Evasion of cell death is one crucial capability acquired by tumour cells to ward-off anti-tumour therapies and represents a fundamental challenge to sustaining clinical efficacy for currently available agents. Inhibitor of apoptosis (IAP) proteins use their ubiquitin E3 ligase activity to promote cancer cell survival by mediating proliferative signalling and blocking cell death in response to diverse stimuli. Using immunoaffinity enrichment and MS, ubiquitination sites on thousands of proteins were profiled upon initiation of cell death by IAP antagonists in IAP antagonist-sensitive and -resistant breast cancer cell lines. Our analyses identified hundreds of proteins with elevated levels of ubiquitin-remnant [K-GG (Lys-Gly-Gly)] peptides upon activation of cell death by the IAP antagonist BV6. The majority of these were observed in BV6-sensitive, but not-resistant, cells. Among these were known pro-apoptotic regulators, including CYC (cytochrome c), RIP1 (receptor-interacting protein 1) and a selection of proteins known to reside in the mitochondria or regulate NF-κB (nuclear factor κB) signalling. Analysis of early time-points revealed that IAP antagonist treatment stimulated rapid ubiquitination of NF-κB signalling proteins, including TRAF2 [TNF (tumour necrosis factor) receptor-associated factor 2], HOIL-1 (haem-oxidized iron-regulatory protein 2 ubiquitin ligase-1), NEMO (NF-κB essential modifier), as well as c-IAP1 (cellular IAP1) auto-ubiquitination. Knockdown of several NF-κB pathway members reduced BV6-induced cell death and TNF production in sensitive cell lines. Importantly, RIP1 was found to be constitutively ubiquitinated in sensitive breast-cancer cell lines at higher basal level than in resistant cell lines. Together, these data show the diverse and temporally defined roles of protein ubiquitination following IAP-antagonist treatment and provide critical insights into predictive diagnostics that may enhance clinical efficacy.
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February 2015
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Research Article|
February 06 2015
Ubiquitination profiling identifies sensitivity factors for IAP antagonist treatment
Eugene Varfolomeev;
Eugene Varfolomeev
*Departments of Early Discovery Biochemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
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Anita Izrael-Tomasevic;
Anita Izrael-Tomasevic
†Protein Chemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
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Kebing Yu;
Kebing Yu
†Protein Chemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
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Daisy Bustos;
Daisy Bustos
†Protein Chemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
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Tatiana Goncharov;
Tatiana Goncharov
*Departments of Early Discovery Biochemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
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Lisa D. Belmont;
Lisa D. Belmont
‡Discovery Oncology, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
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Alexandre Masselot;
Alexandre Masselot
†Protein Chemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
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Corey E. Bakalarski;
Corey E. Bakalarski
†Protein Chemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
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Donald S. Kirkpatrick;
Donald S. Kirkpatrick
1
†Protein Chemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
1Correspondence may be addressed to either of these authors (email donaldk@gene.com or domagoj@gene.com).
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Domagoj Vucic
Domagoj Vucic
1
*Departments of Early Discovery Biochemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
1Correspondence may be addressed to either of these authors (email donaldk@gene.com or domagoj@gene.com).
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Publisher: Portland Press Ltd
Received:
September 22 2014
Revision Received:
November 10 2014
Accepted:
November 21 2014
Accepted Manuscript online:
November 21 2014
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2015 Biochemical Society
2015
Biochem J (2015) 466 (1): 45–54.
Article history
Received:
September 22 2014
Revision Received:
November 10 2014
Accepted:
November 21 2014
Accepted Manuscript online:
November 21 2014
Citation
Eugene Varfolomeev, Anita Izrael-Tomasevic, Kebing Yu, Daisy Bustos, Tatiana Goncharov, Lisa D. Belmont, Alexandre Masselot, Corey E. Bakalarski, Donald S. Kirkpatrick, Domagoj Vucic; Ubiquitination profiling identifies sensitivity factors for IAP antagonist treatment. Biochem J 15 February 2015; 466 (1): 45–54. doi: https://doi.org/10.1042/BJ20141195
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