Molecular cloning and functional characterization of murine sphinogosine kinase
Molecular cloning and functional characterization of murine sphinogosine kinase
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DOI：
10.1074/jbc.273.37.23722
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发表时间：
1998-09-11
影响因子：
4.8
通讯作者：
Spiegel, S
中科院分区：
文献类型：
--
作者：
Kohama, T;Olivera, A;Spiegel, S
鞘氨醇磷酸（SPP）是一种新型的具有双重功能的脂质信使。在细胞内，它调节增殖和存活，在细胞外，它是G蛋白偶联受体Edp-1的配体。基于从纯化的大鼠肾脏鞘氨醇激酶（调节SPP水平的酶）获得的肽序列，我们在此报告了第一批哺乳动物鞘氨醇激酶的克隆、鉴定和表征（鼠SPHK 1a和SPHK 1b），序列分析表明这些是新的激酶，它们与其他已知的激酶不相似，并且它们在进化上是保守的，与酿酒酵母和秀丽隐杆线虫鞘氨醇激酶序列的比较表明，在所有这些序列中有几个区块是高度保守的。这些块之一包含不变的，带正电荷的基序，GGHGK，这可能是ATP结合位点的一部分。从多个小鼠组织的北方印迹分析，我们观察到在成年肺和脾中表达最高，在骨骼肌和肝中几乎检测不到水平。人胚肾细胞和NIH 3 T3成纤维细胞瞬时转染鞘氨醇激酶表达载体有显着增加（超过100倍）鞘氨醇激酶活性。该酶特异性地磷酸化D-胆-鞘氨醇，并且不催化磷脂酰肌醇、二酰基甘油、神经酰胺、D，L-苏型-二氢鞘氨醇或N，N-二甲基鞘氨醇的磷酸化。后两种鞘脂是鞘氨醇激酶在转染细胞中的竞争性抑制剂，如先前发现的纯化的大鼠肾酶。转染细胞也有一个显着的增加，在质量水平的SPP与鞘氨醇水平的同时减少，在较小程度上，在神经酰胺水平，我们的数据表明，鞘氨醇激酶是一个典型的成员一类新的脂质激酶。鞘氨醇激酶的克隆是证实SPP作为第二信使的细胞内作用的重要步骤。
Sphingosine-l-phosphate (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edp-1. Based on peptide sequences obtained from purified rat kidney sphingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian sphingosine kinases (murine SPHK1a and SPHK1b), Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved, Comparison with Saccharomyces cerevisiae and Caenorhabditis elegans sphingosine kinase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positively charged motif, GGHGK, which may be part of the ATP binding site. From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and Liver. Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) in sphingosine kinase activity. The enzyme specifically phosphorylated D-erythro-sphingosine and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, D,L-threo-dihydrosphingosine or N,N-dimethylsphingosine. The latter two sphingolipids were competitive inhibitors of sphingosine kinase in the transfected cells as was previously found with the purified rat kidney enzyme. Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of sphingosine and, to a lesser extent, in ceramide levels, Our data suggest that sphingosine kinase is a prototypical member of a new class of lipid kinases. Cloning of sphingosine kinase is an important step in corroborating the intracellular role of SPP as a second messenger.