Human histone deacetylase 6 (HDAC6) is the major deacetylase responsible for removing the acetyl group from Lys40 of α-tubulin (αK40), which is located lumenally in polymerized microtubules. Here, we provide a detailed kinetic analysis of tubulin deacetylation and HDAC6/microtubule interactions using individual purified components. Our data unequivocally show that free tubulin dimers represent the preferred HDAC6 substrate, with a K M value of 0.23 µM and a deacetylation rate over 1,500-fold higher than that of assembled microtubules. We attribute the lower deacetylation rate of microtubules to both longitudinal and lateral lattice interactions within tubulin polymers. Using TIRF microscopy, we directly visualized stochastic binding of HDAC6 to assembled microtubules without any detectable preferential binding to microtubule tips. Likewise, indirect immunofluorescence microscopy revealed that microtubule deacetylation by HDAC6 is carried out stochastically along the whole microtubule length, rather than from the open extremities. Our data thus complement prior studies on tubulin acetylation and further strengthen the rationale for the correlation between tubulin acetylation and microtubule age.

Department of Biochemistry Faculty of Natural Science Charles University Albertov 6 Prague 2 Czech Republic

Institute of Biotechnology CAS BIOCEV Prumyslova 595 252 50 Vestec Czech Republic

Institute of Organic Chemistry and Biochemistry of the Academy of Sciences of the Czech Republic Flemingovo n 2 166 10 Prague 6 Czech Republic

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Gundersen GG, Bulinski JC. Distribution of tyrosinated and nontyrosinated alpha-tubulin during mitosis. J Cell Biol. 1986;102:1118–1126. doi: 10.1083/jcb.102.3.1118. PubMed DOI PMC

Edde B, et al. Posttranslational glutamylation of alpha-tubulin. Science. 1990;247:83–85. doi: 10.1126/science.1967194. PubMed DOI

Redeker V, et al. Polyglycylation of tubulin: a posttranslational modification in axonemal microtubules. Science. 1994;266:1688–1691. doi: 10.1126/science.7992051. PubMed DOI

L’Hernault SW, Rosenbaum JL. Chlamydomonas alpha-tubulin is posttranslationally modified by acetylation on the epsilon-amino group of a lysine. Biochemistry. 1985;24:473–478. doi: 10.1021/bi00323a034. PubMed DOI

Song Y, et al. Transglutaminase and polyamination of tubulin: posttranslational modification for stabilizing axonal microtubules. Neuron. 2013;78:109–123. doi: 10.1016/j.neuron.2013.01.036. PubMed DOI PMC

Eipper BA. Properties of rat brain tubulin. J Biol Chem. 1974;249:1407–1416. PubMed

Nogales E, Wolf SG, Downing KH. Structure of the alpha beta tubulin dimer by electron crystallography. Nature. 1998;391:199–203. doi: 10.1038/34465. PubMed DOI

Tran AD, et al. HDAC6 deacetylation of tubulin modulates dynamics of cellular adhesions. Journal of cell science. 2007;120:1469–1479. doi: 10.1242/jcs.03431. PubMed DOI

Kalebic N, et al. alphaTAT1 is the major alpha-tubulin acetyltransferase in mice. Nat Commun. 2013;4:1962. doi: 10.1038/ncomms2962. PubMed DOI

Jeong SG, Cho GW. The tubulin deacetylase sirtuin-2 regulates neuronal differentiation through the ERK/CREB signaling pathway. Biochem Biophys Res Commun. 2017;482:182–187. doi: 10.1016/j.bbrc.2016.11.031. PubMed DOI

Dompierre JP, et al. Histone deacetylase 6 inhibition compensates for the transport deficit in Huntington’s disease by increasing tubulin acetylation. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2007;27:3571–3583. doi: 10.1523/JNEUROSCI.0037-07.2007. PubMed DOI PMC

Reed NA, et al. Microtubule acetylation promotes kinesin-1 binding and transport. Curr Biol. 2006;16:2166–2172. doi: 10.1016/j.cub.2006.09.014. PubMed DOI

Chen S, Owens GC, Makarenkova H, Edelman DB. HDAC6 regulates mitochondrial transport in hippocampal neurons. PloS one. 2010;5:e10848. doi: 10.1371/journal.pone.0010848. PubMed DOI PMC

Akella JS, et al. MEC-17 is an alpha-tubulin acetyltransferase. Nature. 2010;467:218–222. doi: 10.1038/nature09324. PubMed DOI PMC

Hubbert C, et al. HDAC6 is a microtubule-associated deacetylase. Nature. 2002;417:455–458. doi: 10.1038/417455a. PubMed DOI

Matsuyama A, et al. In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation. The EMBO journal. 2002;21:6820–6831. doi: 10.1093/emboj/cdf682. PubMed DOI PMC

North BJ, Marshall BL, Borra MT, Denu JM, Verdin E. The human Sir2 ortholog, SIRT2, is an NAD + -dependent tubulin deacetylase. Molecular cell. 2003;11:437–444. doi: 10.1016/S1097-2765(03)00038-8. PubMed DOI

Bobrowska A, Donmez G, Weiss A, Guarente L, Bates G. SIRT2 ablation has no effect on tubulin acetylation in brain, cholesterol biosynthesis or the progression of Huntington’s disease phenotypes in vivo. PloS one. 2012;7:e34805. doi: 10.1371/journal.pone.0034805. PubMed DOI PMC

Zhang Y, et al. Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally. Molecular and cellular biology. 2008;28:1688–1701. doi: 10.1128/MCB.01154-06. PubMed DOI PMC

Skoge RH, Ziegler M. SIRT2 inactivation reveals a subset of hyperacetylated perinuclear microtubules inaccessible to HDAC6. J Cell Sci. 2016;129:2972–2982. doi: 10.1242/jcs.187518. PubMed DOI

Choudhary C, et al. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science. 2009;325:834–840. doi: 10.1126/science.1175371. PubMed DOI

Chu CW, et al. A novel acetylation of beta-tubulin by San modulates microtubule polymerization via down-regulating tubulin incorporation. Mol Biol Cell. 2011;22:448–456. doi: 10.1091/mbc.E10-03-0203. PubMed DOI PMC

Huan Y, et al. Epigenetic Modification Agents Improve Gene-Specific Methylation Reprogramming in Porcine Cloned Embryos. PLoS One. 2015;10:e0129803. doi: 10.1371/journal.pone.0129803. PubMed DOI PMC

Ouyang H, et al. Protein aggregates are recruited to aggresome by histone deacetylase 6 via unanchored ubiquitin C termini. J Biol Chem. 2012;287:2317–2327. doi: 10.1074/jbc.M111.273730. PubMed DOI PMC

Han Y, et al. Acetylation of histone deacetylase 6 by p300 attenuates its deacetylase activity. Biochem Biophys Res Commun. 2009;383:88–92. doi: 10.1016/j.bbrc.2009.03.147. PubMed DOI

Liu Y, Peng L, Seto E, Huang S, Qiu Y. Modulation of histone deacetylase 6 (HDAC6) nuclear import and tubulin deacetylase activity through acetylation. J Biol Chem. 2012;287:29168–29174. doi: 10.1074/jbc.M112.371120. PubMed DOI PMC

Okuda K, Ito A, Uehara T. Regulation of Histone Deacetylase 6 Activity via S-Nitrosylation. Biol Pharm Bull. 2015;38:1434–1437. doi: 10.1248/bpb.b15-00364. PubMed DOI

Lafarga V, Aymerich I, Tapia O, Mayor F, Jr., Penela P. A novel GRK2/HDAC6 interaction modulates cell spreading and motility. EMBO J. 2012;31:856–869. doi: 10.1038/emboj.2011.466. PubMed DOI PMC

Kovacs JJ, et al. HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor. Mol Cell. 2005;18:601–607. doi: 10.1016/j.molcel.2005.04.021. PubMed DOI

Zhang X, et al. HDAC6 modulates cell motility by altering the acetylation level of cortactin. Mol Cell. 2007;27:197–213. doi: 10.1016/j.molcel.2007.05.033. PubMed DOI PMC

Parmigiani RB, et al. HDAC6 is a specific deacetylase of peroxiredoxins and is involved in redox regulation. Proc Natl Acad Sci U S A. 2008;105:9633–9638. doi: 10.1073/pnas.0803749105. PubMed DOI PMC

Li Y, Zhang X, Polakiewicz RD, Yao TP, Comb MJ. HDAC6 is required for epidermal growth factor-induced beta-catenin nuclear localization. J Biol Chem. 2008;283:12686–12690. doi: 10.1074/jbc.C700185200. PubMed DOI PMC

Kawaguchi Y, et al. The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell. 2003;115:727–738. doi: 10.1016/S0092-8674(03)00939-5. PubMed DOI

Seidel C, Schnekenburger M, Dicato M, Diederich M. Histone deacetylase 6 in health and disease. Epigenomics. 2015;7:103–118. doi: 10.2217/epi.14.69. PubMed DOI

Li G, Jiang H, Chang M, Xie H, Hu L. HDAC6 alpha-tubulin deacetylase: a potential therapeutic target in neurodegenerative diseases. J Neurol Sci. 2011;304:1–8. doi: 10.1016/j.jns.2011.02.017. PubMed DOI

Simoes-Pires C, et al. HDAC6 as a target for neurodegenerative diseases: what makes it different from the other HDACs? Mol Neurodegener. 2013;8:7. doi: 10.1186/1750-1326-8-7. PubMed DOI PMC

Aldana-Masangkay GI, Sakamoto KM. The role of HDAC6 in cancer. J Biomed Biotechnol. 2011;2011:875824. doi: 10.1155/2011/875824. PubMed DOI PMC

Zhao Z, Xu H, Gong W. Histone deacetylase 6 (HDAC6) is an independent deacetylase for alpha-tubulin. Protein and peptide letters. 2010;17:555–558. doi: 10.2174/092986610791112620. PubMed DOI

Du Y, Seibenhener ML, Yan J, Jiang J, Wooten MC. aPKC phosphorylation of HDAC6 results in increased deacetylation activity. PloS one. 2015;10:e0123191. doi: 10.1371/journal.pone.0123191. PubMed DOI PMC

Williams KA, et al. Extracellular signal-regulated kinase (ERK) phosphorylates histone deacetylase 6 (HDAC6) at serine 1035 to stimulate cell migration. The Journal of biological chemistry. 2013;288:33156–33170. doi: 10.1074/jbc.M113.472506. PubMed DOI PMC

Watabe M, Nakaki T. Protein kinase CK2 regulates the formation and clearance of aggresomes in response to stress. Journal of cell science. 2011;124:1519–1532. doi: 10.1242/jcs.081778. PubMed DOI

Zou H, Wu Y, Navre M, Sang BC. Characterization of the two catalytic domains in histone deacetylase 6. Biochemical and biophysical research communications. 2006;341:45–50. doi: 10.1016/j.bbrc.2005.12.144. PubMed DOI

Schultz BE, et al. Kinetics and comparative reactivity of human class I and class IIb histone deacetylases. Biochemistry. 2004;43:11083–11091. doi: 10.1021/bi0494471. PubMed DOI

Lai MJ, et al. Synthesis and biological evaluation of 1-arylsulfonyl-5-(N-hydroxyacrylamide)indoles as potent histone deacetylase inhibitors with antitumor activity in vivo. J Med Chem. 2012;55:3777–3791. doi: 10.1021/jm300197a. PubMed DOI PMC

Wagner FF, et al. Potent and selective inhibition of histone deacetylase 6 (HDAC6) does not require a surface-binding motif. Journal of medicinal chemistry. 2013;56:1772–1776. doi: 10.1021/jm301355j. PubMed DOI

Bergman JA, et al. Selective histone deacetylase 6 inhibitors bearing substituted urea linkers inhibit melanoma cell growth. Journal of medicinal chemistry. 2012;55:9891–9899. doi: 10.1021/jm301098e. PubMed DOI PMC

Giannini G, et al. N-Hydroxy-(4-oxime)-cinnamide: a versatile scaffold for the synthesis of novel histone deacetylase [correction of deacetilase] (HDAC) inhibitors. Bioorganic & medicinal chemistry letters. 2009;19:2346–2349. doi: 10.1016/j.bmcl.2009.02.029. PubMed DOI

Miyake Y, et al. Structural insights into HDAC6 tubulin deacetylation and its selective inhibition. Nat Chem Biol. 2016;12:748–754. doi: 10.1038/nchembio.2140. PubMed DOI

Moores CA, Milligan RA. Visualisation of a kinesin-13 motor on microtubule end mimics. J Mol Biol. 2008;377:647–654. doi: 10.1016/j.jmb.2008.01.079. PubMed DOI PMC

Wolf SG, Mosser G, Downing KH. Tubulin conformation in zinc-induced sheets and macrotubes. J Struct Biol. 1993;111:190–199. doi: 10.1006/jsbi.1993.1049. PubMed DOI

Szyk A, et al. Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferase. Cell. 2014;157:1405–1415. doi: 10.1016/j.cell.2014.03.061. PubMed DOI PMC

Friedmann DR, Aguilar A, Fan J, Nachury MV, Marmorstein R. Structure of the alpha-tubulin acetyltransferase, alphaTAT1, and implications for tubulin-specific acetylation. Proc Natl Acad Sci U S A. 2012;109:19655–19660. doi: 10.1073/pnas.1209357109. PubMed DOI PMC

Taschner M, Vetter M, Lorentzen E. Atomic resolution structure of human alpha-tubulin acetyltransferase bound to acetyl-CoA. Proc Natl Acad Sci U S A. 2012;109:19649–19654. doi: 10.1073/pnas.1209343109. PubMed DOI PMC

Li W, et al. Molecular basis of the acetyltransferase activity of MEC-17 towards alpha-tubulin. Cell Res. 2012;22:1707–1711. doi: 10.1038/cr.2012.154. PubMed DOI PMC

Riester D, Hildmann C, Grunewald S, Beckers T, Schwienhorst A. Factors affecting the substrate specificity of histone deacetylases. Biochem Biophys Res Commun. 2007;357:439–445. doi: 10.1016/j.bbrc.2007.03.158. PubMed DOI

Hai Y, Christianson DW. Histone deacetylase 6 structure and molecular basis of catalysis and inhibition. Nat Chem Biol. 2016;12:741–747. doi: 10.1038/nchembio.2134. PubMed DOI PMC

Zhang Y, et al. HDAC-6 interacts with and deacetylates tubulin and microtubules in vivo. The EMBO journal. 2003;22:1168–1179. doi: 10.1093/emboj/cdg115. PubMed DOI PMC

Li H, DeRosier DJ, Nicholson WV, Nogales E, Downing KH. Microtubule structure at 8 A resolution. Structure. 2002;10:1317–1328. doi: 10.1016/S0969-2126(02)00827-4. PubMed DOI

Meurer-Grob P, Kasparian J, Wade RH. Microtubule structure at improved resolution. Biochemistry. 2001;40:8000–8008. doi: 10.1021/bi010343p. PubMed DOI

Howes SC, Alushin GM, Shida T, Nachury MV, Nogales E. Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure. Molecular biology of the cell. 2014;25:257–266. doi: 10.1091/mbc.E13-07-0387. PubMed DOI PMC

Asthana J, Kapoor S, Mohan R, Panda D. Inhibition of HDAC6 deacetylase activity increases its binding with microtubules and suppresses microtubule dynamic instability in MCF-7 cells. The Journal of biological chemistry. 2013;288:22516–22526. doi: 10.1074/jbc.M113.489328. PubMed DOI PMC

Purev E, Neff L, Horne WC, Baron R. c-Cbl and Cbl-b act redundantly to protect osteoclasts from apoptosis and to displace HDAC6 from beta-tubulin, stabilizing microtubules and podosomes. Molecular biology of the cell. 2009;20:4021–4030. doi: 10.1091/mbc.E09-03-0248. PubMed DOI PMC

Ly N, et al. alphaTAT1 controls longitudinal spreading of acetylation marks from open microtubules extremities. Sci Rep. 2016;6:35624. doi: 10.1038/srep35624. PubMed DOI PMC

Skoge RH, Dolle C, Ziegler M. Regulation of SIRT2-dependent alpha-tubulin deacetylation by cellular NAD levels. DNA Repair (Amst) 2014;23:33–38. doi: 10.1016/j.dnarep.2014.04.011. PubMed DOI

Geuens G, et al. Ultrastructural colocalization of tyrosinated and detyrosinated alpha-tubulin in interphase and mitotic cells. J Cell Biol. 1986;103:1883–1893. doi: 10.1083/jcb.103.5.1883. PubMed DOI PMC

Braun M, et al. Adaptive braking by Ase1 prevents overlapping microtubules from sliding completely apart. Nat Cell Biol. 2011;13:1259–1264. doi: 10.1038/ncb2323. PubMed DOI

Hyman AA. Preparation of marked microtubules for the assay of the polarity of microtubule-based motors by fluorescence. J Cell Sci Suppl. 1991;14:125–127. doi: 10.1242/jcs.1991.Supplement_14.25. PubMed DOI

Schindelin J, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Piperno G, Fuller MT. Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J Cell Biol. 1985;101:2085–2094. doi: 10.1083/jcb.101.6.2085. PubMed DOI PMC

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