The alternative splice variant of DAPK-1, s-DAPK-1, induces proteasome-independent DAPK-1 destabilization.

Death-associated protein kinase 1 (DAPK-1) is a Ca(2+)/CaM-regulated kinase involved in multiple cellular signalling pathways that trigger cell survival, apoptosis, and autophagy. An alternatively spliced product expressed from the dapk1 locus, named s-DAPK-1, does not contain the kinase domain but has part of the DAPK-1 ankyrin-repeat and a novel polypeptide tail extension which is processed proteolytically in vivo. Cleavage of this polypeptide tail from s-DAPK-1 can regulate the ability of the protein to mimic one of the biological functions of DAPK-1 in promoting membrane blebbing. The full-length DAPK-1 protein is a relatively long-lived protein whose half-life is regulated by stress-activated signals from TNFR1 or HSP90 that can promote DAPK-1 protein degradation. Transfection of s-DAPK-1 into cells can also have a direct effect on DAPK-1 protein itself by promoting DAPK-1 de-stabilization. This effect does not require the novel polypeptide tail-extension of s-DAPK-1, as the core ankyrin-repeat containing region of s-DAPK-1 is sufficient to promote DAPK-1 protein de-stabilization. Conversely, the minimal domain on full-length DAPK-1 that responds to the effect of s-DAPK-1 is not the ankyrin-repeat domain but the core kinase domain of DAPK-1. The de-stabilization of DAPK-1 by s-DAPK-1 is not dependent upon the proteasome. However, s-DAPK-1 itself is a very short-lived protein which is regulated by a proteasomal-dependent pathway. Together, these data identify a novel function of s-DAPK-1 in controlling the half-life of DAPK-1 protein itself and indicate that the degradation of each gene product is controlled by two distinct degradation pathways.

An alternative transcript from the death-associated protein kinase 1 locus encoding a small protein selectively mediates membrane blebbing.

Death-associated protein kinase 1 (DAPK-1) is a multidomain protein kinase with diverse roles in autophagic, apoptotic and survival pathways. Bioinformatic screens were used to identify a small internal mRNA from the DAPK-1 locus (named s-DAPK-1). This encodes a 295 amino acid polypeptide encompassing part of the ankyrin-repeat domain, the P-loop motifs, part of the cytoskeletal binding domain of DAPK-1, and a unique C-terminal 'tail' extension not present in DAPK-1. Expression of s-DAPK-1 mRNA was detected in a panel of normal human tissues as well as primary colorectal cancers, indicating that its expression occurs in vivo. s-DAPK-1 gene transfection into cells produces two protein products: one with a denatured mass of 44 kDa, and a smaller product of 40 kDa. Double alanine mutation of the C-terminal tail extension of s-DAPK-1 (Gly296/Arg297) prevented production of the 40 kDa fragment, suggesting that the smaller product is generated by in vivo proteolytic processing. The s-DAPK-1 gene cannot substitute for full-length DAPK-1 in an mitogen-activated protein kinase kinase/extracellular signal-regulated kinase-dependent apoptotic transfection assay. However, the transfection of s-DAPK-1 was able to mimic full-length DAPK-1 in the induction of membrane blebbing. The 44 kDa protease-resistant mutant s-DAPK-1G296A/R297A had very low activity in membrane blebbing, whereas the 40 kDa s-DAPK-1Deltatail protein exhibited the highest levels of membrane blebbing. Deletion of the tail extension of s-DAPK-1 increased its half-life, shifted the equilibrium of the protein from cytoskeletal to soluble cytosolic pools, and altered green fluorescent protein-tagged s-DAPK-1 protein localization as observed by confocal microscopy. These data highlight the existence of an alternative product of the DAPK-1 locus, and suggest that proteolytic removal of the C-terminal tail of s-DAPK-1 is required to stimulate maximally its membrane-blebbing function.