LRRK2 phosphorylates moesin at Thr558; characterisation 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


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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    • "Among its candidate substrates are, LRRK2 itself [21] and a broad number of cytoskeleton-related proteins [22] [23] [24] [25]. The exact function of this kinase is still unknown. "
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    ABSTRACT: We have used the human neuroblastoma cell line SH-SY5Y overexpressing Bcl-xL (SH-SY5Y/ Bcl-xL ) to clarify the effects of this mitochondrial protein on the control of mitochondrial dynamics and the autophagic processes which occur after the inhibition of leucine-rich repeat kinase 2 (LRRK2) with GSK2578215A. In wild type (SH-SY5Y/Neo) cells, GSK2578215A (1nM) caused a disruption of mitochondrial morphology and an imbalance in intracellular reactive oxygen species (ROS) as indicated by an increase in dichlorofluorescein fluorescence and 4-hydroxynonenal. However, SH-SY5Y/ Bcl-xL cells under GSK2578215A treatment, unlike the wild type, preserved a high mitochondrial membrane potential and did not exhibit apoptotical chromatins. In contrast to wild type cells, in SH-SY5Y/Bcl-xL cells, GSK2578215A did not induce mitochondrial translocation of neither dynamin related protein-1 or the proapoptotic protein, Bax. In SH-SY5Y/Neo, but not SH-SY5Y/Bcl-xL cells, mitochondrial fragmentation elicited by GSK2578215A precedes an autophagic response. Furthermore, the overexpression of Bcl-xL protein restores the autophagic flux pathway disrupted by this inhibitor. SH-SY5Y/Neo, but not SH-SY5Y/Bcl-xL cells, responded to LRRK2 inhibition by an increase in the levels of acetylated tubulin, indicating that this was abrogated by Bcl-xL overexpression. This hyperacetylation of tubulin took place earlier than any of the above-mentioned events suggesting that it is involved in the autophagic flux interruption. Pre-treatment with tempol prevented the GSK2578215A-induced mitochondrial fragmentation, autophagy and the rise in acetylated tubulin in SH-SY5Y/Neo cells. Thus, these data support the notion that ROS act as a second messenger connexion between LRRK2 inhibition and these deleterious responses, which are markedly alleviated by the Bcl-xL-mediated ROS generation blockade.
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    • "LRRK2 encodes a large multi-domain protein characterized by leucine-rich repeats, a GTPase domain and a kinase domain (Bosgraaf and Van Haastert, 2003). The cellular functions of LRRK2 remain unclear because it has been linked to multiple diverse cellular processes, including mitochondrial function (Smith et al., 2005), regulation of transcription (Kanao et al., 2010) and translation (Gehrke et al., 2010; Imai et al., 2008; Martin et al., 2014), Golgi protein sorting (Sakaguchi-Nakashima et al., 2007), apoptosis (Ho et al., 2009), and regulation of the dynamics of actin (Jaleel et al., 2007; Parisiadou et al., 2009) and microtubules (Gandhi et al., 2008; Gillardon, 2009; Kett et al., 2012; Lin et al., 2009). Understanding the normal cellular functions of LRRK2 is vital because the mechanisms mediating the pathogenicity of mutant forms of LRRK2 are likely to be related to the physiological functions of the wild-type protein. "
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    ABSTRACT: Mutations in LRRK2 cause a dominantly inherited form of Parkinson's disease (PD), and are the most common known genetic determinant of PD. As inhibitor-based therapies targeting LRRK2 have emerged as a key therapeutic strategy in PD, understanding the consequences of inhibiting the normal cellular functions of LRRK2 is vital. Despite much interest, the physiologic functions of LRRK2 remain unclear. Several recent studies have linked the toxicity caused by overexpression of pathogenic mutant forms of LRRK2 to defects in the endolysosomal and autophagy pathways, raising the question of whether endogenous LRRK2 might play a role in these processes. Here we report the characterization of multiple novel nonsense alleles in the Drosophila LRRK2 homolog lrrk. Using these alleles, we show that lrrk loss-of-function causes striking defects in the endolysosomal and autophagy pathways, including accumulation of markedly enlarged lysosomes that are laden with undigested contents, consistent with a defect in lysosomal degradation. lrrk loss-of-function also results in accumulation of autophagosomes, as well as enlarged early endosomes laden with mono-ubiquitinated cargo proteins, suggesting an additional defect in lysosomal substrate delivery. Interestingly, the lysosomal abnormalities in these lrrk mutants can be suppressed by a constitutively active form of the small GTPase rab9, which promotes retromer-dependent recycling from late endosomes to the Golgi. Collectively, our data provides compelling evidence of a vital role for lrrk in lysosomal function and endolysosomal membrane transport in vivo, and suggests a link between lrrk and retromer-mediated endosomal recycling.
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    • "Furthermore, dopamine neuron loss has been demonstrated in invertebrate organisms and through virally mediated G2019S LRRK2 expression in rodent brain (Lee et al. 2010; Dusonchet et al. 2011). Numerous studies on primary neuronal cultures and intact rodent brain have demonstrated that LRRK2 is important in maintaining neurite length and branching (Smith et al. 2005; MacLeod et al. 2006; West et al. 2007) and may be important in regulating neurite outgrowth, possibly via phosphorylation of ERM (ezrin/radixin/moesin) proteins and consequent maintenance of F-actin homeostasis in filopodia necessary for proper neurite outgrowth (Jaleel et al. 2007; Parisiadou et al. 2009). Expression of pathogenic LRRK2 variants results in neurite dystrophy or loss and ultimately leads to cell death. "
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