Dhekne Herschel S, Tonelli Francesca, Yeshaw Wondwossen M, Chiang Claire Y, Limouse Charles, Jaimon Ebsy, Purlyte Elena, Alessi Dario R, Pfeffer Suzanne R
Department of Biochemistry, Stanford University School of Medicine, Stanford, United States.
Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Stanford, United States.
Elife. 2023 Oct 24;12:e87098. doi: 10.7554/eLife.87098.
Activating mutations in the leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases, particularly Rab10 and Rab8A, and we showed previously that these phosphoRabs play an important role in LRRK2 membrane recruitment and activation (Vides et al., 2022). To learn more about LRRK2 pathway regulation, we carried out an unbiased, CRISPR-based genome-wide screen to identify modifiers of cellular phosphoRab10 levels. A flow cytometry assay was developed to detect changes in phosphoRab10 levels in pools of mouse NIH-3T3 cells harboring unique CRISPR guide sequences. Multiple negative and positive regulators were identified; surprisingly, knockout of the gene was especially effective in decreasing phosphoRab10 levels in multiple cell types and knockout mouse tissues. Rab-driven increases in phosphoRab10 were specific for Rab12, LRRK2-dependent and PPM1H phosphatase-reversible, and did not require Rab12 phosphorylation; they were seen with wild type and pathogenic G2019S and R1441C LRRK2. As expected for a protein that regulates LRRK2 activity, Rab12 also influenced primary cilia formation. AlphaFold modeling revealed a novel Rab12 binding site in the LRRK2 Armadillo domain, and we show that residues predicted to be essential for Rab12 interaction at this site influence phosphoRab10 and phosphoRab12 levels in a manner distinct from Rab29 activation of LRRK2. Our data show that Rab12 binding to a new site in the LRRK2 Armadillo domain activates LRRK2 kinase for Rab phosphorylation and could serve as a new therapeutic target for a novel class of LRRK2 inhibitors that do not target the kinase domain.
富含亮氨酸重复激酶2（LRRK2）中的激活突变会导致帕金森病。LRRK2使一部分Rab GTP酶磷酸化，尤其是Rab10和Rab8A，并且我们之前表明这些磷酸化的Rab在LRRK2的膜募集和激活中发挥重要作用（维德斯等人，2022年）。为了更多地了解LRRK2通路的调控，我们进行了一项基于CRISPR的无偏见全基因组筛选，以鉴定细胞磷酸化Rab10水平的调节因子。我们开发了一种流式细胞术检测方法，以检测携带独特CRISPR引导序列的小鼠NIH-3T3细胞池中磷酸化Rab10水平的变化。我们鉴定出了多个负调控因子和正调控因子；令人惊讶的是，敲除该基因在降低多种细胞类型和敲除小鼠组织中的磷酸化Rab10水平方面特别有效。Rab驱动的磷酸化Rab10增加对Rab12具有特异性，依赖于LRRK2且可被PPM1H磷酸酶逆转，并且不需要Rab12磷酸化；在野生型以及致病性G2019S和R1441C LRRK2中均观察到这种增加。正如对调节LRRK2活性的蛋白质所预期的那样，Rab12也影响初级纤毛的形成。AlphaFold建模揭示了LRRK2犰狳结构域中一个新的Rab12结合位点，并且我们表明在该位点预测对Rab12相互作用至关重要的残基以不同于Rab29对LRRK2激活的方式影响磷酸化Rab10和磷酸化Rab12水平。我们的数据表明，Rab12与LRRK2犰狳结构域中的一个新位点结合可激活LRRK2激酶以进行Rab磷酸化，并且可以作为一类不靶向激酶结构域的新型LRRK2抑制剂的新治疗靶点。
