Nuclear localization of 5-lipoxygenase as a determinant of leukotriene B4 synthetic capacity
Nuclear localization of 5-lipoxygenase as a determinant of leukotriene B4 synthetic capacity
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DOI：
10.1073/pnas.2133253100
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发表时间：
2003-10-14
影响因子：
11.1
通讯作者：
Brock, TG
中科院分区：
文献类型：
--
作者：
Luo, M;Jones, SM;Brock, TG
酶5-脂氧合酶（5-LO）起始从花生四烯酸合成白三烯。在静息细胞中，5-LO可以积聚在细胞质或核质中，并且在细胞刺激时，易位到膜以启动白三烯合成。在这里，我们使用突变体的5-LO改变亚细胞定位，以评估的作用，核定位在确定白三烯B-4（LTB 4）的合成。核定位序列或磷酸化位点的突变使LTB 4的合成减少60%，与5-LO的核定位减少平行。两个位点一起突变或5-LO上所有三个核定位序列的突变抑制LTB 4合成90%，并取消核定位。突变体中LTB 4生成减少不能归因于5-LO量、酶活性或膜结合的差异。相反，细胞核内的5-LO作用于不同的位点，即核膜，而细胞溶质5-LO作用于细胞质和核周膜。外源性花生四烯酸与活化的核5-LO共定位的证据表明，这种差异的意义。这些结果明确地表明，5-LO在静息细胞核内的定位是随后激活时产生LTB 4的能力的有力决定因素。
The enzyme 5-lipoxygenase (5-LO) initiates the synthesis of leukotrienes from arachidonic acid. In resting cells, 5-LO can accumulate in either the cytoplasm or the nucleoplasm and, upon cell stimulation, translocates to membranes to initiate leukotriene synthesis. Here, we used mutants of 5-LO with altered subcellular localization to assess the role that nuclear positioning plays in determining leukotriene B-4 (LTB4) synthesis. Mutation of either a nuclear localization sequence or a phosphorylation site reduced LTB4 synthesis by 60%, in parallel with reduced nuclear localization of 5-LO. Mutation of both sites together or mutation of all three nuclear localization sequences on 5-LO inhibited LTB4 synthesis by 90% and abolished nuclear localization. Reduced LTB4 generation in mutants could not be attributed to differences in 5-LO amount, enzymatic activity, or membrane association. Instead, 5-LO within the nucleus acts at a different site, the nuclear envelope, than does cytosolic 5-LO, which acts at cytoplasmic and perinuclear membranes. The significance of this difference was suggested by evidence that exogenously derived arachidonic acid colocalized with activated nuclear 5-LO. These results unequivocally demonstrate that the positioning of 5-LO within the nucleus of resting cells is a powerful determinant of the capacity to generate LTB4 upon subsequent activation.