Prohibitin RegulatesTGF-beta Induced Apoptosis as a Downstream Effector of Smad-Dependent and -independent Signaling

BACKGROUND. Prohibitin (PHB), a protein located oil the inner mitochondrial membrane and nuclei, is all 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. METHOD. TGF-beta activation of Raf-Erk intracellular signaling, led to PHB phosphorylation, decreased inner mitochondrial permeability, and increased Cell survival. Calcein-based immonofluorescence Studies revealed the functional involvement of PHB in maintaining inner mitochondrial membrane permeability as all integral component of TGF-beta induced apoptosis in prostate cancer cells. RESULTS. 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. CONCLUSION. 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). Prostate 70: 17-26, 2010. (c) 2009 Wiley-Liss, Inc.