Deactylase Inhibitors Disrupt Cellular Complexes Containing Protein Phosphatases and Deacetylases

Abstract

Affinity isolation of protein serine/threonine phosphatases on the immobilized phosphatase inhibitor microcystin-LR identified histone deacetylase 1(HDAC1), HDAC6, and HDAC10 as novel components of cellular phosphatase complexes. Other HDACs, specifically HDAC2, -3, -4, and -5, were excluded from such complexes. In vitro biochemical studies showed that recombinant HDAC6, but not HDAC4, bound directly to the protein phosphatase (PP)1 catalytic subunit. No association was observed between HDAC6 and PP2A, another major protein phosphatase. PP1 binding was mapped to the second catalytic domain and adjacent C-terminal sequences in HDAC6, and treatment of cells with trichostatin A (TSA) disrupted endogenous HDAC6⅐PP1 complexes. Consistent with the inhibition of tubulin deactylase activity of HDAC6, TSA enhanced cellular tubulin acetylation, and acetylated tubulin was present in the PP1 complexes from TSA-treated cells. Trapoxin B, a weak HDAC6 inhibitor, and calyculin A, a cell-permeable phosphatase inhibitor, had no effect on the stability of the HDAC6⅐PP1 complexes or on tubulin acetylation. Mutations that inactivated HDAC6 prevented its incorporation into cellular PP1 complexes and suggested that when bound together both enzymes were active. Interestingly, TSA disrupted all the cellular HDAC⅐phosphatase complexes analyzed. This study provided new insight into the mechanism by which HDAC inhibitors elicited coordinate changes in cellular protein phosphorylation and acetylation and suggested that changes in these protein modifications at multiple subcellular sites may contribute to the known ability of HDAC inhibitors to suppress cell growth and transformation.Reversible protein phosphorylation is the most prevalent protein modification regulating eukaryotic cell physiology. Identification of numerous acetyltransferases and deacetylases suggests that protein acetylation also controls many physiological events (1). Studies of histone acetylation and phosphorylation show that both protein modifications play key roles in chromatin remodeling and gene transcription (2). The cellular mechanisms that coordinate the acetylation and phosphorylation of histones and other cellular proteins are still poorly understood.The precise orchestration of protein phosphorylation and acetylation is best exemplified during growth factor-stimulated phosphorylation and acetylation of histone H3 (3). Histone H3 is phosphorylated on serine 10 in fibroblasts in response to mitogens or following oncogenic transformation (4). Several kinases (5-8) catalyze the phosphorylation of histone H3 at serine 10 to generate an improved substrate for acetyltransferases that modify the adjacent lysine 14 (9). Conversely, serine 10 phosphorylation inhibits the acetylation of histone H3 at lysine 4 (10). The changes in phosphorylation and acetylation of histone H3 create novel platforms that recruit nuclear factors and/or stabilize pre-existing nuclear complexes required for chromatin remodeling and gene expression (2). How cells orchestrate the ordered phosp...

Histone deacetylase inhibitors: molecular mechanisms of action

“…Recombinant HDAC6 binds directly to protein phosphatase 1 (PP1) catalytic subunit. TSA disrupts endogenous HDAC6-PP1 complexes (Brush et al, 2004). HDAC1 and 10 are also components of cellular phosphatase complexes.…”

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

“…However, at least one report shows that both proteins interact with the major cellular phosphatase, PP1, and could therefore be involved in the same regulatory networks (Brush et al, 2004). A very recent study also led to the identification of two factors involved in the pre-mRNA 3 0 -end processing, the cleavage factor CFIm25 and poly-A polymerase, as in vivo HDAC10 substrates, suggesting a probable function for this protein in the regulation of the 3 0 -end processing machinery (Shimazu et al, 2007).…”

Lymphocyte Chemotaxis Is Regulated by Histone Deacetylase 6, Independently of Its Deacetylase Activity

“…These findings indicate that TSA negatively affects lymphocyte extravasation, mainly their firm adhesion to endothelium under flow stress conditions, an effect that is not evident under static conditions. This could be due to blockage of deacetylase activity and/or disruption of HDAC6 molecular complexes by TSA (Brush et al, 2004).…”

“…TSA is a broad HDACs inhibitor, whereas tubacin is specific for the tubulin deacetylase activity of HDAC6 (Hubbert et al, 2002;Matsuyama et al, 2002;Haggarty et al, 2003;Zhang et al, 2003). These compounds inhibit the HDAC6 deacetylase activity by chelating a Zn 2ϩ cation (Furumai et al, 2001;Jose et al, 2004) and may also alter the formation of dynamic molecular complexes of HDAC6 with other intracellular proteins such PP1, HSP90, Dia2, dynein, or tubulin (Kawaguchi et al, 2003;Brush et al, 2004;Destaing et al, 2005;Hideshima et al, 2005;Kovacs et al, 2005). In this regard, it has been reported that TSA disrupts HDAC6/PP1 association, affecting the enzymatic activity of both molecules (Brush et al, 2004).…”