Sli2 (Ypk1), a Homologue of Mammalian Protein Kinase SGK, Is a Downstream Kinase in the Sphingolipid-Mediated Signaling Pathway of Yeast

ISP-1 is a new type of immunosuppressant, the structure of which is homologous to that of sphingosine. In a previous study, ISP-1 was found to inhibit mammalian serine palmitoyltransferase, the primary enzyme involved in sphingolipid biosynthesis, and to reduce the intracellular pool of sphingolipids. ISP-1 induces the apoptosis of cytotoxic T cells, which is triggered by decreases in the intracellular levels of sphingolipids. In this study, the inhibition of yeast (Saccharomyces cerevisiae) proliferation by ISP-1 was observed. This ISP-1-induced growth inhibition was also triggered by decreases in the intracellular levels of sphingolipids. In addition, DNA duplication without cytokinesis was detected in ISP-1-treated yeast cells on flow cytometry analysis. We have cloned multicopy suppressor genes of yeast which overcome the lethal sphingolipid depletion induced by ISP-1. One of these genes, SLI2, is synonymous with YPK1, which encodes a serine/threonine kinase. Kinase-dead mutants of YPK1 did not show any resistance to ISP-1, leading us to predict that the kinase activity of the Ypk1 protein should be essential for this resistance to ISP-1. Ypk1 protein overexpression had no effect on sphingolipid biosynthesis by the yeast. Furthermore, both the phosphorylation and intracellular localization of the Ypk1 protein were regulated by the intracellular sphingolipid levels. These data suggest that the Ypk1 protein is a downstream kinase in the sphingolipid-mediated signaling pathway of yeast. The Ypk1 protein was reported to be a functional homologue of the mammalian protein kinase SGK, which is a downstream kinase of 3-phosphoinositide-dependent kinase 1 (PDK1). PDK1 phosphotidylinositol (PI) is regulated by PI-3,4,5-triphosphate and PI-3,4-bisphosphate through the pleckstrin homology (PH) domain. Overexpression of mammalian SGK also overcomes the sphingolipid depletion in yeast. Taking both the inability to produce PI-3,4,5-triphosphate and PI-3,4-bisphosphate and the lack of a PH domain in the yeast homologue of PDK1, the Pkh1 protein, into account, these findings further suggest that yeast may use sphingolipids instead of inositol phospholipids as lipid mediators.

ISP-1是一种新型免疫抑制剂，其结构与鞘氨醇（sphingosine）具有同源性。既往研究表明，ISP-1可抑制哺乳动物丝氨酸棕榈酰转移酶（serine palmitoyltransferase）——鞘脂生物合成的核心关键酶，并降低细胞内鞘脂池水平。ISP-1可诱导细胞毒性T细胞（cytotoxic T cells）凋亡，该效应由细胞内鞘脂水平下降所触发。本研究观察到ISP-1可抑制酿酒酵母（Saccharomyces cerevisiae）的增殖，且该生长抑制效应同样由细胞内鞘脂水平降低所介导。此外，流式细胞术分析显示，经ISP-1处理的酵母细胞出现了胞质分裂受阻伴DNA复制异常的表型。我们已克隆出可克服ISP-1诱导的酵母鞘脂耗竭致死效应的多拷贝抑制基因（multicopy suppressor genes）。其中一个基因SLI2与YPK1为等位基因，后者编码一种丝氨酸/苏氨酸激酶（serine/threonine kinase）。YPK1的激酶失活突变体（kinase-dead mutants）未表现出对ISP-1的抗性，提示Ypk1蛋白的激酶活性是其介导ISP-1抗性的必要条件。Ypk1蛋白的过表达并未对酵母鞘脂生物合成产生影响。进一步研究发现，Ypk1蛋白的磷酸化与细胞内定位均受细胞内鞘脂水平调控。上述数据表明，Ypk1蛋白是酵母鞘脂介导信号通路中的下游激酶。已有研究显示，Ypk1蛋白与哺乳动物血清和糖皮质激素调节激酶（SGK）为功能同源物，而SGK是3-磷酸肌醇依赖性蛋白激酶1（3-phosphoinositide-dependent kinase 1, PDK1）的下游激酶。PDK1可通过其pleckstrin同源结构域（PH结构域），受PI-3,4,5-三磷酸（PI-3,4,5-triphosphate）与PI-3,4-二磷酸（PI-3,4-bisphosphate）的调控。哺乳动物SGK的过表达同样可逆转酵母中的鞘脂耗竭表型。结合酵母PDK1同源物Pkh1蛋白无法合成PI-3,4,5-三磷酸与PI-3,4-二磷酸，且缺乏PH结构域这一事实，上述发现进一步提示：酵母可能以鞘脂替代肌醇磷脂作为脂质信号介导分子。