Expression of a catalytically inactive sphingosine kinase mutant blocks agonist-induced sphingosine kinase activation - A dominant-negative sphingosine kinase
Expression of a catalytically inactive sphingosine kinase mutant blocks agonist-induced sphingosine kinase activation - A dominant-negative sphingosine kinase
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DOI：
10.1074/jbc.m006176200
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发表时间：
2000-10-27
影响因子：
4.8
通讯作者：
Wattenberg, BW
中科院分区：
文献类型：
--
作者：
Pitson, SM;Moretti, PAB;Wattenberg, BW
鞘氨醇激酶（SK）催化鞘氨醇1-磷酸（S1 P）的形成，鞘氨醇1-磷酸（S1 P）是在多种哺乳动物细胞过程（包括抑制细胞凋亡和刺激细胞增殖）中起重要作用的脂质信使。细胞中SIP的基础水平通常较低，但当细胞暴露于各种激动剂时，通过SK活性的快速和瞬时激活，SIP的基础水平可迅速增加。迄今为止，受这些升高的S1 P水平影响的确切信号传导途径的阐明依赖于使用已知对细胞中的其他酶具有直接影响的SK抑制剂。此外，这些抑制剂阻断基础SK活性，其被认为在细胞中具有管家功能。为了产生一个特定的抑制剂的SK激活，我们试图产生一个无催化活性，显性负SK。这是通过定点突变的Gly（82）的人SK，一个残基确定通过序列相似性推定的催化结构域的二酰甘油激酶的天冬氨酸。当在HEK 293 T细胞中高水平表达时，该突变体没有检测到SK活性。HEK 293 T细胞中肿瘤坏死因子-α（TNF α）、白细胞介素-β和佛波醇酯对内源性SK活性的激活被这种失活的鞘氨醇激酶（hSK（G82 D））的表达阻断。基础SK活性不受hSR（G82 D）表达的影响。hSK（G82 D）的表达对TNF α诱导的蛋白激酶C和鞘磷脂酶活性的激活没有影响。因此，hSK（G82 D）作为一个特定的显性负SK阻断SK激活。这一发现为阐明SK激活后S1 P水平升高影响的确切信号通路提供了有力的工具。为此，我们采用显性阴性SK来证明细胞外信号调节激酶1和2（ERK 1，2）的TNF α激活依赖于SK激活。
Sphingosine kinase (SK) catalyzes the formation of sphingosine 1-phosphate (S1P), a lipid messenger that plays an important role in a variety of mammalian cell processes, including inhibition of apoptosis and stimulation of cell proliferation. Basal levels of SIP in cells are generally low but can increase rapidly when cells are exposed to various agonists through rapid and transient activation of SK activity. To date, elucidation of the exact signaling pathways affected by these elevated S1P levels has relied on the use of SK inhibitors that are known to have direct effects on other enzymes in the cell. Furthermore, these inhibitors block basal SK activity, which is thought to have a housekeeping function in the cell. To produce a specific inhibitor of SK activation we sought to generate a catalytically inactive, dominant-negative SK. This was accomplished by site-directed mutagenesis of Gly(82) to Asp of the human SK, a residue identified through sequence similarity to the putative catalytic domain of diacylglycerol kinase. This mutant had no detectable SK activity when expressed at high levels in HEK293T cells. Activation of endogenous SK activity by tumor necrosis factor-alpha (TNF alpha), interleukin-lp, and phorbol esters in HEK293T cells was blocked by expression of this inactive sphingosine kinase (hSK(G82D)), Basal SK activity was unaffected by expression of hSR(G82D). Expression of hSK(G82D) had no effect on TNF alpha -induced activation of protein kinase C and sphingomyelinase activities. Thus, hSK(G82D) acts as a specific dominant-negative SK to block SK activation. This discovery provides a powerful tool for the elucidation of the exact signaling pathways affected by elevated S1P levels following SK activation. To this end we have employed the dominant-negative SK to demonstrate that TNF alpha activation of extracellular signal-regulated kinases 1 and 2 (ERK1,2) is dependent on SK activation.