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Probing phosphorylation‐dependent protein interactions within functional domains of histone deacetylase 5 ( HDAC 5)
Author(s) -
Guise Amanda J.,
Mathias Rommel A.,
Rowland Elizabeth A.,
Yu Fang,
Cristea Ileana M.
Publication year - 2014
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201400092
Subject(s) - corepressor , histone deacetylase 5 , nuclear localization sequence , histone deacetylase , phosphorylation , nls , microbiology and biotechnology , biology , nuclear transport , nuclear export signal , histone , acetylation , nuclear protein , mitosis , transcription factor , cell nucleus , biochemistry , cytoplasm , repressor , gene
Class IIa histone deacetylases ( HDAC s) are critical transcriptional regulators, shuttling between nuclear and cytoplasmic cellular compartments. Within the nucleus, these HDAC s repress transcription as components of multiprotein complexes, such as the nuclear corepressor and beclin‐6 corepressor ( BC o R ) 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 A urora B modulates the localization and function of the class IIa HDAC 5 during mitosis. While we identified numerous additional HDAC 5 phosphorylations, their regulatory functions remain unknown. Here, we studied phosphorylation sites within functional HDAC 5 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 HDAC 5 localization, enzymatic activity, and protein interactions. Combining molecular biology and quantitative MS , we have defined the interactions and HDAC 5‐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.