Cai (NIH Bethesda) provided LRRK2-/- mice

Cai (NIH Bethesda) provided LRRK2-/- mice. LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain name. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD. DOI: (Martin et al., 2014). In accordance with our previous observations (Physique 2G), phosphorylation levels of RAB7L1 were barely detectable, and even lower than those of Msn and Rps15. Strikingly, levels of pRab8a were about ten occasions higher as compared to Rps15 and Msn, two of the best in?vitro LRRK2 substrates known to date, demonstrating that Rabs with Thr sites in the switch II domain name are primary LRRK2 targets (Physique 2H,I). Open in a separate window Physique 2. Phosphorylation of Rab GTPases by LRRK2 in?vitro.(A) Position of threonine 72 in the switch II region of Rab8a (PDB: 4HLY). (B) Sequence alignment of Rab10 and other indicated Rab-family members. (C) Phosphorylation of Rab10 (1 M) by wt-, G2019S- or kinase inactive LRRK2-D1994A. Inhibition of LRRK2-G2019S by GSK2578215A or HG-10-102-01 prevents phosphorylation. (D) Time course of LRRK2 (wt) mediated Rab8a (4 M) phosphorylation and (E) quantification of phosphorylation stoichiometry (n=3). (F) Time course of LRRK2-wt-mediated pT75-Rab1a phosphorylation and MS-based label-free quantification (n=3). (G) In?vitro phosphorylation of recombinant Rab proteins (4 M) by LRRK2-wt. (H) Phosphorylation of recombinant Rab7L1, Rab8a, moesin, and Rps15 by LRRK2 and (I) quantification of the signals. For all those reactions LRRK2 inhibitors= 2 M and LRRK2= 100 ng. Error bars indicate mean SEM of replicates.?MS, mass spectrometry; SEM, standard error of the mean; wt, wild type. DOI: Figure 2figure supplement 1. Open in a separate windows Phosphorylation of Rab GTPases by LRRK2 in?vitro.(A) Superposition of the crystal structures of 14 Rab isoforms (Rab1a, 1b, 2, 3, 4, 6, 7, Rabbit Polyclonal to MOS 9, 12, 18, 27, 30, 31, 43). All potential LRRK2 phosphorylation sites (in grey) cluster in the same region. (B) MS analysis of in?vitro phosphorylated Rab10 identified three LRRK2-specific sites (note that phosphorylation is prevented completely by HG-10-102-01) and pT73 as the one with the highest intensity. The collosion-induced dissociation (CID) fragmentation spectrum and the Andromeda?score (score) Tulobuterol (Cox et al., 2011 ) for the tryptic pT73-Rab10 peptide are shown. (C) Phosphorylation of Rab8a and Rab1b by LRRK2-wt. Inhibition of LRRK2 by HG-10-102-01 prevents phosphorylation. (D) HPLC trace of tryptic peptides of Rab8a and Rab1b (E) after in?vitro phosphorylation by LRRK2-wt and sequence analysis of tryptic peptides. Y axis models are relative Cherenkov counts per minute.?MS, mass spectrometry; wt, wild type. DOI: A subset of Rabs are physiological LRRK2 substrates Tulobuterol Because of the high conservation of T73-Rab10 (Figure 2B) and the ability of LRRK2 to phosphorylate multiple Rabs in?vitro, we inspected our quantitative MS data further to determine whether all sequence and structurally equivalent sites are targets of LRRK2. This turned out not to be the case as pS72-Rab7a was not regulated in either of our screens. LRRK2 thus phosphorylates only a subset of Rab GTPases in mouse fibroblasts. Surprisingly, we noticed that pS105-Rab12, which is not phosphorylated by LRRK2 in?vitro (Physique 2G), was among the significantly?modulated sites in PS1 and also downregulated upon MLI-2 treatment in wt cells as compared to the inhibitor-resistant A2016T mutant in PS2 (Physique 3A,B). However, because of elevated intergroup variability and stringent FDR cut-offs, it was Tulobuterol not selected in our first analysis. LRRK2 is found also in lower eukaryotes such as and (Liu et al., 2011) and T73-Rab10 is usually conserved in these organisms as well. Also, S105-Rab12 is present throughout the vertebrates (Physique 3A,B). We identified both pT73-Rab10 and pS105-Rab12 multiple occasions with high identification and phosphosite localization scores (Supplementary file 1) and the MS/MS fragmentation spectra of the corresponding synthetic peptides Tulobuterol independently validated the MS results (Physique 3figure health supplement 1A,B). Total proteins degrees of Rab10 and Rab12 didn’t modification appreciably in the A2016T knock-in model as judged by quantitative MS evaluation, ruling out that how the observed phospho-level adjustments are because of differential protein manifestation (Shape 3figure health supplement 2A). Open up in another window Shape 3. A genuine amount of Rab GTPases are physiological LRRK2 substrates.(A) MS-quantified pT73-Rab10 peptide intensities in PS1 and PS2. Series alignment from the T73-Rab10 region.