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A Direct Interaction between Leucine-rich Repeat Kinase 2 and Specific β-Tubulin Isoforms Regulates Tubulin Acetylation*

  • Bernard M.H. Law
    Footnotes
    Affiliations
    Department of Pharmacology, UCL School of Pharmacy, University College London 29-39 Brunswick Square, London WC1N 1AX, United Kingdom
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  • Victoria A. Spain
    Footnotes
    Affiliations
    Department of Pharmacology, UCL School of Pharmacy, University College London 29-39 Brunswick Square, London WC1N 1AX, United Kingdom
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  • Veronica H.L. Leinster
    Affiliations
    Department of Pharmacology, UCL School of Pharmacy, University College London 29-39 Brunswick Square, London WC1N 1AX, United Kingdom
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  • Ruth Chia
    Affiliations
    Cell Biology and Gene Expression Unit, Laboratory of Neurogenetics
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  • Alexandra Beilina
    Affiliations
    Cell Biology and Gene Expression Unit, Laboratory of Neurogenetics
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  • Hyun J. Cho
    Affiliations
    Transgenics Section, Laboratory of Neurogenetics, NIA, National Institutes of Health, Bethesda, MD 20892
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  • Jean-Marc Taymans
    Affiliations
    Department of Neurosciences, Laboratory for Neurobiology and Gene Therapy, and Leuven Research Institute for Neuroscience & Disease (LIND), Katholieke Universiteit Leuven, 3000 Leuven, Belgium
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  • Mary K. Urban
    Affiliations
    Cell Biology and Gene Expression Unit, Laboratory of Neurogenetics
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  • Rosa M. Sancho
    Footnotes
    Affiliations
    Department of Pharmacology, UCL School of Pharmacy, University College London 29-39 Brunswick Square, London WC1N 1AX, United Kingdom
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  • Marian Blanca Ramírez
    Footnotes
    Affiliations
    Department of Pharmacology, UCL School of Pharmacy, University College London 29-39 Brunswick Square, London WC1N 1AX, United Kingdom
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  • Saskia Biskup
    Affiliations
    Hertie Institute for Clinical Brain Research and German Center for Neurodegenerative Diseases, 72076 Tübingen, Germany
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  • Veerle Baekelandt
    Affiliations
    Department of Neurosciences, Laboratory for Neurobiology and Gene Therapy, and Leuven Research Institute for Neuroscience & Disease (LIND), Katholieke Universiteit Leuven, 3000 Leuven, Belgium
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  • Huaibin Cai
    Affiliations
    Transgenics Section, Laboratory of Neurogenetics, NIA, National Institutes of Health, Bethesda, MD 20892
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  • Mark R. Cookson
    Affiliations
    Cell Biology and Gene Expression Unit, Laboratory of Neurogenetics
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  • Daniel C. Berwick
    Affiliations
    Department of Pharmacology, UCL School of Pharmacy, University College London 29-39 Brunswick Square, London WC1N 1AX, United Kingdom
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  • Kirsten Harvey
    Correspondence
    To whom correspondence should be addressed: Kirsten Harvey MD PhD, Department of Pharmacology, UCL School of Pharmacy, University College London, London WC1N 1AX, United Kingdom. Tel.: 0207-753-5888; Fax: 0207-753-5902
    Affiliations
    Department of Pharmacology, UCL School of Pharmacy, University College London 29-39 Brunswick Square, London WC1N 1AX, United Kingdom
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  • Author Footnotes
    * This work was supported, in whole or in part, by the National Institutes of Health Intramural Research Program of the National Institutes of Health, NIA (to M. R. C. and H. C.), Wellcome Trust Grants WT088145AIA and WT095010MA (to K. H.), grants from the Michael J. Fox Foundation (to K. H., V. B., and J. M. T.), a Vera Down British Medical Association Research grant (to K. H.), an Ibercaja Obra Social award (to M. B. R.), and the Fund Druwé-Eerdekens managed by the King Baudouin Foundation (to J. M. T.).
    7 D. C. Berwick and K. Harvey, unpublished data.
    1 Both authors contributed equally to the study.
    2 Present address: Dept. of Biology and Biochemistry, University of Bath, Claverton Down, Bath, Bath and NE Somerset, BA2 7AY, United Kingdom.
    3 Present address: Alzheimer's Research UK, 3 Riverside, Granta Park, Great Abington CB21 6AD, United Kingdom.
    4 Present address: Instituto de Parasitología y Biomedicina (López-Neyra), Consejo Superior do Investigaciones Cientificas (CSIC), 18100 Granada, Spain.
Open AccessPublished:November 25, 2013DOI:https://doi.org/10.1074/jbc.M113.507913
      Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease.

      Introduction

      Mutations in LRRK2, encoding leucine-rich repeat kinase 2 (LRRK2)
      The abbreviations used are:
      LRRK2
      leucine-rich repeat kinase 2
      PD
      Parkinson disease
      ANOVA
      analysis of variance
      YTH
      yeast two-hybrid
      Q-YTH
      quantitative YTH
      MT
      microtubule
      MEF
      mouse embryonic fibroblast
      EGFP
      enhanced green fluorescent protein
      BisTris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
      are a common cause of inherited Parkinson disease (PD) (
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      Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease.
      ,
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      Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology.
      ). Because LRRK2 mutation carriers present symptoms and brain pathology very similar to idiopathic PD (
      • Paisán-Ruíz C.
      • Jain S.
      • Evans E.W.
      • Gilks W.P.
      • Simón J.
      • van der Brug M.
      • López de Munain A.
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      • Lees A.
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      • Pérez-Tur J.
      • Wood N.W.
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      Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease.
      ,
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      ), understanding the biological role of LRRK2 could help to uncover new therapeutic strategies for both inherited and sporadic cases. LRRK2 belongs to the ROCO family of proteins, which are characterized by the unique combination of a Roc (Ras of complex proteins) domain with intrinsic GTPase activity and a COR (C-terminal of Roc) domain. The combination of a GTPase domain and a kinase domain suggests a complex role for LRRK2 in cell signaling (
      • Berwick D.C.
      • Harvey K.
      LRRK2 signaling pathways. The key to unlocking neurodegeneration?.
      ). Additional protein-protein interaction domains, such as leucine-rich repeat (LRR) and WD40 propeller motifs suggest that LRRK2 has multiple protein interactors that potentially localize LRRK2 to different subcellular compartments (
      • Paisán-Ruíz C.
      • Jain S.
      • Evans E.W.
      • Gilks W.P.
      • Simón J.
      • van der Brug M.
      • López de Munain A.
      • Aparicio S.
      • Gil A.M.
      • Khan N.
      • Johnson J.
      • Martinez J.R.
      • Nicholl D.
      • Carrera I.M.
      • Pena A.S.
      • de Silva R.
      • Lees A.
      • Martí-Massó J.F.
      • Pérez-Tur J.
      • Wood N.W.
      • Singleton A.B.
      Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease.
      ,
      • Zimprich A.
      • Biskup S.
      • Leitner P.
      • Lichtner P.
      • Farrer M.
      • Lincoln S.
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      • Vieregge P.
      • Asmus F.
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      • Meitinger T.
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      • Wszolek Z.K.
      • Gasser T.
      Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology.
      ,
      • Kumari U.
      • Tan E.K.
      LRRK2 in Parkinson's disease. Genetic and clinical studies from patients.
      ,
      • Berwick D.C.
      • Harvey K.
      LRRK2 signaling pathways. The key to unlocking neurodegeneration?.
      ). Although many sequence variants have been reported in LRRK2, dominant mutations clearly segregating with PD are only found in the RocCOR tandem domain or the kinase domain (
      • Paisán-Ruíz C.
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      • Evans E.W.
      • Gilks W.P.
      • Simón J.
      • van der Brug M.
      • López de Munain A.
      • Aparicio S.
      • Gil A.M.
      • Khan N.
      • Johnson J.
      • Martinez J.R.
      • Nicholl D.
      • Carrera I.M.
      • Pena A.S.
      • de Silva R.
      • Lees A.
      • Martí-Massó J.F.
      • Pérez-Tur J.
      • Wood N.W.
      • Singleton A.B.
      Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease.
      ,
      • Zimprich A.
      • Biskup S.
      • Leitner P.
      • Lichtner P.
      • Farrer M.
      • Lincoln S.
      • Kachergus J.
      • Hulihan M.
      • Uitti R.J.
      • Calne D.B.
      • Stoessl A.J.
      • Pfeiffer R.F.
      • Patenge N.
      • Carbajal I.C.
      • Vieregge P.
      • Asmus F.
      • Müller-Myhsok B.
      • Dickson D.W.
      • Meitinger T.
      • Strom T.M.
      • Wszolek Z.K.
      • Gasser T.
      Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology.
      ,
      • Kumari U.
      • Tan E.K.
      LRRK2 in Parkinson's disease. Genetic and clinical studies from patients.
      ,
      • Berwick D.C.
      • Harvey K.
      LRRK2 signaling pathways. The key to unlocking neurodegeneration?.
      ). LRRK2 kinase and GTPase activity are clearly important for the cytotoxicity and neurite changes observed with LRRK2 mutants in cellular models (
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      Parkinson's disease-associated mutations in LRRK2 link enhanced GTP-binding and kinase activities to neuronal toxicity.
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      Kinase activity is required for the toxic effects of mutant LRRK2/dardarin.
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      Leucine-rich repeat kinase 2 mutations and Parkinson's disease. Three questions.
      ,
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      GTPase activity plays a key role in the pathobiology of LRRK2.
      ,
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      • Ribe E.
      • Troy C.M.
      • Dauer W.T.
      The Parkinson disease protein leucine-rich repeat kinase 2 transduces death signals via Fas-associated protein with death domain and caspase-8 in a cellular model of neurodegeneration.
      ,
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      Role of autophagy in G2019S-LRRK2-associated neurite shortening in differentiated SH-SY5Y cells.
      ,
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      The familial Parkinsonism gene LRRK2 regulates neurite process morphology.
      ). However, the precise mechanisms by which LRRK2 mediates these events remain elusive.
      One newly emerging theme is the interaction between LRRK2 and the cytoskeleton. For example, LRRK2 has been shown to interact with microtubules (MTs) (
      • Gandhi P.N.
      • Wang X.
      • Zhu X.
      • Chen S.G.
      • Wilson-Delfosse A.L.
      The Roc domain of leucine-rich repeat kinase 2 is sufficient for interaction with microtubules.
      ,
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      Leucine-rich repeat kinase 2 phosphorylates brain tubulin-β isoforms and modulates microtubule stability. A point of convergence in parkinsonian neurodegeneration?.
      ,
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      Interaction of elongation factor 1-α with leucine-rich repeat kinase 2 impairs kinase activity and microtubule bundling in vitro.
      ,
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      • Terada M.
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      LRRK2 Parkinson disease mutations enhance its microtubule association.
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      LRRK2 phosphorylates tubulin-associated tau but not the free molecule. LRRK2-mediated regulation of the Tau-tubulin association and neurite outgrowth.
      ,
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      • Deak M.
      • Hentati F.
      • Reith A.D.
      • Prescott A.R.
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      Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization.
      ,
      • Sancho R.M.
      • Law B.M.
      • Harvey K.
      Mutations in the LRRK2 Roc-COR tandem domain link Parkinson's disease to Wnt signalling pathways.
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      Localization of LRRK2 to membranous and vesicular structures in mammalian brain.
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      ,
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      LRRK2 function on actin and microtubule dynamics in Parkinson disease.
      ) and influence MAPK and Wnt signaling pathways (
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      Mutations in the LRRK2 Roc-COR tandem domain link Parkinson's disease to Wnt signalling pathways.
      ,
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      The importance of Wnt signalling for neurodegeneration in Parkinson's disease.
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      ), is seen in several animal models expressing LRRK2 mutants (
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      LRRK2 G2019S mutation induces dendrite degeneration through mislocalization and phosphorylation of tau by recruiting autoactivated GSK3β.
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      Axonal transport and the delivery of pre-synaptic components.
      ).
      Here, we demonstrate a specific and direct interaction between LRRK2 and three β-tubulin isoforms that is mediated by the LRRK2 Roc domain and β-tubulin C termini. We demonstrate that this interaction is dependent on guanidine nucleotide binding and modulated by Roc domain autophosphorylation and disrupted by the pathogenic LRRK2 mutation R1441G. We also show that lysine 362 (Lys-362) and alanine 364 (Ala-364) in TUBB and TUBB4 underlie the isoform specificity of the LRRK2-β-tubulin interaction. Molecular modeling indicates that Lys-362 is present on an interaction surface in the lumen of MT filaments close to the lysine 40 (Lys-40) acetylation site in α-tubulin. Corroborating this finding, LRRK2 knock-out mouse embryonic fibroblasts (MEFs) show increased tubulin acetylation. Last, we demonstrate that LRRK2 co-localizes with highly dynamic cytoskeletal structures in dopaminergic cells, and that LRRK2 overexpression and mutation impacts upon the morphology of growth cones. Taken together, these data suggest a role for LRRK2 in the regulation of cytoskeletal dynamics with implications for the pathogenesis of PD.

      DISCUSSION

      This study defines the interaction between LRRK2 and the cytoskeleton, demonstrating that the LRRK2 Roc domain binds specifically to three neuronally expressed β-tubulin isoforms, TUBB, TUBB4, and TUBB6, but not the other common isoforms, TUBB1, TUBB2A/B/C, and TUBB3. This suggests that LRRK2 distribution along MTs is determined by the tubulin composition of MTs, in particular by the types of β-tubulin present. This specificity might also account for differences in LRRK2 association with MTs in different brain regions or cell types. We mapped the LRRK2-β-tubulin interaction surface to a site centered around Lys-362, which is located on the same surface of α/β-tubulin heterodimers as two sites important for the modulation of MT stability: the binding site for the MT-stabilizing drug taxol on β-tubulin (Fig. 4, A and B) and the MT acetylation site on α-tubulin at Lys-40 (Fig. 7, A and B). These observations suggested that LRRK2 binding to MTs could modulate MT stability. Consistent with this view, LRRK2 knock-out MEF cells show a marked increase in MT acetylation at the key α-tubulin residue Lys-40 (Fig. 7, C–E), indicative of greater MT stability. Whether this represents increased acetylation or decreased deacetylation (or a combination of both mechanisms) cannot yet be determined. Nonetheless, it is interesting to speculate as to the mechanism by which the LRRK2 protein might decrease Lys-40 acetylation. In mammals, the major enzyme catalyzing tubulin acetylation is the α-tubulin Lys-40 acetyltransferase (also known as MEC-17) (
      • Shida T.
      • Cueva J.G.
      • Xu Z.
      • Goodman M.B.
      • Nachury M.V.
      The major α-tubulin K40 acetyltransferase αTAT1 promotes rapid ciliogenesis and efficient mechanosensation.
      ,
      • Akella J.S.
      • Wloga D.
      • Kim J.
      • Starostina N.G.
      • Lyons-Abbott S.
      • Morrissette N.S.
      • Dougan S.T.
      • Kipreos E.T.
      • Gaertig J.
      MEC-17 is an α-tubulin acetyltransferase.
      ), whereas deacetylation is carried out by two enzymes, histone deacetylase 6 (HDAC6) (
      • Hubbert C.
      • Guardiola A.
      • Shao R.
      • Kawaguchi Y.
      • Ito A.
      • Nixon A.
      • Yoshida M.
      • Wang X.F.
      • Yao T.P.
      HDAC6 is a microtubule-associated deacetylase.
      ,
      • Matsuyama A.
      • Shimazu T.
      • Sumida Y.
      • Saito A.
      • Yoshimatsu Y.
      • Seigneurin-Berny D.
      • Osada H.
      • Komatsu Y.
      • Nishino N.
      • Khochbin S.
      • Horinouchi S.
      • Yoshida M.
      In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation.
      ,
      • Zhang Y.
      • Li N.
      • Caron C.
      • Matthias G.
      • Hess D.
      • Khochbin S.
      • Matthias P.
      HDAC-6 interacts with and deacetylates tubulin and microtubules in vivo.
      ) and sirtuin2 (SIRT2) (
      • North B.J.
      • Marshall B.L.
      • Borra M.T.
      • Denu J.M.
      • Verdin E.
      The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase.
      ). In principle LRRK2 could alter the activity of any one of these enzymes, for example, via protein-protein interaction, or alternatively via phosphorylation. However, given the large size of LRRK2 (see following paragraph), we would suggest that the most likely explanation is that LRRK2 simply prevents α-tubulin Lys-40 acetyltransferase from accessing Lys-40, thereby keeping α-tubulin in a non-acetylated state. Clearly, these ideas require further testing. Nonetheless, we conclude that LRRK2-tubulin interactions affect MT acetylation and thereby promote MT destabilization.
      We also note that Lys-362 has been reported to be a site of ubiquitination in two proteomic studies (TUBB2B, TUBB3, and TUBB6 (
      • Wagner S.A.
      • Beli P.
      • Weinert B.T.
      • Schölz C.
      • Kelstrup C.D.
      • Young C.
      • Nielsen M.L.
      • Olsen J.V.
      • Brakebusch C.
      • Choudhary C.
      Proteomic analyses reveal divergent ubiquitylation site patterns in murine tissues.
      ); TUBB, TUBB2C, TUBB3, TUBB4, and TUBB6 (
      • Sarraf S.A.
      • Raman M.
      • Guarani-Pereira V.
      • Sowa M.E.
      • Huttlin E.L.
      • Gygi S.P.
      • Harper J.W.
      Landscape of the Parkin-dependent ubiquitylome in response to mitochondrial depolarization.
      )). Ubiquitinated Lys-362 would be predicted to block LRRK2 association with MTs. Therefore, a dynamic regulation of LRRK2-β-tubulin interactions and by extension MT acetylation via Lys-362 ubiquitination in TUBB, TUBB4, and TUBB6 is plausible.
      Our hypothesis is that LRRK2-β-tubulin binding interferes with tubulin acetylation and occurs predominantly in dynamic pools of MTs with a more open, flexible conformation (
      • Cueva J.G.
      • Hsin J.
      • Huang K.C.
      • Goodman M.B.
      Posttranslational acetylation of α-tubulin constrains protofilament number in native microtubules.
      ). Consistent with this idea, we found a preferential localization of LRRK2 to MTs within growth cones, rather than adjacent stable MTs in axons (Fig. 6, B and F). Growth cone function relies on the dynamic instability of MTs and dynamic MTs display weaker interprotofibril associations. This creates greater luminal space, allowing for the binding of a large molecule such as LRRK2. LRRK2-β-tubulin interactions are likely to be of functional relevance to growth cone biology, because over-expression of LRRK2 during differentiation results in a reduction in growth cone width (Fig. 6I) and number of filopodia per growth cone (Fig. 6J). Nonetheless, the location of the LRRK2 binding site on β-tubulins does raise the question of whether this protein might interact with MTs within mature, stable MT tracts, such as those in axons. It should be noted that the presence of protein complexes within MT filaments is well supported in the literature. Indeed, the MAP Tau has been demonstrated to partially reside inside this compartment (
      • Kar S.
      • Fan J.
      • Smith M.J.
      • Goedert M.
      • Amos L.A.
      Repeat motifs of tau bind to the insides of microtubules in the absence of taxol.
      ). However, whereas LRRK2 seems able to access the lumen of dynamic MT structures at growth cones, we would suggest that it is less likely that LRRK2 can access the β-tubulin binding site along the entire length of neurites within the MT lumen. An electron microscopic study of cross-sectioned MT filaments in Drosophila revealed an average internal area of 244 nm2, indicating that MTs with an internal diameter greater than 19 nm are extremely rare in nature (
      • Cueva J.G.
      • Hsin J.
      • Huang K.C.
      • Goodman M.B.
      Posttranslational acetylation of α-tubulin constrains protofilament number in native microtubules.
      ). Assuming a tubulin width of ∼6.5 nm (
      • Cueva J.G.
      • Hsin J.
      • Huang K.C.
      • Goodman M.B.
      Posttranslational acetylation of α-tubulin constrains protofilament number in native microtubules.
      ), this internal diameter can be considered equivalent to three tubulin monomers at most. The crystal structure of LRRK2 has not been resolved and thus the physical size of this protein is unknown. Nonetheless, at 2527 amino acids in length, LRRK2 is over five times the size of a tubulin monomer. Thus it seems likely that LRRK2 is too large to enter this compartment. In conclusion, LRRK2 appears to bind directly to the lumen of MTs interfering with tubulin acetylation in vivo. This binding is likely restricted to locations where MT protofibrils are held in an “open” confirmation.
      We also demonstrated that the Roc-domain R1441G (and the less frequent familial R1441H) mutant with proven pathogenicity disrupted LRRK2-β-tubulin interactions, whereas the R1441C variant increased LRRK2-β-tubulin interactions. A trend toward an increased LRRK2-β-tubulin interaction for the R1441C mutant was previously reported (
      • Gandhi P.N.
      • Wang X.
      • Zhu X.
      • Chen S.G.
      • Wilson-Delfosse A.L.
      The Roc domain of leucine-rich repeat kinase 2 is sufficient for interaction with microtubules.
      ). By contrast, the non-segregating R1514Q variant (
      • Healy D.G.
      • Falchi M.
      • O'Sullivan S.S.
      • Bonifati V.
      • Durr A.
      • Bressman S.
      • Brice A.
      • Aasly J.
      • Zabetian C.P.
      • Goldwurm S.
      • Ferreira J.J.
      • Tolosa E.
      • Kay D.M.
      • Klein C.
      • Williams D.R.
      • Marras C.
      • Lang A.E.
      • Wszolek Z.K.
      • Berciano J.
      • Schapira A.H.
      • Lynch T.
      • Bhatia K.P.
      • Gasser T.
      • Lees A.J.
      • Wood N.W.
      • International LRRK2 Consortium
      Phenotype, genotype, and worldwide genetic penetrance of LRRK2-associated Parkinson's disease. A case-control study.
      ), showed no influence on LRRK2-β-tubulin interactions (Fig. 9). This observation suggests that altered LRRK2-β-tubulin interactions are likely to occur in patients with these LRRK2 mutations. This suggests that the interaction between LRRK2 and MTs requires fine regulation, and that both decreased and increased interaction strength could affect the dynamic instability of MTs. Both increased and decreased MT stability are detrimental, well illustrated by the effect of anticancer chemotherapeutic medication increasing (e.g. taxol) or decreasing (e.g. vincristine) MT stability. Interestingly, interaction strength was also markedly weakened by two mutations (T1343D, T1491D) mimicking LRRK2 autophosphorylation within the LRRK2 Roc domain (Fig. 8, B and C). These phosphomimetic mutants serve as a proxy for increased LRRK2 kinase activity, which cannot be tested directly in the RocCOR tandem domain constructs used in our YTH assays. Importantly, elevated kinase activity has been a consistent observation for the most common G2019S LRRK2 mutation, and thus these data suggest the G2019S mutation will also lead to decreased luminal MT binding. Thus, our data indicate that three pathogenic LRRK2 mutations, G2019S, R1441G, and R1441H, are likely to cause reduced LRRK2-β-tubulin interactions. Interestingly, effects of over-expression of wild-type LRRK2, the R1441G and G2019S mutants on the expression and phosphorylation of the MAP Tau were shown previously (
      • Melrose H.L.
      • Dächsel J.C.
      • Behrouz B.
      • Lincoln S.J.
      • Yue M.
      • Hinkle K.M.
      • Kent C.B.
      • Korvatska E.
      • Taylor J.P.
      • Witten L.
      • Liang Y.Q.
      • Beevers J.E.
      • Boules M.
      • Dugger B.N.
      • Serna V.A.
      • Gaukhman A.
      • Yu X.
      • Castanedes-Casey M.
      • Braithwaite A.T.
      • Ogholikhan S.
      • Yu N.
      • Bass D.
      • Tyndall G.
      • Schellenberg G.D.
      • Dickson D.W.
      • Janus C.
      • Farrer MJ.
      Impaired dopaminergic neurotransmission and microtubule-associated protein tau alterations in human LRRK2 transgenic mice.
      ,
      • Li Y.
      • Liu W.
      • Oo T.F.
      • Wang L.
      • Tang Y.
      • Jackson-Lewis V.
      • Zhou C.
      • Geghman K.
      • Bogdanov M.
      • Przedborski S.
      • Beal M.F.
      • Burke R.E.
      • Li C.
      Mutant LRRK2(R1441G) BAC transgenic mice recapitulate cardinal features of Parkinson's disease.
      ), whereas in R1441C knock-in mice no effect on Tau expression or phosphorylation was observed (
      • Tong Y.
      • Pisani A.
      • Martella G.
      • Karouani M.
      • Yamaguchi H.
      • Pothos E.N.
      • Shen J.
      R1441C mutation in LRRK2 impairs dopaminergic neurotransmission in mice.
      ). This might be an effect of R1441G over-expression in comparison to endogenous expression levels of the R1441C mutant in knock-in mice, but might also correspond to different effects of these mutants on the interaction between LRRK2 and MTs as suggested in this study (Fig. 8, A and B).
      Intuitively, one would expect processes relevant to PD pathogenesis to be modulated similarly by all LRRK2 mutants segregating with the disease. However, different effects of LRRK2 mutants have been described previously in numerous cellular and biochemical assays. These include opposite effects on measurements of LRRK2 kinase activity and protein-protein interaction strength, for example, with 14-3-3 and DVL proteins (
      • Greggio E.
      • Cookson M.R.
      Leucine-rich repeat kinase 2 mutations and Parkinson's disease. Three questions.
      ,
      • Sancho R.M.
      • Law B.M.
      • Harvey K.
      Mutations in the LRRK2 Roc-COR tandem domain link Parkinson's disease to Wnt signalling pathways.
      ,
      • Nichols R.J.
      • Dzamko N.
      • Morrice N.A.
      • Campbell D.G.
      • Deak M.
      • Ordureau A.
      • Macartney T.
      • Tong Y.
      • Shen J.
      • Prescott A.R.
      • Alessi D.R.
      14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization.
      ). As such, it is perhaps more likely that PD-relevant processes perturbed by LRRK2 mutants are under fine regulation, with “too much” and “too little” equally able to elicit neurodegeneration. It is thus fascinating that MT overstabilization and destabilization will have detrimental effects on numerous MT-dependent processes. Taken together therefore, our data shed light on the cells biological role of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 could contribute to the pathogenesis of PD.
      Mutations in human tubulin genes have been found in a number of genetic disorders. These include lissencephaly/SBH (TUBA1A) (
      • Keays D.A.
      • Tian G.
      • Poirier K.
      • Huang G.J.
      • Siebold C.
      • Cleak J.
      • Oliver P.L.
      • Fray M.
      • Harvey R.J.
      • Molnár Z.
      • Piñon M.C.
      • Dear N.
      • Valdar W.
      • Brown S.D.
      • Davies K.E.
      • Rawlins J.N.
      • Cowan N.J.
      • Nolan P.
      • Chelly J.
      • Flint J.
      Mutations in α-tubulin cause abnormal neuronal migration in mice and lissencephaly in humans.
      ,
      • Kumar R.A.
      • Pilz D.T.
      • Babatz T.D.
      • Cushion T.D.
      • Harvey K.
      • Topf M.
      • Yates L.
      • Robb S.
      • Uyanik G.
      • Mancini G.M.
      • Rees M.I.
      • Harvey R.J.
      • Dobyns W.B.
      TUBA1A mutations cause wide spectrum lissencephaly (smooth brain) and suggest that multiple neuronal migration pathways converge on α-tubulins.
      ), asymmetric polymicrogyria (TUBB2B) (
      • Jaglin X.H.
      • Poirier K.
      • Saillour Y.
      • Buhler E.
      • Tian G.
      • Bahi-Buisson N.
      • Fallet-Bianco C.
      • Phan-Dinh-Tuy F.
      • Kong X.P.
      • Bomont P.
      • Castelnau-Ptakhine L.
      • Odent S.
      • Loget P.
      • Kossorotoff M.
      • Snoeck I.
      • Plessis G.
      • Parent P.
      • Beldjord C.
      • Cardoso C.
      • Represa A.
      • Flint J.
      • Keays D.A.
      • Cowan N.J.
      • Chelly J.
      Mutations in the β-tubulin gene TUBB2B result in asymmetrical polymicrogyria.
      ), and the ocular motility disorder CFEOM3 (TUBB3) (
      • Demer J.L.
      • Clark R.A.
      • Tischfield M.A.
      • Engle E.C.
      Evidence of an asymmetrical endophenotype in congenital fibrosis of extraocular muscles type 3 resulting from TUBB3 mutations.
      ,
      • Tischfield M.A.
      • Baris H.N.
      • Wu C.
      • Rudolph G.
      • Van Maldergem L.
      • He W.
      • Chan W.M.
      • Andrews C.
      • Demer J.L.
      • Robertson R.L.
      • Mackey D.A.
      • Ruddle J.B.
      • Bird T.D.
      • Gottlob I.
      • Pieh C.
      • Traboulsi E.I.
      • Pomeroy S.L.
      • Hunter D.G.
      • Soul J.S.
      • Newlin A.
      • Sabol L.J.
      • Doherty E.J.
      • de Uzcátegui C.E.
      • de Uzcátegui N.
      • Collins M.L.
      • Sener E.C.
      • Wabbels B.
      • Hellebrand H.
      • Meitinger T.
      • de Berardinis T.
      • Magli A.
      • Schiavi C.
      • Pastore-Trossello M.
      • Koc F.
      • Wong A.M.
      • Levin A.V.
      • Geraghty M.T.
      • Descartes M.
      • Flaherty M.
      • Jamieson R.V.
      • Møller H.U.
      • Meuthen I.
      • Callen D.F.
      • Kerwin J.
      • Lindsay S.
      • Meindl A.
      • Gupta Jr., M.L.
      • Pellman D.
      • Engle E.C.
      Human TUBB3 mutations perturb microtubule dynamics, kinesin interactions, and axon guidance.
      ,
      • Poirier K.
      • Saillour Y.
      • Bahi-Buisson N.
      • Jaglin X.H.
      • Fallet-Bianco C.
      • Nabbout R.
      • Castelnau-Ptakhine L.
      • Roubertie A.
      • Attie-Bitach T.
      • Desguerre I.
      • Genevieve D.
      • Barnerias C.
      • Keren B.
      • Lebrun N.
      • Boddaert N.
      • Encha-Razavi F.
      • Chelly J.
      Mutations in the neuronal β-tubulin subunit TUBB3 result in malformation of cortical development and neuronal migration defects.
      ). Most intriguingly, however, an R2G amino acid substitution in TUBB4 has recently been reported as causing the rare movement disorder, dystonia type 4 (also known as whispering dysphonia) (
      • Hersheson J.
      • Mencacci N.E.
      • Davis M.
      • Macdonald N.
      • Trabzuni D.
      • Ryten M.
      • Pittman A.
      • Paudel R.
      • Kara E.
      • Fawcett K.
      • Plagnol V.
      • Bhatia K.P.
      • Medlar A.J.
      • Stanescu H.C.
      • Hardy J.
      • Kleta R.
      • Wood N.W.
      • Houlden H.
      Mutations in the autoregulatory domain of β-tubulin 4a cause hereditary dystonia.
      ,
      • Lohmann K.
      • Wilcox R.A.
      • Winkler S.
      • Ramirez A.
      • Rakovic A.
      • Park J.S.
      • Arns B.
      • Lohnau T.
      • Groen J.
      • Kasten M.
      • Brüggemann N.
      • Hagenah J.
      • Schmidt A.
      • Kaiser F.J.
      • Kumar K.R.
      • Zschiedrich K.
      • Alvarez-Fischer D.
      • Altenmüller E.
      • Ferbert A.
      • Lang A.E.
      • Münchau A.
      • Kostic V.
      • Simonyan K.
      • Agzarian M.
      • Ozelius L.J.
      • Langeveld A.P.
      • Sue C.M.
      • Tijssen M.A.
      • Klein C.
      Whispering dysphonia (DYT4 dystonia) is caused by a mutation in the TUBB4 gene.
      ). This mutation was found to elicit a decrease in TUBB4 expression (
      • Hersheson J.
      • Mencacci N.E.
      • Davis M.
      • Macdonald N.
      • Trabzuni D.
      • Ryten M.
      • Pittman A.
      • Paudel R.
      • Kara E.
      • Fawcett K.
      • Plagnol V.
      • Bhatia K.P.
      • Medlar A.J.
      • Stanescu H.C.
      • Hardy J.
      • Kleta R.
      • Wood N.W.
      • Houlden H.
      Mutations in the autoregulatory domain of β-tubulin 4a cause hereditary dystonia.
      ,
      • Lohmann K.
      • Wilcox R.A.
      • Winkler S.
      • Ramirez A.
      • Rakovic A.
      • Park J.S.
      • Arns B.
      • Lohnau T.
      • Groen J.
      • Kasten M.
      • Brüggemann N.
      • Hagenah J.
      • Schmidt A.
      • Kaiser F.J.
      • Kumar K.R.
      • Zschiedrich K.
      • Alvarez-Fischer D.
      • Altenmüller E.
      • Ferbert A.
      • Lang A.E.
      • Münchau A.
      • Kostic V.
      • Simonyan K.
      • Agzarian M.
      • Ozelius L.J.
      • Langeveld A.P.
      • Sue C.M.
      • Tijssen M.A.
      • Klein C.
      Whispering dysphonia (DYT4 dystonia) is caused by a mutation in the TUBB4 gene.
      ). Because our work has established LRRK2 as a direct interactor of TUBB4, one might expect dystonia type 4 patients to display decreased LRRK2 localization to MTs. In light of the likely decrease in LRRK2 MT binding in R1441G and G2019S carriers, this is an intriguing possibility, suggesting that LRRK2 might be connected to the pathogenesis of an additional movement disorder. This also suggests the possibility that mutations in human tubulin genes might be a genetic risk factor for PD, and we would contend that a genetic analysis of selected tubulin genes (particularly TUBB, TUBB4, and TUBB6) in PD patients is warranted. In any case, it is fascinating that proteins encoded by two genes linked to familial movement disorders should interact directly, suggesting the possibility of a common pathomechanism, and highlighting the potential importance of the LRRK2-β-tubulin interaction.

      Acknowledgments

      We thank Prof. Robert J. Harvey for constructive comments on the manuscript and Dr. Victoria James for assistance with molecular modeling. We also thank Fangye Gao for assistance in generating cell lines stably expressing EGFP-LRRK2.

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