DAP-kinase is a Ca2+ calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity
DAP-kinase is a Ca2+ calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity
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DOI：
10.1093/emboj/16.5.998
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发表时间：
1997-03-03
期刊：
影响因子：
11.4
通讯作者：
Kimchi, A
中科院分区：
文献类型：
--
作者：
Cohen, O;Feinstein, E;Kimchi, A
DAP激酶最初被鉴定为一种基因，其反义介导的表达减少保护HeLa细胞免受干扰素-γ诱导的程序性细胞死亡。它是在我们实验室通过功能基因选择方法克隆的。根据其氨基酸序列，这160 kDa的蛋白被预测为一种新型的钙调素调节的丝氨酸/苏氨酸激酶，携带锚蛋白重复序列和死亡结构域。在这项工作中，我们已经表明，该激酶是自磷酸化，并能够磷酸化的外源性底物中的钙离子/钙调蛋白依赖性的方式。我们证明了钙调素直接结合到重组激酶，并产生了一个组成型活性激酶突变体的钙调素调节结构域的删除。通过免疫染色和生化分级，我们证明了激酶是本地化的细胞骨架，与微丝系统，并映射的蛋白质内的一个区域，负责结合到细胞骨架。几项试验将细胞死亡功能归因于该基因。野生型DAP激酶的异位表达诱导靶细胞死亡，并且杀伤性质严格依赖于内源激酶活性的状态。相反，一个催化失活的突变体，进行赖氨酸丙氨酸取代内的激酶结构域，显示显性负功能和保护细胞从干扰素-γ诱导的细胞死亡。因此，DAP-激酶是一种新的细胞骨架相关的细胞死亡丝氨酸/苏氨酸激酶，其通过Ca 2 +/钙调蛋白的激活可能与细胞死亡期间发生的细胞骨架改变的生化机制有关。
DAP-kinase was initially identified as a gene whose anti-sense-mediated reduced expression protected HeLa cells from interferon-gamma-induced programmed cell death. It was cloned in our laboratory by a functional gene selection approach. According to its amino acid sequence, this 160 kDa protein was predicted to be a novel type of calmodulin-regulated serine/threonine kinase which carries ankyrin repeats and the death domain. In this work we have shown that the kinase was autophosphorylated and capable of phosphorylating an exogenous substrate in a Ca2+/calmodulin-dependent manner. We proved that calmodulin binds directly to the recombinant kinase, and generated a constitutively active kinase mutant by the deletion of the calmodulin-regulatory domain. By immunostaining and biochemical fractionations we demonstrated that the kinase is localized to the cytoskeleton, in association with the microfilament system, and mapped a region within the protein which is responsible for binding to the cytoskeleton. Several assays attributed a cell death function to the gene. Ectopic expression of wild-type DAP-kinase induced the death of target cells, and the killing property depended strictly on the status of the intrinsic kinase activity. Conversely, a catalytically inactive mutant that carried a lysine to alanine substitution within the kinase domain, displayed dominant-negative features and protected cells from interferon-gamma-induced cell death. DAP-kinase is therefore a novel cytoskeletal-associated cell death serine/threonine kinase whose activation by Ca2+/calmodulin may be linked to the biochemical mechanism underlying the cytoskeletal alterations that occur during cell death.