Control of DAPK-1 degradation

Abstract

DAPK-1 is calcium-calmodulin regulated protein kinase involved in multiple cellular pathways including apoptosis, autophagy, cell survival and motility. The cytokine TNF-α has been reported to induce the degradation of DAPK-1. Here I identified the protease cathepsin B as a novel binding partner of DAPK-1 that protects DAPK-1 from TNF-α induced degradation. Using deletion mutants of DAPK-1, I mapped the cathepsin B binding domain on DAPK-1 to amino acids 836-947. Overexpression of this mini-protein DAPK-1(836-947) facilitated degradation of full-length DAPK-1 and apoptosis induced by TNFR-1. Moreover, siRNA mediated knock-down of DAPK-1 enhanced TNF-α induced apoptosis, confirming the role of DAPK-1 as a survival factor in the TNF-α signalling pathway. In addition, a splice variant of DAPK-1, which I have called s-DAPK-1, was discovered. s-DAPK-1 shares part of DAPK-1’s ankyrin repeats region and cytoskeletal binding domain, and possesses an unique tail region, which contains a cleavage site at its first two amino acids. Unlike DAPK-1, s-DAPK-1 does not contribute to apoptosis induced by high level of MEK/ERK signalling, but it does mimic DAPK-1’s function to induce membrane blebbing. The proteolytically processed form of s-DAPK-1 is more active in the induction of membrane blebbing, which may be due to its higher stability compared to that of full-length s-DAPK-1, suggesting that the tail region can control s-DAPK-1 stability and activity. Co-transfection of s-DAPK-1 and DAPK-1 leads to reduction in DAPK-1 expression level, suggesting a role for s-DAPK-1 to regulate DAPK-1 stability. The kinase domain of DAPK-1 is the region required for s-DAPK-1 to promote DAPK-1 degradation. Surprisingly, s-DAPK-1 does not bind directly to DAPK-1, suggesting that the interaction is indirect and mediated by as yet unidentified accessory proteins. Finally, the experiments with proteasome and lysosome inhibitors indicated that s-DAPK-1 induces DAPK-1 degradation via both lysosome and proteasome pathways.