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

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BackgroundApoptosis 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.MethodsCaspase‐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.Results2‐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.ConclusionCombined 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.