title: 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 multiprotein complexes, such as the nuclear corepressor and beclin-6 corepressor (BCoR) complexes. Cytoplasmic relocalization relieves 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, Ser1108), 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 MS, we have defined the interactions and HDAC5-containing complexes mediated by site-specific phosphorylation and quantified selected changes using parallel reaction monitoring. These results expand the current understanding of HDAC regulation, and the functions of this critical family of proteins within human cells.
authors.affiliation: Princeton University, Department of Molecular Biology, Princeton, NJ, USA.
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Guise
authors.forename: Rommel A
authors.initials: RA
authors.lastname: Mathias
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Rowland
authors.forename: Fang
authors.initials: F
authors.lastname: Yu
authors.forename: Ileana M
authors.initials: IM
authors.lastname: Cristea
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D006655:Histone Deacetylases / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D064326:MEF2 Transcription Factors / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D060066:Protein Interaction Maps / Q000502:physiology*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D064326:MEF2 Transcription Factors; D010750:Phosphoproteins; C404500:HDAC5 protein, human; D006655:Histone Deacetylases
keywords: Cell biology; Class IIa HDAC; HDAC3; Histone deacetylase 5; MEF2D; Phosphorylation