MK2 Phosphorylates Caspase‐3, Facilitates Nuclear Translocation of Caspase 3, and Regulates Apoptosis

Apoptosis is a necessary and key pathologic feature in the development of acute lung injury (ALI). Previous research has identified MAPK Activated Protein Kinase 2 (MK2) as a potential regulator in the development of apoptosis, by facilitating the nuclear translocation of active caspase-3, the executioner of apoptosis. Specifically, loss of MK2 results in cytoplasmic sequestration of active caspase-3. Since MK2 is a kinase, we hypothesize that MK2 phosphorylates caspase-3 leading to its nuclear translocation and ultimately executing apoptosis.Caspase-3 phosphorylation was determined by 2-D gel electrophoresis with and without phosphatases, in addition to direct protein kinase assays using radiolabelled phosphate. Caspase-3 localization was determined by performing nuclear and cytoplasmic extractions, and subsequent measurement of caspase-3 activity. Apoptosis was determined by flow cytometry. NCI-H23 (non-small cell cancer, NSLC) cells were used as a model due to their ability to resist apoptosis, as well as their ability to sequester active-caspase-3 in the cytoplasm.2-D gel electrophoresis showed that the p-19 fragment of cleaved caspase-3, the fragment that translocates to the nucleus, has a shift in isoelectric focus that is absent with the loss of MK2. In addition, 2-D gel analyses of lung homogenates from WT mice exposed to LPS and treated with phosphatase have similar isoelectric foci for caspase 3 as lung homogenates from MK2-/- mice exposed to LPS. Furthermore, phosphor-imaging shows that MK2 directly phosphorylates caspase-3. Interestingly, immunoblots of NCI-H23 cells (which sequester active-caspase-3 in the cytoplasm) have lower MK2 expression than the small-cell lung cancer cell line, H446. Flow cytometry with DAPI staining demonstrated increased apoptosis following etoposide treatment in cells over-expressing MK2 compared to control transfections. Over-expression of MK2 led to an increase in nuclear caspase-3 activity. Additionally, overexpression of nuclear localization sequence (NLS) tagged caspase 3 led to increased etoposide induced apoptosis compared to WT caspase 3 or control transfections.Combined these results strongly suggest that MK2 phosphorylates caspase-3, which may facilitate the nuclear translocation of caspase-3, thereby promoting the execution of apoptosis. Further work is needed to understand the mechanisms by which MK2 regulates the trafficking of active caspase-3.