Probing phosphorylation‐dependent protein interactions within functional domains of histone deacetylase 5 (HDAC5)

Abstract

Class IIa histone deacetylases (HDACs) are critical transcriptional regulators, shuttling between nuclear and cytoplasmic cellular compartments. Within the nucleus, these HDACs repress transcription as components of multi-protein complexes, such as the nuclear co-repressor (NCoR) and beclin-6 co-repressor (BCoR) complexes. Cytoplasmic relocalization releases this transcriptional repressive function. Class IIa HDAC shuttling is controlled, in part, by phosphorylations flanking the nuclear localization signal (NLS). Furthermore, we have reported that phosphorylation within the NLS by the kinase Aurora B modulates the localization and function of the class IIa HDAC5 during mitosis. While we identified numerous additional HDAC5 phosphorylations, their regulatory functions remain unknown. Here we studied phosphorylation sites within functional HDAC5 domains, including the deacetylation domain (DAC, Ser755), nuclear export signal (NES, S1108), and an acidic domain (AD, Ser611). We have generated phosphomutant cell lines to investigate how absence of phosphorylation at these sites impacts HDAC5 localization, enzymatic activity, and protein interactions. Combining molecular biology and quantitative mass spectrometry, we have defined the interactions and HDAC5-containing complexes mediated by site-specific phosphorylation and quantified selected changes using parallel reaction monitoring (PRM). These results expand the current understanding regarding HDAC regulation, and the functions of this critical family of proteins within human cells.

Post-translational Modifications Regulate Class IIa Histone Deacetylase (HDAC) Function in Health and Disease

“…Loss of phosphorylation within an acidic region, the DAC domain, and the NES was shown to modulate HDAC5 protein interactions, including associations necessary for transcriptional repression (12). Interestingly, mass spectrometry-based quantification of HDAC5 phosphorylations within these functional domains showed substantial variation in their relative abundances (12).…”

“…Previous HDAC5 structure prediction indicated that the majority of phosphorylations map to the external surface of the protein, suggesting that these modifications may be primarily important for mediating protein interactions (12). Loss of phosphorylation within an acidic region, the DAC domain, and the NES was shown to modulate HDAC5 protein interactions, including associations necessary for transcriptional repression (12).…”

“…Loss of phosphorylation within an acidic region, the DAC domain, and the NES was shown to modulate HDAC5 protein interactions, including associations necessary for transcriptional repression (12). Interestingly, mass spectrometry-based quantification of HDAC5 phosphorylations within these functional domains showed substantial variation in their relative abundances (12). Prominent modifications could point to sites necessary for protein folding or structural stability, whereas modifications with low abundances may reflect dynamic or transient events that are spatially and temporally modulated.…”

“…Specifically, S611 mutation led to changes in the association of HDAC5 with the transcription factor MEF2D (12). Therefore, future studies investigating additional PTMs could shed light on their impact on HDAC localizations and interactions within different regions of the same subcellular compartments (e.g.…”

“…Class I (HDAC1, -2, -3, and -8) are related to Rpd3, class IIa (HDAC4, -5, -7, and -9) and class IIb (HDAC6 and 10) are similar to Hda1, and class IV (HDAC11) has homology to both Rpd3 and Hda1 (11). These HDAC classes are zinc-dependent enzymes, in contrast to class III HDACs, commonly known as sirtuins (SIRT1-7), which require the cofactor NADϩ for activity (12,13).…”