Department of Cogno-Mechatronics Engineering, Pusan National University, Busan, South Korea.
Department of Cellular and Molecular Medicine, University of Ottawa Brain and Mind Research Institute, University of Ottawa, Ottawa, ON, Canada.
J Neurochem. 2019 Aug;150(3):312-329. doi: 10.1111/jnc.14683. Epub 2019 Apr 2.
Loss of function mutations in the PTEN-induced putative kinase 1 (Pink1) gene have been linked with an autosomal recessive familial form of early onset Parkinson's disease (PD). However, the underlying mechanism(s) responsible for degeneration remains elusive. Presently, using co-immunoprecipitation in HEK (Human embryonic kidney) 293 cells, we show that Pink1 endogenously interacts with FK506-binding protein 51 (FKBP51 or FKBP5), FKBP5 and directly phosphorylates FKBP5 at Serine in an in vitro kinase assay. Both FKBP5 and Pink1 have been previously associated with protein kinase B (AKT) regulation. We provide evidence using primary cortical cultured neurons from Pink1-deficient mice that Pink1 increases AKT phosphorylation at Serine 473 (Ser473) challenged by 1-methyl-4-phenylpyridinium (MPP ) and that over-expression of FKBP5 using an adeno-associated virus delivery system negatively regulates AKT phosphorylation at Ser473 in murine-cultured cortical neurons. Interestingly, FKBP5 over-expression promotes death in response to MPP in the absence of Pink1. Conversely, shRNA-mediated knockdown of FKBP5 in cultured cortical neurons is protective and this effect is reversed with inhibition of AKT signaling. In addition, shRNA down-regulation of PH domain leucine-rich repeat protein phosphatase (PHLPP) in Pink1 WT neurons increases neuronal survival, while down-regulation of PHLPP in Pink1 KO rescues neuronal death in response to MPP . Finally, using co-immunoprecipitation, we show that FKBP5 interacts with the kinase AKT and phosphatase PHLPP. This interaction is increased in the absence of Pink1, both in Mouse Embryonic Fibroblasts (MEF) and in mouse brain tissue. Expression of kinase dead Pink1 (K219M) enhances FKBP5 interaction with both AKT and PHLPP. Overall, our results suggest a testable model by which Pink1 could regulate AKT through phosphorylation of FKBP5 and interaction of AKT with PHLPP. Our results suggest a potential mechanism by which PINK1-FKBP5 pathway contributes to neuronal death in PD. OPEN SCIENCE BADGES: This article has received a badge for Open Materials because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
PTEN 诱导的假定激酶 1（Pink1）基因的功能丧失突变与早发性帕金森病（PD）的常染色体隐性家族形式有关。然而，导致退化的潜在机制仍不清楚。目前，我们使用人胚肾 293 细胞中的共免疫沉淀，证明 Pink1 内源地与 FK506 结合蛋白 51（FKBP51 或 FKBP5）相互作用，并在体外激酶测定中直接使 FKBP5 在丝氨酸处磷酸化。FKBP5 和 Pink1 先前都与蛋白激酶 B（AKT）的调节有关。我们使用 Pink1 缺陷型小鼠的原代皮质培养神经元提供证据表明，Pink1 增加了 1-甲基-4-苯基吡啶（MPP）挑战下 AKT 在丝氨酸 473（Ser473）的磷酸化，并且使用腺相关病毒传递系统过表达 FKBP5 会负调控在鼠皮质培养神经元中 AKT 在 Ser473 的磷酸化。有趣的是，FKBP5 的过表达在没有 Pink1 的情况下促进了对 MPP 的死亡反应。相反，在培养的皮质神经元中用 shRNA 介导的 FKBP5 敲低可提供保护作用，而 AKT 信号的抑制可逆转这种作用。此外，在 Pink1 WT 神经元中下调 PH 结构域富含亮氨酸重复蛋白磷酸酶（PHLPP）可增加神经元存活，而在 Pink1 KO 中下调 PHLPP 可挽救 MPP 引起的神经元死亡。最后，通过共免疫沉淀，我们表明 FKBP5 与激酶 AKT 和磷酸酶 PHLPP 相互作用。在缺乏 Pink1 的情况下，这种相互作用在小鼠胚胎成纤维细胞（MEF）和小鼠脑组织中均增加。激酶失活的 Pink1（K219M）的表达增强了 FKBP5 与 AKT 和 PHLPP 的相互作用。总的来说，我们的结果提出了一个可测试的模型，即 Pink1 可以通过磷酸化 FKBP5 并通过 AKT 与 PHLPP 相互作用来调节 AKT。我们的结果表明，PINK1-FKBP5 途径在 PD 中的神经元死亡中可能具有潜在的机制。
