Prohibitin regulates TGF-beta induced apoptosis as a downstream effector of Smad-dependent and -independent signaling.
Prohibitin regulates TGF-beta induced apoptosis as a downstream effector of Smad-dependent and -independent signaling.
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DOI：
10.1002/pros.21033
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发表时间：
2010-01-01
期刊：
影响因子：
2.8
通讯作者：
Kyprianou, Natasha
中科院分区：
文献类型：
--
作者：
Zhu, Brian;Zhai, Jianjun;Zhu, Haining;Kyprianou, Natasha
Prohibitin（PHB）是一种位于线粒体内膜和细胞核上的蛋白质，是前列腺癌细胞中转化生长因子-β（TGF-β）信号转导的细胞内效应物。本研究探讨了转化生长因子-β（TGF-β）诱导人前列腺癌细胞凋亡和生存的过程中，聚羟基丁酸（PHB）的作用。前列腺癌细胞中的shRNA PHB功能丧失通过Smad依赖性机制导致对TGF-β的凋亡反应增强。TGF-β激活Raf-Erk细胞内信号传导，导致PHB磷酸化，降低内部线粒体通透性，并增加细胞存活。基于钙黄绿素的免疫荧光研究揭示了在前列腺癌细胞中，作为TGF-β诱导的细胞凋亡的组成部分，在维持线粒体内膜通透性中，PHB的功能参与。我们的研究结果表明，诱导TGF-β凋亡是由Smad依赖性和Smad非依赖性信号转导（MAPK）汇聚在聚羟基丁酸作为下游效应调节线粒体内通透性。通过使TGF-β处理的细胞与抗-PHB进行免疫沉淀和质谱分析来鉴定推定的PHB相关蛋白。筛选的激酶特异性磷酸化位点的PHB揭示了三个蛋白激酶（PKC）结合位点。我们的研究结果表明，TGF-β导致PKC抑制剂14-3-3蛋白的上调，并促进其与PHB的关联，而PHB与PKC-δ的关联被MEK 1抑制剂抑制，证明了MEK-ERK信号传导和prohibitin磷酸化之间的关键相互依赖性。这些发现表明，PHB作为通过Smad依赖性途径（凋亡）和MAPK细胞内信号传导（存活）对TGF-β的细胞应答的下游决定因素的双重作用。
Prohibitin (PHB), a protein located on the inner mitochondrial membrane and nuclei, is an intracellular effector of transforming growth factor-β (TGF-β) signaling in prostate cancer cells. This study investigated the involvement of PHB in the apoptosis and survival outcomes of human prostate cancer cell to TGF-β. shRNA PHB loss of function in prostate cancer cells led to enhanced apoptotic response to TGF-β via Smad-dependent mechanism. TGF-β activation of Raf-Erk intracellular signaling, led to PHB phosphorylation, decreased inner mitochondrial permeability, and increased cell survival. Calcein-based immunofluorescence studies revealed the functional involvement of PHB in maintaining inner mitochondrial membrane permeability as an integral component of TGF-β induced apoptosis in prostate cancer cells. Our results demonstrate that induction of TGF-β apoptosis is mediated by Smad-dependent and Smad-independent signaling (MAPK) converging at PHB as a downstream effector regulating inner mitochondrial permeability. Putative PHB associated proteins were identified by subjecting TGF-β treated cells to immunoprecipitation with anti-PHB, and mass spectrometry. A screen for the kinase specific phosphorylation sites of PHB revealed three protein kinase (PKC) binding sites. Our results demonstrate that TGF-β led to upregulation of the PKC inhibitor 14-3-3 protein and promoted its association with PHB, while PHB association with PKC-δ, was inhibited by the MEK1 inhibitor, documenting a critical interdependence between the MEK-ERK signaling and prohibitin phosphorylation. These findings suggest a dual role for PHB as a downstream determinant of the cellular response to TGF-β via Smad-dependent pathway (apoptosis) and MAPK intracellular signaling (survival).
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