Advances in Biological Chemistry, 2011, 1, 109-118
doi:10.4236/abc.2011.13013 Published Online November 2011 (
Published Online November 2011 in SciRes.
The interaction between Fe65 and Tip60 is regulated by
S-nitrosylation on 440 cystein residue of Fe65
Eun Jeoung Lee1, Sung Hwa Shin2, Sunghee Hyun1, Jaesun Chun3, Sang Sun Kang2,4*
1Department of Pre-Medicine, Eulji University School of Medicine, Daejeon, Republic of Korea;
2Department of Biology Education, Chungbuk National University, Cheongju, Republic of Korea;
3Department of Biology Education, Korea National University of Education, Cheongwon, Republic of Korea;
4Biotechnology Research Institute, Chungbuk National University, Cheongju, Republic of Korea.
Email: *
Received 27 August 2011; revised 30 September 2011; accepted 9 October 2011.
The S-Nitrosylation of protein thiol groups by NO is
a widely recognized protein modification. The treat-
ment of cells with NOBF4 induces the S-nitrosylation
of FE65. In this study, we present evidence showing
that FE65 modified by NO (Nitric Oxide) via S-ni-
trosylation induces functional changes in the protein
that inhibits the HAT activity of Tip60. The results of
mutational analysis of FE65 demonstrated further
that the cysteine residue of FE65 (Cys440) is critical
to the process of S-nitrosylation. The mutation of the
cysteine residue which completely ablated the S-ni-
trosylation of FE65 also lost its inhibitory effects on
Tip60 HAT activity. Thus, our findings show, for the
first time, that the novel regulation mechanism of
Tip60 activity may operate via FE65 binding, which
is enhanced by S-nitrosylation on the FE65 Cys440
residue. This study describes the interaction between
FE65 and Tip60, which is en hanced by a posttrans la-
tional modification of FE65 (through S-nitrosylation)
by NO, promoting the association of the FE65-Tip60
protein complex and inhibiting both the HAT activity
of Tip60 and cell death.
Keywords: S-Nitrosylation; NO (Nitric Oxide); FE65;
HAT Activity; Tip60
FE65 functions as an adaptor protein within the cell, the
identification of ligand proteins for the PID1/PID2 do-
main or the WW domain may provide us with some
clues as to its biological roles [1-3]. A few proteins ha-
ve been identified as ligand proteins to the PID1 domain
of FE65. FE65 interacts with the transcription factor (CP2/
LSF/LBP1), Tip60 (Histone acetyltransferase; HA-T),
Teashirt, and calsyntenin and low-density lipoprotein re-
ceptor-related protein through its PI-D1 domain [4,5].
Further, because the protein is regulated by posttransla-
tional modifications, including protein S-nitrosylation,
glycosylation, or nitrosylation, it is also crucial to deter-
mine the manner in which the modification affects bind-
ing with each of its ligand proteins [2,3].
NO is a gaseous signal mediator that evidences a nu-
mber of important biological effects [6]. NO has been
shown, in many instances, to exert its effects via protein
S-nitrosylation, in which the binding of NO to Cys resi-
dues regulates protein function [7,8]. S-nitrosylation re-
gulates protein function via covalent attachment me-
chanisms that control the addition or removal of NO
from a cysteine thiol.
It has been previously reported that the targets of S-
nitrosylation modification, among others, include bovine
serum albumin, tissue-type plasminogen activator, XIAP,
the N-methyl-D-aspartate receptor, oncogenic p21ras, TR-
PC1, and transcriptional activators [9-15]. After analysis
of the S-nitrosylation proteins harboring more than one
Cys residue, a specific consensus motif for S-nitro-syla-
tion (Lys /Arg/Hi /Asp/Glu) Cys (Asp/Glu) (sites –1, 0,
and +1, respectively) is determined [7,8,16]. After reco-
gnizing the putative S-nitrosylation Cys residue in FE65
with the consensus sequence information, we evaluated
the S-nitrosylation of FE65 and determined its biological
significance [1,17]. The results of our sitedirected mu-
genesis analysis (C440A) and in vitro Snitrosylation as-
y revealed that S-nitrosylation occurs on FE65 and the
440 cysteine residue (439Arg/Cys/Glu441) of FE65 as its
spefic modulation site. Additionally, we noted that FE65
netively modulates Tip60 HAT activity via protein-rotein
interactions, which are enhanced by S-nitrosylation. He-
in, we reported for the first time that the S-nitrosylation
of FE65 appears to perform a crucial function in the
E. J. Lee et al. / Advances in Biological Chemistry 1 (2011) 109-118
regulation of Tip60 activity via the modulation of their
protein interactions.
2.1. Materials
NOBF4 was acquired from Aldrich. Mouse anti-FE65
monoclonal antibody was obtained from Chemikon. Ra-
bbit muscle FE65 (80 units/mg) was purchased from
Boeer Mannheim. Other chemicals were of the highest
grade of purity, and were obtained from Sigma. All
chemicals were purchased from Sigma-Aldrich, unless
stated otherwise. HEK293T and HEK 293 cells were
maintained in DMEM (Gibco) supplemented with 10%
FBS and 1% penicillin/streptomycin (Gibco) at 37˚C
with 5% CO2. Transfection was conducted with Lipo-
fectamine and Plus Reagent (Invitrogen) in accordance
with the manufacturer’s instructions.
2.2. DNA Constructions
FE65 and Tip60 were cloned from the SuperScript hu-
man brain cDNA library. Full-length FE65 was cloned
into pRK5-GFP and HA vectors for the cell culture study
and into the pGEX4T-2 vector for the production of re-
combinant protein. The truncated fragments of FE65
encoding for amino acids 1-470 (WW and PID1), 1-265
(PID1-2) were generated via PCR, using the full-length
FE65 as a template, then cloned into pRK5-GFP vector.
The FE65 fragment (1-470 aa C470A) was also cloned
into pGEX4T.2 vector for recombinant protein produc-
tion. The sequence integrity of all constructs was con-
firmed via sequencing.
2.3. Overexpression and Purification of
Recombinant Proteins
GST, GST-FE65, and GST-FE65 C440A were expressed
in Rosetta (DH5α) pLys Escherichia coli (RBC). Over-
expression of bacterial cultures in the linear growth
phase (0.6 OD) were induced by 1 mM IPTG at 37˚C
overnight, and the recombinant proteins were subse-
quently purified by GSH-Sepharose (GE Healthcare).
GFP-tagged recombinant FE65 was generated in accor-
dance with the methods developed by Stennicke and Sa-
lvesen, and was purified using Ni-NTA Sepharose (GE
Healthcare). Concentrations of the recombinant proteins
were quantified via SDS/PAGE, using BSA as a stan-
2.4. In Vitro S-Nitrosylation Assay
The biotin switch assay was conducted according to the
method described by Jaffrey and Snyder, with some mo-
difications. Nitrosylated cell lysates or recombinant pro-
teins in HENT buffer (250 mM Hepes, 1 mM EDTA, 0.1
mM Neocupoine, 1% Triton X-100) were incubated for
20 min with 10 mM methyl methanethiosulfonate (MM-
TS) (Thermo Scientific) at 50˚C, and then excess MM-
TS was removed via three passages through a G25 Se-
phadex spin column. The samples were then incubated
with 5 mM ascorbate and 0.4 mM biotin-HPDP (Thermo
Scientific) for 1 h at room temperature with rotation.
Unreacted biotin-HPDP was then removed with a G25
Sephadex spin column and the biotinylated samples
were then incubated for 1 h with 50 μL of Neutravidin-
agarose (Thermo Scientific). The pellets were then
washed 5 times with neutralization buffer [20 mM Hepes
(pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% Triton X-
100] with 0.6 M NaCl and eluted with an SDS sample
buffer and subjected to Western blot analysis.
2.5. Interaction Between FE65 and Tip60
HEK293T lysates with or without GFP-FE65 expression
were treated for 15 min with GSH (500 μM), NOBF4
(500 μM), or DTT (1 mM) at 37˚C as indicated. The
lysates were then passed once through a G25 Sephadex
spin column and the recombinant FE65 (8 μg) was
added to the lysates, and the co-IP protocol was con-
ducted as described. Tip60 was detected with Tip60 an-
tibody (Sigma).
2.6. Cell Death Analysis
HEK293T cells were transfected with 0.25 μg of GFP-
FE65 or control vector. Thirty hours after transfection,
the cells were pretreated for 6 h with 100 μM NO donor
NOBF4 (Calbiochem). The cells were then treated with
the selected drugs and under the indicated conditions: 50
ng/mL TNF-α and 0.1 μg/mL actinomycin D for 24 h; 50
μM rotenone for 16 h; 2 mM dopamine for 24 h; 3 μM
MG132 for 24 h. In the FE65-induced cell death assay,
in addition to GFP-FE65 C470A, or (1-470 aa) and con-
trol vector, the cells were also cotransfected with GFP as
a control. Cell death was analyzed 60 h after transfection
via a Trypan blue exclusion assay.
2.7. Tip60 Binding Assay
Tip60 (1 µg/assay) and up to 200 µM individual NO
donor were incubated for 5 min in 50 mM triethylam-
monium buffer (pH 7.5) in a total volume of 50 µl at
37˚C. The samples were diluted into 950 µl of 50 mM
triethylammonium buffer (pH 7.5) containing 50 µM
arsenate, 2.4 mM glutathione, and 100 µg/ml of gly-
ceraldehyde-3-phosphate at 37˚C. The enzymatic reduc-
tion of NAD+ to NADH was initiated via the addition of
250 µM NAD+. FE65 activity was monitored by re-
cording fluorescence emissions above 430 nm after ex-
citation at 313 and 366 nm, respectively. Samples with-
out NO donors were used as controls.
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E. J. Lee et al. / Advances in Biological Chemistry 1 (2011) 109-118
Copyright © 2011 SciRes.
2.8. ELISA for Histone Acetyltransferase (HAT) crylamidegel.
3. Results
Extracts were immunoprecipitat-ed as above, except that
the high salt wash was omitted. The immunoprecipitates
were prepared test samples for each assay in 96-well
plate. Mix the prepared assay mix, add 65 µl of assay
mix to each well, mix to start the reaction. Incubation
plates at 37 for 3hr depending on the color develop-
ment. Read sample in a plate reader at 440 nm (biovi-
sion). In some of the assays, HAT activity was analyzed
using Tip60 immobilized on NiTA-agarose beads.
3.1. S-Nitrosylation of FE65 by NO on its 440
Cysteine Residue
After noting the presence of 440 cysteine residue (439RCE441)
in FE65 with the consensus sequences for S-nitrosy-
lation(Lys/Arg/His/Asp/Glu)Cys(Asp/Glu) (sites –1, 0,
and +1, respectively (Figure 1(a)); we attempted to de-
termine whether FE65 was S-Nitrosylated by NO in
HEK293 cells [7,8]. In order to detect the thiol modifi-
cations of the FE65, we employed an S-nitrocystein rab-
bit antibody that binds to the modified sulfhydryl groups.
We detected that the human FE65 in HEK293 is modi
fied with NO (Figure 1(b)). To further confirm the FE65
S-notrosylation, we treated Nitrosonium tetrafluorobo-
rate (NOBF4) which is an exclusively NO+ releasing NO
donor which forces protein-S-nitrosothiol generation via
a transnitrosation reaction to enhance the effect. In the
first set of experiments, we incubated the HEK293 cell
transfected with rat GFP FE65 for 5 min with NOBF4.
Following protein precipitation, the enzyme was resus-
pended, exposed, and electrophoretically separated on a
nonreducing 11% SDS gel (Figure 1(c)). GFP-rat FE65
exposed to NOBF4 was found to harbor the high amount
S-nitrosylated state (Figure 1(c) right lane). HEK293
2.9. Immunoprecipitation
Total cell lysates of EC were denatured via incubation
with 0.5% SDS, 50 mM sodium phosphate, pH 8.0, and
2 mM EDTA at 90˚C, to allow for immunoprecipitation.
The samples were then supplemented to obtain a compo-
sition of 50 mM sodium phosphate, pH 7.2, 1% sodium
deoxycholate, 1% Triton X-100, 0.5% SDS, 150 mM
NaCl, 2 mM EDTA, 5 mM NaF, 2 mM Na4P2O7, 2 mM
Na3VO4, 1% aprotinin, and 200 µg/ml leupeptin at 4˚C.
Mouse anti-FE65 monoclonal antibody (usually at a 1:
100 dilution) was added, followed 18 h later by protein
A-Sepharose. The washed immunoprecipitates were then
analyzed via western bolt with rabbit anti-FE65 or
mouse anti-GFP (Santacruze) after separation in nonre-
ducing or reducing 11% sodium dodecyl sulfate-polya-
Figure 1. S-nitrosylation of FE65.
E. J. Lee et al. / Advances in Biological Chemistry 1 (2011) 109-118
cell transfected with GFP vector alone was used as the
negative control (left lane). Thus, this result also indi-
cates that FE65 is an S-Nitrosylation protein that is
modified by NO.
Next, in order to map the potential S-nitrosylation
sites of FE65, we constructed the FE65 mutant (C440A)
which is predicted with the consensus S-nitrosylation
site motif in Figure 1(a) and subjected it to immuno-
blotting with an S-nitrocystein rabbit antibody (Figure
1(d)) or the biotin switch assay (Figure 1(e)). As shown
in Figure 1(d), GFP FE65 C440A mutant was not de-
tected with S-nitrocystein Ab, while GFP FE65 WT was
recognized by the antibody (Figure 1(d) upper lane). To
monitor the rat GFP FE65 in HEK293 cell, the im-
munoblotting was performed with ant-FE65 Ab (Figure
1(d) bottom lane), after protein immune-precipitation
with GFP Ab. Next, we isolated GST-FE65 WT or C440A
mutant protein form E. coli and subjected them to the
biotin switch assay. Consistent with Figure 1(c), GST
FE65 WT was recognized in the biotin switch assay,
while GST FE65 C440A mutant was not reacted (Figure
1(e)). Therefore, together with the biochemical data, the
440 cysteine residue of (439RCE441) in FE65 is the S-
nitrosylation site, as predicted in Figure 1(a) .
3.2. S-Nitrosylation of FE65 Enhanced its
Binding to Tip60
A number of physiological functions of FE65 are associ-
ated with its PID1 domains, including Tip60, CP2, LSF,
Teashirt, and calsyntenin; thus, we suspected that the S-
nitrosylation of FE65 PID1 domain could affect its
Tip60 binding activity in cells exposed to a variety of
cell death stimuli [4,5]. We investigated whether NO
modification on FE65 influences its binding to Tip60.
With NOBF4 treatment concentration, we observed that
FE65 increases its binding to Tip60 (Figure 2(b) left
lane). To compare the effect of S-nitrosylation on FE65,
FE65 C440A (which is mutated the S-nitrosylated site)
was also treated with NOBF4. As shown in Fig. 2B right
lane, the NOBF4 treatment FE65 C440A does not influ-
ence on its binding to Tip60 in HEK293 cell. To monitor
S-nitrosylation of FE65 and FE65 C440A with treatment
of NOBF4, the western blot using S-nitrosylation spe-
cific antibody was performed (Figure 2(c)). As we ex-
pected, FE65 WT with NOBF4 treatment was shown the
strong western result (Figure 2(c) second lane). Thus, S-
nitrosylation on the Cys 440 residue of FE65 seems to
increase its binding affinity to Tip60. Further, we moni
Figure 2. NO infusion promoted the binding between FE65 and Tip60 but inhibited the HAT activity of Tip60.
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E. J. Lee et al. / Advances in Biological Chemistry 1 (2011) 109-118 113
tored each HAT activity of the total cell lysates with
ELISA (Figure 2(d)). The HAT activity of FE65 WT
with NOBF4 treatment was reduced significantly, com-
paring FE65 C440A. Therefore, it seems to be that
S-nitrosylation on the Cys 440 residue of FE65 reduced
it HAT activity (Figure 2(d)). Together, these results
suggested that the binding enhancement between FE65
and Tip60 by S-nitrosylation on the Cys 440 residue of
FE65 decreases the HAT activity of Tip60.
3.3. The Reduction of Tip60 HAT Activi ty by the
Increase of FE65 Expression
Activity ELISA assay of HEK293 cells transfected with
EGFP FE65 or FE65 C440A, and then monitored the
FE65, Tip60, or actin expression by using their own spe-
cific antibody. The transfection of EGFP FE65 WT in
HEK293 lysate resulted in the reduction of Tip60 HAT
activity (Figure 3(a)). By way of contrast, the transfec-
tion of EGFP FE65 C440A in HEK293 lysate did not
effect on Tip60 HAT activity (Figure 3(b)). A variety of
previous studies have suggested that FE65’s anti-Tip60
activity depends on direct physical interactions between
FE65 and Tip60 [18-20]. Thus, FE65 C440 residue may
result in losses in the anti-Tip60 activity of FE65 by in-
terfering with the direct interaction between FE65 and
Tip60. Taken together, these results demonstrated that
the S-nitrosylation on C440 residue of FE65 increases its
binding with Tip60, resulting in the inhibition HAT ac-
tivity of Tip60.
3.4. S-Nitrosylation of FE65 Does Not Affect its
Subcellular Localization
To determine whether NO could affect FE65 subcellular
localization and Tip60 interaction, HEK293 cells trans-
fected with GFP-FE65 were treated with the NO donor,
NOBF4, which indicated that the scaffolding activity was
affected by FE65 S-nitrosylation (Figure 4). The trans-
fected EGFP-FE65 wt or its FE65 mutant (C440A) (all
constructs were shown as green color) were detected by
fluorescence microscopy and the FE65 position appeared
as red (Figure 4). We noted that the FE65 modification
proteins were detected primarily in the nuclear speckle
(Figure 4 middle lane). EGFP-FE65 wt was merged
with FE65 in the nuclear speckles surrounding the nu-
clear pore (Figure 4(a)). EGFP-FE65 wt, which was
observed as a fibrous form in the cytoplasm (similar to
Figure 1(b)), was not merged (yellow color) with FE65
C440A. Thus, these results also implicated FE65 as one
of the FE65 S-nitrosylation proteins. Because FE65 WT
was shown to have merged with Tip60 (yellow color) in
the nuclear speckles (dots) around the nuclear pore but
not in the cytoplasm (Figure 4(a) right lane), the S-ni-
trosylation of FE65 appears to be related to its nuclear
subcellular localization.
Figure 3. The increase of FE65 expression promoted its binding with Tip60 but inhibited the HAT activity of Tip60.
opyright © 2011 SciRes. ABC
E. J. Lee et al. / Advances in Biological Chemistry 1 (2011) 109-118
Figure 4. The subcellular localization of FE65 by S-nitrosylatio.
Similar to the confocal results of FE65 wt, its FE65C-
440A mutant was also detected primarily in the nucleus
(Figure 4(b)). However, the FE65 C440A mutant (which
evidenced a diffuse, rather than a dot form) did not
merge well with Tip60 at the nuclear pore (Figure 4(b)
right). These results indicated that S-nitrosylation on the
C440 of FE65 is required for its nuclear speckle local-
ization around the nuclear pore, consistent with the re-
sults shown in Figure 4(a). Further, the results (Figure
4(b)) again verified that the C440A of FE65 is the FE65
S-nitrosylation site, based on the results of confocal mi-
croscopic analysis.
3.5. Increase in FE65 Protein Stability Induced
by S-Nitrosylation
In order to compare the protein stability of FE65WT or
C440A in HEK293 cells, DNA were transfected in the
cells. Then, the transfected cells were treated with cyclo-
hexamide, and the protein stabilities were assessed and
compared. The FE65 proteins were chased for the indi-
cated time periods. The EGFP FE65 proteins were im-
munoprecipitated with a polyclonal anti-GFP antibody
and subjected to SDS-PAGE followed by Western blot-
ting with a monoclonal FE65 antibody (Figure 5(a)). An
equal quantity of cell lysate was subjected to Western
blotting using an actin antibody to monitor protein levels.
As shown in Figure 4(b), we noted that FE65 WT was
more stable than the C440A mutant. In order to eliminate
the mutational effect of the C440A mutant on its protein
stability, the protein stability of the FE65 C440A mutant
was also evaluated using the same procedure. It was ob-
served that the protein stability of the FE65 C440A mu-
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E. J. Lee et al. / Advances in Biological Chemistry 1 (2011) 109-118 115
tant was not altered as profoundly as that of the C440A
mutant (Figure 5(b)). Thus, these data also demon-
strated that the 440 Cys residue of FE65 appears to be
required for its protein stability.
Taken together, we observed that S-nitrosylation on
Cys440 residue of Fe65 increases its binding affinity to
Tip60, which affects HAT activity and protein stability
of Tip60.
4. Figure Legends
4.1. Figure 1. S-Nitrosylation of FE65.
1) Rat FE65 contains 666 amino acids, and functional
domains and key amino acids are indicated. C440 is the
PID1, where specific modifications (S-nitrosylation, sul-
phonation) occur as the result of NO. FE65 binds to sev-
eral proteins with its PID, including Tip60. The putative
S-nitrosylation site was noted in human FE65 (Cys853).
Cys853 (852KCD855) was replaced with alanine (C440A).
Among 14 Cys residues in FE65, the Cys853 residue is
immediately followed by the acidic amino residue (Lys/
Arg/His/Asp/Glu) Cys (Asp/Glu) (sites –1, 0, and +1, re-
spectively) which is required for S-nitrosylation by nitric
acid (NO). The FE65 Cys 853 that is replaced with ala-
nine is marked with an arrow.
2) HEK293 cell lysate was immunoprecipitated with
FE65 Ab or a normal serum (as a negative control). To
detect the thiol modifications of the FE65, the im-
munoblotting was performed with an S-nitrocystein rab-
bit antibody.
3) HEK293 cells transfected with GFP-tagged FE65
WT with 0.5 mM NOBF4. After immnoprecipitating
with S-nitrocystein Ab the precipitant subjected to im-
munoblot with an S-nitrocystein rabbit antibody. The un-
transfected HEK293 cells were used as a negative con-
4) After transfected GFP-FE65 or C440A mutant in
HEK293 cells. The immnoprecipitation with a rabbit anti-
GFP Ab was conducted. The immunoprecipitant was
immune blotted with an S-nitrocystein rabbit antibody
(upper lane), or FE65 antibody (bottom lane). The trans-
fected DNA was indicated above, and the immunoblot
antibody was shown in left.
5) GST fusion proteins with FE65 fragments contain-
ing Cys440A (as shown in Figure 1) were prepared from
E. coli, and subjected to a biotin-switch assay. The GST
fusion proteins with FE65 fragments harboring C440A
were prepared from E. coli and subjected to the biotin-
switch assay. These results were replicated at least in
Figure 5. Protein stability of FE65 or C440A mutant.
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E. J. Lee et al. / Advances in Biological Chemistry 1 (2011) 109-118
4.2. Figure 2. NO Infusion Promoted the Binding
Between FE65 and Tip60 but Inhibited the
HAT Activity of Tip60.
HEK293 cells were transiently transfected with GFP-
FE65 WT or the C440A plasmid. After 48 h, the cells
were lysed, the total proteins were recollected, and im-
munoprecipitation was conducted using nickel beads.
Western blot assays were subsequently conducted with a
rabbit FE65 Ab (a), an S-nitrocystein rabbit antibody (b)
or an anti-Tip60 antibody (c) in HEK293 cells. (d) The
effects of FE65 S-nitrosylation on Tip60 HAT activity
were assessed v ia an ELISA assay with the same sample.
The ELISA assays revealed an inverse correlation be-
tween levels of Tip60 and magnitudes of FE65 dependent.
4.3. Figure 3. The Increase of FE65 Expression
Promoted its Binding with Tip60 but
Inhibited the HAT Activity of Tip60.
After transfection with GFP-FE65 WT DNA as indicated
above (0, 2, 4 ug), the quantification of the Tip60 (a) or
FE65 (b) expression level in western blot was performed
with anti-Tip60 Ab or anti-FE65 Ab (c) The effects of
FE65 on Tip60 HAT activity were assessed via an ELI-
SA assay with the same sample in Figure 3 (a) and (b).
The ELISA assays revealed an inverse correlation be-
tween levels of Tip60 and magnitudes of FE65-depend-
ent transfection. (d) Amount of cell protein was analyzed
via Western blot with anti-actin antibody for the negative
control. These results were replicated at least 3 times.
4.4. Figure 4. The Subcellular Localization of
FE65 by S-Nitrosylation
S-nitrosylation of FE65 by NO inhibits nuclear localiza-
tion (a and b). HEK293 cells transfected with GFP-
FE65 and GFP-FE65 C440A were incubated with either
100 μM NOBF4 for 6 h. GFP-FE65 WT was observed to
merge with Tip60 (yellow color) in the nuclear speckles
(dots) around the nuclear pore but not in the cytoplasm
(a). However, the transfected GFP-FE65 C440A mutant
was not merged with Tip60 (b) in the treatment of 100
μM NOBF4 for 6 h. The localization of GFP-FE65 WT
or C440A was compared via direct immunofluorescence
microscopy (X 400). S-nitrosylation of FE65 appears to
be related to its nuclear subcellular localization. These
results were replicated at least 3 times.
4.5. Figure 5. Protein Stability of FE65 or
C440A Mutant.
1) EGFP-FE65 or the FE65 C440A mutant was trans-
fected into HEK293 cells and the cells were treated with
cyclohexamide. The FE65 proteins were chased for the
indicated time periods. Western blotting with a mono-
clonal FE65 antibody of SDS-PAGE subjected EGFP-
FE65 proteins is shown. In order to monitor the amount
of protein, equal quantities of cell lysates were subjected
to Western blotting using an actin antibody. The results
shown are one representative of five repeated experi-
2) Quantification of the pulse-chase experiment is
shown by image analysis with Fuji Image Quant soft-
5. Discussion
The results of this study demonstrated, for the first time,
that FE65 can be S-nitrosylated on its C440 residue. The
mechanism by which the S-nitrosylation of FE65 im-
pairs its protective functions appears to occur through
the inhibition of its Tip60 activity. This idea contrasts
sharply with the mechanism underlying the S-nitrosyla-
tion-mediated impairment of FE65’s Tip60 binding
function. FE65’s binding function to Tip60 is increased
by the S-nitrosylation of FE65 via an enhancement of
the binding affinity of FE65 to Tip60.
S-nitrosylation is a nitric oxide (NO)-induced post-
translational modification in which a cysteinyl thiol (R-
SH) is converted to a nitrosothiol; this modification is
known to function as a regulatory mechanism of various
classes of proteins, including ion channels such as
TRPA1, TRPC5, the skeletal muscle-type ryanodine re-
captor (ryanodine receptor type 1) channel, the N-me-
thyl-D-aspartate receptor channel, the cardiac L-type
Ca2+channel, and the cardiac Na+ channel. Thus, protein
S-nitrosylation may convey a broad spectrum of cellular
signals. NO is a pleiotropic cell signaling molecule that
controls a variety of biological processes [16]. Accord-
ing to the classical view, cyclic GMP mediates NO sig-
naling [7,8]. The importance of a cGMP- independent
pathway occurring via protein S-nitrosylation is being
increasingly recognized by researchers in the field of NO
signal transduction. Thus, protein direct S-nitrosylation
appears to be an important NO-mediated regulatory me-
chanism relevant to various classes of proteins. However,
the direct link between protein S-nitrosylation and its
functional relevance has been demonstrated only for
limited examples. Our result which S-nitrosylation regu-
lates FE65 function seems to be the good example for
this researcher field, even the mechanism and the func-
tional significance underlying the preferential S-nitrosy-
lation of the target Cys remain incompletely understood.
Protein S-nitrosylation, like phosphorylation, seems to
be emerging as an important player in regulating protein
activity and signal transduction in response to DNA
damage. Tip60 appears to be quite relevant to acetylation,
and activates several key DNA damage-responsive pro-
teins, such as H2AX, ATM, and p53. Interestingly, the
S-nitrosylation of FE65 occurs somewhat later, thus
opyright © 2011 SciRes. ABC
E. J. Lee et al. / Advances in Biological Chemistry 1 (2011) 109-118 117
suggesting that FE65 S-nitrosylation may be directly
regulated in some fashion. We are pursuing now the pos-
sibility that both FE65 is also modified by NO as the
target proteins to respond DNA damage. Further, be-
cause we also notice S-nitrosylation site in Tip60 and
observed this modification, we do not rule out the possi-
bility that our result in Figure 4 is due to the direct
S-nitrosylation on Tip 60 by NOBF4 treatment.
Because we determined that FE65 S-nitrosylation was
increased in both an MPTP animal model of AD and in
AD patients, these results indicate that neurons may be
more susceptible to cell death in the face of unfavorable
conditions such as proteasomal dysfunction and protein
aggregation-induced toxicity. Our findings also indicate
that a more thorough understanding of how nitrosative
stress can contribute to AD will help us to develop new
therapeutic approaches for this disease or for senescence.
In addition to its ability to impair neuroprotection pro-
teins, S-nitrosylation has also been shown to be involved
in mediating cell death via FE65 (our unpublished data).
Our studies also demonstrated that the S-nitrosylation of
FE65 can mediate the translocation of the FE65-Tip60
protein complex to the nucleus, and initiate apoptosis
(Figure 5). Our finding that the S-nitrosylation of FE65
can affect its Tip60 binding function further suggests
that nitrosative stress can affect neuronal survival via the
targeting of a number of pathways.
FE65 is also known to form dimers in the cell, and
this dimerization is important for its physiological func-
tions and scaffolding activity with its WW domain. The
results of our study did not reveal whether the S-nitro-
sylation of FE65 induces its monomerization for a less
active form. In addition to FE65, several other proteins,
including actin, creatine kinase, glycogen phosphorylase,
and the homodimeric HIV-1 protease are targets for S-
nitrosylation in intact cells after exposure to oxidative
stress. Several recent studies have also shown that other
neuroprotective proteins, including peroxiredoxin and
protein-disulphide isomerase, are modified by S-nitrosy-
lation, and this modification compromises their normal
protective functions. Thus, the repertoire of activities via
which S-nitrosylation can compromise cellular survival
is rather diverse. Along this line, it has been recently
demonstrated that the treatment of endothelial cells with
the combination of NOBF4 and cysteine, a system that
generates GSSG and nitric oxide, resulted in the loss of
intracellular glutathione, probably as the result of the
formation of protein-mixed disulfides. NO enhances the
effects of FE65’s Tip60 binding ability against a variety
of cell death stimuli.
In summary, this study describes the interaction be-
tween FE65 and Tip60, which is enhanced by a post-
translational modification of FE65 (through S-nitrosy-
lation) by NO, promoting the association of the FE65-
Tip60 protein complex and inhibiting both the HAT ac-
tivity of Tip60 and cell death. Greater insight into the
FE65 and Tip60 signal cascade is expected to contribute
to our molecular understanding of a role of both FE65
and Tip60 in neurodegenerative disorders, and is also
expected to help in the development of novel strategies
for the treatment of such disorders.
This work was supported by National Research Foundation of Korea
(NRF) grants (2009-0076024 and 2009- 0069007) funded by the Korea
government (MEST) to S S Kang. We also appreciated The Core Facil-
ity of Chungbuk National University.
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