Abstract
Histone deacetylase 2 (HDAC2) is a member of a large family of enzymes that alter gene expression by catalyzing the removal of acetyl groups from core histones. Originally isolated as a transcriptional co-repressor, HDAC2 possesses extensive amino acid sequence homology to HDAC1 (the founding member and most extensively studied HDAC enzyme). Because of this high degree of sequence similarity between HDAC1 and HDAC2, coupled with the fact that the two always co-exist in the same complexes, it is difficult to assess whether different properties exist between these two proteins. We report here that HDAC2 is a phosphoprotein similar to HDAC1. In addition, like HDAC1, the phospho-acceptor sites in HDAC2 are located in the C-terminal portion of the protein. However, unlike HDAC1, which can be phosphorylated by protein kinase CK2, cAMP-dependent protein kinase, and protein kinase G, HDAC2 is phosphorylated uniquely by protein kinase CK2 in vitro. Studies using unfractionated cell extracts with CK2 inhibitors suggest that protein kinase CK2 is the major source of HDAC2 kinase. Finally, and perhaps most interesting, HDAC2 phosphorylation promotes enzymatic activity, selectively regulates complex formation, but has no effect on transcriptional repression. Together, our data indicate that like many HDACs, HDAC2 is regulated by post-translational modification, particularly phosphorylation. Furthermore, we demonstrate for the first time that there are similarities and differences in the regulation of HDAC1 and HDAC2 by phosphorylation.
Highlights
In eukaryotes, DNA is tightly bound to histones, forming repeating units of DNA-protein particles called nucleosomes
Histone deacetylase 2 (HDAC2) Is Phosphorylated at Serine Residues in Vivo—To determine whether HDAC2 is subject to phosphorylation modification, we prepared an extract from metabolically 32P-labeled HeLa cells, immunoprecipitated the endogenous HDAC2 protein, and resolved the product on an SDS-polyacrylamide gel
By using partial acid hydrolysis in HCl followed by twodimensional thin layer electrophoresis of the labeled phosphoamino acid, the presence of phosphoserine but not phosphothreonine or phosphotyrosine was unambiguously identified in HDAC2 (Fig. 1B)
Summary
DNA is tightly bound to histones, forming repeating units of DNA-protein particles called nucleosomes. For experiments that involved the labeling of FLAG-HDAC2 proteins in vivo, expression plasmids were transfected into cells using a standard calcium phosphate co-precipitation method.
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