Proteomic Analysis Reveals a Role for Bcl2-associated Athanogene 3 and Major Vault Protein in Resistance to Apoptosis in Senescent Cells by Regulating ERK1/2 Activation

Martina P Pasillas,Sarah Shields,Rebecca Reilly,Jan Strnadel,Christian Behl,Robin Park,John R Yates,Richard Klemke,Steven L Gonias,Judith A Coppinger

doi:10.1074/mcp.m114.037697

Abstract

Senescence is a prominent solid tumor response to therapy in which cells avoid apoptosis and instead enter into prolonged cell cycle arrest. We applied a quantitative proteomics screen to identify signals that lead to therapy-induced senescence and discovered that Bcl2-associated athanogene 3 (Bag3) is up-regulated after adriamycin treatment in MCF7 cells. Bag3 is a member of the BAG family of co-chaperones that interacts with Hsp70. Bag3 also regulates major cell-signaling pathways. Mass spectrometry analysis of the Bag3 Complex revealed a novel interaction between Bag3 and Major Vault Protein (MVP). Silencing of Bag3 or MVP shifts the cellular response to adriamycin to favor apoptosis. We demonstrate that Bag3 and MVP contribute to apoptosis resistance in therapy-induced senescence by increasing the level of activation of extracellular signal-regulated kinase1/2 (ERK1/2). Silencing of either Bag3 or MVP decreased ERK1/2 activation and promoted apoptosis in adriamycin-treated cells. An increase in nuclear accumulation of MVP is observed during therapy-induced senescence and the shift in MVP subcellular localization is Bag3-dependent. We propose a model in which Bag3 binds to MVP and facilitates MVP accumulation in the nucleus, which sustains ERK1/2 activation. We confirmed that silencing of Bag3 or MVP shifts the response toward apoptosis and regulates ERK1/2 activation in a panel of diverse breast cancer cell lines. This study highlights Bag3-MVP as an important complex that regulates a potent prosurvival signaling pathway and contributes to chemotherapy resistance in breast cancer.

Highlights

From the ‡Department of Pathology, University of California San Diego, La Jolla, CA 92093; §Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland; ¶Institute for Pathobiochemistry, University Medical Center of the Johannes Gutenberg-University Mainz, Duesbergweg 6, D-55099 Mainz, Germany; ʈDepartment of Chemical Physiology, the Scripps Research Institute, La, Jolla, California 92037
We show that Bcl2 associated athanogene 3 (Bag3) and Major Vault Protein (MVP) contribute to apoptosis resistance by regulating extracellular signal-regulated kinase1/2 (ERK1/2) signaling in senescent MCF7 and ZR751 cells
MVP and Bag3 Promote Resistance to Apoptosis by Regulating ERK1/2 in MCF7 Cells—As Bag3 and MVP have been shown to stabilize and scaffold different signaling pathways, we examined their role in regulating signaling pathways implicated in therapy-induced senescence (TIS)

Summary

Introduction

From the ‡Department of Pathology, University of California San Diego, La Jolla, CA 92093; §Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland; ¶Institute for Pathobiochemistry, University Medical Center of the Johannes Gutenberg-University Mainz, Duesbergweg 6, D-55099 Mainz, Germany; ʈDepartment of Chemical Physiology, the Scripps Research Institute, La, Jolla, California 92037. We show that Bag3 and MVP contribute to apoptosis resistance by regulating ERK1/2 signaling in senescent MCF7 and ZR751 cells. We observed increased levels of the cleaved PARP protein fragment (89 kDa) as well as cleaved caspase 7 in Bag3 gene-silenced cells after 3 days of adriamycin treatment, confirming the increase in apoptosis (Fig. 4B).

Results

Conclusion