Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1

Abstract

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 (Ser65) 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 (Ser111) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser111 of each of the Rabs, we demonstrate that Rab Ser111 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 Ser111 phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser111 phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser65. We further show mechanistically that phosphorylation at Ser111 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 Ser111 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.

PINK1 and Parkin: emerging themes in mitochondrial homeostasis

“…Recently published phosphoproteomic analyses demonstrated that Rab8A, 8B and 13 are indirect substrates of PINK1 [28 ]. The temporal dynamics of Rab phosphorylation following mitochondrial insult is consistent with their potential contribution to the later stages of mitophagy [28 ].…”

Miro phosphorylation sites regulate Parkin recruitment and mitochondrial motility

“…Recruitment and activation of Parkin, although distinct processes, are thus tightly linked (35,36) by the positive feed-forward effect of ubiquitination by Parkin causing additional binding sites for Parkin, and additional binding causing further ubiquitination and allosteric activation of Parkin. Parkin substrates can also be de-ubiquitinated by Usp30 (37) and Usp15 (38).PINK1, however, can phosphorylate other proteins, including the motor-adaptor protein Miro (also called RhoT1/2), Mitofusin 1/2, and Hsp75 (also known as Trap1) (15,(39)(40)(41)(42), and the functional role of those modifications in regulating Parkin is unclear. Given that some of these proteins are also substrates of Parkin (15, 39), it is possible that modification of these proteins can act as a layer of regulation to modulate the overall levels of Parkin on mitochondria or to modulate specific aspects of mitochondrial dynamics, such as motility and fusion.…”

“…PINK1, however, can phosphorylate other proteins, including the motor-adaptor protein Miro (also called RhoT1/2), Mitofusin 1/2, and Hsp75 (also known as Trap1) (15,(39)(40)(41)(42), and the functional role of those modifications in regulating Parkin is unclear. Given that some of these proteins are also substrates of Parkin (15,39), it is possible that modification of these proteins can act as a layer of regulation to modulate the overall levels of Parkin on mitochondria or to modulate specific aspects of mitochondrial dynamics, such as motility and fusion.…”

A modification switch on a molecular switch: Phosphoregulation of Rab7 during endosome maturation

“…Given that S72 site is conserved among many other Rab proteins, it would be interesting to further test the presence of phosphorylation/dephosphorylation switch in global regulation of membrane localization and activity of other Rab proteins as well. In fact, a recent study showing phosphorylation mediated regulation of Rab8 activity 19 strengthens the argument that phosphorylation might be an important regulatory mechanism of different Rab proteins.…”

“…18 Lately, Lai et al, reported PINK1 mediated phosphorylation of Rab8A at Ser111 and its implication on Rab8A activation. 19 These studies underline the fact that posttranslational modifications such as phosphorylation indeed plays critical role in regulation of Rab GTPase activity. Interestingly, our recent studies have unraveled dephosphorylation of Rab7 by a tumor suppressor phosphatase PTEN.…”