Cullin3-Based Polyubiquitination and p62-Dependent Aggregation of Caspase-8 Mediate Extrinsic Apoptosis Signaling

Abstract

Cell-surface death receptors such as DR4 and DR5 trigger apoptosis through a death-inducing signaling complex (DISC) that recruits the apical protease caspase-8. Apoptosis commitment requires efficient activation and autocatalytic release of caspase-8 into the cytoplasm to engage executioner caspases. While DISC recruitment initiates caspase-8 stimulation, full activation of the protease depends on further molecular aggregation events that are not fully understood. Here, we show that death receptor ligation induces polyubiquitination of caspase-8, through a previously unknown interaction of the DISC with a cullin3 (CUL3)-based E3 ligase. CUL3-mediated caspase-8 polyubiquitination required the RING box protein RBX1, whereas the deubiquitinase A20 reversed this modification. The ubiquitin-binding protein p62/sequestosome-1 promoted aggregation of CUL3-modified caspase-8 within p62-dependent foci, leading to full activation and processing of the enzyme and driving commitment to cell death. These results identify a mechanism that positively controls apoptosis signaling by polyubiquitination and aggregation of a key initiator caspase.

DR5‐Cbl‐b/c‐Cbl‐TRAF2 complex inhibits TRAIL‐induced apoptosis by promoting TRAF2‐mediated polyubiquitination of caspase‐8 in gastric cancer cells

“…The E3 ubiquitin ligase cullin 3 (CUL3)‐based polyubiquitination and p62‐dependent aggregation of caspase‐8 mediated extrinsic apoptosis signaling (Jin et al ., 2009). In prostate cancer cells, TRAIL induced caspase‐8 polyubiquitination, and the inhibition of caspase‐8 polyubiquitination restored caspase‐8 activation and apoptotic signaling (Fiandalo et al ., 2013).…”

Regulation of death receptor signaling by the ubiquitin system

“…Jin et al 103 recently reported that DR4/5 stimulation by Apo2L/TRAIL induces K63-linked ubiquitination of caspase-8. Caspase-8 is polyubiquitinated on the p10 subunit, which stabilizes the processed caspase-8 heterotetramer by recruiting the ubiquitin-binding protein p62.…”

“…As caspase-8 is modified by both K48-and K63-linked polyubiquitin chains after Apo2L treatment, 103 it will be interesting to identify the physiological E2 used by the CUL3 ligase complex and to determine how the E2 binds ROC1 and orients the acceptor ubiquitin such that K63-linked ubiquitin chains are polymerized. A20 is recruited to the complex and mediates caspase-8 deubiquitination, 103 thus it is also possible that A20 facilitates caspase-8 K48 polyubiquitination. In addition, the identity of the adaptor protein that recruits caspase-8 to the CUL3/ROC1 complex is of intense interest.…”

“…K63-polyubiquitinated caspase-8 has more potent enzymatic activity and more readily promotes apoptosis, and RNAi of CUL3, ROC1, and p62 attenuate Apo2L/TRAIL-induced apoptosis. 103 The report of K63-linked ubiquitin chain polymerization by a cullin-containing ubiquitin ligase complex is novel. As caspase-8 is modified by both K48-and K63-linked polyubiquitin chains after Apo2L treatment, 103 it will be interesting to identify the physiological E2 used by the CUL3 ligase complex and to determine how the E2 binds ROC1 and orients the acceptor ubiquitin such that K63-linked ubiquitin chains are polymerized.…”

“…103 The report of K63-linked ubiquitin chain polymerization by a cullin-containing ubiquitin ligase complex is novel. As caspase-8 is modified by both K48-and K63-linked polyubiquitin chains after Apo2L treatment, 103 it will be interesting to identify the physiological E2 used by the CUL3 ligase complex and to determine how the E2 binds ROC1 and orients the acceptor ubiquitin such that K63-linked ubiquitin chains are polymerized. A20 is recruited to the complex and mediates caspase-8 deubiquitination, 103 thus it is also possible that A20 facilitates caspase-8 K48 polyubiquitination.…”