The Rockefeller University Press $30.00
J. Cell Biol. Vol. 200 No. 3 241–247
Correspondence to Natasha Snider: email@example.com
Abbreviations used in this paper: AcK, acetyl-lysine; AU, acid urea; HMM,
high molecular mass; HSE, high salt extract; IF, intermediate filament protein;
K8, keratin 8; SIRT2, sirtuin 2; WT, wild type.
Protein lysine acetylation is a reversible process involving the
modification of -amino groups of lysine residues with an ace-
tyl moiety from acetyl-CoA (Yang and Seto, 2008; Kim and
Yang, 2011). The dynamics of this process are regulated by
specific enzymes carrying out lysine acetylation and deacety-
lation in response to different stimuli (Aka et al., 2011). For ex-
ample, acetylation has a major role regulating metabolism, as
most metabolic enzymes are acetylated in response to the type
and abundance of cellular energy source (Zhao et al., 2010).
Further, a global proteome analysis identified acetylation as a
highly pervasive modification able to alter functional protein
networks, thereby modulating cell cycle regulation, DNA re-
pair, nuclear transport, and cytoskeletal dynamics (Choudhary
et al., 2009).
Although microtubule cytoskeletal dynamics have long
been known to be regulated by the reversible acetylation of
-tubulin at Lys-40 (L’Hernault and Rosenbaum, 1985), the
exact functional significance of this process is unclear. However,
-tubulin acetylation is associated with microtubule stabiliza-
tion (Janke and Bulinski, 2011). Similarly, actin is acetylated at
Lys-61 and its acetylation is linked to the formation of stabi-
lized actin stress fibers (Kim et al., 2006). In stark contrast,
there is lack of insight into the extent to which lysine acetyla-
tion plays a role in the function of intermediate filament pro-
teins (IFs). A better understanding of IF regulation by acetylation
should provide important insight into the role of acetylation on
cytoskeletal dynamics in general.
The IF cytoskeleton of simple-type epithelia consists of
heteropolymers between keratin 8 (K8) and K18. Mutations in
human K8 (e.g., G62C and R341H) predispose their carriers
to acute and chronic liver disease progression (Omary et al.,
2009; Strnad et al., 2010). A properly functioning IF cytoskel-
eton is critical for the ability of cells to cope with stress
(Toivola et al., 2010). Stress-mediated posttranslational modi-
fications in normal and diseased human liver, including phos-
phorylation (Omary et al., 2006), transamidation (Kwan et al.,
2012), sumoylation (Snider et al., 2011), and their cross talk,
have important consequences on keratin filament organiza-
tion. Recent proteomic studies revealed multiple potential
acetylated sites on K8 and K18 (Leech et al., 2008; Choudhary
(IFs) are unknown. We investigated the regulation of kera
tin 8 (K8), a type II simple epithelial IF, by lysine acetylation.
K8 was basally acetylated and the highly conserved Lys207
was a major acetylation site. K8 acetylation regulated fila
ment organization and decreased keratin solubility. Acety
lation of K8 was rapidly responsive to changes in glucose
levels and was upregulated in response to nicotinamide
adenine dinucleotide (NAD) depletion and in diabetic mouse
ysine acetylation is an important posttranslational modi
fication that regulates microtubules and microfila
ments, but its effects on intermediate filament proteins
and human livers. The NADdependent deacetylase sirtuin
2 (SIRT2) associated with and deacetylated K8. Pharmaco
logic or genetic inhibition of SIRT2 decreased K8 solubility
and affected filament organization. Inhibition of K8 Lys
207 acetylation resulted in sitespecific phosphorylation
changes of K8. Therefore, K8 acetylation at Lys207, a
highly conserved residue among type II keratins and
other IFs, is upregulated upon hyperglycemia and down
regulated by SIRT2. Keratin acetylation provides a new
mechanism to regulate keratin filaments, possibly via mod
ulating keratin phosphorylation.
Glucose and SIRT2 reciprocally mediate the
regulation of keratin 8 by lysine acetylation
Natasha T. Snider,1 Jessica M. Leonard,1 Raymond Kwan,1 Nicholas W. Griggs,1 Liangyou Rui,1 and M. Bishr Omary1,2
1Department of Molecular and Integrative Physiology and 2Department of Medicine, University of Michigan Medical School, Ann Arbor, MI 48109
© 2013 Snider et al. This article is distributed under the terms of an Attribution–Noncommercial–
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T H E J O U R N A L O F C E L L B I O L O G Y
JCB • VOLUME 200 • NUMBER 3 • 2013 242
We next performed site-directed mutagenesis (Lys-to-Arg)
or used a C-terminal truncated version of K8 lacking the last
14 amino acids, including Lys-472 and Lys-483 (Ku et al.,
2005), to determine which K8 residues are major acetylation
sites. Relative to wild-type (WT) K8, the acetylation of the
K207R mutant was negligible, suggesting that Lys-207 is a
major acetylation site (Fig. 1 B). Acetylated K8 is also de-
tected after K8/K18 immunoprecipitation, followed by blot-
ting with a mouse anti-AcK antibody (Fig. 1 C). The effect of
the mutation was not a result of a general acetylation defect
because acetylation of -tubulin was unaltered (Fig. 1 D). As
an additional control, we tested whether the K207R mutation
affects K8/K18 tetramer formation and found that it was not
the case (Fig. 1 E). To assess K8 acetylation independently of
the AcK antibody method, we examined K8 migration by
acid urea (AU) gel electrophoresis. Because urea leads to
protein denaturation without altering the charge, the migra-
tion profile of proteins is determined by the number of pro-
tonated groups, as shown for histone acetylation (Shechter
et al., 2007). Notably, two distinct bands for the WT K8 pro-
tein but only one for the K207R mutant are resolved (Fig. 1 F).
Because the phosphorylated form of K8 comigrates with the
top band (Fig. 1 F), we conclude that the second band, lack-
ing in the K207R mutant, most likely contains acetylated K8.
Importantly, the K8 band lacking in the Lys-207 arginine mu-
tant is restored when Lys-207 is mutated to an acetyl-mimetic
glutamine (Fig. 1 G).
K8 acetylation is important for filament
organization and solubility
Given that Lys-207 is highly conserved (Fig. S1 B), we hypoth-
esized that its acetylation status may regulate K8/K18 filament
properties. Therefore, we compared the filament organization of
et al., 2009), but these sites have yet to be validated and their
significance investigated. The goals of the present study were
to investigate the regulation of K8 by lysine acetylation and
identify the specific sites, contexts, and consequences associ-
ated with this modification.
Results and discussion
Identification of Lys-207 as a major
acetylation site on human K8
Using high-resolution mass spectrometry, Choudhary et al.
(2009) identified 1,750 acetylated proteins with 3,600 corre-
sponding lysine acetylation sites. K8 was among the proteins
identified by this method and found to have five acetylation
sites (Lys-117, Lys-207, Lys-295, Lys-325, and Lys-347),
all located within the coiled-coil rod domain region of
the protein (Herrmann et al., 2009). Evidence that lysines
located outside of the rod domain may also be subject to acet-
ylation came from another proteomic study that identified
Lys-472 and Lys-483 in the tail domain of K8 as being acet-
ylated (Leech et al., 2008). The aforementioned studies used
human cell lines of lymphoid (MV4-11 and Jurkat) or epi-
thelial (A549 and HCT116) origin. To determine if these
data can be validated in normal tissue, we performed mass
spectrometry analysis on human K8 derived from livers of
human K8-overexpressing mice (Nakamichi et al., 2005).
Three of the seven lysines identified by prior proteomic stud-
ies were acetylated in normal liver under basal conditions,
i.e., in the absence of injury or deacetylase inhibitors (Fig. S1 A).
We also detected basal K8 acetylation in human colon carci-
noma HT29 cells, as determined by immune isolation using
a rabbit anti-acetyl-lysine (anti-AcK) antibody followed by
blotting with anti-K8 antibody (Fig. 1 A).
Figure 1. Human K8 is basally acetylated at
the highly conserved Lys-207. (A) K8 is basally
acetylated in HT29 cells, as demonstrated by
immunoprecipitation using rabbit anti-acetylated
lysine (AcK) antibody and K8 immunoblot.
(B) Comparison of acetylation status of WT
human K8, three Lys-to-Arg mutants, and a
C-terminal deletion mutant lacking Lys-472
and Lys-483. BHK-21 cells were transfected
with constructs expressing the designated K8
variant together with WT K18, followed by
lysis and immunoprecipitation of acetylated
proteins using a rabbit anti-AcK antibody and
K8 immunoblot. (C) K8/K18 immunoprecipi-
tation from lysates of BHK-21 cells expressing
either WT or K207R K8, followed by immu-
noblot with a mouse (m) anti-AcK antibody.
(D) BHK-21 cells that were untransfected (Con.)
or transfected with WT or K207R K8 were
lysed followed by rabbit anti-AcK immuno-
precipitation and K8 or -tubulin immunoblot-
ting. (E) Native gel electrophoresis of WT and
K207R K8 showing that the mutation does not
affect K8/K18 tetramer formation. (F and G)
Analysis of WT, K207R, and K207Q K8 by
AU gel electrophoresis followed by Coomassie
staining and immune blotting for total or phos-
Lysine acetylation of keratin 8 • Snider et al.
setting of hyperglycemia (Fig. 3 B and Fig. S3 A), whereas
-tubulin acetylation was unchanged (Fig. 3 B). Further, K8
acetylation in diabetic human liver tissue was also dramatically
increased (Fig. 3 C). Thus, K8 acetylation is dynamically regu-
lated in response to physiological and pathological alterations
in glucose concentration.
Sirtuin 2 (SIRT2) functions
as a K8 deacetylase
Nutrient availability also impacts acetylation reactions via
modulating the activity of sirtuins, energy-sensitive NAD-
dependent deacetylases (Schwer and Verdin, 2008; Imai and
Guarente, 2010). Treatment of cells with FK866, an NAD
synthesis inhibitor (Hasmann and Schemainda, 2003), sig-
nificantly augmented K8 acetylation (Fig. 3 D). There are
seven mammalian sirtuins (SIRT1–7), but only SIRT1–3 and
SIRT7 have deacetylase activity (Imai and Guarente, 2010).
SIRT2 is the only isoform found primarily in the cytosol.
Coimmunoprecipitation revealed an interaction between SIRT2
and K8 under glucose-free and low glucose conditions, which
was diminished in the presence of high glucose (Fig. 3 E).
Intracellular cross-linking revealed the existence of an 90-kD
protein complex (Fig. 3 F), corresponding to the combined
molecular sizes of K8 and SIRT2, which is consistent with
a direct association. Further, upon overexpressing SIRT1 or
SIRT2, we detected a significant amount of SIRT2, but not
SIRT1, present in the keratin-rich insoluble high salt ex-
tract (HSE), as seen by both immunoblot and Coomassie
(Fig. 3 G). The partitioning of SIRT2 into the HSE is directly
dependent on the amount of K8/K18 expressed (Fig. 3 H).
SIRT2 levels and K8/K18 filament organization do not differ
between WT and ob/ob mice (Fig. S3, B and C). In contrast,
we found decreased SIRT2 expression in cirrhotic human
liver explants, which was associated with an increase in K8
acetylation (Fig. S3 D).
WT K8 with the acetylation-deficient K207R and acetylation-
mimetic K207Q mutants. We observed differences in filament
density surrounding the nuclei (Fig. 2 A) and quantitative
analysis of the image data (Fig. S2, A and B) showed an in-
crease in the signal intensity for K207Q and a decrease for
K207R within a distance of 2–4 µm from the nuclei relative
to WT K8 (Fig. 2 B). We next assessed the effect of these mu-
tations on K8 solubility by a sequential fractionation method
using a detergent-free buffer, followed by nonionic (NP-40)
and ionic (Empigen) detergent solubilization and compared
these fractions to the remaining pellet and total K8 by immune
blotting (Fig. 2 C). There was no significant difference be-
tween the amounts of K8 monomer in each fraction relative to
total K8 (Fig. 2 D). However, one major difference was in the
presence of high molecular mass (HMM) K8 species in the in-
soluble pellet. Compared with WT K8, the abundance of these
K8 complexes, which we observed under reducing and nonre-
ducing (not depicted) conditions, was diminished by 80% in
the K207R mutant and partially restored in the K207Q mutant
(Fig. 2 E). This was confirmed using two independent K8 anti-
bodies (Fig. S1 C). These findings indicate that acetylation
regulates K8 filament organization and solubility properties.
K8 acetylation is regulated by glucose
concentration in vitro and in vivo
Because acetyl-CoA is a donor of the acetyl group in lysine
acetylation reactions, we investigated the dynamics of K8 acet-
ylation in response to glucose availability. As shown in Fig. 3 A,
K8 acetylation at Lys-207 is minimal after transient (6-h) glu-
cose deprivation, but is rapidly (within 1 h) and dose depend-
ently increased in response to glucose addition. To confirm that
this occurs in normal tissues, we investigated mouse K8 acety-
lation in vivo under normoglycemic and hyperglycemic condi-
tions by comparing livers from WT and ob/ob (leptin-deficient)
mice. Basal mouse liver K8 acetylation was up-regulated in the
Figure 2. K8 acetylation affects keratin fila-
ment organization and solubility properties.
(A) Representative images from NIH-3T3 cells
expressing WT, K207R, and K207Q K8. Bar,
10 µm. (B) Quantification of the K8 signal
intensity as a function of distance from the
cell nucleus (n = 5). (C) Western blot analysis
under reducing conditions of WT, K207R, and
K207Q-K8 from sequential subcellular frac-
tions (long and short exposures of the same
membrane are shown at the top and bottom,
respectively). (D) Quantification of the K8
monomer band from three separate experi-
ments. (E) Quantification of the K8 HMM sig-
nal from three separate experiments. ***, P <
0.001; *, P < 0.05; relative to WT; one-way
analysis of variance. The results are presented
as the mean and the standard deviation.
JCB • VOLUME 200 • NUMBER 3 • 2013 244
is involved in K8 deacetylation under both glucose-deficient
and -replete conditions (Fig. 4 D). AGK2 caused a dose-dependent
decrease in soluble K8 (Fig. 4 E) and formation of HMM
K8/K18 complexes (Fig. 4 F and Fig. S3 F) and increased
K8/K18 acetylation (Fig. 4 F). There was a general filament
reorganization and an appearance of perinuclear K8/K18
aggregates in 5–10% of the cells treated with AGK2 (Fig. 4 G).
These data indicate a functional connection between SIRT2
and K8 deacetylation with significant consequences to fila-
Acetylation modulates K8 site-specific
K8 serine phosphorylation is known to promote solubility
(Omary et al., 1998, 2006), whereas we show here that acet-
ylation decreases K8 solubility. Therefore, we investigated a
potential effect of K8 acetylation on K8 phosphorylation. As
shown in Fig. 5 (A and B), K8 Ser-74 (but not Ser-432)
phosphorylation is significantly diminished in the K207R
mutant and restored to near baseline WT levels in the K207Q
acetylation mimetic. These data suggest that acetylation of
K8 at Lys-207 may exert some of its effects on keratin filament
organization by modulation of site-specific K8 phosphoryla-
tion. This is supported by prior evidence showing that phos-
phorylation of K8 at Ser-74 plays an important role in the
ability of keratin filaments to reorganize (Ku et al., 2002). To
that end, glucose starvation followed by restimulation leads
to reorganization of K8/K18 filaments and appearance of strong
perinuclear K8/K18 staining in association with increased K8
Ser-74 phosphorylation (Fig. 5 C).
In the present study we demonstrated a functional link be-
tween cellular metabolic status and site-specific K8 acetylation,
with Lys-207 being a major site. The highly conserved nature of
Lys-207 in K8 suggests that lysine acetylation may be an evolu-
tionarily conserved process important for the regulation of other
IFs. The sequence context of K8 Lys-207 (K-V/A-D/E-L-E)
is also conserved among the type II keratins. We show that
acetylation regulates perinuclear keratin filament organization
(Fig. 2). Acetylation also promotes the formation of insoluble
tightly associated K8 complexes, which may indicate a simi-
larity to its role in promoting microtubule and microfilament
stabilization (Kim et al., 2006; Janke and Bulinski, 2011).
Although the mechanism of how K8 acetylation modulates kera-
tin filament organization remains to be explored, the dramatic
and site-specific effect of K8 Lys-207 acetylation on K8 Ser-74
phosphorylation (Fig. 5) suggests that modulation of keratin
phosphorylation and possibly other posttranslational modifica-
tions could mediate such effects.
The increased K8 acetylation in diabetic animals may
have important pathophysiological consequences during liver
injury associated with the metabolic syndrome. SIRT2 levels
are significantly diminished in adipose tissue from obese sub-
jects and SIRT2 is transcriptionally repressed in diet-induced
obesity (Krishnan et al., 2012). Collectively, our findings show
that K8 Lys-207 is a highly conserved and dynamically acety-
lated residue that is targeted by SIRT2 and serves as a sensor of
the cellular metabolic environment. This sensor effect could
Alteration of keratin acetylation, solubility,
and filament organization in response to
changes in SIRT2 levels and activity
Upon overexpression, SIRT2 displayed a cytoplasmic distri-
bution and colocalized with endogenous K8 (Fig. 4 A). The
multinucleation is consistent with the known role of SIRT2
in regulating mitotic exit and genome integrity (Dryden
et al., 2003; Kim et al., 2011). Overexpression of SIRT2 sig-
nificantly decreased acetylated K8 (Fig. 4 B), whereas in-
creased K8 acetylation was detected after SIRT2 knockdown
(Fig. 4 C and Fig. S3 E). Use of the SIRT2-selective inhibitor
AGK2 (Outeiro et al., 2007) further demonstrated that SIRT2
Figure 3. K8 acetylation is dynamically up-regulated by glucose in vitro
and in vivo. (A) BHK-21 cells expressing WT or K207R K8 were cultured
in glucose-free medium for 6 h followed by 1-h incubation in glucose-free
(0 mM), low (7 mM), or high (25 mM) glucose medium. The cell lysates
were immunoprecipitated with a rabbit anti-AcK antibody followed by K8
immunoblotting. (B) Liver lysates from three individual WT or ob/ob mice
(see Materials and methods) were immunoprecipitated with rabbit AcK
antibody followed by immunoblotting for K8. Total and acetylated -tubulin
blots were done for comparison. (C) K8 acetylation in normal and dia-
betic human liver tissue. (D) K8 acetylation in control, vehicle-, and FK866-
treated (NAD-depleted) cells. Two isoforms of SIRT2 protein migrate at 37
and 43 kD. (E) Immunoprecipitation using rabbit anti-SIRT2 antibody from
lysates of HepG2 cells cultured under different glucose conditions followed
by K8 immunoblotting demonstrates a glucose-sensitive K8–SIRT2 inter-
action. (F) Glucose-starved (3 h) HepG2 cells were incubated in the
absence (–) or presence (+) of the cell-permeable cross-linker DSP and
lysed, and the K8/K18 immunoprecipitates were analyzed by blotting for
SIRT2 under nonreducing or reducing conditions. Arrows highlight the
90-kD species. (G) Flag-tagged human SIRT1 or SIRT2 were coexpressed
with WT K8/K18 in BHK-21 cells and the Triton X-100 (soluble) and HSE
fractions were analyzed by Flag immunoblot or Coomassie stain. (H) Same
as G, except that different amounts of K8/K18 cDNA were cotransfected.
245 Lysine acetylation of keratin 8 • Snider et al.
Cell cultures and transfection
BHK-21 (baby hamster kidney), NIH3T3 (mouse fibroblast), HepG2 (human
hepatoma), and HT29 (human colon carcinoma) cells were obtained from
American Type Culture Collection and maintained as recommended by
the supplier. Lipofectamine 2000 (for BHK-21) or Lipofectamine LTX (for
NIH3T3, HepG2, and HT29; Invitrogen) were used for transfections. Control
and SIRT2 siRNA were obtained from Thermo Fisher Scientific and trans-
fected into HT29 or HepG2 cells using RNAiMAX (Invitrogen) per the man-
ufacturer’s instructions. Plasmid encoding flag-tagged SIRT1 was provided
by M. Greenberg (Harvard Medical School, Boston, MA) via Addgene
(plasmid 1791) and SIRT2 plasmid was provided by E. Verdin (University
of California, San Francisco, San Francisco, CA) via Addgene (plasmid
13813). Biochemical and immunofluorescence staining analyses were per-
formed 18–48 h posttransfection.
Preparation of cell and tissue lysates, immunoprecipitation,
Cultured cells or liver tissues were homogenized in ice-cold NP-40 buffer
(150 mM sodium chloride, 1% NP-40, and 50 mM Tris, pH 8.0) supple-
mented with protease inhibitors. Immunoprecipitation was performed using
antibodies to AcK or K8/K18 (DC10 or TS1) conjugated to Dynabeads–
Protein G (Invitrogen) for 2–3h at 4°C with shaking. To induce intracellular
cross-linking before cell lysis, the cell-permeable cross-linker DSP was added
to HepG2 cells for 30 min at room temperature following manufacturer rec-
ommendations. 2-Mercaptoethanol was added to some samples to reverse
cross-linking before analysis. To induce NAD depletion, cells were treated
with 10 nM FK866 for 18 h. Total lysates and immunoprecipitates were
resolved on gradient 4–20% or 10% SDS-PAGE gels and were transferred
onto polyvinylidene difluoride membranes, which were then blocked
provide a critical link in the chain of cellular events surrounding
K8/K18-mediated protection during metabolic stress in hepa-
tocytes (Loranger et al., 1997; Toivola et al., 2010) by, for ex-
ample, linking nutrient availability to K8 phosphorylation.
Materials and methods
Antibodies and chemicals
The antibodies used in the study were rabbit anti-AcK (Abcam) and mouse
anti-AcK Ac-K-103 (Cell Signaling Technology). Other antibodies were
directed to acetylated -tubulin (6-11B-1; Abcam); SIRT2 (EP1668Y;
Epitomics); Flag (OriGene); -tubulin, human K18 (DC10), and human
K8 (mouse TS1 and rabbit EP1628Y [Thermo Fisher Scientific]); mouse
K8 (Troma I; Developmental Studies Hybridoma Bank); K8 pSer74 (Liao et al.,
1997); and K8 pS432 (Ku and Omary, 1997). The chemicals used were
AGK2 (Tocris), dithiobis[succinimidylpropionate] (DSP; Thermo Fisher Scien-
tific), and FK866 (Cayman Chemical).
The human K8 cDNA in vector pcDNA3.1 was mutated to generate single-
point lysine to arginine or glutamine mutations using the QuikChange site-
directed mutagenesis kit (Agilent Technologies). Generation of the truncated
mutant of K8 was performed as described previously (Ku et al., 2005). In
brief, a frame-shift mutation at Ile465 (ATC→ATCC) generates a truncated
468–amino acid protein (instead of 482). The WT and mutant keratin con-
structs were confirmed by DNA sequencing.
Figure 4. K8 acetylation is dependent on SIRT2 activity and expression levels. (A) Immunofluorescence staining of K8/K18 and Flag-SIRT2 in HepG2 cells
shows significant colocalization. Bars, 20 µm. (B) Rabbit anti-AcK immunoprecipitation of untransfected and Flag-SIRT2–transfected HT29 cell lysates fol-
lowed by K8 immunoblotting. (C) HT29 cells were transfected with control or SIRT2 siRNA for 18 h, followed by 6-h incubation in glucose-free medium. The
lysates were analyzed by rabbit anti-AcK immunoprecipitation followed by K8 immunoblotting. (D) HT29 cells were cultured in the absence or presence of
25 mM glucose and 10 µM AGK2 for 6 h. The lysates were immunoprecipitated with rabbit anti-AcK antibody followed by K8 immunoblot. (E) HepG2 cells
were untreated () or treated with DMSO vehicle (0) or different concentrations of AGK2 for 18 h and the Triton X-100–soluble fractions were assessed for
the presence of K8 and -tubulin (soluble fraction marker). (F) Immunoblot of K8 and AcK in HSEs of HT29 cells after treatment with vehicle (0) or AGK2 for
18 h. (G) Filament reorganization and perinuclear aggregate formation (arrows) in HepG2 cells treated with 10 µM AGK2 for 18 h. Bars, 20 µm.
JCB • VOLUME 200 • NUMBER 3 • 2013 246
Human and animal liver experiments
Human liver tissues were used under an approved human subjects proto-
col. Non-diseased and diabetic human liver tissues were obtained from the
National Disease Research Interchange. Cirrhotic liver explants were ob-
tained from patients who underwent liver transplantation for end-stage liver
disease (Ku et al., 2005). Animal use was approved by, and performed in
accordance with, the University Committee on Use and Care of Animals at
the University of Michigan. Livers were isolated from four different male
8-wk-old human K8-overexpressing transgenic mice (FVB/N background)
for mass spectrometry analysis (performed by MS Bioworks) of human
K8 acetylation sites. For the mass spectrometry analysis, K8 was enriched
by high salt extraction followed by SDS-PAGE electrophoresis and exci-
sion of the protein band. Livers from female 12-wk-old leptin-deficient
ob/ob mice and corresponding age- and sex-matched WT C57BL/6J
mice were used to assess K8 acetylation under hyperglycemic and normo-
glycemic conditions (three mice/group). The total body weights for the
WT and ob/ob mice, respectively, were 18.7 ± 0.3 and 42.7 ± 1.3 g
(P < 0.0001), with corresponding blood glucose values of 230 ± 22
and 535 ± 84 mg/dl (P = 0.02).
The graph data were presented and statistically analyzed using Prism 5
software (GraphPad Software). Photoshop (CS2; Adobe) was used for
immunoblot densitometry (Fig. 2, D and E; Fig. 5 B; and Fig. S3 A).
National Institutes of Health ImageJ software was used to create the sig-
nal intensity plots shown in Fig. 2 B (raw data numbers plotted) by
drawing 10-µm-long lines originating from the nuclei and performing a
plot profile analysis, which provides the signal intensity as a function
distance (as shown in Fig. S2).
Online supplemental material
Fig. S1 supports the data presented in Figs. 1 and 2 and shows the lysine
residues selected for the analysis based on mass spectrometry data, con-
servation of the major K8 acetylation site (Lys-207) in all IFs (Type I–VI),
and immunoblots of K8 and its acetylation site mutants. Fig. S2 supports
data presented in Fig. 2 (A and B) and shows additional examples of fila-
ment organization of WT K8 and its acetylation-site mutants and an example
of signal intensity measurement for perinuclear K8 filaments. Fig. S3 sup-
ports the data presented in Figs. 3 and 4 and shows quantification of the
K8 AcK signal in WT and ob/ob mouse livers and corresponding SIRT2
levels and keratin filament organization; SIRT2 expression and acetylation
of K8 in cirrhotic human livers; immunostaining for AcK and K8 upon SIRT2
knockdown; and Coomassie stain and K18 blot upon pharmacologic
SIRT2 inhibition. Online supplemental material is available at http://www
This work was supported by National Institutes of Health grants R01 DK52951
(M.B. Omary), K01 DK093776 (N.T. Snider), and P30 DK34933 (University
Submitted: 6 September 2012
Accepted: 29 December 2012
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(5% milk in PBS and 0.1% Tween 20) and incubated with the designated
antibodies. Nondenaturing gel electrophoresis was performed using the
NativePAGE Bis-Tris gel system (Invitrogen) following the manufacturer’s
protocol. High salt extracts were performed using an established proce-
dure (Ku et al., 2004).
AU gel electrophoresis
The AU gel method was adopted from a published protocol (Shechter
et al., 2007). Upon solubilization of cells in NP-40 buffer, the insoluble
pellets were dissolved by vortexing and homogenization in an appropriate
volume of AU sample buffer (0.36 g urea, 100 µl of 0.2% Pyronin Y, 50 µl
glacial acetic acid, and 500 µl of 25mg/ml protamine sulfate) and sep-
arated on 8% AU gels. Each 8% AU mini-gel was prepared by mixing
3.6 g urea, 1.3 ml acrylamide/bis-acrylamide (60:0.4), 0.5 ml of ace-
tic acid, 4.4 ml of water, 60 µl TEMED, and 140 µl of ammonium persul-
fate (10%). The gels were run at 200 V in 5% acetic acid (running buffer)
for 60–90 min, and then transferred onto PVDF membranes (25 min, 500 mA)
in 0.7% acetic acid (transfer buffer) followed by Coomassie blue stain-
ing or immunoblotting.
Immunofluorescence staining and confocal imaging
The cells were fixed in methanol for 10 min at 20°C, air dried for 15 min,
and incubated in blocking solution (PBS/2.5% wt/vol BSA/2% goat serum).
Primary antibodies were added for 1 h, followed by three PBS washes and
incubation with Alexa Fluor–conjugated secondary antibodies for 30 min.
All incubations were performed at room temperature. After overnight mount-
ing in ProLong Gold containing DAPI (Invitrogen), the cells were imaged on
a laser-scanning confocal microscope (FluoView 500; Olympus) using a 60×
oil immersion (1.4 NA) objective. Images were magnified using FluoView
software (version 5.0; Olympus). An 405-nm laser diode, 488-nm argon
laser, and 543-nm HeNe green laser were used to excite DAPI, Alexa Fluor
488, and Alexa Fluor 594, respectively. Signal separation was maximized
by sequential scanning.
Figure 5. Acetylation and glucose stimulation modulate K8 site-specific
phosphorylation. (A) BHK-21 cells were transfected with the indicated
plasmids followed by analysis of the total cell lysates by immunoblotting
for total and phospho-K8 (pS74 and pS432). (B) Quantification of the
immunoblot data shown in A from three separate experiments. **, P <
0.01; one-way analysis of variance. The results are presented as the mean
and the standard deviation. (C) HepG2 cells were grown under normal
(4.5g/l glucose) culture conditions (Control) or were glucose starved for 5 h,
and then restimulated with glucose-containing medium (4.5g/l) for 1 h
before fixation and immunostaining for total and K8 pS74. Bars, 20 µm.
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