Identification of DAPK as a scaffold protein for the LIMK/cofilin complex in TNF-induced apoptosis

The role of cytoskeleton-associated proteins during TNF-induced apoptosis is not fully understood. A potential candidate kinase that might connect TNF signaling to actin reorganization is the death-associated protein kinase (DAPK). To identify new DAPK interaction partners in TNF-induced apoptosis, we performed a peptide array screen. We show that TNF-treatment enhanced the phosphorylation of LIMK at threonine508 and its downstream target cofilin at serine3 (p-cofilinSer3). Modulation of DAPK activity and expression by DAPK inhibitor treatment, siRNA knockdown, and overexpression affected the phosphorylation of both proteins. We propose a 3D structural model where DAPK functions as a scaffold for the LIMK/cofilin complex and triggers a closer interaction of both proteins under TNF stimulation. Upon TNF a striking redistribution of LIMK, DAPK, and cofilin to the perinuclear compartment was observed. The pro-apoptotic DAPK/LIMK/cofilin multiprotein complex was abrogated in detached cells, indicating that its signaling was no longer needed if cells committed to apoptosis. P-cofilinSer3 was strongly accumulated in cells with condensed chromatin, pronounced membrane blebs and Annexin V up-regulation. From studying different cofilinSer3 mutants we suggest that p-cofilinSer3 is an indicator of TNF-induced apoptosis. Collectively, our findings identify a novel molecular cytoskeleton-associated mechanism in TNF-induced DAPK-dependent apoptosis.