Functional consequences of mutations in CDKL5, an X-linked gene involved in infantile spasms and mental retardation
Functional consequences of mutations in CDKL5, an X-linked gene involved in infantile spasms and mental retardation
复制标题
DOI：
10.1074/jbc.m606325200
复制
发表时间：
2006-10-20
影响因子：
4.8
通讯作者：
Kilstrup-Nielsen, Charlotte
中科院分区：
文献类型：
--
作者：
Bertani, Ilaria;Rusconi, Laura;Kilstrup-Nielsen, Charlotte
已在 Rett 综合征、West 综合征和 X 连锁婴儿痉挛症患者中发现 X 连锁细胞周期蛋白依赖性激酶样 5 (CDKL5) 基因突变，这些患者具有一般顽固性早期癫痫发作和智力低下的共同特征。致病突变分布在催化结构域和大COOH末端。在本报告中，我们研究了 CDKL5 的一些 Rett 突变以及一些有助于强调该蛋白质功能域的合成设计衍生物的功能后果。对突变的 CDKL5 衍生物进行了体外激酶测定，并分析了激活环内 TEY (Thr-Glu-Tyr) 基序的磷酸化、其亚细胞定位以及 CDKL5 与其自身相互作用的能力。野生型 CDKL5 在体外自动磷酸化并介导甲基 CpG 结合蛋白 2 (MeCP2) 的磷酸化，而 Rett 突变蛋白表现出催化活性受损和增强，这表明正确的大脑功能需要严格调节 CDKL5。此外，我们发现 CDKL5 可以自我关联并介导其自身 TEY（Thr-Glu-Tyr）基序的磷酸化。最终，我们证明 COOH 末端调节 CDKL5 特性；特别是，它会对催化活性产生负面影响，并且是其适当的亚核定位所必需的。我们提出了一个模型，其中 CDKL5 磷酸化是其进入细胞核所必需的，而 COOH 末端结构域的一部分可能通过蛋白质-蛋白质相互作用负责在该细胞区室中的稳定驻留。
Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with Rett syndrome, West syndrome, and X-linked infantile spasms sharing the common features of generally intractable early seizures and mental retardation. Disease-causing mutations are distributed in both the catalytic domain and in the large COOH terminus. In this report, we examine the functional consequences of some Rett mutations of CDKL5 together with some synthetically designed derivatives useful to underline the functional domains of the protein. The mutated CDKL5 derivatives have been subjected to in vitro kinase assays and analyzed for phosphorylation of the TEY (Thr-Glu-Tyr) motif within the activation loop, their subcellular localization, and the capacity of CDKL5 to interact with itself. Whereas wild-type CDKL5 autophosphorylates and mediates the phosphorylation of the methyl-CpG-binding protein 2 (MeCP2) in vitro, Rett-mutated proteins show both impaired and increased catalytic activity suggesting that a tight regulation of CDKL5 is required for correct brain functions. Furthermore, we show that CDKL5 can self-associate and mediate the phosphorylation of its own TEY (Thr-Glu-Tyr) motif. Eventually, we show that the COOH terminus regulates CDKL5 properties; in particular, it negatively influences the catalytic activity and is required for its proper sub-nuclear localization. We propose a model in which CDKL5 phosphorylation is required for its entrance into the nucleus whereas a portion of the COOH-terminal domain is responsible for a stable residency in this cellular compartment probably through protein-protein interactions.