Phosphopeptide Analysis Reveals Two Discrete Clusters of Phosphorylation in the N-Terminus and the Roc Domain of the Parkinson-Disease Associated Protein Kinase LRRK2
Phosphopeptide Analysis Reveals Two Discrete Clusters of Phosphorylation in the N-Terminus and the Roc Domain of the Parkinson-Disease Associated Protein Kinase LRRK2
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DOI：
10.1021/pr9008578
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发表时间：
2010-04-01
影响因子：
4.4
通讯作者：
Ueffing, Marius
中科院分区：
文献类型：
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作者：
Gloeckner, Christian Johannes;Boldt, Karsten;Ueffing, Marius
富含亮氨酸重复序列激酶2（LRRK2）的突变增加了其激酶活性，与家族性帕金森病（PD）有关。由于磷酸化决定了大多数蛋白激酶的功能状态，我们通过质谱系统地绘制了LRRK2磷酸化位点。我们的分析显示，在LRR结构域之前的一个狭窄的富含丝氨酸的区域中存在高度的组成性磷酸化。在质谱分析之前，允许在体外自激酶测定中对纯化的LRRK2进行de nova自磷酸化，我们发现了多个自磷酸化位点。只有丝氨酸和苏氨酸残基被发现磷酸化，表明LRRK2作为一个真正的丝氨酸苏氨酸激酶。自磷酸化主要靶向ROC的GTP结合域，其聚集在ROC的GTP结合口袋周围，表明激酶和ROC域之间的交叉调节活性。总之，磷蛋白LRRK2作为一种自催化活性的丝氨酸苏氨酸激酶发挥作用。在两个离散的结构域内的磷酸化位点的聚类表明，磷酸化可能以一种未知的方式调节其生物学功能。
Mutations in leucine-rich repeat kinase 2 (LRRK2) that increase its kinase activity associate with familial forms of Parkinson disease (PD). As phosphorylation determines the functional state of most protein kinases, we systematically mapped LRRK2 phosphorylation sites by mass spectrometry. Our analysis revealed a high degree of constitutive phosphorylation in a narrow serine-rich region preceding the LRR-domain. Allowing de nova autophosphorylation of purified LRRK2 in an in vitro autokinase assay prior to mass spectrometric analysis, we discovered multiple sites of autophosphorylation. Solely serine and threonine residues were found phosphorylated suggesting LRRK2 as a true serine threonine kinase. Autophosphorylation mainly targets the ROC GTPase domain and its clustering around the GTP binding pocket of ROC suggests cross-regulatory activity between kinase and Roc domain. In conclusion, the phosphoprotein LRRK2 functions as an autocatalytically active serine threonine kinase. Clustering of phosphosites within two discrete domains suggest that phosphorylation may regulate its biological functions in a yet unknown fashion.