yeast cell growth. This effect depends on PI3K kinase activity and is reversed partially by a PI3K inhibitor (LY294002) and reversed fully by co-expression of catalytically active PTEN (but not its purported yeast orthologue, Tepl). Growth arrest by PI3K correlates with loss of PIP2 (phosphatidyfinositol 4,5-bisphosphate) and its conversion into PIP, (phosphatidylinositol 3,4,5-trisphosphate). PIP2 depletion causes severe rearrangements of actin and septin architecture, defects in secretion and endocytosis, and activation of the mitogen-activated protein kinase, Slt2. In yeast producing PIP3, PKB/c-Akt localizes to the plasma membrane and its phosphorylation is enhanced. Phospho-specific antibodies show that both active and kinase-dead PKB/c-Akt are phosphorylated at Thr(308) and Ser(473) Thr(301) phosphorylation, but not Ser(473) phosphorylation, requires the yeast orthologues of mammalian PDK1 (3-phosphoinositide-dependent protein kinase-1): Pkh1 and Pkh2. Elimination of yeast Tor1 and Tor2 function, or of the related kinases (Tel1, Mec1 and Tra1), did not block Ser(473) phosphorylation,implicating another kinase(s). Reconstruction of the PI3K/PTEN/Akt pathway in yeast permits incisive study of these enzymes and analysis of their functional interactions in a simplified context, establishes a new tool to screen for novel agonists and antagonists and provides a method to deplete PIP2 uniquely in the yeast cell.