Phosphorylation of the Retinoblastoma protein (Rb) on serine-807 is required for association with Bax.
Phosphorylation of the Retinoblastoma protein (Rb) on serine-807 is required for association with Bax.
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DOI：
10.4161/15384101.2014.964093
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Krucher NA
中科院分区：
文献类型：
--
作者：
Antonucci LA;Egger JV;Krucher NA
最近的发现，视网膜母细胞瘤蛋白（Rb）是能够直接在线粒体通过与促凋亡蛋白Bax的相互作用，以非转录的方式调节细胞凋亡，促使Rb和Bax之间形成的复合物的调查。由于Rb在细胞增殖、凋亡、衰老和分化过程中的功能是通过磷酸化来调节的，因此我们有必要阐明Rb的磷酸化状态及其与Bax的关系及其在凋亡中的作用。在这项研究中，我们发现Rb磷酸化的至少4个C-末端磷酸化位点（S608，S795，S807/S811，和T821）是目前在线粒体在非应激细胞条件下。体外结合实验表明，Bax与3种癌细胞中S807/S811磷酸化的Rb结合。Bax和Rb在S807/S811上磷酸化之间的生理相关关联通过使用对Rb在S807/S811和Bax上磷酸化具有特异性的抗体的相互免疫共沉淀实验来证明。在Rb缺失的C33 A细胞中表达的突变型Rb蛋白表明，Rb的S807磷酸化促进了与Bax的结合，并且模拟Rb的S807磷酸化可以阻止由于PNUTS下调而诱导的细胞凋亡。最后，使用siRNA激活MCF 7细胞中的磷酸酶活性，Rb在包括S807/S811的几个位点被去磷酸化，与Bax解离并触发凋亡。这些研究表明，Rb的磷酸化调节其与Bax的关联及其在凋亡中的作用。
The recent finding that the Retinoblastoma protein (Rb) is able to regulate apoptosis in a non-transcriptional manner directly at the mitochondria by interaction with the pro-apoptotic protein Bax prompted this investigation of the complex formed between Rb and Bax. Because the function of Rb in the cellular processes of proliferation, apoptosis, senescence and differentiation is regulated by phosphorylation we endeavored to elucidate the phosphorylation status of Rb with respect to its association with Bax and its role in apoptosis. In this study we found that Rb phosphorylated on at least 4 C-terminal phosphorylation sites (S608, S795, S807/S811, and T821) is present at the mitochondria under non-stressed cellular conditions. An in vitro binding assay showed that Bax binds to Rb phosphorylated at S807/S811 in 3 cancer cell types. Physiologically relevant association between Bax and Rb phosphorylated on S807/S811 was demonstrated by reciprocal co-immunoprecipitation experiments using antibodies specific for Rb phosphorylated on S807/S811 and Bax. Mutant Rb proteins expressed in Rb-null C33A cells showed that phosphorylation of S807 of Rb promotes association with Bax and that mimicking phosphorylation at S807 of Rb can block the induction of apoptosis due to PNUTS downregulation. Finally using siRNA to activate phosphatase activity in MCF7 cells, Rb is dephosphorylated at several sites including S807/S811, dissociates from Bax and apoptosis is triggered. These studies show that phosphorylation of Rb regulates its association with Bax and its role in apoptosis.