Nuclear Import of Histone Deacetylase 5 by Requisite Nuclear Localization Signal Phosphorylation

Abstract

Histone deacetylase 5 (HDAC5), a class IIa deacetylase, is a prominent regulator of cellular and epigenetic processes that underlie the progression of human disease, ranging from cardiac hypertrophy to cancer. Although it is established that phosphorylation mediates 14 -3-3 protein binding and provides the essential link between HDAC5 nucleo-cytoplasmic shuttling and transcriptional repression, thus far only four phospho-acceptor sites have been functionally characterized. Here, using a combinatorial proteomics approach and phosphomutant screening, we present the first evidence that HDAC5 has at least 17 in vivo phosphorylation sites within functional domains, including Ser278 and Ser279 within the nuclear localization signal (NLS), Ser1108 within the nuclear export signal, and Ser755 in deacetylase domain. Global and targeted MS/MS analyses of NLS peptides demonstrated the presence of single (Ser278 and Ser279) and double (Ser278/Ser279) phosphorylations. The double S278/279A mutation showed reduced association with HDAC3, slightly decreased deacetylation activity, and significantly increased cytoplasmic localization compared with wild type HDAC5, whereas the S278A and S1108A phosphomutants were not altered. Live cell imaging revealed a deficiency in nuclear import of S278/279A HDAC5. Phosphomutant stable cell lines confirmed the cellular redistribution of NLS mutants and revealed a more pronounced cytoplasmic localization for the single S279A mutant. Proteomic analysis of immunoisolated S278/279A, S279A, and S259/498A mutants linked altered cellular localization to changes in protein interactions. S278/279A and S279A HDAC5 showed reduced association with the NCoR-HDAC3 nuclear corepressor complex as well as protein kinase D enzymes, which were potentiated in the S259/498A mutant. These results provide the first link between phosphorylation outside the known 14 -3-3 sites and downstream changes in protein interactions. Together these studies identify Ser279 as a critical phosphorylation within the NLS involved in the nuclear import of

Dephosphorylation at a Conserved SP Motif Governs cAMP Sensitivity and Nuclear Localization of Class IIa Histone Deacetylases*

“…Ser-279 of HDAC5 was just reported to be a target of cAMP signaling and PKA phosphorylation (81) and was independently identified as a phosphorylation site by mass spectrometry (79). Together, these findings provide further support for the importance of phosphorylation at Ser-266 of HDAC4 and Ser-279 of HDAC5.…”

“…As for phosphatases, PP2A forms stable complexes with class IIa HDACs and inhibits their phosphorylation at 14-3-3 binding sites (10,35,36,79). Okadaic acid, an inhibitor of PP2A, did not have any effect on Ser-266 phosphorylation in response to cAMP treatment (supplemental Fig.…”

“…Additional phosphorylation sites have been identified in class IIa HDACs, including Ser-298 of HDAC4 (36), Ser-253 of the HDAC9 splice variant (78,82), and multiple residues of HDAC4 and HDAC5 reported by phosphoproteomic studies (see the Human Protein Reference Database) (79,80,83,84), suggesting that complex multisite phosphorylation programs may respond to cAMP and other upstream signaling cues (Fig. 8).…”

Functional Proteomics Establishes the Interaction of SIRT7 with Chromatin Remodeling Complexes and Expands Its Role in Regulation of RNA Polymerase I Transcription

“…Point mutations in the sirtuin-core domain related to deacetylase function have been shown to result in reduction of rDNA transcription (9). However, several studies could not detect in vitro deacetylase activity of SIRT7 from human cells (7,9), suggesting that SIRT7 may carry out its functions via recruitment of proteins similar to the deacetylation-deficient class IIa HDACs (14). Still, SIRT7 protein interactions remain poorly characterized.…”

“…Isolation of SIRT7-containing Protein Complexes-SIRT7 and control immunoaffinity purifications on magnetic beads were performed via EGFP, as previously described (13,14). HEK293 cell lines stably expressing EGFP alone, SIRT7WT-EGFP-FLAG (WT), SIRT7S111A-EGFP-FLAG (S111A), and SIRT7dE2-EGFP-FLAG (where dE2 indicates deletion of exon 2; see Fig.…”