Distinct classes of phosphatidylinositol 3′-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells
Distinct classes of phosphatidylinositol 3′-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells
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DOI：
10.1074/jbc.275.2.992
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发表时间：
2000-01-14
影响因子：
4.8
通讯作者：
Codogno, P
中科院分区：
文献类型：
--
作者：
Petiot, A;Ogier-Denis, E;Codogno, P
3-甲基腺嘌呤在哺乳动物细胞的隔离步骤中停止巨自噬，也抑制磷酸肌醇 3-激酶 (PI3K) 活性，提高了 PI3K 信号传导控制巨自噬途径的可能性（Blommaart, E. F. C., Krause, U., Schellens, J. P. M., Vreeling-Sindelarova, H., 和 Meijer, A. J. (1997) 毛皮。 J，生物化学。 243、240-246）。本研究的目的是鉴定参与控制人结肠癌 HT-29 细胞巨自噬隔离的 PI3K。 I 类 PISK 产品（磷脂酰肌醇 3,4-二磷酸和磷脂酰肌醇 3,4,5-三磷酸）的增加是由于用合成脂质（二棕榈酰磷脂酰肌醇 3,4-二磷酸和二棕榈酰磷脂酰肌醇 3,4,5-三磷酸）喂养细胞或由 通过白细胞介素 13 刺激酶活性可减少巨自噬，相反，通过用合成脂质喂养细胞或通过过度表达 p150 接头，增加 III 类 PISK 产物（磷脂酰肌醇 3-磷酸）会刺激巨自噬。特定的 III 类 PISK 反义寡核苷酸的转染极大地抑制了巨自噬的速率。根据 III 类 PISK（渥曼青霉素 PI3K 抑制剂）的作用，在低纳摩尔范围（IC50 5-15 nM）抑制巨自噬隔离和蛋白质降解。进一步的体外酶测定表明，3-甲基腺嘌呤抑制 III 类 PISK 活性。二棕榈酰磷脂酰肌醇 3-磷酸补充剂或 p150 过表达可挽救过表达 G α(i3) 蛋白 GTPase 缺陷突变体的 HT-29 细胞中的巨自噬途径，表明在 HT-29 细胞的对照巨自噬中需要 III 类 PISK 和三聚体 G(i3) 蛋白信号传导。总之，我们的结果表明不同类别的 PI3K 以相反的方向控制巨自噬途径。在膜回收的背景下讨论了 PI3K 在巨自噬中的作用。
3-Methyladenine which stops macroautophagy at the sequestration step in mammalian cells also inhibits the phosphoinositide 3-kinase (PI3K) activity raising the possibility that PI3K signaling controls the macroautophagic pathway (Blommaart, E. F. C., Krause, U., Schellens, J. P. M., Vreeling-Sindelarova, H., and Meijer, A. J. (1997) fur. J, Biochem. 243, 240-246). The aim of this study was to identify PI3Ks involved in the control of macroautophagic sequestration in human colon cancer HT-29 cells. An increase of class I PISK products (phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate) caused by either feeding cells with synthetic lipids (dipalmitoyl phosphatidylinositol 3,4-bisphosphate and dipalmitoyl phosphatidylinositol 3,4,5-triphosphate) or by stimulating the enzymatic activity by interleukin-13 reduced macroautophagy, In contrast, an increase in the class III PISK product (phosphatidylinositol 3-phosphate), either by feeding cells with a synthetic lipid or by overexpressing the p150 adaptor, stimulates macroautophagy. Transfection of a specific class III PISK antisense oligonucleotide greatly inhibited the rate of macroautophagy. In accordance with a role of class III PISK, wortmannin tan inhibitor of PI3Ks) inhibits macroautophagic sequestration and protein degradation in the low nanomolar range (IC50 5-15 nM), Further in vitro enzymatic assay showed that 3-methyladenine inhibits the class III PISK activity. Dipalmitoyl phosphatidylinositol 3-phosphate supplementation or p150 overexpression rescued the macroautophagic pathway in HT-29 cells overexpressing a GTPase-deficient mutant of the G alpha(i3) protein suggesting that both class III PISK and trimeric G(i3) protein signaling are required in the control macroautophagy in HT-29 cells. In conclusion, our results demonstrate that distinct classes of PI3K control the macroautophagic pathway in opposite directions. The roles of PI3Ks in macroautophagy are discussed in the context of membrane recycling.