Cellular IAP proteins and LUBAC differentially regulate necrosome-associated RIP1 ubiquitination.

M C De Almagro,K Newton,T Goncharov,D Vucic

doi:10.1038/cddis.2015.158

Abstract

Necroptosis is a caspase-independent regulated type of cell death that relies on receptor-interacting protein kinases RIP1 (receptor-interacting protein kinases 1) and RIP3. Tumor necrosis factor-α (TNFα)-stimulated assembly of the TNFR1 (TNF receptor 1)-associated signaling complex leads to the recruitment of RIP1, whose ubiquitination is mediated by the cellular inhibitors of apoptosis (c-IAPs). Translocation of RIP1 to the cytoplasm and association of RIP1 with the necrosome is believed to correlate with deubiquitination of RIP1. However, we found that RIP1 is ubiquitinated with K63 and linear polyubiquitin chains during TNFα, IAP antagonist BV6 and caspase inhibitor zVAD-fmk-induced necroptotic signaling. Furthermore, ubiquitinated RIP1 is associated with the necrosome, and RIP1 ubiquitination in the necrosome coincides with RIP3 phosphorylation. Both cellular IAPs and LUBAC (linear ubiquitin chain assembly complex) modulate RIP1 ubiquitination in IAP antagonist-treated necrotic cells, but they use different mechanisms. c-IAP1 regulates RIP1 recruitment to the necrosome without directly affecting RIP1 ubiquitination, whereas HOIP and HOIL1 mediate linear ubiquitination of RIP1 in the necrosome, but are not essential for necrosome formation. Knockdown of the E3 ligase c-IAP1 decreased RIP1 ubiquitination, necrosome assembly and necroptosis induced by TNFα, BV6 and zVAD-fmk. c-IAP1 deficiency likely decreases necroptotic cell death through the activation of the noncanonical NF-κB pathway and consequent c-IAP2 upregulation. The ability to upregulate c-IAP2 could determine whether c-IAP1 absence will have a positive or negative impact on TNFα-induced necroptotic cell death and necrosome formation. Collectively, these results reveal unexpected complexity of the roles of IAP proteins, IAP antagonists and LUBAC in the regulation of necrosome assembly.

Highlights

RIPK1) and RIP3 and has distinct cellular features, which include early loss of plasma membrane integrity, organelle swelling and inflammation.[4,5,6]
To investigate the ubiquitination status of RIP1 during necroptosis, human colon carcinoma HT29 cells were induced to undergo necroptosis with TNFα, IAP antagonist BV6 and the pancaspase inhibitor zVADfmk
Deubiquitination allows RIP1 to move from the TNFR1-associated complex to the cell death-promoting complex-II or necrosome.[10,34,37]

Summary

Introduction

RIPK1) and RIP3 and has distinct cellular features, which include early loss of plasma membrane integrity, organelle swelling and inflammation.[4,5,6]. LUBAC is an ubiquitin E3 ligase complex that attaches linear polyubiquitin chains on RIP1 and consists of the catalytic subunit HOIP and the auxiliary proteins HOIL1 and sharpin.[14,15,16,17] Diverse ubiquitination chains assembled on RIP1 are proposed to keep RIP1 within complex-I.10,13 c-IAP deficiency, which can be achieved by gene targeting or by using IAP antagonists that stimulate c-IAP proteasomal degradation, permits non-ubiquitinated RIP1 to leave the TNFR1-associated complex and join FADD, caspase-8/10 and FLIP in cytoplasmic complex-II.[10,18,19,20,21] If caspase activation is inhibited, RIP1 engages RIP3 to form the necrosome signaling complex and switches cell death from apoptosis to necroptosis.[22] The kinase activities of RIP1 and RIP3 are essential for necroptosis and the RIP1 kinase inhibitor, necrostatin-1, blocks necroptotic cell death.[23,24,25] Following autophosphorylation of RIP1 and RIP3, phosphorylated RIP3 recruits its substrate, the pseudokinase MLKL (mixed lineage kinase domain-like).

Methods

Results

Conclusion