TY - JOUR
T1 - Probing phosphorylation-dependent protein interactions within functional domains of histone deacetylase 5 (HDAC5)
AU - Guise, Amanda J.
AU - Mathias, Rommel A.
AU - Rowland, Elizabeth A.
AU - Yu, Fang
AU - Cristea, Ileana M.
N1 - Publisher Copyright:
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
PY - 2014/10
Y1 - 2014/10
N2 - 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 reactionmonitoring. These results expand the current understanding ofHDAC regulation, and the functions of this critical family of proteins within human cells.
AB - 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 reactionmonitoring. These results expand the current understanding ofHDAC regulation, and the functions of this critical family of proteins within human cells.
KW - Cell biology
KW - Class iia HDAC
KW - Hdac3
KW - Histone deacetylase 5
KW - Mef2d
KW - Phosphorylation
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U2 - 10.1002/pmic.201400092
DO - 10.1002/pmic.201400092
M3 - Article
C2 - 24920159
AN - SCOPUS:84908073840
SN - 1615-9853
VL - 14
SP - 2156
EP - 2166
JO - Proteomics
JF - Proteomics
IS - 19
ER -