Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson’s disease (PD), with growing importance also for Crohn’s disease and cancer. LRRK2 is a large and complex protein possessing both GTPase and kinase activity. Moreover, LRRK2 activity and function can be influenced by its phosphorylation status. In this regard, many LRRK2 PD-associated mutants display decreased phosphorylation of the constitutive phosphorylation cluster S910/S935/S955/S973, but the role of these changes in phosphorylation status with respect to LRRK2 physiological functions remains unknown. Here, we propose that the S910/S935/S955/S973 phosphorylation sites act as key regulators of LRRK2-mediated autophagy under both basal and starvation conditions. We show that quadruple LRRK2 phosphomutant cells (4xSA; S910A/S935A/S955A/S973A) have impaired lysosomal functionality and fail to induce and proceed with autophagy during starvation. In contrast, treatment with the specific LRRK2 kinase inhibitors MLi-2 (100 nM) or PF-06447475 (150 nM), which also led to decreased LRRK2 phosphorylation of S910/S935/S955/S973, did not affect autophagy. In explanation, we demonstrate that the autophagy impairment due to the 4xSA LRRK2 phospho-dead mutant is driven by its enhanced LRRK2 kinase activity. We show mechanistically that this involves increased phosphorylation of LRRK2 downstream targets Rab8a and Rab10, as the autophagy impairment in 4xSA LRRK2 cells is counteracted by expression of phosphorylation-deficient mutants T72A Rab8a and T73A Rab10. Similarly, reduced autophagy and decreased LRRK2 phosphorylation at the constitutive sites were observed in cells expressing the pathological R1441C LRRK2 PD mutant, which also displays increased kinase activity. These data underscore the relation between LRRK2 phosphorylation at its constitutive sites and the importance of increased LRRK2 kinase activity in autophagy regulation and PD pathology.

富含亮氨酸重复激酶 2 ( LRRK2 ) 基因的突变是帕金森病 (PD) 最常见的遗传原因，对于克罗恩病和癌症也越来越重要。 LRRK2 是一种大而复杂的蛋白质，具有 GTP 酶和激酶活性。此外，LRRK2 的活性和功能可能受到其磷酸化状态的影响。在这方面，许多 LRRK2 PD 相关突变体表现出组成型磷酸化簇 S910/S935/S955/S973 的磷酸化降低，但这些磷酸化状态变化相对于 LRRK2 生理功能的作用仍不清楚。在此，我们提出 S910/S935/S955/S973 磷酸化位点在基础条件和饥饿条件下充当 LRRK2 介导的自噬的关键调节因子。我们发现四重 LRRK2 磷酸突变细胞（4xSA；S910A/S935A/S955A/S973A）的溶酶体功能受损，并且在饥饿期间无法诱导和进行自噬。相比之下，用特异性 LRRK2 激酶抑制剂 MLi-2 (100 nM) 或 PF-06447475 (150 nM) 治疗也会导致 S910/S935/S955/S973 的 LRRK2 磷酸化降低，但不会影响自噬。在解释中，我们证明 4xSA LRRK2 磷酸化死亡突变体导致的自噬损伤是由其增强的 LRRK2 激酶活性驱动的。我们从机制上表明，这涉及 LRRK2 下游靶标 Rab8a 和 Rab10 的磷酸化增加，因为 4xSA LRRK2 细胞中的自噬损伤被磷酸化缺陷突变体 T72A Rab8a 和 T73A Rab10 的表达所抵消。 同样，在表达病理性 R1441C LRRK2 PD 突变体的细胞中观察到自噬减少和组成位点 LRRK2 磷酸化减少，该突变体也表现出激酶活性增加。这些数据强调了 LRRK2 组成位点的磷酸化与 LRRK2 激酶活性增加在自噬调节和 PD 病理学中的重要性之间的关系。