Tài liệu Báo cáo khoa học: An alternative transcript from the death-associated protein kinase 1

An alternative transcript from the death-associated

protein kinase 1 locus encoding a small protein selectively

mediates membrane blebbing

Yao Lin1

, Craig Stevens1

, Roman Hrstka2

, Ben Harrison1

, Argyro Fourtouna1

, Suresh Pathuri1

,

Borek Vojtesek2 and Ted Hupp1

1 Institute of Genetics and Molecular Medicine, Cell Signalling Unit, CRUK p53 Signal Transduction Group, University of Edinburgh, UK

2 Masaryk Memorial Cancer Institute, Brno, Czech Republic

Death-associated protein kinase 1 (DAPK-1) is a

Ca2+ ⁄ calmodulin-regulated serine ⁄threonine kinase

composed of multiple functional domains, including a

kinase domain, a calmodulin-binding domain, eight

ankyrin repeats, two P-loop motifs, a cytoskeletal

binding domain, a death domain, and a C-terminal

regulatory tail [1]. It has been shown that DAPK-1

is involved in the regulation of distinct processes,

Keywords

DAPK-1; ERK; membrane blebbing; p53;

proteolysis

Correspondence

T. Hupp, Institute of Genetics and Molecular

Medicine, Cell Signalling Unit, CRUK p53

Signal Transduction Group, University of

Edinburgh, Edinburgh EH4 2XR, UK

Fax: +44 131 7773542

Tel: +44 131 7773583

E-mail: [email protected]

(Received 14 January 2008, revised 11

March 2008, accepted 14 March 2008)

doi:10.1111/j.1742-4658.2008.06404.x

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. Dou￾ble 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-depen￾dent apoptotic transfection assay. However, the transfection of s-DAPK-1

was able to mimic full-length DAPK-1 in the induction of membrane bleb￾bing. The 44 kDa protease-resistant mutant s-DAPK-1G296A ⁄R297A had

very low activity in membrane blebbing, whereas the 40 kDa s-DAPK￾1Dtail protein exhibited the highest levels of membrane blebbing. Deletion

of the tail extension of s-DAPK-1 increased its half-life, shifted the equilib￾rium 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 mem￾brane-blebbing function.

Abbreviations

GFP, green fluorescent protein; GST, glutathione S-transferase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein

kinase kinase; TM, tail mutant.

2574 FEBS Journal 275 (2008) 2574–2584 ª 2008 The Authors Journal compilation ª 2008 FEBS