Caspase-mediated Specific Cleavage of Human Histone Deacetylase 4

Abstract

Histone deacetylase 4 (HDAC4) is a class II HDAC implicated in controlling gene expression important for diverse cellular functions, but little is known about how its expression and stability are regulated. We report here that this deacetylase is unusually unstable, with a half-life of less than 8 h. Consistent with the instability of HDAC4 protein, its mRNA was also highly unstable (with a half-life of less than 4 h). The degradation of HDAC4 could be accelerated by exposure of cells to ultraviolet irradiation. HDAC4 degradation was not dependent on proteasome or CRM1-mediated export activity but instead was caspase-dependent and was detectable in diverse human cancer lines. Of two potential caspase consensus motifs in HDAC4, both lying within a region containing proline-, glutamic acid-, serine-, and threonine-rich (PEST) sequences, we identified, by site-directed mutagenesis, Asp-289 as the prime cleavage site. Notably, this residue is not conserved among other class IIa members, HDAC5, -7, and -9. Finally, the induced expression of caspase-cleavable HDAC4 led to markedly increased apoptosis. These results therefore unexpectedly link the regulation of HDAC4 protein stability to caspases, enzymes that are important for controlling cell death and differentiation.Histone deacetylases (HDACs) 1 have been increasingly implicated in mediating diverse fundamental cellular activities. Based on sequence homology with their yeast orthologs, mammalian HDACs have been divided into three classes. Class I HDACs include HDAC1, -2, -3, -7, -8, and -11, whereas class II HDACs contain HDAC4, -5, -6, -7, -9, and -10 (for recent reviews, see Refs. 1-4). Among class II, HDAC4, -5, -7, and -9 form a subclass known as class IIa, whereas HDAC6 and -10 constitute class IIb. A third class of mammalian HDACs includes the Sir2-like proteins Sirt1-7 (5, 6). The most well characterized function of HDACs is the deacetylation of core histones, which in turn leads to the compaction of nucleosomes to repress gene transcription. HDACs have also been implicated in the deacetylation of nonhistone targets. For example, HDAC6 regulates the deacetylation of tubulin, which may in turn promote cell motility (7-9).Despite the increasing repertoire of cellular activities that have been found to involve HDACs, relatively little is known regarding mechanisms regulating their expression. For example, HDAC1 binding to the CCAAT/enhancer-binding protein ␣-promoter increased upon treatment of cells with a proteasome inhibitor, but the protein levels were not directly assessed (10). HDAC5 and HDAC6 are ubiquitinated, but it is unclear how their stability is regulated (11). Interestingly, HDAC1 and HDAC4 undergo sumoylation, a post-translational modification that is reminiscent of ubiquitination but does not appear to regulate protein degradation (12, 13).To assess how the levels of HDAC4 and other HDACs might be controlled, we measured their protein stability following the inhibition of de novo synthesis. HDAC4 was found to be exceptionally unstable, with a h...

HDAC4 Promotes Growth of Colon Cancer Cells via Repression of p21

“…We then determined the subcellular localization of endogenous HDAC4 in colon cancer cells by immunofluorescence analysis. Consistent with reports in other cell types (Wang and Yang, 2001;Liu et al, 2004), HDAC4 expression in the HCT116 colon cancer cell line was observed in both the nucleus and cytoplasm (Supplemental Figure 1B). In contrast, the class I HDAC HDAC1 exhibited an exclusively nuclear staining pattern, whereas localization of the class IIb HDAC HDAC6 was predominantly cytoplasmic, consistent with previous studies (Hubbert et al, 2002;Waltregny et al, 2004).…”

“…The HDAC4 protein comprises 1084 amino acids, with a nuclear localization signal (NLS) located at the amino terminus (amino acids [aa] 244 -279), a catalytic HDAC domain (aa 621-1040), and a nuclear export signal (NES) (aa 1044 -1069) located at the carboxy terminus (Wang and Yang, 2001;Liu et al, 2004). A schematic diagram is shown in Figure 7A.…”

Crystal structure of a conserved N-terminal domain of histone deacetylase 4 reveals functional insights into glutamine-rich domains

“…There is little structural change in the four-helix bundle between the wild type and the His97Phe mutant. Previous studies have shown that the N-terminal domain of HDAC4 containing the glutamine-rich sequence can repress MEF2-dependent transcription independently of the C-terminal catalytic domain (8)(9)(10). Whereas the His97Phe mutant similarly repressed MEF2-dependent transcription, the Phe93Asp mutant showed decreased ability to repress MEF2-dependent transcription (L.G.…”

“…Class II HDACs, which include HDAC4, HDAC5, and HDAC9, contain an additional N-terminal extension that confers responsiveness to calcium signals and mediates interactions with transcription factors and cofactors (4)(5)(6)(7). A prominent feature of this Nterminal region is a highly conserved glutamine-rich domain that can repress transcription independently of the C-terminal catalytic domain (8)(9)(10).…”

Class IIa histone deacetylases: regulating the regulators

“…The adapter domain of class IIa HDACs contains specific residues that are subjected to various posttranslational modifications, such as proteolytic cleavage (Bakin and Jung, 2004;Liu et al, 2004;Paroni et al, 2004), ubiquitination , sumoylation (Kirsh et al, 2002;Petrie et al, 2003) and most importantly phosphorylation. In response to various stimuli, a set of serine residues in the adapter domain of class IIa HDACs is phosphorylated, creating docking sites for the 14-3-3 proteins (Grozinger and Schreiber, 2000;Wang et al, 2000).…”