Dependence of Leucine-rich Repeat Kinase 2 (LRRK2) Kinase Activity on Dimerization
Dependence of Leucine-rich Repeat Kinase 2 (LRRK2) Kinase Activity on Dimerization
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DOI：
10.1074/jbc.m109.025437
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发表时间：
2009-12-25
影响因子：
4.8
通讯作者：
West, Andrew B.
中科院分区：
文献类型：
--
作者：
Sen, Saurabh;Webber, Philip J.;West, Andrew B.
富含亮氨酸重复激酶 2 (LRRK2) 基因的显性错义突变是帕金森病最常见的已知遗传原因。 LRRK2 编码丝氨酸/苏氨酸蛋白激酶，致病性突变可能会增加激酶活性。 GTPase 结构域中的内在 GTP 结合可能通过内部信号转导级联控制激酶活性。与许多蛋白激酶一样，LRRK2 通过可能调节酶活性的机制进行自我相互作用。我们发现 GTP 酶或激酶活性的破坏会增强高分子量寡聚体的形成，并阻止 LRRK2 二聚体结构的形成。此外，短暂应用广谱激酶抑制剂十字孢菌素可消除 LRRK2 二聚体并促进 LRRK2 高分子量寡聚体。 LRRK2 与细胞系中其他蛋白质的相互作用不依赖于激酶，并且包括分子伴侣和细胞骨架成分，这表明 LRRK2 自组装主要决定了复杂的大小。为了进一步探索激酶激活的机制，我们将编码致病性 G2019S 突变的可溶性 LRRK2 蛋白分离成高分子量寡聚体、二聚体和单体，并发现激酶活性存在于二聚体 LRRK2 中。一些在体外增加激酶活性的 PD 相关突变显着增加了二聚体结构相对于 LRRK2 总蛋白的比例，为致病性突变如何改变正常的酶调节提供了额外的见解。靶向和跟踪 LRRK2 二聚化可能提供观察活细胞中 LRRK2 激酶活性的清晰方法，并且通过激酶抑制或其他方式破坏二聚体 LRRK2 可能会减弱 LRRK2 酶输出的致病性增加。
Dominant missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known genetic cause of Parkinson disease. LRRK2 encodes a serine/threonine protein kinase, and pathogenic mutations may increase kinase activity. Intrinsic GTP binding in the GTPase domain may govern kinase activity through an internal signal transduction cascade. As with many protein kinases, LRRK2 self-interacts through mechanisms that may regulate enzymatic activity. We find that the disruption of either GTPase or kinase activity enhances the formation of high molecular weight oligomers and prevents the formation of LRRK2 dimer structures. In addition, brief application of the broad spectrum kinase inhibitor staurosporine ablates LRRK2 dimers and promotes LRRK2 high molecular weight oligomers. LRRK2 interactions with other proteins in cell lines are kinase-independent and include chaperones and cell cytoskeleton components, suggesting that LRRK2 self-assembly principally dictates complex size. To further explore the mechanics of kinase activation, we separate soluble LRRK2 protein that encodes the pathogenic G2019S mutation into high molecular weight oligomers, dimers, and monomers and find that kinase activity resides with dimeric LRRK2. Some PD-associated mutations that increase kinase activity in vitro significantly increase the proportion of dimer structures relative to total LRRK2 protein, providing additional insight into how pathogenic mutations may alter normal enzymatic regulation. Targeting and tracking LRRK2 dimerization may provide a clear way to observe LRRK2 kinase activity in living cells, and disruption of dimeric LRRK2 through kinase inhibition or other means may attenuate pathogenic increases in LRRK2 enzymatic output.