Mitogen Activated Protein Kinase Activated Protein Kinase 2 (MK2) Interacts with the Apoptotic Cascade in Two Distinct Ways to Promote 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 initiating the apoptotic cascade. Activation of the apoptotic cascade culminates in activation of caspase-3, an executioner caspase, and the purported terminal step of apoptosis. How MK2 directly interacts with the apoptotic pathway, however, is not fully understood, and the focus of these studies. METHODS: Two separate cell culture models were used: H23 cells (low endogenous MK2 expression) and human lung microvascular endothelial cells (HLMVECs). Cells were infected with various adenoviral constructs containing: eGFP, wildtype (WT) MK2 or MK2 mutant constructs. Cell-free phosphorylation assays containing recombinant MK2, caspase-3, and radio-labeled phosphate were then performed. Separate non-radio-labeled samples were analyzed via mass spectrometry. Caspase-3 activity was measured using a commercially available kit. Apoptosis was measured using flow cytometry. RESULTS: In H23 cells, cells infected with the constitutively active MK2 mutant construct had higher caspase-3 activity compared to WT MK2 and eGFP. In HLMVECs, cells infected with the constitutively active MK2 construct similarly had higher caspase-3 activity compared to WT MK2, and cells infected with a dominant-negative MK2 activity construct had lower caspase-3 activity than WT MK2. Use of cell-free assays demonstrated MK2-dependent phosphorylation of caspase-3 which was confirmed via mass spectrometry. Proximity-based biotinylation assays and co-immunoprecipitation demonstrated binding of MK2 and caspase-3. Interestingly, mutation of the nuclear localization sequence of MK2 still allowed for binding to caspase-3 but prevented nuclear translocation of the MK2-caspase-3 complex and prevented apoptosis. CONCLUSION: Increased MK2 kinase activity potentiated caspase-3 activity in vitro in HLMVECs and a model cell line. The exact mechanism of increased caspase-3 activation by MK2 activity is not clear but may occur via direct phosphorylation of caspase-3 by MK2. Additionally, MK2 binds with caspase-3 during nuclear translocation and execution of apoptosis. Disrupting the binding between MK2 and caspase-3 or preventing nuclear translocation of this complex may attenuate apoptosis. In total, our data shows that MK2 interacts with caspase-3 in two distinct ways to promote apoptosis. Research reported was supported by the National Heart, Lung, And Blood Institute of the National Institutes of Health under Award Number T32HL007534. This abstract was presented at the American Physiology Summit 2025 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.