Lin Yao, Stevens Craig, Hrstka Roman, Harrison Ben, Fourtouna Argyro, Pathuri Suresh, Vojtesek Borek, Hupp Ted
Institute of Genetics and Molecular Medicine, Cell Signalling Unit, CRUK p53 Signal Transduction Group, University of Edinburgh, UK.
FEBS J. 2008 May;275(10):2574-84. doi: 10.1111/j.1742-4658.2008.06404.x. Epub 2008 Apr 15.
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.
死亡相关蛋白激酶1（DAPK-1）是一种多结构域蛋白激酶，在自噬、凋亡和生存途径中发挥多种作用。利用生物信息学筛选从DAPK-1基因座鉴定出一个小的内部mRNA（命名为s-DAPK-1）。它编码一个295个氨基酸的多肽，包含锚蛋白重复结构域的一部分、P环基序、DAPK-1细胞骨架结合结构域的一部分，以及DAPK-1中不存在的独特C末端“尾巴”延伸。在一组正常人体组织以及原发性结直肠癌中检测到s-DAPK-1 mRNA的表达，表明其表达在体内发生。将s-DAPK-1基因转染到细胞中产生两种蛋白质产物：一种变性质量为44 kDa，另一种较小产物为40 kDa。s-DAPK-1 C末端尾巴延伸的双丙氨酸突变（Gly296/Arg297）阻止了40 kDa片段的产生，表明较小的产物是由体内蛋白水解加工产生的。在丝裂原活化蛋白激酶激酶/细胞外信号调节激酶依赖性凋亡转染试验中，s-DAPK-1基因不能替代全长DAPK-1。然而，s-DAPK-1的转染能够在诱导膜泡形成方面模拟全长DAPK-1。44 kDa蛋白酶抗性突变体s-DAPK-1G296A/R297A在膜泡形成方面活性非常低，而40 kDa的s-DAPK-1缺失尾巴蛋白表现出最高水平的膜泡形成。缺失s-DAPK-1的尾巴延伸增加了其半衰期，使蛋白质的平衡从细胞骨架池转移到可溶性胞质池，并通过共聚焦显微镜观察改变了绿色荧光蛋白标记的s-DAPK-1蛋白的定位。这些数据突出了DAPK-1基因座存在一种替代产物，并表明蛋白水解去除s-DAPK-1的C末端尾巴是最大程度刺激其膜泡形成功能所必需。
