Abstract 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 is funded by: NIH. Award Number T32HL007534. Award Number 1F32HL182184-01.
Kamal et al. (Fri,) studied this question.