Zhu Brian, Zhai Jianjun, Zhu Haining, Kyprianou Natasha
Department of Surgery, Division of Urology, University of Kentucky College of Medicine, Lexington, Kentucky, USA.
Prostate. 2010 Jan 1;70(1):17-26. doi: 10.1002/pros.21033.
Prohibitin (PHB), a protein located on the inner mitochondrial membrane and nuclei, is an intracellular effector of transforming growth factor-beta (TGF-beta) 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-beta. shRNA PHB loss of function in prostate cancer cells led to enhanced apoptotic response to TGF-beta via Smad-dependent mechanism.
TGF-beta 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-beta induced apoptosis in prostate cancer cells.
These finding indicates that induction of TGF-beta 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-beta 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-beta led to upregulation of the PKC inhibitor 14-3-3 protein and promoted its association with PHB, while PHB association with PKC-delta, 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-beta via Smad-dependent pathway (apoptosis) and MAPK intracellular signaling (survival).
抑制素（PHB）是一种位于线粒体内膜和细胞核的蛋白质，是前列腺癌细胞中转化生长因子-β（TGF-β）信号传导的细胞内效应器。本研究调查了PHB在人前列腺癌细胞对TGF-β的凋亡和生存结果中的作用。前列腺癌细胞中PHB功能丧失的短发夹RNA（shRNA）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，线粒体内膜通透性降低，细胞存活率增加。基于钙黄绿素的免疫荧光研究揭示了PHB作为TGF-β诱导前列腺癌细胞凋亡的一个组成部分，在维持线粒体内膜通透性方面的功能作用。
这些发现表明，TGF-β诱导的凋亡是由Smad依赖性和Smad非依赖性信号传导（MAPK）介导的，它们在PHB处汇聚，PHB作为调节线粒体内膜通透性的下游效应器。通过用抗PHB抗体对TGF-β处理的细胞进行免疫沉淀和质谱分析，鉴定了推定的与PHB相关的蛋白质。对PHB的激酶特异性磷酸化位点的筛选揭示了三个蛋白激酶（PKC）结合位点。
我们的结果表明，TGF-β导致PKC抑制剂14-3-3蛋白上调，并促进其与PHB的结合，而MEK1抑制剂抑制了PHB与PKC-δ的结合，证明了MEK-ERK信号传导与抑制素磷酸化之间的关键相互依赖性。这些发现表明PHB在通过Smad依赖性途径（凋亡）和MAPK细胞内信号传导（生存）对TGF-β的细胞反应中具有双重作用，作为下游决定因素。
