Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, UK.
Biochem Pharmacol. 2010 Jun 1;79(11):1562-72. doi: 10.1016/j.bcp.2010.01.019. Epub 2010 Jan 25.
BNIP3 is a hypoxia-inducible BH3-only member of the Bcl-2 family of proteins that regulate apoptosis and autophagy. However the role of BNIP3 in the hypoxia response has proved difficult to define and remains controversial. In this study we show that in cancer cells, knockdown or forced expression of BNIP3 fails to modulate cell survival under hypoxic or normoxic conditions. However, we demonstrate that BNIP3 is regulated post-translationally, existing as multiple monomeric and dimeric phosphorylated forms. Upon treatment with microtubule inhibitors, but not other classes of chemotherapeutics, BNIP3 becomes hyperphosphorylated. We demonstrate that the phosphorylation of BNIP3 occurs in synchrony with phosphorylation of its binding partners Bcl-2 and Bcl-xL. Microtubule inhibitor-induced phosphorylation of these proteins occurs independently of the AKT/mTor and JNK kinase pathways and requires Mps1 mitotic checkpoint kinase activity. Inhibition of mitotic arrest in the presence of paclitaxel blocks the phosphorylation of BNIP3, Bcl-2 and Bcl-xL, demonstrating that these proteins are phosphorylated by a mitochondrially active mitotic kinase. We show that phosphorylation increases the stability of BNIP3 and that BNIP3 predominantly interacts with the phosphorylated form of Bcl-2. This study provides new insight into the post-translational functional control of these Bcl-2 family members.
BNIP3 是 Bcl-2 家族蛋白中的一种缺氧诱导型 BH3 仅成员，可调节细胞凋亡和自噬。然而，BNIP3 在缺氧反应中的作用很难确定，并且仍然存在争议。在这项研究中，我们表明在癌细胞中，BNIP3 的敲低或强制表达都不能调节缺氧或常氧条件下的细胞存活。然而，我们证明 BNIP3 是通过翻译后修饰来调节的，存在多种单体和二聚体磷酸化形式。在用微管抑制剂处理时，但不是用其他类别的化疗药物处理时，BNIP3 会发生过度磷酸化。我们证明 BNIP3 的磷酸化与其结合伙伴 Bcl-2 和 Bcl-xL 的磷酸化同步发生。这些蛋白的微管抑制剂诱导的磷酸化独立于 AKT/mTor 和 JNK 激酶途径，并且需要 Mps1 有丝分裂检查点激酶活性。在紫杉醇存在下抑制有丝分裂阻滞会阻止 BNIP3、Bcl-2 和 Bcl-xL 的磷酸化，表明这些蛋白是由线粒体活跃的有丝分裂激酶磷酸化的。我们表明磷酸化增加了 BNIP3 的稳定性，并且 BNIP3 主要与 Bcl-2 的磷酸化形式相互作用。这项研究为这些 Bcl-2 家族成员的翻译后功能控制提供了新的见解。
