Birchwood C J, Saba J D, Dickson R C, Cunningham K W
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
J Biol Chem. 2001 Apr 13;276(15):11712-8. doi: 10.1074/jbc.M010221200. Epub 2001 Jan 19.
In mammalian cells, intracellular sphingosine 1-phosphate (S1P) can stimulate calcium release from intracellular organelles, resulting in the activation of downstream signaling pathways. The budding yeast Saccharomyces cerevisiae expresses enzymes that can synthesize and degrade S1P and related molecules, but their possible role in calcium signaling has not yet been tested. Here we examine the effects of S1P accumulation on calcium signaling using a variety of yeast mutants. Treatment of yeast cells with exogenous sphingosine stimulated Ca(2+) accumulation through two distinct pathways. The first pathway required the Cch1p and Mid1p subunits of a Ca(2+) influx channel, depended upon the function of sphingosine kinases (Lcb4p and Lcb5p), and was inhibited by the functions of S1P lyase (Dpl1p) and the S1P phosphatase (Lcb3p). The biologically inactive stereoisomer of sphingosine did not activate this Ca(2+) influx pathway, suggesting that the active S1P isomer specifically stimulates a calcium-signaling mechanism in yeast. The second Ca(2+) influx pathway stimulated by the addition of sphingosine was not stereospecific, was not dependent on the sphingosine kinases, occurred only at higher doses of added sphingosine, and therefore was likely to be nonspecific. Mutants lacking both S1P lyase and phosphatase (dpl1 lcb3 double mutants) exhibited constitutively high Ca(2+) accumulation and signaling in the absence of added sphingosine, and these effects were dependent on the sphingosine kinases. These results show that endogenous S1P-related molecules can also trigger Ca(2+) accumulation and signaling. Several stimuli previously shown to evoke calcium signaling in wild-type cells were examined in lcb4 lcb5 double mutants. All of the stimuli produced calcium signals independent of sphingosine kinase activity, suggesting that phosphorylated sphingoid bases might serve as messengers of calcium signaling in yeast during an unknown cellular response.
在哺乳动物细胞中，细胞内的1-磷酸鞘氨醇（S1P）可刺激细胞内细胞器释放钙，从而激活下游信号通路。出芽酵母酿酒酵母表达能够合成和降解S1P及相关分子的酶，但其在钙信号传导中的可能作用尚未得到验证。在此，我们使用多种酵母突变体研究了S1P积累对钙信号传导的影响。用外源性鞘氨醇处理酵母细胞通过两条不同途径刺激Ca(2+)积累。第一条途径需要Ca(2+)流入通道的Cch1p和Mid1p亚基，依赖于鞘氨醇激酶（Lcb4p和Lcb5p）的功能，并受到S1P裂解酶（Dpl1p）和S1P磷酸酶（Lcb3p）功能的抑制。鞘氨醇的无生物学活性的立体异构体不会激活此Ca(2+)流入途径，这表明活性S1P异构体特异性刺激酵母中的钙信号传导机制。添加鞘氨醇刺激的第二条Ca(2+)流入途径不是立体特异性的，不依赖于鞘氨醇激酶，仅在添加更高剂量的鞘氨醇时发生，因此可能是非特异性的。缺乏S1P裂解酶和磷酸酶的突变体（dpl1 lcb3双突变体）在未添加鞘氨醇的情况下表现出组成型高Ca(2+)积累和信号传导，并且这些效应依赖于鞘氨醇激酶。这些结果表明内源性S1P相关分子也可触发Ca(2+)积累和信号传导。在lcb4 lcb5双突变体中研究了几种先前显示可在野生型细胞中引发钙信号传导的刺激。所有刺激均产生独立于鞘氨醇激酶活性的钙信号，这表明磷酸化的鞘氨醇碱可能在未知的细胞反应过程中作为酵母中钙信号传导的信使。
