Leucine-rich repeat kinase 2 phosphorylates brain tubulin-beta isoforms and modulates microtubule stability - a point of convergence in Parkinsonian neurodegeneration?
Leucine-rich repeat kinase 2 phosphorylates brain tubulin-beta isoforms and modulates microtubule stability - a point of convergence in Parkinsonian neurodegeneration?
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DOI：
10.1111/j.1471-4159.2009.06235.x
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发表时间：
2009-09-01
影响因子：
4.7
通讯作者：
Gillardon, Frank
中科院分区：
文献类型：
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作者：
Gillardon, Frank
富含亮氨酸重复序列激酶2（LRRK 2）的常染色体显性突变是晚发性帕金森病最常见的遗传原因。最普遍的LRRK 2（G2019 S）突变已多次被证明可增强激酶活性和神经毒性，然而，导致神经退行性变的分子机制仍不清楚。在这里，我们表明，重组人LRRK 2优先磷酸化从牛脑中纯化的微管蛋白-β，磷酸化是三倍增强的LRRK 2（G2019 S）突变。通过串联质谱，Thr 107被鉴定为磷酸化位点，其在微管蛋白-β家族成员之间以及不同物种的微管蛋白-β基因之间高度保守。LRRK 2与来自野生型小鼠脑和来自LRRK 2过表达的非神经元人胚肾293细胞的微管蛋白-β共免疫沉淀。然而，LRRK 2对微管蛋白磷酸化和组装的影响仅在小鼠脑样品中可检测到。牛脑微管蛋白与LRRK 2的体外共孵育增加了微管相关蛋白存在下的微管稳定性，这可能解释了培养中LRRK 2缺陷神经元中神经突长度的减少。这些研究结果表明，LRRK 2（G2019 S）诱导的帕金森病患者脑神经变性可能部分介导的微管蛋白-β的磷酸化增加和微管动力学的约束。
Autosomal dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of late-onset Parkinson's disease. The most prevalent LRRK2(G2019S) mutation has repeatedly been shown to enhance kinase activity and neurotoxicity, however, the molecular mechanisms leading to neurodegeneration remain poorly defined. Here we show that recombinant human LRRK2 preferentially phosphorylates tubulin-beta purified from bovine brain and that phosphorylation is three-fold enhanced by the LRRK2(G2019S) mutation. By tandem mass spectrometry, Thr107 was identified as phosphorylation site which is highly conserved between tubulin-beta family members and also between tubulin-beta genes of different species. LRRK2 was co-immunoprecipitated with tubulin-beta both from wild-type mouse brain and from LRRK2 over-expressing, non-neuronal human embryonic kidney 293 cells. However, an effect of LRRK2 on tubulin phosphorylation and assembly was only detectable in mouse brain samples. In vitro co-incubation of bovine brain tubulins with LRRK2 increased microtubule stability in the presence of microtubule-associated proteins which may explain the reduction in neurite length in LRRK2-deficient neurons in culture. These findings suggest that LRRK2(G2019S)-induced neurodegeneration in Parkinsonian brains may be partly mediated by increased phosphorylation of tubulin-beta and constraining of microtubule dynamics.