Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis

Abstract

We previously reported that Parkinson’s disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies.

Rab29 activation of the Parkinson's disease‐associated LRRK2 kinase

“…Members of the Rab GTPase family, including Rab8A, Rab10, and Rab29 (also known as RAB7L1), are substrates for LRRK2 (Steger et al , ). Recent work has defined a subset of 14 Rab proteins (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35, and Rab43) that are potential direct substrates for LRRK2 (Steger et al , ). The LRRK2 phosphorylation site (Thr72 for Rab8A and Thr73 for Rab10) for all of these Rab proteins lies within the effector‐binding, switch II motif (Pfeffer, ; Cherfils & Zeghouf, ).…”

“…The LRRK2 phosphorylation site (Thr72 for Rab8A and Thr73 for Rab10) for all of these Rab proteins lies within the effector‐binding, switch II motif (Pfeffer, ; Cherfils & Zeghouf, ). LRRK2 phosphorylation of Rab8A and Rab10 proteins blocks binding to Rab GDP‐dissociation inhibitor (GDI) that is required for Rab protein membrane delivery and recycling; phosphorylation also inhibits binding of Rab8A to Rabin‐8, its cognate guanine nucleotide exchange factor (GEF) (Steger et al , , ). Pathogenic mutations located within the GTPase (R1441G/C) and COR (Y1699C) domains do not directly stimulate LRRK2 kinase activity in vitro (Jaleel et al , ; Nichols et al , ); nevertheless, they markedly enhance phosphorylation of Rab isoforms to an even greater extent than the G2019S mutation in vivo (Ito et al , ; Steger et al , ).…”

Role of the endolysosomal system in Parkinson’s disease

“…LRRK2’s role in vesicular trafficking, lysosomal homeostasis, and autophagy are well‐accepted. Phosphorylation by LRRK2 is essential for a subset of RAB GTPases’ function of E‐L vesicular sorting and trafficking (Steger et al, ; Steger et al, ). In an over‐expression system, RAB7L1 was noted to interact strongly with LRRK2 at the switch‐II site (Steger et al, ), unlike as seen in in vitro kinase assays (Steger et al, ).…”

“…Phosphorylation by LRRK2 is essential for a subset of RAB GTPases’ function of E‐L vesicular sorting and trafficking (Steger et al, ; Steger et al, ). In an over‐expression system, RAB7L1 was noted to interact strongly with LRRK2 at the switch‐II site (Steger et al, ), unlike as seen in in vitro kinase assays (Steger et al, ). Several LRRK2 mutations significantly affect RAB pathways, where variations in Rab7L1’s interactions with LRRK2 have been identified to increase the risk of sporadic PD.…”

The LRRK2 signalling system

“…These represent probably the most thoroughly validated substrates for LRRK2 to date (other than LRRK2 itself) and yet—as will be discussed further below—despite this the impact of mutations on phosphorylation of these substrates is not yet completely clear. More recent data have highlighted a range of Rab proteins as being potentially phosphorylated by LRRK2, including Rab-3 A/B/C/D, Rab-12, Rab-35 and Rab-43, in addition to Rab-8 and Rab-10 (Steger et al 2017). Of these, phosphoregulation of Rab-10 and Rab-12 has recently been replicated by an independent group (Thirstrup et al 2017).…”