Molecular Cell 23, 607–618, August 18, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.molcel.2006.06.026
Substrate and Functional Diversity
of Lysine Acetylation Revealed
by a Proteomics Survey
Sung Chan Kim,1Robert Sprung,1Yue Chen,1
Yingda Xu,1Haydn Ball,1Jimin Pei,1Tzuling Cheng,3
Yoonjung Kho,1Hao Xiao,4Lin Xiao,5
Nick V. Grishin,1,2Michael White,3Xiang-Jiao Yang,5
and Yingming Zhao1,*
1Department of Biochemistry
2Howard Hughes Medical Institute and
Department of Biochemistry
3Department of Cell Biology
University of Texas Southwestern Medical Center
Dallas, Texas 75390
4ImmuneChem Pharmaceuticals Inc.
204-8678 Greenall Avenue
British Columbia V5J 3M6
5Molecular Oncology Group
Department of Medicine
McGill University Health Centre
Quebec H3A 1A1
posttranslational modification that is known to play
a key role in regulating transcription and other DNA-
dependent nuclear processes. However, the extent of
this modification in diverse cellular proteins remains
largely unknown, presenting a major bottleneck for
lysine-acetylation biology. Here we report the firstpro-
teomic survey of this modification, identifying 388
acetylation sites in 195 proteins among proteins de-
rived from HeLa cells and mouse liver mitochondria.
In addition to regulators of chromatin-based cellular
processes, nonnuclear localized proteins with diverse
functions were identified. Most strikingly, acetyllysine
including many longevity regulators and metabolism
enzymes. Our study reveals previously unappreciated
roles for lysine acetylation in the regulation of diverse
cellular pathways outside of the nucleus. The com-
bined data sets offer a rich source for further charac-
terization of the contribution of this modification to
cellular physiology and human diseases.
Lysine acetylation is a reversible and highly regulated
posttranslational modification. The posttranslational
modification was initially discovered on histones about
four decades ago (Vidali et al., 1968). However, it took
another 30 years before the first nonhistone protein,
p53, was identified to be lysine acetylated (Gu and
Roeder, 1997). The modification regulates diverse pro-
tein properties including DNA-protein interactions, sub-
cellular localization, transcriptional activity, and protein
stability. In addition to its important roles infundamental
biology, lysine acetylation and its regulatory enzymes
(histone acetyltransferases and histone deacetylases
[HDACs]) are intimately linked to aging and several
major diseases such as cancer, neurodegenerative dis-
orders, and cardiovascular diseases (Blander and Guar-
ente, 2004; Carrozza et al., 2003; McKinsey and Olson,
2004; Yang, 2004a). Despite intensive research over the
past decade, fewer than 90 lysine-acetylated proteins
have been identified (Kouzarides, 2000; Yang, 2004a).
The diverse cellular and physiological functions of ly-
sine acetylation cannot be exclusively explained by its
templated processes. This implies a wider influence for
the modification among proteins. In particular, lysine
acetylation has the potential to be connected to calorie
restriction and longevity, because the activity of Sirt1,
a mammalian ortholog of the yeast deacetylase Sir2,
can be modulated by calorie restriction (Bordone and
Guarente, 2005). Sirt1 appears to target diverse nonhis-
to regulate different cellular functions including stress
response, apoptosis, and energy metabolism (Brunet
et al., 2004; Cohen et al., 2004; Hubbert et al., 2002;
Vaziri et al., 2001). Nevertheless, the mechanism under
which Sirt1 regulates various cellular pathways and
physiological functions, including calorie restriction, re-
in epigenetic regulation and in the regulation of onco-
genic processes, HDAC inhibitors have been developed
and are currently under more than 30 clinical trials as
therapeutic agents for both hematologic malignancies
and solid tumors (Egger et al., 2004). Suberoylanilide
hydroxamic acid (SAHA), the most advanced clinical
compound of HDAC inhibitors, affects cell growth,
differentiation, and apoptosis preferentially on a broad
spectrum of transformed cells, but not normal cells
(Kelly et al., 2003). However, effects on histones cannot
explain such specificity, as histones are ubiquitously
expressed in both normal and transformed cells. Unfor-
tunately, the downstream targets of SAHA, as well as
the mechanism of its antitumor activity, are largely un-
known. Identification and functional characterization of
acetyllysine residues in cytosolic proteins and signaling
molecules (Cohen and Yao, 2004; Kaiser and James,
2004), and demonstration of HATs and HDACs in the
cytosol and mitochondria (Cohen and Yao, 2004;
Kovacs et al., 2005; McKinsey and Olson, 2004; Michi-
shita et al., 2005; Schwer et al., 2002) further support
the notion that the modification may be ubiquitous in
cells. Despite this evidence, the scope of lysine acetyla-
tion is largely unclear, creating one of the major black
boxes for its functional studies in DNA-independent
System-wide analysis of lysine acetylation is a poten-
tially powerful approach toward dissecting the complex
networks of such a highly versatile modification. Never-
theless, proteomics of lysine acetylation is no less
challenging than that of other protein modifications.
nonacetylated proteins with a wide range of expression
levels. Enrichment of lysine-acetylated proteins or pep-
tides is therefore necessary prior to proteomics analysis
in order to reduce the complexity and dynamic range of
the cellular proteome. However, no systematic analysis
has been reported for lysine acetylation.
Here we describe a proteomics survey of the acetylly-
chondria by combining immunoaffinity purification of
tification by nano-HPLC/MS/MS analysis (Figure 1A).
This screening identified 388 lysine acetylation sites
from 195 proteins, dramatically extending the known in-
ventory of in vivo acetylation sites and substrates. Most
lysine-acetylated proteins identified in this study do not
have obvious roles in DNA-templated processes. More-
over, we identified 277 lysine acetylation sites in 133
mitochondrial proteins, thereby conclusively establish-
ing that lysine acetylation is an abundant posttransla-
tional modification in the mitochondrion. These results
clearly indicate that regulation of DNA-templated pro-
cess is just one of many roles that lysine acetylation
plays invivo. Integration ofthese data setsoffers a step-
ping stone toward understanding and characterizing
functional consequences of lysine acetylation in diverse
cellular regulatory schemes and in the pathophysiology
of human diseases.
Results and Discussion
Specific Detection of Lysine-Acetylated Proteins
To evaluate the specificity of anti-acetyllysine antibody,
we carried out immunofluorescent staining of HeLa cells
and revealed thattheantibody produced strong positive
signals in the nucleus and the midbody of the cells
two subcellular regions. Western blotting analysis with
nuclear and cytosolic protein extracts of HeLa cells
showed that multiple protein bands spanning a wide
mass range were detected in both extracts (Figure 2B).
Figure 1. Strategy for Proteomics Analysis of Lysine-Acetylated Proteins
(A) Cytosolic and nuclear protein fractions from HeLa cells were digested with trypsin. The resulting peptides were subjected to immunoaffinity
purification using an anti-acetyllysine antibody. The isolated peptides were analyzed by HPLC/MS/MS in an LTQ 2D ion-trap mass spectrometer
for peptide identification.
(B) An example MS spectrum with a peptide targeted for MS/MS analysis marked by an arrow.
(C) MS/MS analysis of the peptide marked in (B) led to identification of an acetylated peptide with sequence MCLVEIEK*APK (K* indicates
an acetylated lysine), unique to the NADH dehydrogenase75kDa subunit. The labels band ydesignatethe N-and C-terminal fragments,respec-
tively, of the peptide produced by breakage at the peptide bond in the mass spectrometer. The number represents the number of N- or
C-terminal residues present in the peptide fragment.
groups. Identification of acetyllysine residues in a large
number of metabolic enzymes and longevity proteins
tory pathway of energy metabolism. Lysine acetylation,
together with the concentrations of acetyl-CoA and
NAD, might serve as a liaison connecting cellular energy
level with HAT/HDAC activities and enzymatic activities
of acetylated substrates.
At the mechanistic level, lysine acetylation not only
neutralizes a positive charge but also increases the hy-
drophobicity and size of lysine’s side chain. As ex-
pected, the modification is likely to induce significant
conformational changes of substrate proteins, possibly
leading to changes in function. Identification of lysine
acetylation sites among proteins with known or pre-
dicted biological function confirms previously estab-
lished mechanisms of functional regulations and sug-
gests new ones. The impact of lysine acetylation on
protein function is reminiscent of the effects of protein
In eukaryotic cells, each protein may be subject to
multiple modifications, which can interact with each
other to form a dynamic regulatory program (Pawson
tionsites, as illustrated for acetylation sitesin this study,
is one of the first steps toward understanding the funda-
mental question how such a program operates in vivo.
Given the critical roles of lysine acetylation in regulating
diverse cellular physiology, it is likely that proteins regu-
lating lysine acetylation (HDACs and HATs) and their
substrates constitute a rich source of candidate pro-
teins relevant to human disease and therapeutic targets
for drug design. In addition, the acetylation data sets
generated in this study could drive experimental efforts
toward functional characterization of lysine-acetylated
proteinsand theanatomy oflysine acetylation pathways
specific to human diseases.
Cell Culture, Transfection, and Immunocytochemistry
Suspension HeLa S3 cells were grown in DMEM supplemented with
5% newborn calf serum, nonessential amino acids, 100 units/ml
penicillin, and 100 mg/ml streptomycin in 2 liter round-bottom side-
arm spinner flasks. Cells were treated with 0.3 mM tricostatin A
(TSA; class I/II HDAC inhibitor) and 50 mM of sirtinol (class III
HDAC inhibitor) at a density of 5 3 105cells/ml for 24 hr before har-
vesting, for preparation of cytosolic and nuclear extracts.
HeLa adhesion cells and NIH 3T3 cells were grown in DMEM sup-
plemented with 10% fetal bovine serum and 10% newborn calf se-
rum, respectively. Transfections were carried out using Superfect
transfection reagent for NIH 3T3 cells and Lipofectamine 2000 for
For subcellular localization of acetyllysine proteins, HeLa cells
were fixed and stained with anti-acetyllysine polyclonal antibody
(0.65 mg/ml) using a 1:50 dilution with or without competition with
50 mg of acetylated BSA. To assess the effects of acetylation on
stress fiber formation, HeLa cells were treated with 1 mM TSA or
0.2 mM resveratrol (class III HDAC agonist) or were left untreated
for 2 hr. Rhodamine-conjugated phalloidin was used to visualize
Cytosolic Extracts, Nuclear Extracts, and Mitochondrial Lysates
Preparation of cytosolic (S-100) and nuclear extracts from HeLa S3
cells, and protein lysate from mouse liver mitochondria, is described
in detail in Supplemental Experimental Procedures.
In-Solution Tryptic Digestion
Ten milligrams of proteins of interest were precipitated with ace-
tone, followed by centrifugation at 22,000 3 g for 10 min. The result-
ing pellet was rinsed twice with cold acetone to remove residual
salts, resuspended in 50 mM NH4HCO3(pH 8.5), and digested with
trypsin at an enzyme-to-substrate ratio of 1:50 for 16 hr at 37?C.
The tryptic peptides were reduced with 5 mM DTT at 50?C for
30 min and then alkylated using 15 mM iodoacetamide at RT for
30minindarkness. The reaction wasquenchedwith 15mMcysteine
at RT for 30 min. To ensure complete digestion, additional trypsin
at an enzyme-to-substrate ratio of 1:100 was added to the peptide
mixture and incubated for an additional 3 hr. The peptides were
dried in a SpeedVac.
Affinity Purification of Lysine-Acetylated Peptides
The affinity-purified anti-acetyllysine antibody (ImmunoChem Phar-
maceuticals Inc., Burnaby, British Columbia, Canada) was immobi-
lized onto protein A-conjugated agarose beads at 3–4 mg/ml by in-
cubation at 4?C for 4 hr. The supernatant was removed and the
beads were washed three times with NETN buffer (50 mM Tris$HCl
[pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.5% NP40).
The tryptic peptides obtained above were resolubilized in NETN
buffer. Insoluble particles were removed by centrifugation at
100,000 3 g for 10 min. Affinity purification was carried out by incu-
immobilized agarose beads at 4?C for 6 hr with gentle shaking. The
beads were washed three times with 1 ml of NETN buffer and twice
with ETN (50 mM Tris$HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA). The
bound peptides were eluted from the beads by washing three times
with 50 ml of 0.1% TFA. The eluates were combined and dried in
a SpeedVac. The resulting peptides were cleaned in mC18 ZipTips
instructions, prior to nano-HPLC/mass spectrometric analysis.
Protein Identification by Nano-HPLC-MS/MS and Manual
Verification of Identified Peptides
The procedures are detailed in Supplemental Experimental Proce-
perimental Procedures, Supplemental Results, and Supplemental
References and can be found with this article online at http://
This work was supported by The Robert A. Welch Foundation
(I-1550 to Y.Z. and I-1414 to M.W.), NCI/NIH’s ‘‘Innovative Technol-
ogies for the Molecular Analysis of Cancer’’ program (CA107943 to
Y.Z.), the National Cancer Institute of Canada (to X.-J.Y.), and the
Canadian Institutes for Health Research (to X.-J.Y.). We thank Steve
McKnight and Shanhai Xie for preparation of liver mitochondria and
Qin, and Helen Yin for their insightful suggestions. We also thank
John Cottrell from MatrixScience for his assistance.
Received: February 6, 2006
Revised: May 3, 2006
Accepted: June 27, 2006
Published: August 17, 2006
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