Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways

Abstract

The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination.An increasing number of histone deacetylases (HDACs) are being characterized in higher eukaryotes. These proteins have been grouped in distinct families according to the similarity of their sequence to a yeast founding member. Class I HDACs are homologous to yeast RPD3, while class II members are related to yeast HDA1 and class III members are related to yeast SIR2 deacetylase (13,20). Class I HDACs are found in various nuclear multiprotein complexes containing either HDAC1/2 or HDAC3. Class II HDACs show the interesting property of being capable of a nucleocytoplasmic shuttling. Indeed, all of the class II HDACs, HDAC4, -5, -6, and -7, are subject to a regulated intracellular localization (20). Although there is evidence for a role for some of these HDACs in transcriptional repression, their possible function in the cytoplasm remains elusive (20,21). Within these enzymes, the endogenous HDAC6 was found to be essentially cytoplasmic (2, 37). A fraction of the murine HDAC6 (mHDAC6) translocates, however, in the nucleus under specific circumstances, such as arrest of cell proliferation (37). In order to gain an insight into the function of cytoplasmic HDACs, cytosolic mHDAC6 was immunopurified from mouse testis cytosolic extracts. The identified mHDAC6-associated proteins showed striking sequence homology to yeast regulatory proteins involved in the control of protein ubiquitination. These proteins are the mammalian homologue of yeast UFD3, known as phospholipase A2-activating protein (PLAP) (12), as well as the homologue of yeast Cdc48p AAA ATPase (p97/VCP/Cdc48p) (11). The UFD pathway was discovered in yeast after the observation that a protein containing a nonremovable N-terminal ubiquitin (Ub) moiety had a short half-life (19). The protein degradation pathway involved was called UFD, for Ub fusion degradation. A ...

Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer

“…HDAC1 and HDAC2 are mainly found associated in the same molecular complexes with Mi2/ NURD (nucleosome remodelling and histone deacetylase), SIN3A and Co-REST, whereas HDAC3 is found in a different complex (the nuclear co-receptor (NCOR)/ silencing mediator for retinoid and thyroid hormone receptors (SMRT) complex) [126][127][128][129][130][131][132][133] . HDAC6 has been found in cytoplasmic complexes with proteins involved in the ubiquitin signalling pathway 134 . Large multi-enzymatic complexes that contain histone acetyltransferase (HAT) or HDAC have also been described, which might represent the biochemical explanation of the dynamic nature of histone acetylation 135 .…”

Class II histone deacetylases: versatile regulators

“…1b). Class IIb HDACs also show some degree of tissue-specific gene expression: HDAC6 is predominantly expressed in testis [13,83], and HDAC10 is expressed in liver, spleen and kidney [8,10,12]. Different splice variants are observed for HDAC10, as shown for HDAC3, -7 and -9, suggesting an additional level of regulation by RNA processing.…”

“…4) [83]. HDAC6 contains a C-terminal zinc finger that shows significant homology to a zinc finger located in several ubiquitin-specific proteases (ZnF-UBP) [83] (Fig.…”

“…4). The ZnF-UBP motif (also identified as a polyubiquitin-associated zinc finger or PAZ domain) [86] mediates the binding of ubiquitin to HDAC6 [83,86]. The specific interaction of proteins involved in protein ubiquitination and the fact that both acetylation and ubiquitination target lysine residues suggest that HDAC6 could participate in the deacetylation of proteins before their ubiquitination.…”

HDAC6, at the crossroads between cytoskeleton and cell signaling by acetylation and ubiquitination

“…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 ubiquitin-binding domain in HDAC6 (Seigneurin-Berny et al, 2001) led to the unraveling of its participation in cellular functions depending on cell signaling through protein ubiquitination (Kawaguchi et al, 2003;Boyault et al, 2006). HDAC6 therefore appears as a protein functioning at the crossroads between at least two cellular signaling systems, respectively, involving protein lysine acetylation and ubiquitination.…”