Abstract
Loss of function mutations in the PTEN-induced kinase 1 (PINK1) kinase are causal for autosomal recessive Parkinson's disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies, we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor and GTPase activating protein. In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2 in vitro. Strikingly, we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 of recombinant nucleotide-bound Rab8A in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in transfected HEK293 cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and nuclear magnetic resonance structure of Ser111-phosphorylated Rab1B. The structures reveal that the phosphorylated SF3 motif does not induce any major changes, but may interfere with effector-Switch II interactions through intramolecular H-bond formation and/or charge effects with Arg79. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A indicating a potential cross-talk between PINK1-regulated mitochondrial homeostasis and LRRK2 signalling that requires further investigation in vivo.
Highlights
Autosomal recessive mutations in PTEN-induced kinase 1 (PINK1) represent the second most frequent cause of early onset Parkinson’s disease (PD) [1]
MST3 and TAK1 were still capable of phosphorylating pSer111-Rab8A:GDP to a similar extent as WT-Rab8A:GDP (Figure 3B). Consistent with these results we found pSer111-Rab1B:GDP has no effect on the ability of MST3 and TAK1 kinases to phosphorylate Thr72 compared with WT-Rab1B:GDP compared with LRRK2 (Supplementary Figure S8B)
Using genetic code expansion technology, we have been able to demonstrate that PINK1-dependent phosphorylation of Rab GTPases at Ser111 impairs the ability of Rabs to interact with Guanosine nucleotide exchange factor (GEF) and GTPase activating protein (GAP)
Summary
Autosomal recessive mutations in PTEN-induced kinase 1 (PINK1) represent the second most frequent cause of early onset Parkinson’s disease (PD) [1]. In response to chemical uncouplers (e.g. carbonyl cyanide m-chlorophenyl hydrazone (CCCP)) that induce mitochondrial depolarization, PINK1 is activated and directly phosphorylates Parkin at Serine 65 (Ser65) within its N-terminal Ubiquitin-like domain [2,3] as well as the equivalent Ser residue of ubiquitin [4,5,6,7]. The phosphorylation of both of these residues is required for maximal activation of Parkin E3 ligase activity that induces ubiquitylation of a myriad of substrates and acts as a signal for the elimination of damaged mitochondria via mitophagy [4,5,6,7]. Of relevance to PD mechanisms, loss of function mutations of PINK1 abolish Rab protein phosphorylation as assessed in primary mouse embryonic fibroblasts from PINK1 knockout mice or human adult fibroblasts derived from patients harbouring homozygous PINK1 Q456X mutations [8]
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