Kamikawaji Shogo, Ito Genta, Iwatsubo Takeshi
Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo, 113-0033 Japan.
Biochemistry. 2009 Nov 24;48(46):10963-75. doi: 10.1021/bi9011379.
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 PARK8 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 PARK8, although the regulatory mechanism that governs the kinase activity of LRRK2 remains unknown. In this study, we expressed the carboxyl-half fragments of LRRK2 (DeltaN-LRRK2) that harbors the kinase as well as the ras-like (ROC) domains in Sf9 cells, subjected them to in vitro phosphorylation reaction, and analyzed the autophosphorylation by matrix assisted laser desorption/ionization- 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.
帕金森病（PD）是一种主要影响锥体外系运动系统的成人起病的神经退行性疾病。一部分患者以常染色体显性遗传的方式患帕金森病，其中由富含亮氨酸重复激酶2（LRRK2）基因突变引起的PARK8因其高发性以及与散发性帕金森病在临床病理上的相似性而备受关注。以往研究表明，错义突变导致的LRRK2过度激活会在PARK8中引发神经元毒性，尽管调控LRRK2激酶活性的机制仍不清楚。在本研究中，我们在Sf9细胞中表达了包含激酶以及类Ras（ROC）结构域的LRRK2羧基端片段（DeltaN-LRRK2），使其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）质谱仪分析自磷酸化情况。我们确定位于ROC结构域内的Ser1403、Thr1404、Thr1410、Thr1491以及激酶结构域中的Thr1967和Thr1969为自磷酸化位点。将位于激酶结构域内的自磷酸化位点Thr1967替换为丙氨酸会导致激酶活性显著降低，这表明Thr1967与LRRK2的激酶活性有关。针对自磷酸化位点的磷酸特异性抗体能够特异性识别经过体外磷酸化反应的全长LRRK2，这表明自磷酸化发生在完整蛋白中。对自磷酸化的进一步分析将阐明LRRK2的激活机制以及与LRRK2过度激活相关的帕金森病发病机制。
