Dual Arginine Recognition of LRRK2 phosphorylated Rab GTPases

Parkinson’s-disease-associated LRRK2 is a multidomain Ser/Thr kinase that phosphorylates a subset of Rab GTPases to control their effector functions. Rab GTPases are the prime regulators of membrane trafficking in eukaryotic cells. Rabs exert their biological effects by recruitment of effector proteins to subcellular compartments via their Rab-binding domain (RBD). Effectors are modular and typically contain additional domains that regulate various aspects of vesicle formation, trafficking, fusion, and organelle dynamics. The RBD of effectors is typically an α-helical coiled coil that recognizes the GTP conformation of the switch 1 and switch 2 motifs of Rabs. LRRK2 phosphorylates Rab8a at T72 (pT72) of its switch 2 α-helix. This post-translational modification enables recruitment of RILPL2, an effector that regulates ciliogenesis in model cell lines. A newly identified RBD motif of RILPL2, termed the X-cap, has been shown to recognize the phosphate via direct interactions between an arginine residue (R132) and pT72 of Rab8a. Here, we show that a second distal arginine (R130) is also essential for phospho-Rab binding by RILPL2. Through structural, biophysical, and cellular studies, we find that R130 stabilizes the primary R132:pT72 salt bridge through favorable enthalpic contributions to the binding affinity. These findings may have implications for the mechanism by which LRRK2 activation leads to assembly of phospho-Rab complexes and subsequent control of their membrane trafficking functions in cells

LRRK2 transport is regulated by its novel interacting partner Rab32

Leucine-rich repeat kinase 2 (LRRK2) is a multi-domain 280 kDa protein that is linked to Parkinson's disease (PD). Mutations especially in the GTPase and kinase domains of LRRK2 are the most common causes of heritable PD and are also found in sporadic forms of PD. Although the cellular function of LRRK2 is largely unknown there is increasing evidence that these mutations cause cell death due to autophagic dysfunction and mitochondrial damage. Here, we demonstrate a novel mechanism of LRRK2 binding and transport, which involves the small GTPases Rab32 and Rab38. Rab32 and its closest homologue Rab38 are known to organize the trans-Golgi network and transport of key enzymes in melanogenesis, whereas their function in non-melanogenic cells is still not well understood. Cellular processes such as autophagy, mitochondrial dynamics, phagocytosis or inflammatory processes in the brain have previously been linked to Rab32. Here, we demonstrate that Rab32 and Rab38, but no other GTPase tested, directly interact with LRRK2. GFP-Trap analyses confirmed the interaction of Rab32 with the endogenous LRRK2. In yeast two-hybrid experiments we identified a predicted coiled-coil motif containing region within the aminoterminus of LRRK2 as the possible interacting domain. Fluorescence microscopy demonstrated a co-localization of Rab32 and LRRK2 at recycling endosomes and transport vesicles, while overexpression of a constitutively active mutant of Rab32 led to an increased co-localization with Rab7/9 positive perinuclear late endosomes/MVBs. Subcellular fractionation experiments supported the novel role of Rab32 in LRRK2 late endosomal transport and sorting in the cell. Thus, Rab32 may regulate the physiological functions of LRRK2

Rab32 interacts with SNX6 and affects retromer-dependent Golgi trafficking

The Rab family of small GTPases regulate various aspects of cellular dynamics in eukaryotic cells. Membrane trafficking has emerged as central to the functions of leucine-rich repeat kinase 2 (LRRK2), which is associated with inherited and sporadic forms of Parkinson’s disease (PD). Rabs act as both regulators of the catalytic activity and targets for serine/threonine phosphorylation by LRRK2. Rab32, Rab38 and Rab29 have been shown to regulate LRRK2 sub-cellular localization through direct interactions. Recently, Rab29 was shown to escort LRRK2 to the Golgi apparatus and activate the phosphorylation of Rab8 and Rab10. Rab32 is linked to multiple cellular functions including endosomal trafficking, mitochondrial dynamics, and melanosome biogenesis. A missense mutation in Rab32 has also recently been linked to PD. Here, we demonstrate that Rab32 directly interacts with sorting nexin 6 (SNX6). SNX6 is a transient subunit of the retromer, an endosome-Golgi retrieval complex whose Vps35 subunit is strongly associated with PD. We could further show that localization of cation-independent mannose-6-phosphate receptors, which are recycled to the trans-Golgi network (TGN) by the retromer, was affected by both Rab32 and SNX6. These data imply that Rab32 is linked to SNX6/retromer trafficking at the Golgi, and also suggests a possible connection between the retromer and Rab32 in the trafficking and biological functions of LRRK2

A New Mint1 Isoform, but Not the Conventional Mint1, Interacts with the Small GTPase Rab6

Small GTPases of the Rab family are important regulators of a large variety of different cellular functions such as membrane organization and vesicle trafficking. They have been shown to play a role in several human diseases. One prominent member, Rab6, is thought to be involved in the development of Alzheimer’s Disease, the most prevalent mental disorder worldwide. Previous studies have shown that Rab6 impairs the processing of the amyloid precursor protein (APP), which is cleaved to β-amyloid in brains of patients suffering from Alzheimer’s Disease. Additionally, all three members of the Mint adaptor family are implied to participate in the amyloidogenic pathway. Here, we report the identification of a new Mint1 isoform in a yeast two-hybrid screening, Mint1 826, which lacks an eleven amino acid (aa) sequence in the conserved C-terminal region. Mint1 826, but not the conventional Mint1, interacts with Rab6 via the PTB domain. This interaction is nucleotide-dependent, Rab6-specific and influences the subcellular localization of Mint1 826. We were able to detect and sequence a corresponding proteolytic peptide derived from cellular Mint1 826 by mass spectrometry proving the absence of aa 495–505 and could show that the deletion does not influence the ability of this adaptor protein to interact with APP. Taking into account that APP interacts and co-localizes with Mint1 826 and is transported in Rab6 positive vesicles, our data suggest that Mint1 826 bridges APP to the small GTPase at distinct cellular sorting points, establishing Mint1 826 as an important player in regulation of APP trafficking and processing

Mapping of Rab32 binding motif within LRRK2 aminoterminus.

<p>After co-transformation of the yeast strain Y190 with the indicated plasmids with and without the hypothetical coiled-coil motif, cells were grown on synthetic media lacking histidine, supplemented with 30 mM 3 AT. Cell growth on these media and blue staining in subsequent β-gal-filter assays indicated direct interaction of the proteins.</p><p>no growth on selection media or staining in ß-galactosidase filter assay, + growth on selection media and blue staining in in ß-galactosidase filter assay. n = 3 independent experiments.</p><p>Mapping of Rab32 binding motif within LRRK2 aminoterminus.</p

LRRK2-GFP subcellular localization in DsRed-Monomer-Rab32 wt and DsRed-Monomer-Rab32 Q85L overexpressing NIH3T3 cells.

<p>(A) Graphical representation of the most common subcellular features of LRRK2-GFP in DsRed-Monomer-Rab32 wt or DsRed-Monomer-Rab32 Q85L expressing cells. 1: pericentriolar endosome; 2: transport vesicles; 3: perinuclear aggregates. (B–D) Microscopic analysis of NIH3T3 cells co-transfected with plasmids encoding DsRed-Monomer-Rab32 wt or DsRed-Monomer-Rab32 Q85L and LRRK2-GFP. In every cell the LRRK2-GFP channel was analyzed for the occurrence of the features transport vesicles, pericentriolar endosomes and perinuclear aggregates. (-) LRRK2-GFP alone: n = 71 cells from 5 independent experiments; LRRK2-GFP and DsRed-Monomer-Rab32 wildtype: n = 15 cells from 3 independent experiments, LRRK2-GFP and DsRed-Monomer-Rab32 Q85L: n = 21 cells from 5 independent experiments.</p

Co-localization analysis of Rab32 wt and the constitutively active mutant Rab32 Q85L with different endosomal markers.

<p>(A) NIH3T3 cells were co-transfected with plasmids encoding the recycling endosome marker GFP-Rab11B and DsRed-Monomer-Rab32 wt or DsRed-Monomer-Rab32 Q85L, fixed and analyzed by fluorescence microscopy. Scale bar = 10 µm. (B) Cells were transfected with plasmids encoding for GFP-Rab32 wt or GFP-Rab32 Q85L followed by fixation and subsequent immunofluorescence staining of Rab7. Scale bar = 10 µm. (C–E) NIH3T3 cells expressing either GFP-Rab32 wt or GFP-Rab32 Q85L were fixed and stained for Rab7. (C) Microscopic analysis of GFP-Rab32 Q85L that co-localized with endogenous Rab7 (arrows) in the perinuclear area. Non co-localizing Rab7 was indicated by arrowheads. The perinuclear area was defined by the red (outlines nucleus) and the yellow line (outer border for perinuclear area). The image illustrates the cellular area used for the following analysis. (D) Rab7 perinuclear aggregates co-localizing with GFP-Rab32 wt or GFP-Rab32 Q85L and non co-localizing ones. ctrl. = control (untransfected IHKE-1 cells) (E) Quantification of perinuclear Rab7-positive structures. ctrl. (control): perinuclear Rab7 in untransfected cells; wt/Q85L: perinuclear Rab7 co-localizing with GFP-Rab32 constructs. control/Rab32 wt/Rab32 Q85L: n = 1660/18/167 structures in 67/18/60 cells, 1/1/3 independent experiments. Statistical significance was tested by a Student's T-test: p_control-wt = 0.02; p_control-Q85L<0.005; p_wt-Q85L = 0.02. Scale bar = 10 µm.</p

Binding of endogenous LRRK2 by the small GTPase Rab32.

<p>(A) GST-Rab32 wt, GST-Rab32 Q85L or GST as control was applied to glutathione agarose beads followed by incubation with NIH3T3 lysate overnight. Samples were analyzed by 6% SDS-PAGE and subsequent Western blot analysis to detect LRRK2. n≥3 independent experiments. (B) Lysates from IHKE-1 cells stably expressing GFP-Rab32 wt were incubated overnight with an anti-LRRK2 antibody (1E11). IP control = no antibody was added. Co-precipitated GFP-Rab32 wt was detected using an anti-Rab32 antibody. n = 3 independent experiments. (C) Lysates from IHKE-1 cells expressing GFP-Rab32 wt, GFP-Rab32 Q85L or GFP as control were subjected to immunoprecipitation by the GFP-Trap kit. Co-precipitated endogenous LRRK2 was detected using an anti LRRK2 antibody. n = 2 independent experiments.</p