An alternative transcript from the death-associated protein kinase 1 locus encoding a small protein selectively mediates membrane blebbing: Functional transcript expressed by DAPK-1 locus

Lin, Yao; Stevens, Craig; Hrstka, Roman; Harrison, Ben; Fourtouna, Argyro; Pathuri, Suresh; Vojtesek, Borek; Hupp, Ted

Abstract

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-1Δtail 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.

Citation

Lin, Y., Stevens, C., Hrstka, R., Harrison, B., Fourtouna, A., Pathuri, S., Vojtesek, B., & Hupp, T. (2008). An alternative transcript from the death-associated protein kinase 1 locus encoding a small protein selectively mediates membrane blebbing: Functional transcript expressed by DAPK-1 locus. FEBS Journal, 275(10), 2574-2584. https://doi.org/10.1111/j.1742-4658.2008.06404.x

• Journal Article Type: Article
• Acceptance Date: Mar 14, 2008
• Online Publication Date: Apr 15, 2008
• Publication Date: Apr 15, 2008
• Deposit Date: Aug 5, 2016
• Journal: FEBS Journal
• Print ISSN: 1742-464X
• Electronic ISSN: 1742-4658
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 275
• Issue: 10
• Pages: 2574-2584
• DOI: https://doi.org/10.1111/j.1742-4658.2008.06404.x
• Keywords: DAPK-1, ERK, membrane blebbing, p53, proteolysis,
• Public URL: http://researchrepository.napier.ac.uk/Output/328374