Oncogene (2007) 26, 5468–5476 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW HDAC6, at the crossroads between cytoskeleton and cell signaling by acetylation and ubiquitination C Boyault, K Sadoul, M Pabion and S Khochbin INSERM, U823, Equipe Epige´ne´tique et Signalisation Cellulaire, Institut Albert Bonniot, Universite´ Joseph Fourier, Domaine de la Merci, Grenoble, La Tronche Cedex, France Histone deacetylase 6 (HDAC6)is a unique enzyme with et al., 2005). Moreover, it is now clear that acetylation is specific structural and functional features. It is actively or not an exclusive modification of nuclear proteins, since stably maintained in the cytoplasm and is the only many cytoplasmic proteins, including a significant member, within the histone deacetylase family, that subset of mitochondrial proteins, have recently been harbors a full duplication of its deacetylase homology shown to bear lysine acetylation (Cohen et al., 2004; region followed by a specific ubiquitin-binding domain at Dihazi et al., 2005; Iwabata et al., 2005; Kovacs et al., the C-terminus end. Accordingly, this deacetylase func- 2005; Hallows et al., 2006; Kim et al., 2006; Schwer tions at the heart of a cellular regulatory mechanism et al., 2006). The regulation of these acetylations and capable of coordinating various cellular functions largely the determination of their functional significance relying on the microtubule network. Moreover, HDAC6 now constitute a real challenge for biologists. In fact, action as a regulator of the HSP90 chaperone activity while the list of cytoplasmic acetylated proteins is adds to the multifunctionality of the protein, and allows us rapidly growing, basic information on the involved to propose a critical role for HDAC6 in mediating and enzymatic machinery, HATs and HDACs, as well as on coordinating various cellular events in response to their functions, is still missing. different stressful stimuli. The identification of HDAC6 as the first HDAC Oncogene (2007) 26, 5468–5476; doi:10.1038/sj.onc.1210614 actively maintained in the cytoplasm (Verdel et al., 2000) opened the way for the identification of its sub- Keywords: microtubule; actin; HSP90; virus; aggresome; strates and cellular functions controlled by its catalytic transcription activity in this compartment. Further analyses of HDAC6 functions revealed the involvement of the protein in cellular processes dependent and independent of its catalytic activity. Indeed, the discovery of an Introduction ubiquitin-binding domain in HDAC6 (Seigneurin-Berny et al., 2001) led to the unraveling of its participation in Protein lysine acetylation is now emerging as a widely cellular functions depending on cell signaling through occurring post-translational modification and constitu- protein ubiquitination (Kawaguchi et al., 2003; Boyault tes a genuine cellular signaling system involved in the et al., 2006). HDAC6 therefore appears as a protein control of various functions in different cellular functioning at the crossroads between at least two compartments (reviewed in Kouzarides, 2000; Sterner cellular signaling systems, respectively, involving protein and Berger, 2000; Caron et al., 2003; Yang, 2004; lysine acetylation and ubiquitination. Glozak et al., 2005). Originally, lysine acetylation, HDAC6 is not the only cytoplasmic deacetylase. mainly that of histones and transcription factors, was In fact, early investigations showed that under specific regarded as a powerful mean of gene expression circumstances, other HDACs of different classes regulation. Rapid progress in the discovery and func- could also be found in the cytoplasm. However, their tional analysis of an increasing number of nuclear cytoplasmic functions remain elusive and except for enzymes involved in protein lysine acetylation and SIRT2, a class III HDAC member (North et al., 2003), deacetylation, histone acetyltransferases (HATs) and none of the HDAC6 activities seems to be shared by histone deacetylases (HDACs), respectively, have these enzymes (Hubbert et al., 2002; Matsuyama et al., demonstrated that these enzymes, independently of 2002). Among them, HDAC10 deserves a special atten- transcription, can control some of the basic cellular tion since it is the closest relative of HDAC6. Indeed, processes such as protein stability (reviewed in Caron the HDAC10 catalytic domain shows the highest homology to those of HDAC6 and, moreover, its pseudo-duplication is reminiscent of HDAC6 cata- Correspondence: Dr S Khochbin, INSERM, U823, Equipe Epige´ ne´ - lytic domain duplication. This characteristic of HDAC10 tique et Signalisation Cellulaire, Institut Albert Bonniot, Universite´ Joseph Fourier, Domaine de la Merci, Grenoble, La Tronche Cedex, justifies a close examination of the known HDAC10 France. activities in the light of data available on HDAC6 E-mail: [email protected] functions. HDAC6, a coordinator of cell responses to stressful stimuli C Boyault et al 5469 This review will therefore essentially consider the HDAC activity of HDAC6 relies either on the integrity important body of data now available on HDAC6 since of both HDAC domains (Zhang et al., 2003, 2006) or its discovery in 1999 (Grozinger et al., 1999; Verdel and is mediated by its second catalytic domain (Zou et al., Khochbin, 1999), aiming to draw a synthetic scheme of 2006). The discovery of a-tubulin as an HDAC6 sub- its cellular functions and highlight its possible involve- strate (Hubbert et al., 2002; Matsuyama et al., 2002; ment in human pathologies. Zhang et al., 2003) allowed researchers to also include acetylated tubulin in these assays (Haggarty et al., 2003; Zhang et al., 2003, 2006; Zou et al., 2006). Here again, some of the investigators found that the integrity Domain organization and structural features of HDAC6 of both HDAC6 catalytic domains was indispensable for the whole tubulin deacetylase (TDAC) activity of HDAC6 is the only member of the cellular deacetylase the enzyme (Zhang et al., 2003, 2006), whereas others family containing a full duplication of the large class I/II showed that the whole TDAC activity could be HDAC-homology domain (Figure 1), first revealed in attributed to the second domain (Haggarty et al., the mouse HDAC6 (Verdel and Khochbin, 1999) and 2003; Zou et al., 2006). then in its human ortholog (Grozinger et al., 1999). This Additional investigations revealed the existence of feature could be considered as a unique signature other structural requirements for an efficient HDAC allowing the identification of HDAC6 orthologs in and TDAC activity of HDAC6. Indeed, the spacer other species including invertebrates, Drosophila mela- region between the two catalytic domains of the protein nogaster and Caenorhabditis elegans as well as in plants was found to play a crucial role in the total activity of such as Arabidopsis thaliana (Barlow et al., 2001; HDAC6, and amino acid addition or deletion in this reviewed in Yang and Gregoire, 2005). region dramatically affected the TDAC activity of The conservation of the double catalytic domain HDAC6. The HDAC activity of HDAC6 seemed, organization during evolution strongly argues in favor however, to be less dependent on the length of this of a critical role for this domain duplication in HDAC6 linker region than its TDAC activity (Zhang et al., functions and prompted the investigators to uncover 2006). These studies also evidenced an important role its role in the whole deacetylase activity of the enzyme. for a conserved region present in HDACs and some One obvious way to tackle this issue consisted in the HATs, known as substrate recognition site or Esa1- measurement of the deacetylase activity of HDAC6 Rpd3 (ER) motif (Adachi et al., 2002). Both HDAC and mutants containing inactivating mutations in each TDAC activities of HDAC6 rely on this motif present in domain individually. Unexpectedly, these experiments each domain, but the ER in the second HDAC domain generated conflicting results. The first attempt showed has a particularly critical role in TDAC activity of the that each domain possessed an independent catalytic enzyme (Zhang et al., 2006). activity (Grozinger et al., 1999). This result was, Overall, despite these contradictory data, the second however, challenged by other groups showing that the domain of HDAC6 seems to have a determinant role in at least the total TDAC activity of the enzyme (Haggarty et al., 2003; Zhang et al., 2006; Zou et al., HDAC6 2006). NES SE14 In addition to HDAC6 catalytic activities, the control of its intra-cellular localization was also found to be an important issue in the understanding of its functions. DD1 DD2 ZnF-UBP Early investigations considering mouse HDAC6 showed that the protein is actively retained in the cytoplasm and Nuclear Cytoplasmic could, only under specific circumstances, be partially exclusion anchoring Catalytic activity found in the nucleus (Verdel et al., 2000). Indeed, a − α-tubulin strong nuclear export signal (NES) located N terminus - HSP90 Ubiquitin to the first catalytic domain prevents the accumulation binding of the protein in the nucleus (Figure 1). Interestingly, although this N-terminus NES is conserved in human Figure 1 Functional domain organization of HDAC6. HDAC6 is the only member of the histone deacetylase family containing HDAC6 (hHDAC6), another region of the protein was tandem catalytic domains. So far two in vivo substrates, a-tubulin found to ensure a stable anchorage of the protein in the and HSP90, have been identified. In human HDAC6, two different