Phosphatidic Acid Binds and Stimulates Arabidopsis Sphingosine Kinases
Phosphatidic Acid Binds and Stimulates Arabidopsis Sphingosine Kinases
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DOI：
10.1074/jbc.m110.190892
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发表时间：
2011-04-15
影响因子：
4.8
通讯作者：
Wang, Xuemin
中科院分区：
文献类型：
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作者：
Guo, Liang;Mishra, Girish;Wang, Xuemin
磷脂酸（PA）和植物鞘氨醇-1-磷酸（phyto-S1 P）都被鉴定为介导植物对脱落酸（阿坝）应答的脂质信使。为了确定这些信使的关系，我们研究了PA与拟南芥鞘氨醇激酶（SPHKs）磷酸化植物鞘氨醇产生植物S1 P的直接相互作用。从拟南芥At 4g 21540基因座克隆了两个SPHK cDNA，这两个转录本在拟南芥组织中差异表达。两种SPHK都具有催化活性，使各种长链鞘氨醇碱（LCB）磷酸化，并与液泡膜相关。它们都与PA相互作用，如通过脂质过滤器结合、脂质体结合和表面等离子体共振（SPR）所证明的。SPHK 1和SPHK 2与18：1/18：1、16：0/18：1和16：0/18：2 PA的结合较强，但与16：0/16：0、8：0/8：0、18：0/18：0和18：2/18：2 PA的结合较差。表面稀释动力学分析表明，PA通过降低Km（B）增加特异性常数来刺激SPHK活性。结果表明，注释的At 4g 21540基因座实际上由两个独立的SPHK基因组成。PA结合两种SPHK，并且相互作用促进脂质底物结合到酶的催化位点。PA-SPHK相互作用取决于PA分子种类。这些数据表明，这两个拟南芥SPHK是PA的分子靶标，PA刺激SPHK是拟南芥信号网络的一部分。
Phosphatidic acid (PA) and phytosphingosine-1-phosphate (phyto-S1P) have both been identified as lipid messengers mediating plant response to abscisic acid (ABA). To determine the relationship of these messengers, we investigated the direct interaction of PA with Arabidopsis sphingosine kinases (SPHKs) that phosphorylate phytosphingosine to generate phyto-S1P. Two unique SPHK cDNAs were cloned from the annotated At4g21540 locus of Arabidopsis, and the two transcripts are differentially expressed in Arabidopsis tissues. Both SPHKs are catalytically active, phosphorylating various long-chain sphingoid bases (LCBs) and are associated with the tonoplast. They both interact with PA as demonstrated by lipid-filter binding, liposome binding, and surface plasmon resonance (SPR). SPHK1 and SPHK2 exhibited strong binding to 18:1/18:1, 16:0/18:1, and 16:0/18:2 PA, but poor binding to 16:0/16:0, 8:0/8:0, 18:0/18:0, and 18:2/18:2 PA. Surface dilution kinetics analysis indicates that PA stimulates SPHK activity by increasing the specificity constant through decreasing K-m(B). The results show that the annotated At4g21540 locus is actually comprised of two separate SPHK genes. PA binds to both SPHKs, and the interaction promotes lipid substrate binding to the catalytic site of the enzyme. The PA-SPHK interaction depends on the PA molecular species. The data suggest that these two Arabidopsis SPHKs are molecular targets of PA, and the PA stimulation of SPHK is part of the signaling networks in Arabidopsis.