Furry promotes acetylation of microtubules in the mitotic spindle by inhibition of SIRT2 tubulin deacetylase

  • Nagai T
  • Ikeda M
  • Chiba S
 et al. 
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The structure and function of microtubules (MTs) are regulated by post-translational modifications of tubulin subunits, such as acetylation of the Lys40 residue of a-tubulin. Regulation of the organization and dynamics of MTs is essential for the precise formation of the mitotic spindle. Spindle MTs are highly acetylated, but the mechanism regulating this acetylation is largely unknown. Furry (Fry) is an evolutionarily conserved protein that binds to MTs and colocalizes with acetylated MTs in the mitotic spindle. In this study, we examined the role of Fry in the acetylation of MTs in the mitotic spindle. Depletion of Fry significantly reduced the level of MT acetylation in the mitotic spindle. Expression of the N-terminal fragment of Fry induced hyperacetylation of MTs in both mitotic and interphase cells. These results indicate that Fry promotes MT acetylation in the mitotic spindle. We also found that Fry binds to the tubulin deacetylase SIRT2, preferentially in mitotic cells. Cell-free experiments revealed that the N-terminal region of Fry is the domain responsible for binding to and inhibiting the tubulin-deacetylase activity of SIRT2. AGK2, a specific inhibitor of SIRT2, increased the level of MT acetylation in the mitotic spindle, indicating that SIRT2 is involved in the deacetylation of spindle MTs. Furthermore, AGK2 reversed the decrease in MT acetylation induced by Fry depletion. In summary, these results suggest that Fry plays a crucial role in promoting the level of MT acetylation in the mitotic spindle by inhibiting the tubulin-deacetylase activity of SIRT2. Introduction Microtubules (MTs) are cytoskeletal filaments that play essential roles in diverse cellular functions, including intracellular transport, organelle positioning, chromosome segregation, neurite outgrowth, ciliogenesis, cell migration and cell morphogenesis. MTs are composed of heterodimers of a-and b-tubulin subunits, which are diversified by post-translational modifications such as acetylation, detyrosination, polyglutamylation and polyglycylation. These post-translational modifications influence the interaction of MTs with a diverse set of microtubule-associated proteins (MAPs) and motor proteins, and thereby alter the dynamic properties and cellular functions of MTs. Thus, post-translational modifications of MTs generate 'tubulin codes' that define the organization and diverse functions of MTs (Janke and Bulinski, 2011; Verhey and Gaertig, 2007). Acetylation of the Lys40 residue in a-tubulin (hereafter referred to as 'tubulin acetylation') is a well-defined MT modification that was originally discovered in Chlamydomonas (L'Hernault and Rosenbaum, 1985). Unlike other post-translational modifications, the acetylation site is positioned on the luminal side of MTs (Nogales et al., 1999). Tubulin acetylation is induced by stabilization of MTs, which can be achieved by treatment with taxol, and is therefore regarded as a marker of long-lived MTs (Piperno et al., 1987). It is currently unknown whether tubulin acetylation itself affects MT stability (Matsuyama et al., 2002; Palazzo et al., 2003; Zilberman et al., 2009). Although neither a defect nor an excess of tubulin acetylation causes a severe phenotype in Tetrahymena, nematodes and mice (Fukushige et al., 1999; Gaertig et al., 1995; Zhang et al., 2008), tubulin acetylation does modulate the ability of MTs to bind to MAPs and motor proteins, and may regulate MT stability and function (Dompierre et al., 2007; Reed et al., 2006; Sudo and Baas, 2010). Recent studies showed that tubulin acetylation restricts the number of protofilaments in nematode touch receptor neurons, suggesting that tubulin acetylation is involved in the organization of MTs (Cueva et al., 2012; Topalidou et al., 2012). However, the existence of a similar regulatory mechanism in other cell types and organisms remains unknown. MTs are highly acetylated in mitotic cells. During metaphase, acetylated tubulin (Ac-tubulin) is enriched at interpolar and kinetochore MTs, but not at astral MTs, and Ac-tubulin becomes concentrated on the midbody during telophase and cytokinesis (Cha et al., 1998; Piperno et al., 1987). However, the lack of current knowledge about regulation of tubulin acetylation during mitosis precludes an understanding of the functional significance of this post-translational modification. The enzymes responsible for tubulin deacetylation and acetylation have been identified; histone deacetylase 6 (HDAC6) and Sirtuin 2 (SIRT2) are tubulin deacetylases, and a-tubulin acetyl transferase (aTAT, also termed

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  • T. Nagai

  • M. Ikeda

  • S. Chiba

  • S.-i. Kanno

  • K. Mizuno

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