PIKfyve regulation of endosome-linked pathways.
PIKfyve regulation of endosome-linked pathways.
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DOI：
10.1111/j.1600-0854.2009.00915.x
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发表时间：
2009-07
期刊：
影响因子：
--
通讯作者：
Clague MJ
中科院分区：
文献类型：
--
作者：
de Lartigue J;Polson H;Feldman M;Shokat K;Tooze SA;Urbé S;Clague MJ
磷酸肌醇5-激酶（PIKfyve）是合成PtdIns（3，5）P2的关键酶，其已经涉及与内吞途径相关的各种运输事件。我们现在已经直接比较了HeLa细胞中siRNA介导的PIKfyve敲低与酶活性的特异性药理学抑制剂的作用。这两种方法都诱导CI-M6 PR和trans-Golgi网络（TGN）-46蛋白分布的变化，这些蛋白在内体和TGN之间循环，导致它们在分散的斑点中积累，而TGN标记物golgin-245保留核周分布。给药后，CD 8-CI-M6 PR（逆转录依赖性）和CD 8-Furin（逆转录非依赖性）嵌合体从细胞表面向TGN的运输延迟，滋贺毒素B亚基的运输也是如此。PIKfyve的siRNA敲除在表皮生长因子受体（EGFR）降解中没有产生缺陷，除非与其激活分子Vac 14的敲除组合，这表明PtdIns（3，5）P2的低阈值对于该途径是必要的和足够的。因此，PIKfyve的药理学抑制导致对活化的表皮生长因子（EGF）和Met受体的溶酶体降解的深刻阻断。免疫荧光显示EGF受体被困在肿胀的内体室的内部。在缺乏氨基酸的细胞中，PIKfyve抑制导致GFP-LC 3的脂化形式的积累，GFP-LC 3是自噬体结构的标志物，其可以被可视化为荧光斑点。我们认为PIKfyve抑制可能使晚期内体/溶酶体隔室难以与自噬体和含EGFR的多泡体融合。
The phosphoinositide 5-kinase (PIKfyve) is a critical enzyme for the synthesis of PtdIns(3,5)P2, that has been implicated in various trafficking events associated with the endocytic pathway. We have now directly compared the effects of siRNA-mediated knockdown of PIKfyve in HeLa cells with a specific pharmacological inhibitor of enzyme activity. Both approaches induce changes in the distribution of CI-M6PR and trans-Golgi network (TGN)-46 proteins, which cycles between endosomes and TGN, leading to their accumulation in dispersed punctae, whilst the TGN marker golgin-245 retains a perinuclear disposition. Trafficking of CD8-CI-M6PR (retromer-dependent) and CD8-Furin (retromer-independent) chimeras from the cell surface to the TGN is delayed following drug administration, as is the transport of the Shiga toxin B-subunit. siRNA knockdown of PIKfyve produced no defect in epidermal growth factor receptor (EGFR) degradation, unless combined with knockdown of its activator molecule Vac14, suggesting that a low threshold of PtdIns(3,5)P2 is necessary and sufficient for this pathway. Accordingly pharmacological inhibition of PIKfyve results in a profound block to the lysosomal degradation of activated epidermal growth factor (EGF) and Met receptors. Immunofluorescence revealed EGF receptors to be trapped in the interior of a swollen endosomal compartment. In cells starved of amino acids, PIKfyve inhibition leads to the accumulation of the lipidated form of GFP-LC3, a marker of autophagosomal structures, which can be visualized as fluorescent punctae. We suggest that PIKfyve inhibition may render the late endosome/lysosome compartment refractory to fusion with both autophagosomes and with EGFR-containing multivesicular bodies.
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影响因子：
64.5
作者：
Knight, Zachary A.;Gonzalez, Beatriz;Shokat, Kevan M.
通讯作者：
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影响因子：
4.8
作者：
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影响因子：
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作者：
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DOI：
10.1083/jcb.200612068
发表时间：
2007-06-18
期刊：
The Journal of cell biology
影响因子：
--
作者：
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通讯作者：
Barr FA
影响因子：
9.2
作者：
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通讯作者：
Clague, MJ