Identification of the Autophosphorylation Sites of LRRK2
Identification of the Autophosphorylation Sites of LRRK2
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DOI：
10.1021/bi9011379
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发表时间：
2009-11-24
期刊：
影响因子：
2.9
通讯作者：
Iwatsubo, Takeshi
中科院分区：
文献类型：
--
作者：
Kamikawaji, Shogo;Ito, Genta;Iwatsubo, Takeshi
帕金森病（PD）是一种影响锥体外系运动系统的主要成人发病的神经退行性疾病。一部分患者发展为常染色体显性遗传的PD，其中富含亮氨酸重复激酶2（LRRK 2）基因突变引起的PARKS由于其与散发性PD的高频率和临床病理相似性而突出显示。先前的研究表明，错义突变引起的LRRK 2过度激活导致PARKS中的神经元毒性，尽管控制LRRK 2激酶活性的调节机制仍然未知。在这项研究中，我们在St 9细胞中表达了含有激酶以及ras样（ROC）结构域的LRRK 2的羧基半片段（Delta N-LRRK 2），对它们进行体外磷酸化反应，并通过基质辅助激光解吸/电离-飞行时间（MALDI-TOF）质谱仪分析了自磷酸化。我们鉴定了位于ROC结构域中的Ser 1403、Thr 1404、Thr 1410、Thr 1491以及激酶结构域中的Thr 1967和Thr 1969为自磷酸化位点。Thr 1967，一个位于激酶结构域内的自磷酸化位点，Ala的取代引起了激酶活性的显着下降，暗示Thr 1967在LRRK 2的激酶活性。磷酸特异性抗体的自磷酸化位点特异性识别全长LRRK 2进行体外磷酸化反应，表明自磷酸化发生在全蛋白。对自身磷酸化的进一步分析将阐明LRRK 2的激活机制，以及与LRRK 2过度激活相关的PD病理机制。
Parkinson's disease (PD) is a major adult-onset neurodegenerative disorder affecting the extrapyramidal motor system. A subset of patients develop PD as an autosomal dominant trait, of which PARKS caused by mutations in the leucine-rich repeat kinase 2 (LRRK2) gene is highlighted because of its high frequency and clinicopathological similarity to sporadic PD. Previous studies have suggested that overactivation of LRRK2 caused by missense mutations leads to neuronal toxicity in PARKS, although the regulatory mechanism that governs the kinase activity of LRRK2 remains unknown. In this study, we expressed the carboxyl-half fragments of LRRK2 (Delta N-LRRK2) that harbors the kinase as well as the ras-like (ROC) domains in St9 cells, subjected them to in vitro phosphorylation reaction, and analyzed the autophosphorylation by matrix assisted laser desorption/lonization- time of flight (MALDI-TOF) mass spectrometer. We identified Ser1403, Thr1404, Thr1410, Thr1491 located within the ROC domain, as well as Thr1967 and Thr1969 in the kinase domain, as the autophosphorylation sites. Substitution of Thr1967, an autophosphorylation site located within the kinase domain, to Ala caused a significant decrease in the kinase activity, implicating Thr1967 in the kinase activity of LRRK2. Phosphospecific antibodies to the autophosphorylation sites specifically recognized full-length LRRK2 subjected to in vitro phosphorylation reaction, indicating that the autophosphorylation takes place in holoproteins. Further analysis of autophosphorylation will clarify the mechanism of activation of LRRK2, as well as the pathomechanism of PD in relation to overactivation of LRRK2.