DAPK2 positively regulates motility of neutrophils and eosinophils in response to intermediary chemoattractants

Abstract

The tight regulation of granulocyte chemotaxis is crucial for initiation and resolution of inflammation. Here, we show that DAPK2, a Ca(2+)/CaM-sensitive serine/threonine kinase known to modulate cell death in various cell types, is a novel regulator of migration in granulocytes. We demonstrate that human neutrophils and eosinophils express DAPK2 but unlike other leukocytes, no DAPK1 or DAPK3 protein. When DAPK activities were blocked by inhibitors, we found that neither granulocyte lifespan nor phagocytosis was affected. However, such pharmacological inactivation of DAPK activity abolished motility of granulocytes in response to intermediary but not end-target chemoattractants ex vivo. The defect in chemotaxis in DAPK2-inactive granulocytes is likely a result of reduced polarization of the cells, mediated by a lack of MLC phosphorylation, resulting in radial F-actin and pseudopod formation. As neutrophils treated with DAPKi also showed reduced recruitment to the site of inflammation in a mouse peritonitis model, DAPK2 may be a novel target for anti-inflammatory therapies.

Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation

“…S4 in the supplemental material). Quantification of the changes in cell-free area over time revealed that ectopic expression of two complementary split YFP-DAPK2 constructs (YFP N -DAPK2 and YFP C -DAPK2), leading to DAPK2 homodimerization, indeed resulted in cells with increased migration compared to levels in cells expressing control constructs, in line with previous reports that inhibition of DAPK2 activity slows down cellular motility (17). Expression of split YFP-DAPK2 and -CaM or -␣-actinin-1, however, caused the cells to move more slowly, resulting in reduced closure of the wound.…”

“…Whereas DAPK1 negatively regulates migration of cells by interfering with integrin signaling (30), DAPK2 positively regulates migration by acting on spreading and polarization (17). Here, we used a scratch assay to investigate whether DAPK2 homodimerization or its interaction with CaM, ␣-actinin-1, or 14-3-3-␤ would have an impact on cellular migration.…”

“…DAPK2 was previously shown to trigger cellular blebbing, programmed cell death, and motility when ectopically expressed (5,6,17). We therefore analyzed whether the newly identified interaction partners might have an impact on any of these properties.…”

“…As DAPK2 was previously shown to mediate neutrophil differentiation and chemotaxis (16,17), differentiated neutrophils were used for this experimental setup. FLAG-tagged DAPK2 protein was overexpressed in NB4 myeloid precursor cells, and the cells were incubated with alltrans-retinoic acid (ATRA) in order to differentiate the cells to neutrophil-like entities.…”

“…We demonstrated that DAPK2 positively regulates the differentiation of innate immune cells (16) and boosts granulocyte chemotaxis by modulating cellular spreading and polarization (17). As granulocytes treated with DAPK2 inhibitors showed reduced migration toward the site of inflammation in a mouse model of peritonitis, DAPK2 may be a novel target for anti-inflammatory therapies (17). For a recent review on the regulatory role of DAPK2 on apoptosis, autophagy, and inflammation, see Geering (18).…”