Lysosomal interaction of Akt with Phafin2: a critical step in the induction of autophagy.
Lysosomal interaction of Akt with Phafin2: a critical step in the induction of autophagy.
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DOI：
10.1371/journal.pone.0079795
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Noguchi M
中科院分区：
文献类型：
--
作者：
Matsuda-Lennikov M;Suizu F;Hirata N;Hashimoto M;Kimura K;Nagamine T;Fujioka Y;Ohba Y;Iwanaga T;Noguchi M
自噬是哺乳动物细胞中细胞内蛋白质总体处置的进化上保守的机制，并且该途径中的功能障碍与人类疾病相关。虽然丝氨酸苏氨酸激酶Akt被认为在这个过程中发挥作用，但对Akt诱导自噬的分子机制知之甚少。利用酵母双杂交筛选，发现一种N端PH（pleckstrin homology）结构域和C端FYVE（Fab 1，YOTB，Vac 1和EEA 1）结构域的独特结构的溶酶体蛋白Phafin 2（EAPF或PLEKHF 2）与Akt相互作用。蔗糖梯度分离实验表明，Akt和Phafin 2在自噬诱导后共存于同一溶酶体富集级分中。共聚焦显微镜分析和BiFC分析表明，Akt和Phafin 2在诱导自噬后在溶酶体中积累。使用PtdIns（3）P相互作用缺陷的Phafin 2突变体的BiFC分析表明，Akt-Phafin 2复合物的溶酶体积累和随后的自噬诱导是溶酶体PtdIns（3）P依赖性事件。此外，在小鼠巨噬细胞中，Akt和Phafin 2都是消化荧光细菌和/或LPS诱导的自噬所必需的。综上所述，这些发现证实了Akt和Phafin 2的溶酶体积累是通过与PtdIns（3）P相互作用诱导自噬的关键步骤。
Autophagy is an evolutionarily conserved mechanism for the gross disposal of intracellular proteins in mammalian cells and dysfunction in this pathway has been associated with human disease. Although the serine threonine kinase Akt is suggested to play a role in this process, little is known about the molecular mechanisms by which Akt induces autophagy. Using a yeast two-hybrid screen, Phafin2 (EAPF or PLEKHF2), a lysosomal protein with a unique structure of N-terminal PH (pleckstrin homology) domain and C-terminal FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain was found to interact with Akt. A sucrose gradient fractionation experiment revealed that both Akt and Phafin2 co-existed in the same lysosome enriched fraction after autophagy induction. Confocal microscopic analysis and BiFC analysis demonstrated that both Akt and Phafin2 accumulate in the lysosome after induction of autophagy. BiFC analysis using PtdIns (3)P interaction defective mutant of Phafin2 demonstrated that lysosomal accumulation of the Akt-Phafin2 complex and subsequent induction of autophagy were lysosomal PtdIns (3)P dependent events. Furthermore, in murine macrophages, both Akt and Phafin2 were required for digestion of fluorescent bacteria and/or LPS-induced autophagy. Taken together, these findings establish that lysosomal accumulation of Akt and Phafin2 is a critical step in the induction of autophagy via an interaction with PtdIns (3)P.
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DOI：
10.1083/jcb.200907014
发表时间：
2009-09-21
期刊：
The Journal of cell biology
影响因子：
--
作者：
Simonsen A;Tooze SA
通讯作者：
Tooze SA
影响因子：
4.8
作者：
Petiot, A;Ogier-Denis, E;Codogno, P
通讯作者：
Codogno, P
影响因子：
4.8
作者：
Katome, T;Obata, T;Ebina, Y
通讯作者：
Ebina, Y
影响因子：
64.8
作者：
Levine, Beth;Mizushima, Noboru;Virgin, Herbert W.
通讯作者：
Virgin, Herbert W.
影响因子：
4.8
作者：
Pehar, Mariana;Jonas, Mary Cabell;Puglielli, Luigi
通讯作者：
Puglielli, Luigi