Reyniers Lauran, Del Giudice Maria Grazia, Civiero Laura, Belluzzi Elisa, Lobbestael Evy, Beilina Alexandra, Arrigoni Giorgio, Derua Rita, Waelkens Etienne, Li Yan, Crosio Claudia, Iaccarino Ciro, Cookson Mark R, Baekelandt Veerle, Greggio Elisa, Taymans Jean-Marc
Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven, Leuven, Belgium.
Department of Biomedical Sciences, University of Sassari, Sassari, Italy.
J Neurochem. 2014 Oct;131(2):239-50. doi: 10.1111/jnc.12798. Epub 2014 Jul 14.
Genetic studies show that LRRK2, and not its closest paralogue LRRK1, is linked to Parkinson's disease. To gain insight into the molecular and cellular basis of this discrepancy, we searched for LRRK1- and LRRK2-specific cellular processes by identifying their distinct interacting proteins. A protein microarray-based interaction screen was performed with recombinant 3xFlag-LRRK1 and 3xFlag-LRRK2 and, in parallel, co-immunoprecipitation followed by mass spectrometry was performed from SH-SY5Y neuroblastoma cell lines stably expressing 3xFlag-LRRK1 or 3xFlag-LRRK2. We identified a set of LRRK1- and LRRK2-specific as well as common interactors. One of our most prominent findings was that both screens pointed to epidermal growth factor receptor (EGF-R) as a LRRK1-specific interactor, while 14-3-3 proteins were LRRK2-specific. This is consistent with phosphosite mapping of LRRK1, revealing phosphosites outside of 14-3-3 consensus binding motifs. To assess the functional relevance of these interactions, SH-SY5Y-LRRK1 and -LRRK2 cell lines were treated with LRRK2 kinase inhibitors that disrupt 14-3-3 binding, or with EGF, an EGF-R agonist. Redistribution of LRRK2, not LRRK1, from diffuse cytoplasmic to filamentous aggregates was observed after inhibitor treatment. Similarly, EGF induced translocation of LRRK1, but not of LRRK2, to endosomes. Our study confirms that LRRK1 and LRRK2 can carry out distinct functions by interacting with different cellular proteins. LRRK1 and LRRK2 (leucine-rich repeat kinase) interaction partners were identified by two different protein-protein interaction screens. These confirmed epidermal growth factor receptor (EGR-R) as a LRRK1-specific interactor, while 14-3-3 proteins were LRRK2-specific. Functional analysis of these interactions and the pathways they mediate shows that LRRK1 and LRRK2 signaling do not intersect, reflective of the differential role of both LRRKs in Parkinson's disease.
基因研究表明，与帕金森病相关的是亮氨酸重复激酶2（LRRK2），而非其最接近的旁系同源物LRRK1。为深入了解这种差异的分子和细胞基础，我们通过鉴定LRRK1和LRRK2各自独特的相互作用蛋白，来寻找它们特异性的细胞过程。我们用重组的3xFlag-LRRK1和3xFlag-LRRK2进行了基于蛋白质芯片的相互作用筛选，同时，从稳定表达3xFlag-LRRK1或3xFlag-LRRK2的SH-SY5Y神经母细胞瘤细胞系中进行免疫共沉淀，随后进行质谱分析。我们鉴定出了一组LRRK1和LRRK2特异性以及共同的相互作用蛋白。我们最显著的发现之一是，两种筛选方法均表明表皮生长因子受体（EGF-R）是LRRK1特异性的相互作用蛋白，而14-3-3蛋白是LRRK2特异性的。这与LRRK1的磷酸化位点图谱一致，该图谱揭示了14-3-3共有结合基序之外的磷酸化位点。为评估这些相互作用的功能相关性，用破坏14-3-3结合的LRRK2激酶抑制剂或EGF-R激动剂EGF处理SH-SY5Y-LRRK1和-LRRK2细胞系。抑制剂处理后，观察到LRRK2（而非LRRK1）从弥漫性细胞质重新分布到丝状聚集体中。同样，EGF诱导LRRK1（而非LRRK2）转位至内体。我们的研究证实，LRRK1和LRRK2可通过与不同的细胞蛋白相互作用来执行不同的功能。通过两种不同的蛋白质-蛋白质相互作用筛选方法鉴定出了LRRK1和LRRK2（富含亮氨酸重复激酶）的相互作用伙伴。这些方法证实表皮生长因子受体（EGR-R）是LRRK1特异性的相互作用蛋白，而14-3-3蛋白是LRRK2特异性的。对这些相互作用及其介导的信号通路的功能分析表明，LRRK1和LRRK2信号传导不相交，这反映了两种LRRKs在帕金森病中的不同作用。
