Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson's Disease

Department of Neurology and Neuroscience, Mount Sinai School of Medicine, New York, New York, United States of America.
PLoS ONE (Impact Factor: 3.23). 03/2011; 6(3):e17153. DOI: 10.1371/journal.pone.0017153
Source: PubMed


Recent studies show that mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the cause of the most common inherited and some sporadic forms of Parkinson's disease (PD). The molecular mechanism underlying the pathogenic role of LRRK2 mutations in PD remains unknown.
Using affinity purification and mass spectrometric analysis, we investigated phosphorylation sites and binding proteins of LRRK2 purified from mouse brain. We identified multiple phosphorylation sites at N-terminus of LRRK2 including S910, S912, S935 and S973. Focusing on the high stoichiometry S935 phosphorylation site, we developed an anti-pS935 specific antibody and showed that LRRK2 is constitutively phosphorylated at S935 in various tissues (including brain) and at different ages in mice. We find that 14-3-3 proteins (especially isoforms γ and η) bind LRRK2 and this binding depends on phosphorylation of S935. The binding of 14-3-3, with little effect on dimer formation of LRRK2, confers protection of the phosphorylation status of S935. Furthermore, we show that protein kinase A (PKA), but not LRRK2 kinase itself, can cause the phosphorylation of LRRK2 at S935 in vitro and in cell culture, suggesting that PKA is a potential upstream kinase that regulates LRRK2 function. Finally, our study indicates that the common PD-related mutations of LRRK2, R1441G, Y1699C and G2019S, decrease homeostatic phosphorylation levels of S935 and impair 14-3-3 binding of LRRK2.
LRRK2 is extensively phosphorylated in vivo, and the phosphorylation of specific sites (e.g. S935) determines 14-3-3 binding of LRRK2. We propose that 14-3-3 is an important regulator of LRRK2-mediated cellular functions. Our study suggests that PKA, a cAMP-dependent kinase involved in regulating dopamine physiology, is a potential upstream kinase that phosphorylates LRRK2 at S935. Furthermore, the reduction of phosphorylation/14-3-3 binding of LRRK2 due to the common familial PD-related mutations provides novel insight into the pathogenic mechanism of LRRK2-linked PD.

  • Source
    • "A cluster of serines, including Ser910, Ser935, Ser955, and Ser973, found preceding the namesake LRR domain, appears to be constitutively phosphorylated on LRRK2. It has been proposed that these sites are phosphorylated by kinases other than LRRK2 itself (West et al., 2007; Gloeckner et al., 2010; Nichols et al., 2010; Li et al., 2011; Doggett et al., 2012) and are referred to as the cellular phosphorylation sites herein. These sites are dynamically regulated, becoming rapidly dephosphorylated in cells and tissues after inhibition of LRRK2 with small molecule kinase inhibitors. "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Jun 2014 · Frontiers in Molecular Neuroscience
  • Source
    • "LRRK2 overexpression, both in vitro and in vivo, impairs UPS function. This leads to an accumulation of diverse substrates , including SNCA and ubiquitin (Lichtenberg et al., 2011). LRRK2 and other PD-linked proteins: common pathways "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Jun 2014 · Experimental Neurology
  • Source
    • "Binding of LRRK2 to purified synaptic vesicles further revealed direct interactions with syntaxin-1 and NSF via the WD40 domain of LRRK2 (Fig. 4) (Piccoli et al., 2014). Studies of this type have led to the suggestion that LRRK2 regulates exocytosis, possibly by modulating vesicle pool mobilization driven by activity (Piccoli et al., 2011; reviewed in Belluzzi et al., 2012). However, since inhibiting synaptic vesicle reclustering after endocytosis can reduce neurotransmission (Nemani et al., 2010), and since SNARE proteins and NSF are important for both exo-and endocytosis (Xu et al., 2013; Zhang et al., 2013), the observed phenotypes are equally consistent with a LRRK2-mediated effect on endocytosis (Fig. 4). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mutations in LRRK2 (leucine-rich repeat kinase 2) are found associated with both sporadic and familial Parkinson´s disease (PD). Pathogenic mutations are localized to the catalytic domains of LRRK2, including kinase and GTPase domains. Altered catalytic activity correlates with neurotoxicity, indicating that targeting those activities may provide clues as to novel therapeutic strategies for LRRK2-linked PD. However, the cellular readout of such altered catalytic activities remains largely unknown. Recent cell biological studies have started to highlight possible early cellular events which are altered in the presence of pathogenic LRRK2 and may ultimately lead to neuronal demise, and these studies link altered LRRK2 function to various abnormal endolysosomal vesicular trafficking events. This review examines our current knowledge of LRRK2 neurobiology and how pathogenic mutations may lead to neurodegeneration in PD.
    Full-text · Article · May 2014 · Neuropharmacology
Show more