Laboratory for Neurobiology and Gene Therapy, Katholieke Universiteit Leuven, Leuven, Belgium.
PLoS One. 2011;6(8):e23207. doi: 10.1371/journal.pone.0023207. Epub 2011 Aug 12.
Leucine rich repeat kinase 2 (LRRK2) is a Parkinson's disease (PD) gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC) GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.
富含亮氨酸重复激酶 2（LRRK2）是帕金森病（PD）基因，它编码一个包含 GTPase 和激酶结构域的大型多功能蛋白。GTPases 通常在信号转导级联中调节激酶，其中 GTPases 作为分子开关，在与 GDP 结合的“关闭”状态和与 GTP 结合的“开启”状态之间循环。据推测，LRRK2 激酶活性可能在 LRRK2 Ras 相关复合蛋白（ROC）GTPase 结构域与 GTP 结合时增加。在这里，我们通过测量 GDP、GTP 或非水解 GTP 类似物 GTPγS 或 GMPPCP 对 LRRK2 磷酸化活性的影响，广泛地检验了这一假设。我们表明，当在磷酸化反应中与 LRRK2 一起孵育时，重组 LRRK2 蛋白的自身磷酸化和 lrrktide 磷酸化活性不受鸟嘌呤核苷酸的影响。此外，当 LRRK2 的鸟嘌呤核苷酸结合口袋先前被各种核苷酸饱和时，LRRK2 的磷酸化活性不变，与测量到的 G 核苷酸结合位点突变体 T1348N 的活性大大降低形成对比。有趣的是，当核苷酸在 LRRK2 纯化之前与细胞裂解物孵育时，与 GDP 相比，激酶活性被 GTPγS 或 GMPPCP 略微增强，这表明存在一种上游鸟嘌呤核苷酸结合蛋白，它可能以 GTP 依赖的方式激活 LRRK2。使用代谢标记，我们还发现核苷酸对细胞内 LRRK2 的磷酸化没有显著调节，尽管鸟嘌呤核苷酸结合位点突变体显著降低了标记。我们得出的结论是，尽管 LRRK2 的激酶活性需要一个完整的 ROC-GTPase 结构域，但它不依赖于 ROC 与 GDP 或 GTP 的结合。
