The evolutionarily conserved domain of Beclin 1 is required for Vps34 binding, autophagy and tumor suppressor function
The evolutionarily conserved domain of Beclin 1 is required for Vps34 binding, autophagy and tumor suppressor function
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DOI：
10.4161/auto.1.1.1542
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发表时间：
2005-04-01
期刊：
影响因子：
13.3
通讯作者：
Levine, Beth
中科院分区：
文献类型：
--
作者：
Furuya, Norihiko;Yu, Jie;Levine, Beth
Atg6/Beclin 1是一个进化上保守的蛋白家族，已被证明在酵母的液泡蛋白分选（VPS）中起作用；酵母、果蝇、盘基骨菌、秀丽隐杆线虫和哺乳动物的自噬；在小鼠的肿瘤抑制中。Atg6/Beclin 1被认为作为VPS和自噬蛋白的一部分，与III类磷脂酰肌醇t激酶(PI3K)/Vps34复合物起作用。然而，目前尚不清楚Atg6/Beclin 1的哪些结构域是其功能活性和与Vps34结合所必需的。我们假设人类Beclin 1中最高度保守的区域横跨244-337氨基酸，是Vps34结合、自噬和肿瘤抑制功能所必需的。为了验证这一假设，我们评估了野生型和突变型beclin 1基因在自噬缺陷MCF7人乳腺癌细胞中的转移效果。我们发现，与野生型Beclin 1不同，缺乏aa 244-337 （Beclin 1 Delta ECD）的Beclin 1突变体不能增强低Beclin 1表达MCF7的人乳腺癌细胞中饥饿诱导的自噬。与野生型Beclin 1相比，在MCF7 scid小鼠异种移植肿瘤模型中，突变型Beclin 1 δ ECD不能免疫沉淀Vps34，没有Beclin 1相关的Vps34激酶活性，并且缺乏肿瘤抑制功能。组织蛋白酶D的成熟需要完整的vps34依赖性VPS功能，在低表达Beclin 1的自噬缺陷MCF7细胞、转染Beclin 1 δ ECD的自噬缺陷MCF7细胞和转染野生型Beclin 1的自噬能力MCF7细胞中，组织蛋白酶D的成熟是相似的。这些发现确定了Beclin 1的一个进化保守结构域，该结构域对Vps34相互作用、自噬功能和肿瘤抑制功能至关重要。此外，他们认为Beclin 1相关的III类PI3K/ vps34依赖性自噬与人乳腺癌细胞中Beclin 1抑瘤作用的功能和机制有关，而与VPS无关。
Atg6/Beclin 1 is an evolutionarily conserved protein family that has been shown to function in vacuolar protein sorting (VPS) in yeast; in autophogy in yeast, Drosophila, Dictyostelium, C.elegans, and mammals; and in tumor suppression in mice. Atg6/Beclin 1 is thought to function as a VPS and autophagy protein as part of a complex with Class III phosphatidylinositol T-kinase (PI3K)/Vps34. However, nothing is known about which domains of Atg6/Beclin 1 are required for its functional activity and binding to Vps34. We hypothesized that the most highly conserved region of human Beclin 1 spanning from amino acids 244-337 is essential for Vps34 binding, autophagy, and tumor suppressor function. To investigate this hypothesis, we evaluated the effects of wild-type and mutant beclin 1 gene transfer in autophagy-deficient MCF7 human breast carcinoma cells. We found that, unlike wild-type Beclin 1, a Beclin 1 mutant lacking aa 244-337 (Beclin 1 Delta ECD), is unable to enhance starvation-induced autophogy in low Beclin 1-expressing MCF7 human breast carcinoma cells. In contrast to wild-type Beclin 1, mutant Beclin 1 Delta ECD is unable to immunoprecipitate Vps34, has no Beclin 1-associated Vps34 kinase activity, and lacks tumor suppressor function in an MCF7 scid mouse xenograft tumor model. The maturation of cathepsin D, which requires intact Vps34-dependent VPS function, is comparable in autophagy-deficient low-Beclin 1 expressing MCF7 cells, autophagy-deficient MCF7 cells transfected with Beclin 1 Delta ECD, and autophagy-competent MCF7 cells transfected with wild-type Beclin 1. These findings identify an evolutionarily conserved domain of Beclin 1 that is essential for Vps34 interaction, autophagy function, and tumor suppressor function. Furthermore, they suggest a connection between Beclin 1-associated Class III PI3K/Vps34-dependent autophagy, but not VPS, function and the mechanism of Beclin 1 tumor suppressor action in human breast cancer cells.