The ANKosome: a cytoplasmic activation platform for caspase-2

Project Summary Caspase activation platforms (CAPs) are multi-oligomeric complexes essential for initiating cell death or inflammation through the activation of initiator caspases. CAPs are pivotal players in these processes; they are responsible for both sensing lethal/pathogenic stresses and, in turn, engaging caspase activity in the cell. CAPs have come to define entire signaling pathways and include the apoptosome (cyt-c/APAF1/caspase-9), DISCs (DR/FADD/caspase-8), inflammasomes (NLR,AIM2/ASC/caspase-1) and the CAP for caspase-2 (C2), the PIDDosome (PIDD1/RAIDD/C2; the main focus of our lab for the past decade). Recent evidence points to the existence to at least one additional CAP for caspase-2 which, in contrast to the DNA damage-sensing, nucleolar PIDDosome, assembles in the cytoplasm in response to cytoskeletal injury. This RAIDD-dependent but PIDD1-independent C2 CAP may also be responsible for C2 activation and ensuing neuronal cell death in response to beta-amyloid (Ab), as suggested by elegant experiments in an in vitro model of Alzheimer’s disease (Carol Troy, Columbia University, collaborator). However, the molecular identity of this novel C2 CAP distinct from the PIDDosome has remained unknown. Through a genetic screen and additional preliminary data, we identify a strong candidate for this complex, in which the cytoskeletal adaptor, ankyrin-G (ANKG), substitutes for PIDD1 as scaffold, thus physically coupling cytoskeletal integrity to C2. In ongoing experiments, the newly discovered ANKG/RAIDD/C2 complex, which we designate ‘ANKosome’, assembles and drives cell death in mammalian cells exposed to cytoskeleton-targeted anticancer drugs. In this R21 application, the first to study the ANKosome, we will identify the precise ANKG isoform which nucleates the complex and, through structure/function analyses, elucidate the mechanism by which cytoskeletal injury triggers ANKosome assembly via ANKG. We will also delineate the biologic function(s) of the ANKosome, with two main hypotheses. First, we will test whether the complex truly transduces cytoskeletal damage into an apoptotic response in both mammalian cells and live zebrafish embryos. Second, we will test whether the ANKosome mediates apoptosis in murine neurons exposed to Ab using the Troy lab’s Alzheimer’s disease model discussed above. In further support of this latter hypothesis, high levels of ANKG have been reported to accumulate at Ab plaques in Alzheimer’s disease, contributing to the neurodegenerative phenotype. This short term, high risk/reward project seeks to lay the foundations for a new field in apoptotic signaling, that of the ANKosome, with potentially broad impact ranging from AD to cancer treatment, that is, two of the major public health challenges facing our aging population.