Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser(65)) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser(111)) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser(111) of each of the Rabs, we demonstrate that Rab Ser(111) phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser(111) phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser(111) phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser(65). We further show mechanistically that phosphorylation at Ser(111) significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser(111) may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.
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http://dx.doi.org/10.15252/embj.201591593 | DOI Listing |
J Cell Biol
April 2025
Department of Genetics and Cell Biology, College of Life Sciences, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China.
TBC1D20 deficiency causes Warburg Micro Syndrome in humans, characterized by multiple eye abnormalities, severe intellectual disability, and abnormal sexual development, but the molecular mechanisms remain unknown. Here, we identify TBC1D20 as a novel Rab11 GTPase-activating protein that coordinates vesicle transport and actin remodeling to regulate ciliogenesis. Depletion of TBC1D20 promotes Rab11 vesicle accumulation and actin deconstruction around the centrosome, facilitating the initiation of ciliogenesis even in cycling cells.
View Article and Find Full Text PDFJ Extracell Vesicles
January 2025
Vascular Biology Program, Boston Children's Hospital, Boston, Massachusetts, USA.
Extracellular vesicles (EVs) from brain-seeking breast cancer cells (Br-EVs) breach the blood-brain barrier (BBB) via transcytosis and promote brain metastasis. Here, we defined the mechanisms by which Br-EVs modulate brain endothelial cell (BEC) dynamics to facilitate their BBB transcytosis. BEC treated with Br-EVs show significant downregulation of Rab11fip2, known to promote vesicle recycling to the plasma membrane and significant upregulation of Rab11fip3 and Rab11fip5, which support structural stability of the endosomal compartment and facilitate vesicle recycling and transcytosis, respectively.
View Article and Find Full Text PDFInt J Mol Sci
January 2025
Department of Experimental Medicine, University of Salento, Via Provinciale Lecce-Monteroni n. 165, 73100 Lecce, Italy.
Peripherin belongs to heterogeneous class III of intermediate filaments, and it is the only intermediate filament protein selectively expressed in the neurons of the peripheral nervous system. It has been previously discovered that peripherin interacts with proteins important for the endo-lysosomal system and for the transport to late endosomes and lysosomes, such as RAB7A and AP-3, although little is known about its role in the endocytic pathway. Here, we show that peripherin silencing affects lysosomal abundance but also positioning, causing the redistribution of lysosomes from the perinuclear area to the cell periphery.
View Article and Find Full Text PDFJ Exp Clin Cancer Res
January 2025
Department of Neurosurgery, Children's Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Background: Ovarian cancer (OC) progression is one of the commonest cause of female cancer death. While treatments in clinic includes primary surgery and targeted chemotherapy, curative and survival trends in OC have not significantly improved. Thus, further investigation of the mechanisms regarding OC carcinogenesis and discovery of novel targets is of great importance.
View Article and Find Full Text PDFBeijing Da Xue Xue Bao Yi Xue Ban
February 2025
Department of Stomatology, The Fifth People's Hospital of Qinghai Province & Qinghai Cancer Hospital, Xining 810001, China.
Objective: To investigate the effects of LncRNA SNHG20 on epithelial mesenchymal transition (EMT) and microtubule formation in human oral squamous cell carcinoma (OSCC) cells through targeted regulation of the miR-520c-3p/ pathway.
Methods: After real-time fluorescence quantitative detection of LncRNA SNHG20, miR-520c-3p, mRNA expression levels in OSCC tissues and cells, dual luciferase reporter assay was used to detect the relationship between the three. OSCC cells were randomly separated into control group, sh-NC group, sh-SNHG20 group, sh-SNHG20+anti NC group, and sh-SNHG20+anti miR-520c-3p group.
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