Abstract
Death-associated protein kinase 1 (DAPK1, DAPk, DAPK) is known for its involvement in apoptosis and autophagy-associated cell death. Here, we identified an unexpected function of DAPK1 in suppressing necroptosis. DAPK1-deficiency renders macrophages and dendritic cells susceptible to necroptotic death. We also observed an inhibitory role for DAPK1 in necroptosis in HT-29 cells, since knockdown or knockout of DAPK1 in such cells increased their sensitivity to necroptosis. Increased necroptosis was associated with enhanced formation of the RIPK1–RIPK3–MLKL complex in these DAPK1-deficient cells. We further found that DAPK1-deficiency led to decreased MAPK activated kinase 2 (MK2) activation and reduced RIPK1 S321 phosphorylation, with this latter representing a critical step controlling necrosome formation. Most TNF signaling pathways, including ERK, JNK, and AKT, were not regulated by DAPK. In contrast, DAPK bound p38 MAPK and selectively promoted p38 MAPK activation, resulting in enhanced MK2 phosphorylation. Our results reveal a novel role for DAPK1 in inhibiting necroptosis and illustrate an unexpected selectivity for DAPK1 in promoting p38 MAPK-MK2 activation. Importantly, our study suggests that modulation of necroptosis and p38/MK2-mediated inflammation may be achieved by targeting DAPK1.
Highlights
Necroptosis is typically induced in cells via activation of the receptor-interacting protein kinase 1 (RIPK1)-RIPK3 cascade and concomitant inactivation of the Fas-associated protein with death domain (FADD)–caspase-8–c-FLIP complex[1,2,3,4,5,6]
DAPK1-deficiency sensitizes myeloid cells to necroptosis We used bone marrow-derived macrophages (BMDMs) from wild-type control (WT) and Dapk−/− mice to examine the possible role of DAPK1 in necroptosis
DAPK1 knockout did not affect the development of myeloid cells in bone marrow or spleen (Supplementary Fig. 1), nor did DAPK1 deficiency affect the protein expression of RIPK1, RIPK3, mixed lineage kinase domain-like (MLKL), or FADD in BMDMs (Fig. 1a)
Summary
Necroptosis is typically induced in cells via activation of the receptor-interacting protein kinase 1 (RIPK1)-RIPK3 cascade and concomitant inactivation of the FADD–caspase-8–c-FLIP complex[1,2,3,4,5,6]. Binding of TNF to TNF receptor 1 (TNFR1) leads to assembly of complex I8 at the plasma membranes by recruitment of TNF receptorassociated protein with death domain (TRADD) and RIPK1, followed by TNFR-associated factor 2 (TRAF2), TRAF5, cellular inhibitors of apoptosis 1 (cIAP1) and cIAP2, and the linear ubiquitin chain assembly complex (LUBAC). Inactivation or depletion of caspase-8 converts pro-apoptotic complex II into a pro-necroptotic complex, with RIPK1 binding and activating RIPK39–11, resulting in RIPK3 autophosphorylation and activation of mixed lineage kinase domain-like (MLKL)[12,13].
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