DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death

TLDR

DAP kinase (DAPk) and DAPk‑related protein kinase (DRP‑1) are Ca²⁺/calmodulin‑regulated death kinases whose specific functions in programmed cell death remain largely undefined. This study investigates the subcellular events mediated by DAPk and DRP‑1 during cell death. Activation of DAPk or DRP‑1 induces caspase‑independent membrane blebbing and extensive autophagy; dominant‑negative mutants diminish blebbing in TNF‑α–induced apoptosis and reduce autophagy triggered by antiestrogens, starvation, or IFN‑γ, indicating that both kinases are rate‑limiting for these cytoplasmic events, with DRP‑1 localizing to autophagic vesicles.

Abstract

Death-associated protein kinase (DAPk) and DAPk-related protein kinase (DRP)-1 proteins are Ca+2/calmodulin–regulated Ser/Thr death kinases whose precise roles in programmed cell death are still mostly unknown. In this study, we dissected the subcellular events in which these kinases are involved during cell death. Expression of each of these DAPk subfamily members in their activated forms triggered two major cytoplasmic events: membrane blebbing, characteristic of several types of cell death, and extensive autophagy, which is typical of autophagic (type II) programmed cell death. These two different cellular outcomes were totally independent of caspase activity. It was also found that dominant negative mutants of DAPk or DRP-1 reduced membrane blebbing during the p55/tumor necrosis factor receptor 1–induced type I apoptosis but did not prevent nuclear fragmentation. In addition, expression of the dominant negative mutant of DRP-1 or of DAPk antisense mRNA reduced autophagy induced by antiestrogens, amino acid starvation, or administration of interferon-γ. Thus, both endogenous DAPk and DRP-1 possess rate-limiting functions in these two distinct cytoplasmic events. Finally, immunogold staining showed that DRP-1 is localized inside the autophagic vesicles, suggesting a direct involvement of this kinase in the process of autophagy.