Caspase-3 directly binds to MK2 and this binding is required for nuclear transport of caspase-3 and execution of apoptosis

RATIONALE: Lung endothelial cell apoptosis is an irreversible determinant of endothelial barrier dysfunction in acute respiratory distress syndrome (ARDS). Mitogen activated protein kinase activated protein kinase 2 (MK2) is a kinase that is part of a critical stress-activated protein kinase pathway implicated in activation of the apoptotic cascade. The nuclear localization sequence (NLS) on MK2 is required for caspase-3 to enter the nucleus and subsequent execution of apoptosis. How caspase-3 utilizes the NLS of MK2, however, is poorly understood. We hypothesize that caspase-3 directly binds to MK2 and that this binding is required for nuclear translocation into the nucleus. METHODS: Cell-free binding assays were performed with recombinant proteins: MK2, wildtype (WT) caspase-3, and caspase-3 with a mutated putative binding domain (BD-Casp3). Direct binding was assessed via GST-pulldown assays as well as His-Nickel column pulldown assays with immunoblotting. Endothelial cells isolated from global caspase-3 knockout mice (MEC) were infected with adenoviral constructs encoding eGFP, WT caspase-3, or BD-Casp3 for 48 hours. Following exposure to TNFα, apoptosis was measured via Hoechst staining. In separate parallel experiments, cell lysates were generated to assess caspase 3 activity and for immunoblotting. RESULTS: Direct binding of WT MK2 and caspase-3 was demonstrated in a bidirectional manner with both GST- and His-Nickel column pulldown methods, and this binding was attenuated between WT MK2 and BD-Casp3. There was no difference in caspase activity between the recombinant WT caspase-3 and BD-Casp3. Cells infected with WT caspase-3 had higher rates of apoptosis following exposure to TNFα compared to eGFP and BDCasp3. There was no significant difference in overall caspase-3 activity between cells infected with WT caspase-3 and cells infected with BD-Casp3. CONCLUSION: We have demonstrated direct binding between recombinant MK2 and caspase-3 in multiple cell-free pulldown assays. Mutation of a putative binding domain of caspase-3 led to less direct binding and reduced apoptotic response to TNFα. Future experiments will evaluate the effect of mutating the putative binding domain of caspase-3 on its nuclear localization and endothelial barrier function. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.