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An alternative transcript from the death‐associated protein kinase 1 locus encoding a small protein selectively mediates membrane blebbing
Author(s) -
Lin Yao,
Stevens Craig,
Hrstka Roman,
Harrison Ben,
Fourtouna Argyro,
Pathuri Suresh,
Vojtesek Borek,
Hupp Ted
Publication year - 2008
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2008.06404.x
Subject(s) - protein kinase a , microbiology and biotechnology , biology , transfection , mutant , kinase , gene , biochemistry
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.