LRRK2 phosphorylates moesin at threonine-558: Characterization of how Parkinson's disease mutants affect kinase activity

MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK.
Biochemical Journal (Impact Factor: 4.4). 08/2007; 405(2):307-17. DOI: 10.1042/BJ20070209
Source: PubMed

ABSTRACT Mutations in the LRRK2 (leucine-rich repeat kinase-2) gene cause late-onset PD (Parkinson's disease). LRRK2 contains leucine-rich repeats, a GTPase domain, a COR [C-terminal of Roc (Ras of complex)] domain, a kinase and a WD40 (Trp-Asp 40) motif. Little is known about how LRRK2 is regulated, what its physiological substrates are or how mutations affect LRRK2 function. Thus far LRRK2 activity has only been assessed by autophosphorylation and phosphorylation of MBP (myelin basic protein), which is catalysed rather slowly. We undertook a KESTREL (kinase substrate tracking and elucidation) screen in rat brain extracts to identify proteins that were phosphorylated by an activated PD mutant of LRRK2 (G2019S). This led to the discovery that moesin, a protein which anchors the actin cytoskeleton to the plasma membrane, is efficiently phosphorylated by LRRK2, at Thr558, a previously identified in-vivo-phosphorylation site that regulates the ability of moesin to bind actin. LRRK2 also phosphorylated ezrin and radixin, which are related to moesin, at the residue equivalent to Thr558, as well as a peptide (LRRKtide: RLGRDKYKTLRQIRQ) encompassing Thr558. We exploited these findings to determine how nine previously reported PD mutations of LRRK2 affected kinase activity. Only one of the mutations analysed, namely G2019S, stimulated kinase activity. Four mutations inhibited LRRK2 kinase activity (R1941H, I2012T, I2020T and G2385R), whereas the remainder (R1441C, R1441G, Y1699C and T2356I) did not influence activity. Therefore the manner in which LRRK2 mutations induce PD is more complex than previously imagined and is not only caused by an increase in LRRK2 kinase activity. Finally, we show that the minimum catalytically active fragment of LRRK2 requires an intact GTPase, COR and kinase domain, as well as a WD40 motif and a C-terminal tail. The results of the present study suggest that moesin, ezrin and radixin may be LRRK2 substrates, findings that have been exploited to develop the first robust quantitative assay to measure LRRK2 kinase activity.

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Available from: Axel Knebel, Sep 28, 2015
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    • "Intriguingly, the majority of the pathogenic mutations lie in the catalytic domains of LRRK2. The most common mutation results in the substitution of glycine to serine (G2019S) in the activation loop of the protein kinase domain resulting in a constitutive threefold increase in LRRK2 kinase activity (West et al., 2005; Jaleel et al., 2007). The next most common mutations, substitution of arginine to either histidine (R1141H), cysteine (R1441C), or glycine (R1441G) lie in the GTPase domain. "
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    ABSTRACT: Substantial evidence implicates abnormal protein kinase function in various aspects of Parkinson's disease (PD) etiology. Elevated phosphorylation of the PD-defining pathological protein, α-synuclein, correlates with its aggregation and toxic accumulation in neurons, whilst genetic missense mutations in the kinases PTEN-induced putative kinase 1 and leucine-rich repeat kinase 2, increase susceptibility to PD. Experimental evidence also links kinases of the phosphoinositide 3-kinase and mitogen-activated protein kinase signaling pathways, amongst others, to PD. Understanding how the levels or activities of these enzymes or their substrates change in brain tissue in relation to pathological states can provide insight into disease pathogenesis. Moreover, understanding when and where kinase dysfunction occurs is important as modulation of some of these signaling pathways can potentially lead to PD therapeutics. This review will summarize what is currently known in regard to the expression of these PD-implicated kinases in pathological human postmortem brain tissue.
    Frontiers in Molecular Neuroscience 06/2014; 7:57. DOI:10.3389/fnmol.2014.00057 · 4.08 Impact Factor
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    • "The Arg1628Pro risk factor mutation falls within the COR domain (Ross et al., 2008; Tan et al., 2008). The most common mutation in inherited and idiopathic PD encodes Gly2019Ser, and is located in subdomain VII of the kinase domain and has consistently been shown to increase kinase activity 2–3 fold (Paisan-Ruiz et al., 2004; Zimprich et al., 2004; West et al., 2005; Jaleel et al., 2007). Other pathogenic substitutions , Arg1441Cys, Arg1441Gly, Arg1441His, Tyr1699Cys, and Ile2020Thr, modestly increase kinase activity and GTPase activity (West et al., 2005, 2007; Greggio and Cookson, 2009; Nichols et al., 2010; Webber et al., 2011). "
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    ABSTRACT: Missense mutations in the Leucine Rich Repeat protein Kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson’s disease (PD) LRRK2 is a large multi-domain phosphoprotein with a GTPase domain and a serine/threonine protein kinase domain whose activity is implicated in neuronal toxicity; however the precise mechanism is unknown. LRRK2 autophosphorylates on several serine/threonine residues across the enzyme and is found constitutively phosphorylated on Ser910, Ser935, Ser955 and Ser973, which are proposed to be regulated by upstream kinases. Here we investigate the phosphoregulation at these sites by analyzing the effects of disease-associated mutations Arg1441Cys, Arg1441Gly, Ala1442Pro, Tyr1699Cys, Ile2012Thr, Gly2019Ser, and Ile2020Thr. We also studied alanine substitutions of phosphosite serines 910, 935, 955 and 973 and specific LRRK2 inhibition on autophosphorylation of LRRK2 Ser1292, Thr1491, Thr2483 and phosphorylation at the cellular sites. We found that mutants in the Roc-COR domains, including Arg1441Cys, Arg1441His, Ala1442Pro and Tyr1699Cys, can positively enhance LRRK2 kinase activity while concomitantly inducing the dephosphorylation of the cellular sites. Mutation of the cellular sites individually did not affect LRRK2 intrinsic kinase activity; however, Ser910/935/955/973Ala mutations trended toward increased kinase activity of LRRK2. Increased cAMP levels did not lead to increased LRRK2 cellular site phosphorylation, 14-3-3 binding or kinase activity. In cells, inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser1292 by Calyculin A and okadaic acid sensitive phosphatases, while the cellular sites are dephosphorylated by Calyculin A sensitive phosphatases. These findings indicate that comparative analysis of both Ser1292 and Ser910/935/955/973 phosphorylation sites will provide important and distinct measures of LRRK2 kinase and biological activity in vitro and in vivo.
    Frontiers in Molecular Neuroscience 06/2014; 7:54. DOI:10.3389/fnmol.2014.00054 · 4.08 Impact Factor
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    • "Other mutations, including the R1441C mutation in the ROC domain as well as the Y1699C and I2020T mutations, have only variably been found to increase kinase activity. In these studies, discrepancies may have arisen due to the use of different substrates in the kinase assays, or because of the differences in the way the enzyme was collected (Jaleel et al., 2007; Seol, 2010; West et al., 2007). Overall, though, in experimental models, changes in LRRK2 kinase activity appear to be toxic, and seem to induce degeneration of dopamine neurons (Greggio et al., 2006; Iaccarino et al., 2007; Jorgensen et al., 2009; Smith et al., 2006; West et al., 2007). "
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    ABSTRACT: Leucine-rich repeat kinase 2 (LRRK2) is a large, ubiquitous protein of unknown function. Mutations in the gene encoding LRRK2 have been linked to familial and sporadic Parkinson disease (PD) cases. The LRRK2 protein is a single polypeptide that displays GTPase and kinase activity. Kinase and GTPase domains are involved in different cellular signalling pathways. Despite several experimental studies associating LRRK2 protein with various intracellular membranes and vesicular structures such as endosomal/lysosomal compartments, the mitochondrial outer membrane, lipid rafts, microtubule-associated vesicles, the golgi complex, and the endoplasmic reticulum its broader physiologic function(s) remain unidentified. Additionally, the cellular distribution of LRRK2 may indicate its role in several different pathways, such as the ubiquitin-proteasome system, the autophagic-lysosomal pathway, intracellular trafficking, and mitochondrial dysfunction. This review discusses potential mechanisms through which LRRK2 may mediate neurodegeneration and cause PD.
    Experimental Neurology 06/2014; 261. DOI:10.1016/j.expneurol.2014.05.025 · 4.70 Impact Factor
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