New Role for hPar-1 Kinases EMK and C-TAK1 in Regulating Localization and Activity of Class IIa Histone Deacetylases

Abstract

Class IIa histone deacetylases (HDACs) are found both in the cytoplasm and in the nucleus where they repress genes involved in several major developmental programs. In response to specific signals, the repressive activity of class IIa HDACs is neutralized through their phosphorylation on multiple N-terminal serine residues and 14-3-3-mediated nuclear exclusion. Here, we demonstrate that class IIa HDACs are subjected to signal-independent nuclear export that relies on their constitutive phosphorylation. We identify EMK and C-TAK1, two members of the microtubule affinity-regulating kinase (MARK)/Par-1 family, as regulators of this process. We further show that EMK and C-TAK1 phosphorylate class IIa HDACs on one of their multiple 14-3-3 binding sites and alter their subcellular localization and repressive function. Using HDAC7 as a paradigm, we extend these findings by demonstrating that signal-independent phosphorylation of the most N-terminal serine residue by the MARK/Par-1 kinases, i.e., Ser 155 , is a prerequisite for the phosphorylation of the nearby 14-3-3 site, Ser 181 . We propose that this multisite hierarchical phosphorylation by a variety of kinases allows for sophisticated regulation of class IIa HDACs function.

Class IIa histone deacetylases: regulating the regulators

“…In vitro, PKD is much less effective at phosphorylating Ser 259 than Ser 498 of HDAC5 (Huynh and McKinsey, 2006). In contrast, MARK2 and -3 were shown to uniquely phosphorylate Ser 155 but not other 14-3-3 sites of HDAC7 (Dequiedt et al, 2006). CaMKI and -II target different sites in HDAC4 .…”

“…Surprisingly, the corresponding motif is also conserved in HDAC5 and -9 ( Figure 1b). In addition, we recently identified a cryptic 14-3-3 binding site (Ser 181 ) in HDAC7 (Dequiedt et al, 2005(Dequiedt et al, , 2006. This site was overlooked in previous mutational analyses because its phosphorylation seems dependent on prior phosphorylation of the most N-terminal serine residue, Ser 155 .…”

“…However, depending on cell lines examined, a significant portion of the molecules can also be found in the cytoplasm, suggesting that these enzymes may shuttle between the nucleus and the cytoplasm Fischle et al, 2001). Early confocal microscopy experiments with leptomycin B and recently using the fluorescence loss in photobleaching technology confirmed that class IIa HDACs are subject to CRM1-dependent nuclear export Grozinger and Schreiber, 2000;Wang et al, 2000;Kao et al, 2001;Dequiedt et al, 2006). Cytoplasmic accumulation of class IIa HDACs renders them unable to impact on transcription since it sequesters them away from their histone substrates and renders them enzymatically inactive as HDACs (Fischle et al, 2001(Fischle et al, , 2002.…”

Protein phosphatase 2A controls the activity of histone deacetylase 7 during T cell apoptosis and angiogenesis

“…1C, we observed a time-dependent dephosphorylation of HDAC7 on incubation with purified PP2A. To determine whether all four 14-3-3 binding motifs in HDAC7 were similarly targeted by PP2A, aliquots of the dephosphorylation reaction were taken at various time points and phosphorylation of Ser 155 , Ser 181 , Ser 321 , and Ser 449 was followed by HPLC analysis (7). A time-dependent reduction of phosphorylation was observed for each serine residue (Fig.…”

“…Based on the above observations, we reasoned that binding of 14-3-3 proteins might prevent PP2A from gaining access to and dephosphorylating HDAC7. To test this, total lysate from HDAC7-expressing cells was incubated at 30°C in a buffer compatible with dephosphorylation and immunoblotted with antibodies specific for phosphorylated Ser 155 and Ser 181 (7). No significant dephosphorylation of either serine residues was observed under these conditions ( Fig.…”