Cleavage of RIP3 inactivates its caspase-independent apoptosis pathway by removal of kinase domain.
RIP3 (Receptor Interacting Protein 3), a member of the Ser/Thr kinase family, is able to induce apoptosis and activate NF-kappaB in various cell types. However, the detailed mechanism of RIP3-induced apoptosis is largely unknown. In this study, we show that RIP3 is cleaved at Asp328 by caspase-8 under apoptotic stimuli, which is blocked by pan-caspase inhibitor Z-VAD-FMK. In addition, full-length RIP3 induces both caspase-dependent and-independent apoptosis, as well as activates NF-kappaB. However, after cleavage, the C-terminus of RIP3 (aa 329-518) that lacks the kinase domain can form punctuate or filaments-like structures in cytoplasm, which induces only caspase-dependent apoptosis and exhibits a markedly higher NF-kappaB-activating activity than full-length RIP3. More importantly, the cleaved product of RIP3 (aa 329-518) displays better stability than wild type RIP3. Additionally, RIP3(K50A), a kinase-dead RIP3 mutant, also induces only caspase-dependent apoptosis along with an increased NF-kappaB-activating activity compared to RIP3, which further demonstrates that kinase activity of RIP3 is essential for its caspase-independent apoptotic activity. These results will help us to understand the mechanism underlying RIP3-induced apoptosis and the different roles of kinase domain and unique domain of RIP3.
Feng
Shanshan
S
Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, People's Republic of China.
Yang
Yonghui
Y
Mei
Yide
Y
Ma
Li
L
Zhu
De-e
DE
Hoti
Naseruddin
N
Castanares
Mark
M
Wu
Mian
M
D000818:Animals; D017209:Apoptosis; D053181:Caspase 8 / Q000378:metabolism; D020169:Caspases / Q000378:metabolism*; D020134:Catalytic Domain; D002460:Cell Line; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction
D016328:NF-kappa B; C119705:RIPK3 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D053181:Caspase 8; D020169:Caspases