ArfGAP1 is a GTPase activating protein for LRRK2: reciprocal regulation of ArfGAP1 by LRRK2

Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 03/2012; 32(11):3877-86. DOI: 10.1523/JNEUROSCI.4566-11.2012
Source: PubMed


Both sporadic and autosomal dominant forms of Parkinson's disease (PD) have been causally linked to mutations in leucine-rich repeat kinase 2 (LRRK2), a large protein with multiple domains. The kinase domain plays an important role in LRRK2-mediated toxicity. Although a number of investigations have focused on LRRK2 kinase activity, less is known about the GTPase function of LRRK2. The activity of GTPases is regulated by GTPase activating proteins (GAPs) and GTP exchange factors. Here, we identify ArfGAP1 as the first GAP for LRRK2. ArfGAP1 binds LRRK2 predominantly via the WD40 and kinase domain of LRRK2, and it increases LRRK2 GTPase activity and regulates LRRK2 toxicity both in vitro and in vivo in Drosophila melanogaster. Unexpectedly, ArfGAP1 is an LRRK2 kinase substrate whose GAP activity is inhibited by LRRK2, whereas wild-type and G2019S LRRK2 autophosphorylation and kinase activity are significantly reduced in the presence of ArfGAP1. Overexpressed ArfGAP1 exhibits toxicity that is reduced by LRRK2 both in vitro and in vivo. Δ64-ArfGAP1, a dominant-negative ArfGAP1, and shRNA knockdown of ArfGAP1 reduce LRRK2 toxicity. Thus, LRRK2 and ArfGAP1 reciprocally regulate the activity of each other. Our results provide insight into the basic pathobiology of LRRK2 and indicate an important role for the GTPase domain and ArfGAP1 in LRRK2-mediated toxicity. These data suggest that agents targeted toward regulation of LRRK2 GTP hydrolysis might be therapeutic agents for the treatment of PD.

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    • "Roc domain autophosphorylation and resultant effects on GTPase activity require more detailed examination since one possible scenario, a decrease in hydrolysis of GTP to GDP promoted by autophosphorylation and therefore increased via kinase-enhancing mutations , could suggest a common biochemical effect of both kinase domain and Roc domain mutations, which would reinforce the potential importance of GTP-binding/GTPase activity in disease pathogenesis. Indeed, work from our group indicates a significant decrease in GTP hydrolysis rates for G2019S and non-significant decrease for I2020T LRRK2 relative to WT (Xiong et al. 2010, 2012), while another group also reported a 20% decrease in GTPase activity for N-terminally truncated G2019S LRRK2 with no significant effect on GTP binding (Liu et al. 2011a). Whether these effects are mediated through LRRK2 self-regulation (i.e., autophosphorylation), via phosphorylation of exogenous substrates that affect GTPase activity such as ArfGAP1 or a combination of both remains to be determined. "
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    ABSTRACT: Mutations in the catalytic Roc-COR and kinase domains of leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial Parkinson's disease (PD). LRRK2 mutations cause PD with age-related penetrance and clinical features identical to late-onset sporadic PD. Biochemical studies support an increase in LRRK2 kinase activity and a decrease in GTPase activity for kinase domain and Roc-COR mutations, respectively. Strong evidence exists that LRRK2 toxicity is kinase-dependent leading to extensive efforts to identify selective and brain-permeable LRRK2 kinase inhibitors for clinical development. Cell and animal models of PD indicate that LRRK2 mutations affect vesicular trafficking, autophagy, protein synthesis and cytoskeletal function. Although some of these biological functions are affected consistently by most disease-linked mutations, others are not and it is currently unclear how mutations that produce variable effects on LRRK2 biochemistry and function all commonly result in the degeneration and death of dopamine neurons. LRRK2 is typically present in Lewy bodies and its toxicity in mammalian models appears to be dependent on the presence of α-synuclein, which is elevated in human iPS-derived dopamine neurons from patients harboring LRRK2 mutations. Here, we summarize biochemical and functional studies of LRRK2 and its mutations and focus on aberrant vesicular trafficking and protein synthesis as two leading mechanisms underlying LRRK2-linked disease.This article is protected by copyright. All rights reserved.
    Journal of Neurochemistry 09/2014; 131(5). DOI:10.1111/jnc.12949 · 4.28 Impact Factor
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    • "RGS proteins are a family of proteins characterized by a GAP domain of ~130 amino acids, the RGS domain. Other GAP proteins have also been associated with LRRK2 function, including ArfGAP1 and ArhGEF7 (Stafa et al., 2012; Xiong et al., 2012; Habig et al., 2013). Although GAPs and GTPase exchange factors (GEFs) are classically considered to function in regulating signaling of G-protein coupled receptors, these GAPs exhibit a strong role in regulating LRRK2 GTPase activity. "
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    ABSTRACT: LRRK2 is a protein that interacts with a plethora of signaling molecules, but the complexity of LRRK2 function presents a challenge for understanding the role of LRRK2 in the pathophysiology of Parkinson's disease (PD). Studies of LRRK2 using over-expression in transgenic mice have been disappointing, however, studies using invertebrate systems have yielded a much clearer picture, with clear effects of LRRK2 expression, knockdown or deletion in Caenorhabditis elegans and Drosophila on modulation of survival of dopaminergic neurons. Recent studies have begun to focus attention on particular signaling cascades that are a target of LRRK2 function. LRRK2 interacts with members of the mitogen activated protein kinase (MAPK) pathway and might regulate the pathway action by acting as a scaffold that directs the location of MAPK pathway activity, without strongly affecting the amount of MAPK pathway activity. Binding to GTPases, GTPase-activating proteins and GTPase exchange factors are another strong theme in LRRK2 biology, with LRRK2 binding to rac1, cdc42, rab5, rab7L1, endoA, RGS2, ArfGAP1, and ArhGEF7. All of these molecules appear to feed into a function output for LRRK2 that modulates cytoskeletal outgrowth and vesicular dynamics, including autophagy. These functions likely impact modulation of α-synuclein aggregation and associated toxicity eliciting the disease processes that we term PD.
    Frontiers in Molecular Neuroscience 07/2014; 7:64. DOI:10.3389/fnmol.2014.00064 · 4.08 Impact Factor
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    • "Moreover, LRRK2 interacts and phosphorylates a GAP, ADP-ribosylation factor GTPase-activating protein 1 (ArfGAP1). Silencing ArfGAP1 expression was shown to protect against mutant LRRK2 induced neurite shortening, suggesting that ArfGAP1 might also be a potential target in the Parkinson’s disease (Stafa et al., 2012; Xiong et al., 2012). "
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    ABSTRACT: Neurological diseases have placed heavy social and financial burdens on modern society. As the life expectancy of humans is extended, neurological diseases, such as Parkinson's disease, have become increasingly common among senior populations. Although the enigmas of Parkinson's diseases await resolution, more vivid pictures on the cause, progression, and control of the illness are emerging after years of research. On the molecular level, GTPases are implicated in the etiology of Parkinson's disease and are rational pharmaceutical targets for their control. However, targeting individual GTPases, which belong to a superfamily of proteins containing multiple members with a conserved guanine nucleotide binding domain, has proven to be challenging. In contrast, pharmaceutical pursuit of inhibition of kinases, which constitute another superfamily of proteins with more than 500 members, has been fairly successful. We reviewed the breakthroughs in the history of kinase drug discovery to provide guidance for the GTPase field. We summarize recent progress made in the regulation of GTPase activity. We also present an efficient and cost effective approach to drug screening, which uses multiplex flow cytometry and mixture-based positional scanning libraries. These methods allow simultaneous measurements of both the activity and the selectivity of the screened library. Several GTPase activator clusters were identified which showed selectivity against different GTPase subfamilies. While the clusters need to be further deconvoluted to identify individual active compounds, the method described here and the structure information gathered create a foundation for further developments to build upon.
    Frontiers in Molecular Neuroscience 06/2014; 7:52. DOI:10.3389/fnmol.2014.00052 · 4.08 Impact Factor
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