Abstract
Caspase‐2 has been shown to initiate apoptotic cell death in response to specific intracellular stressors such as DNA damage. However, the molecular mechanisms immediately upstream of its activation are still poorly understood. We combined a caspase‐2 bimolecular fluorescence complementation (BiFC) system with fluorophore‐specific immunoprecipitation to isolate and study the active caspase‐2 dimer and its interactome. Using this technique, we found that tumor necrosis factor receptor‐associated factor 2 (TRAF2), as well as TRAF1 and 3, directly binds to the active caspase‐2 dimer. TRAF2 in particular is necessary for caspase‐2 activation in response to apoptotic cell death stimuli. Furthermore, we found that dimerized caspase‐2 is ubiquitylated in a TRAF2‐dependent manner at K15, K152, and K153, which in turn stabilizes the active caspase‐2 dimer complex, promotes its association with an insoluble cellular fraction, and enhances its activity to fully commit the cell to apoptosis. Together, these data indicate that TRAF2 positively regulates caspase‐2 activation and consequent cell death by driving its activation through dimer‐stabilizing ubiquitylation.
Highlights
Protein–protein interactions often result in multimeric complexes that induce signaling to significantly alter cellular biology
We examined if GFP-Trap could immunoprecipitate low levels of caspase-2 bimolecular fluorescence complementation (BiFC) dimers induced by known caspase-2 apoptotic stimuli (Bouchier-Hayes et al, 2009)
RAIDD knockdown affected neither caspase-2 dimerization nor apoptosis in response to cisplatin (Fig EV2D–F). These results suggest that the PIDDosome is not involved in caspase-2 dimerization in this context
Summary
Protein–protein interactions often result in multimeric complexes that induce signaling to significantly alter cellular biology. As such, these protein complexes are tightly controlled by a number of factors such as protein expression or post-translational modifications like phosphorylation or ubiquitylation. Its emerging role as a tumor suppressor (Ho et al, 2009; Dorstyn et al, 2012; Manzl et al, 2012; Parsons et al, 2013; Puccini et al, 2013; Terry et al, 2015; Shalini et al, 2016) and a promoter of metabolic disorders (Johnson et al, 2013; Machado et al, 2015, 2016) indicate that modulating its activity could have therapeutic potential. The molecular regulation of caspase-2 activation has remained largely obscure
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