A feed-forward pathway drives LRRK2 kinase membrane recruitment and apparent activation

Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson’s disease and activated LRRK2 phosphorylates a subset of Rab GTPases. Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360-450 and show that this site, termed “Site #1”, can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed “Site #2”, that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments and mutation analysis demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab reaction products. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and apparent membrane activation of LRRK2 kinase activity.

富亮氨酸重复激酶2 (Leucine Rich Repeat Kinase 2, LRRK2) 的激活突变可引发帕金森病，且激活态的LRRK2可磷酸化一组Rab鸟苷三磷酸酶 (Rab GTPases)。此外，定位于高尔基体的Rab29可将LRRK2招募至高尔基体膜表面，并在该处激活LRRK2，使其既能发生自身磷酸化，又能磷酸化Rab底物。本研究明确了LRRK2的Armadillo结构域 (Armadillo domain) 中位于360-450位氨基酸残基间的Rab29精确结合区域，并发现该被命名为"位点1 (Site #1)"的区域还可结合另外两种LRRK2底物：Rab8A与Rab10。此外，本研究还鉴定出一个位于LRRK2磷酸化Rab8与Rab10之间的、具有更高亲和力的独特N端相互作用界面，被命名为"位点2 (Site #2)"，该界面可将LRRK2锚定在细胞的膜结构上，从而催化后续的多步磷酸化反应。激酶抑制剂洗脱实验与突变分析结果表明，细胞内激酶活性的快速恢复依赖于LRRK2与磷酸化Rab反应产物的结合能力。将纯化的LRRK2招募至经Rab10蛋白修饰的平面脂质双层的重构实验证实，仅激活态的LRRK2可与含磷酸化Rab10的膜表面发生协同结合。上述实验揭示了一条前馈通路，该通路可实现LRRK2激酶活性的空间调控，并使其在膜表面实现表观激活。