Proteomic identification and functional characterization of MYH9, Hsc70, and DNAJA1 as novel substrates of HDAC6 deacetylase activity

Linlin Zhang,Dengwen Li,Jun Zhou,Wenqing Shui,Edward Seto,Shanshan Liu,Yong Zhang,Min Liu,Ningning Liu,Tso-Pang Yao

doi:10.1007/s13238-014-0102-8

Abstract

Histone deacetylase 6 (HDAC6), a predominantly cytoplasmic protein deacetylase, participates in a wide range of cellular processes through its deacetylase activity. However, the diverse functions of HDAC6 cannot be fully elucidated with its known substrates. In an attempt to explore the substrate diversity of HDAC6, we performed quantitative proteomic analyses to monitor changes in the abundance of protein lysine acetylation in response to HDAC6 deficiency. We identified 107 proteins with elevated acetylation in the liver of HDAC6 knockout mice. Three cytoplasmic proteins, including myosin heavy chain 9 (MYH9), heat shock cognate protein 70 (Hsc70), and dnaJ homolog subfamily A member 1 (DNAJA1), were verified to interact with HDAC6. The acetylation levels of these proteins were negatively regulated by HDAC6 both in the mouse liver and in cultured cells. Functional studies reveal that HDAC6-mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1. These findings consolidate the notion that HDAC6 serves as a critical regulator of protein acetylation with the capability of coordinating various cellular functions.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-014-0102-8) contains supplementary material, which is available to authorized users.

Highlights

Reversible lysine acetylation, as an evolutionarily conserved protein posttranslational modification, regulates a variety of cellular processes including gene expression, enzyme activity, and protein-protein interactions
To identify cytoplasmic proteins with acetylation levels changed in response to Histone deacetylase 6 (HDAC6) deficiency, we prepared cytoplasmic fractions from the liver tissues of wild-type and HDAC6 knockout mice (Fig. 1A)
Given that physical interactions are required for enzymesubstrate pairs as reported for HDAC6 and its known substrates (Hubbert et al, 2002; Zhang et al, 2003; Kovacs et al, 2005; Zhang et al, 2007), we examined by immunoprecipitation and immunofluorescence microscopy whether HDAC6 interacts with the new substrates

Summary

Introduction

Reversible lysine acetylation, as an evolutionarily conserved protein posttranslational modification, regulates a variety of cellular processes including gene expression, enzyme activity, and protein-protein interactions. Lysine acetylation was initially reported to regulate nuclear proteins such as histones and transcription factors (Allfrey et al, 1964; Gershey et al, 1968; Gu and Roeder, 1997), largescale proteomic surveys have demonstrated that this modification is prevalent outside the nucleus. The first global proteomic survey of reversible lysine acetylation described 37 acetylated proteins from the cytoplasmic fraction of HeLa cells and 133 from the mitochondria of the mouse liver (Kim et al, 2006). Two studies have extensively characterized the mitochondrial acetylome from the mouse liver via isotope labeling-based or label-free proteomic quantification (Hebert et al, 2013; Rardin et al, 2013).

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