ARHGEF7 (BETA-PIX) Acts as Guanine Nucleotide Exchange Factor for Leucine-Rich Repeat Kinase 2

Background: Mutations within the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of familial and sporadic Parkinson’s disease. The multidomain protein LRRK2 exhibits overall low GTPase and kinase activity in vitro. Methodology/Principal Findings: Here, we show that the rho guanine nucleotide exchange factor ARHGEF7 and the small GTPase CDC42 are interacting with LRRK2 in vitro and in vivo. GTPase activity of full-length LRRK2 increases in the presence of recombinant ARHGEF7. Interestingly, LRRK2 phosphorylates ARHGEF7 in vitro at previously unknown phosphorylation sites. We provide evidence that ARHGEF7 might act as a guanine nucleotide exchange factor for LRRK2 and that R1441C mutant LRRK2 with reduced GTP hydrolysis activity also shows reduced binding to ARHGEF7. Conclusions/Significance: Downstream effects of phosphorylation of ARHGEF7 through LRRK2 could be (i) a feedback control mechanism for LRRK2 activity as well as (ii) an impact of LRRK2 on actin cytoskeleton regulation. A newly identified familial mutation N1437S, localized within the GTPase domain of LRRK2, further underlines the importance of the GTPas

Signal Transduction Protein Array Analysis Links LRRK2 to Ste20 Kinases and PKC Zeta That Modulate Neuronal Plasticity

substrate phosphorylation..Ste20 kinases and PKC zeta contribute to neuronal Tau phosphorylation, neurite outgrowth and synaptic plasticity under physiological conditions. Our data suggest that these kinases may also be involved in synaptic dysfunction and neurite fragmentation in transgenic mice and in human PD patients carrying toxic gain-of-function LRRK2 mutations

The LRRK2 G2385R variant is a partial loss-of-function mutation that affects synaptic vesicle trafficking through altered protein interactions.

Mutations in the Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial Parkinson's disease (PD). LRRK2 protein contains several functional domains, including protein-protein interaction domains at its N- and C-termini. In this study, we analyzed the functional features attributed to LRRK2 by its N- and C-terminal domains. We combined TIRF microscopy and synaptopHluorin assay to visualize synaptic vesicle trafficking. We found that N- and C-terminal domains have opposite impact on synaptic vesicle dynamics. Biochemical analysis demonstrated that different proteins are bound at the two extremities, namely \u3b23-Cav2.1 at N-terminus part and \u3b2-Actin and Synapsin I at C-terminus domain. A sequence variant (G2385R) harboured within the C-terminal WD40 domain increases the risk for PD. Complementary biochemical and imaging approaches revealed that the G2385R variant alters strength and quality of LRRK2 interactions and increases fusion of synaptic vesicles. Our data suggest that the G2385R variant behaves like a loss-of-function mutation that mimics activity-driven events. Impaired scaffolding capabilities of mutant LRRK2 resulting in perturbed vesicular trafficking may arise as a common pathophysiological denominator through which different LRRK2 pathological mutations cause diseas

Medin aggregation causes cerebrovascular dysfunction in aging wild-type mice

Medin is the most common amyloid known in humans, as it can be found in blood vessels of the upper body in virtually everybody over 50 years of age. However, it remains unknown whether deposition of Medin plays a causal role in age-related vascular dysfunction. We now report that aggregates of Medin also develop in the aorta and brain vasculature of wild-type mice in an age-dependent manner. Strikingly, genetic deficiency of the Medin precursor protein, MFG-E8, eliminates not only vascular aggregates but also prevents age-associated decline of cerebrovascular function in mice. Given the prevalence of Medin aggregates in the general population and its role in vascular dysfunction with aging, targeting Medin may become a novel approach to sustain healthy aging

A mechanism for the inhibition of DNA-PK-mediated DNA sensing by a virus

The innate immune system is critical in the response to infection by pathogens and it is activated by pattern recognition receptors (PRRs) binding to pathogen associated molecular patterns (PAMPs). During viral infection, the direct recognition of the viral nucleic acids, such as the genomes of DNA viruses, is very important for activation of innate immunity. Recently, DNA-dependent protein kinase (DNA-PK), a heterotrimeric complex consisting of the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs was identified as a cytoplasmic PRR for DNA that is important for the innate immune response to intracellular DNA and DNA virus infection. Here we show that vaccinia virus (VACV) has evolved to inhibit this function of DNA-PK by expression of a highly conserved protein called C16, which was known to contribute to virulence but by an unknown mechanism. Data presented show that C16 binds directly to the Ku heterodimer and thereby inhibits the innate immune response to DNA in fibroblasts, characterised by the decreased production of cytokines and chemokines. Mechanistically, C16 acts by blocking DNA-PK binding to DNA, which correlates with reduced DNA-PK-dependent DNA sensing. The C-terminal region of C16 is sufficient for binding Ku and this activity is conserved in the variola virus (VARV) orthologue of C16. In contrast, deletion of 5 amino acids in this domain is enough to knockout this function from the attenuated vaccine strain modified vaccinia virus Ankara (MVA). In vivo a VACV mutant lacking C16 induced higher levels of cytokines and chemokines early after infection compared to control viruses, confirming the role of this virulence factor in attenuating the innate immune response. Overall this study describes the inhibition of DNA-PK-dependent DNA sensing by a poxvirus protein, adding to the evidence that DNA-PK is a critical component of innate immunity to DNA viruses

GTPase Activity and Neuronal Toxicity of Parkinson's Disease–Associated LRRK2 Is Regulated by ArfGAP1

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 encodes a large multi-domain protein with GTPase and kinase activity. Initial data indicates that an intact functional GTPase domain is critically required for LRRK2 kinase activity. PD–associated mutations in LRRK2, including the most common G2019S variant, have variable effects on enzymatic activity but commonly alter neuronal process morphology. The mechanisms underlying the intrinsic and extrinsic regulation of LRRK2 GTPase and kinase activity, and the pathogenic effects of familial mutations, are incompletely understood. Here, we identify a novel functional interaction between LRRK2 and ADP-ribosylation factor GTPase-activating protein 1 (ArfGAP1). LRRK2 and ArfGAP1 interact in vitro in mammalian cells and in vivo in brain, and co-localize in the cytoplasm and at Golgi membranes. PD–associated and functional mutations that alter the GTPase activity of LRRK2 modulate the interaction with ArfGAP1. The GTP hydrolysis activity of LRRK2 is markedly enhanced by ArfGAP1 supporting a role for ArfGAP1 as a GTPase-activating protein for LRRK2. Unexpectedly, ArfGAP1 promotes the kinase activity of LRRK2 suggesting a potential role for GTP hydrolysis in kinase activation. Furthermore, LRRK2 robustly and directly phosphorylates ArfGAP1 in vitro. Silencing of ArfGAP1 expression in primary cortical neurons rescues the neurite shortening phenotype induced by G2019S LRRK2 overexpression, whereas the co-expression of ArfGAP1 and LRRK2 synergistically promotes neurite shortening in a manner dependent upon LRRK2 GTPase activity. Neurite shortening induced by ArfGAP1 overexpression is also attenuated by silencing of LRRK2. Our data reveal a novel role for ArfGAP1 in regulating the GTPase activity and neuronal toxicity of LRRK2; reciprocally, LRRK2 phosphorylates ArfGAP1 and is required for ArfGAP1 neuronal toxicity. ArfGAP1 may represent a promising target for interfering with LRRK2-dependent neurodegeneration in familial and sporadic PD

Identification and Characterization of a Leucine-Rich Repeat Kinase 2 (LRRK2) Consensus Phosphorylation Motif

Mutations in LRRK2 (leucine-rich repeat kinase 2) have been identified as major genetic determinants of Parkinson's disease (PD). The most prevalent mutation, G2019S, increases LRRK2's kinase activity, therefore understanding the sites and substrates that LRRK2 phosphorylates is critical to understanding its role in disease aetiology. Since the physiological substrates of this kinase are unknown, we set out to reveal potential targets of LRRK2 G2019S by identifying its favored phosphorylation motif. A non-biased screen of an oriented peptide library elucidated F/Y-x-T-x-R/K as the core dependent substrate sequence. Bioinformatic analysis of the consensus phosphorylation motif identified several novel candidate substrates that potentially function in neuronal pathophysiology. Peptides corresponding to the most PD relevant proteins were efficiently phosphorylated by LRRK2 in vitro. Interestingly, the phosphomotif was also identified within LRRK2 itself. Autophosphorylation was detected by mass spectrometry and biochemical means at the only F-x-T-x-R site (Thr 1410) within LRRK2. The relevance of this site was assessed by measuring effects of mutations on autophosphorylation, kinase activity, GTP binding, GTP hydrolysis, and LRRK2 multimerization. These studies indicate that modification of Thr1410 subtly regulates GTP hydrolysis by LRRK2, but with minimal effects on other parameters measured. Together the identification of LRRK2's phosphorylation consensus motif, and the functional consequences of its phosphorylation, provide insights into downstream LRRK2-signaling pathways

An organelle-specific protein landscape identifies novel diseases and molecular mechanisms

Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine

mTOR independent regulation of macroautophagy by Leucine Rich Repeat Kinase 2 via Beclin-1

Leucine rich repeat kinase 2 is a complex enzyme with both kinase and GTPase activities, closely linked to the pathogenesis of several human disorders including Parkinson’s disease, Crohn’s disease, leprosy and cancer. LRRK2 has been implicated in numerous cellular processes; however its physiological function remains unclear. Recent reports suggest that LRRK2 can act to regulate the cellular catabolic process of macroautophagy, although the precise mechanism whereby this occurs has not been identi ed. To investigate the signalling events through which LRRK2 acts to in uence macroautophagy, the mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) and Beclin-1/phosphatidylinositol 3-kinase (PI3K) pathways were evaluated in astrocytic cell models in the presence and absence of LRRK2 kinase inhibitors. Chemical inhibition of LRRK2 kinase activity resulted in the stimulation of macroautophagy in a non-canonical fashion, independent of mTOR and ULK1, but dependent upon the activation of Beclin 1-containing class III PI3-kinase

Extensive rewiring of the EGFR network in colorectal cancer cells expressing transforming levels of KRASG13D

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.This work was supported by European Union FP7 Grant No. 278568 “PRIMES” and Science Foundation Ireland Investigator Program Grant 14/IA/2395 to W.K. B.K. is supported by SmartNanoTox (Grant no. 686098), NanoCommons (Grant no. 731032), O.R. by MSCA-IF-2016 SAMNets (Grant no. 750688). D.M. is supported by Science Foundation Ireland Career Development award 15-CDA-3495. I.J. is supported by the Canada Research Chair Program (CRC #225404), Krembil Foundation, Ontario Research Fund (GL2-01-030 and #34876), Natural Sciences Research Council (NSERC #203475), Canada Foundation for Innovation (CFI #225404, #30865), and IBM. O.S. is supported by ERC investigator Award ColonCan 311301 and CRUK. I.S. is supported by the Canadian Cancer Society Research Institute (#703889), Genome Canada via Ontario Genomics (#9427 & #9428), Ontario Research fund (ORF/ DIG-501411 & RE08-009), Consortium Québécois sur la Découverte du Médicament (CQDM Quantum Leap) & Brain Canada (Quantum Leap), and CQDM Explore and OCE (#23929). T.C. was supported by a Teagasc Walsh Fellowshi