A potential role for protein palmitoylation and zDHHC16 in DNA damage response
Cao et al. BMC Molecular Biol
A potential role for protein palmitoylation and zDHHC16 in DNA damage response
Na Cao 3
Jia‑Kai Li 1
Yu‑Qing Rao 1
Huijuan Liu 3
Ji Wu 3
Baojie Li 3
Peiquan Zhao 1
Li Zeng 0 2
Jing Li 1
0 Neural Stem Cell Research Lab, Research Department, National Neuroscience Institute , Singapore 308433 , Singapore
1 Department of Ophthalmology, Xin Hua Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai , China
2 Neural Stem Cell Research Lab, Research Department, National Neuroscience Insti‐ tute , Singapore 308433 , Singapore
3 BioX‐ Institutes, Key Laboratory for the Genetics of Developmental and Neu‐ ropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University , Shanghai 200240 , China
Background: Cells respond to DNA damage by activating the phosphatidylinositol‑ 3 kinase‑ related kinases, p53 and other pathways to promote cell cycle arrest, apoptosis, and/or DNA repair. Here we report that protein palmitoylation, a modification carried out by protein acyltransferases with zinc‑ finger and Asp‑ His‑ His‑ Cys domains (zDHHC), is required for proper DNA damage responses. Results: Inhibition of protein palmitoylation compromised DNA damage‑ induced activation of Atm, induction and activation of p53, cell cycle arrest at G2/M phase, and DNA damage foci assembly/disassembly in primary mouse embryonic fibroblasts. Furthermore, knockout of zDHHC16, a palmitoyltransferase gene identified as an interacting protein for c‑ Abl, a non‑ receptor tyrosine kinase involved in DNA damage response, reproduced most of the defects in DNA damage responses produced by the inhibition of protein palmitoylation. Conclusions: Our results revealed critical roles for protein palmitoylation and palmitoyltransferase zDHHC16 in early stages of DNA damage responses and in the regulation of Atm activation.
Protein palmitoylation; DNA damage response; zDHHC16
Protein palmitoylation, or protein S-acylation, is a
posttranslational modification that adds a palmitate moiety to
specific Cys residues by a family of proteins named
protein acyltransferases (PATs) [1–4]. All PAT proteins
contain a DHHC domain, a 51-amino acid Cys-rich domain
with a highly conserved Asp-His-His-Cys sequence. The
other regions of the PATs are variable. The zDHHC genes
are numerically named zDHHC1-24 . Unlike
N-myristoylation or C-prenylation, S-acylation can be reversed
by protein palmitoyl thioesterases and acyl protein
thioesterases, making it a reversible lipid modification .
A number of cellular proteins have been reported to
be palmitoylated, which are involved in different cellular
activities such as cell signaling, protein trafficking, and
cell adhesion [6–8]. Most of the palmitoylated proteins
are membrane or peripheral membrane proteins, yet
it is noteworthy that some non-membrane associated
proteins are also palmitoylated. The importance of
protein palmitoylation is manifested in zDHHC deficient
mouse models. For example, mice deficient for zDHHC8
gene have increased risk of schizophrenia . Mice with
zDHHC13 mutation show alopecia, osteoporosis, and
amyloidosis . A spontaneous mutation of zDHHC21
gene in mice leads to hair loss due to defective epidermal
homeostasis and hair follicle differentiation . We have
recently reported that mice null of zDHHC16 gene are
neonatal lethal with severe heart and eye defects .
Recent studies have also implicated protein
palmitoylation and PATs in cancer development. The expression of
some zDHHC genes was found altered in various
cancer tissues [13, 14]. Yet how PATs participate in cancer
development remains unclear. Cancer development is
driven by the accumulation of gene mutations, especially
loss-of-function mutations of tumor suppressors and
gain-of-function mutations of oncoproteins .
However, cell has a protective system, the DNA damage
response (DDR), to monitor DNA damage and to repair
the damage or eliminate the cells with irreparable DNA
lesions [16–18]. Upon DNA damage, the cell
activates the phosphatidylinositol-3 kinase-related kinases
(PIKKs) such as Atm and Atr at the DNA break sites. A
large number of proteins, including γH2AX and BRCT
domain-containing proteins such as Brca1, TopBP1, and
Mdc1 are recruited to the DNA break sites, forming
transient nuclear structures named DNA damage foci, which
are thought to be the centers for signal propagation and
DNA repair [19–21]. Atm phosphorylates many
substrates including Smad1, p53 and Chk2, which eventually
cause cell cycle arrest and/or apoptosis [22–25]. Thus, a
functional DDR is critical for maintaining genome
integrity and preventing tumor development. On the other
hand, tumor cells usually have disrupted DDR . Up
to date, it is not known whether protein palmitoylation
plays a role in DNA damage response.
In the present study, we investigated the roles of
protein palmitoylation in DNA damage response. We found
defective DDR including Atm activation, p53 induction
and activation, cell cycle arrest at G2/M phase, and
assembly/disassembly of DNA damage foci in primary mouse
embryonic fibroblasts (MEFs) in the presence of
2-bromopalmitate (2BP), a general PAT inhibitor [26–28]. These
results were also observed in MEFs deficient of zDHHC16
gene which encodes a palmitoyltransferase. These findings,
for the first time, unravel an important function of PATs,
in particular zDHHC16, in DNA damage response and in
Atm activation, and provide a possible explanation on how
zDHHC proteins participate in tumorigenesis.
Mice and cells
Mice were housed, bred and used in a specific pathogen
free (SPF) animal facility at the Bio-X Institute,
Shanghai Jiao Tong University. Specifically, no more than five
adult mice were housed in one individually ventilated
cage with sterilized food, water and woodchip bedding.
The animal facility was maintained by professional care
takers 7 days a week on a 12 h light/12 h dark cycle. The
study was approved by the Institutional Animal Care
and Use Committee of Shanghai Jiao Tong University
[SYXK(SH)2011-0112]. Timed pregnant female mice
were euthanized on embryonic E13.5 by intraperitoneal
injection of over-dosed pentobarbital. The time of
pregnancy was determined by visual examination of the
vaginal plug in the early morning. Embryos were dissected
and fibroblasts were isolated as described previously .
The generation and characterization of the zDHHC16
knockout mice were described in detail in our previous
paper . One pregnant C57Bl/6 wildtype and three
zDHHC16 knockout mice were used to obtain all MEFs
used in this study. The knockout mice were in mixed
C57BL/6 and CBA background. All efforts were made to
minimize the suffering of mice.
Cells were cultured in Dulbecco’s modified Eagle’s
medium (Thermo Fisher Scientific Inc./Life
Technologies, Grand island, NY, USA) containing 10 % fetal calf
serum (Excell Biology Inc., Shanghai, China). They were
plated at 106 cells per 6 cm dish and allowed to grow
overnight before any treatment. To inhibit cellular PAT
activity, 2BP (2-bromopalmitate, Sigma-Aldrich, China)
was used at 50 or 100 μM for 24 h as indicated. To induce
DNA damage response, doxorubicin (Dox) (Selleck
Chemicals, Houston, TX, USA) was used at 1 μM for
different time as indicated in each experiment.
Western blot analysis
Standard RIPA buffer containing 1 mM PMSF, 1 μg/mL
aprotonin, leupeptin, and pepstatin was used for protein
extraction. Protein concentration was measured using
Bio-Rad DC protein assay kit (Bio-Rad Inc., Hercules,
CA, USA). Western blot analysis was carried out
according to the standard procedure. We used polyvinylidene
fluoride membrane for protein transfer, and 5 %
nonfat dried milk in PBS as the blocking agent. All primary
antibodies were incubated overnight at 4 °C.
Chemiluminescent detection method (ECL kit, GE Healthcare,
Buckinghamshire, UK) coupled with Bio-Rad ChemiDoc
XRS imaging system were used for the detection,
visualization and quantitation of the proteins. All primary
antibodies were purchased from cell signaling technology
and used according to the provider’s instruction except
for the following: anti-Atm antibody was purchased from
ECM Biosciences (AM3611), anti-p-Atm antibody was
purchased from Millipore (
) and anti-β-Actin was
purchased from Santa Cruz (SC81178).
Cells were digested with 0.25 % trypsin, washed with cold
PBS and fixed in 70 % ethanol at −20 °C overnight. At
the next day, cells were washed with cold PBS again and
incubated in PBS containing 50 μg/mL propidium iodide
(PI) and 100 μg/mL RNase A for 40 min in the dark at
room temperature. The fixed and labeled cells were
analyzed with Becton–Dickinson FACSCalibur (BD
Bioscience, San Jose, CA, USA).
In vitro analysis of DNA damage foci positive for γH2AX,
TopBP1, and BRCA1
Cells cultured on glass slides were fixed in 4 %
paraformaldehyde/PBS for 30 min followed by 0.1 %
TritonX-100/PBS incubation for 40 min at room
temperature. The standard immunostaining procedure
was used. Specifically, 10 % goat serum was used as the
blocking agent. Primary antibody incubation was
carried at 4 °C overnight. Anti-p-H2AX antibody was
purchased from Millipore (
), anti-TopBP1 antibody
was purchased from BD Bioscience (611875), and
antiBrca1 antibody was purchased from Abcam (ab191042).
Reverse transcription (RT)‑polymerase chain reaction (PCR)
Trizol reagent (Invitrogen, Thermo Fisher Scientific,
Grand island, NY, USA) was used to extract whole RNA
and reverse transcribed using SuperScript III reverse
transcriptase and random primer (Invitrogen, ThermoFisher
Scientific, USA). Relative quantitative PCR was performed
using the primers listed in Table 1 and the FastStart
Universal SYBR Green Master from Roche (Roche Diagnostic
GmbH, Mannheim, Germany). β-actin and glyceraldehyde
3-phosphate (GAPDH) were used as internal controls.
Each experiment was repeated at least three times or
using cells isolated from three mutant and control mice.
The results were analyzed using analysis of variance
(ANOVA) with Fisher’s LSD (least significant
difference) test when more than two groups were compared
or unpaired t test when two groups were compared
(SPSS version 18). p Value of equal or less than 0.05 was
accepted as statistically significant.
Most of the zDHHC genes are expressed in MEFs
We chose to use primary MEFs for this study as most
of the immortal cell lines show disrupted DNA
damage response . We first wanted to document which of
the 23 protein-coding zDHHC genes were expressed in
MEFs and whether their expression was altered by DNA
damage. Relative quantitative PCR analysis showed that
MEFs expressed 20 zDHHC genes (Fig. 1). The mRNA
for zDHHC19, 22 and 23 was not detectable in MEFs.
However, they were found in mouse retina tissues (data
not shown). We then treated MEFs with Dox, a
chemotherapeutic drug that generates double-stranded and
single-stranded DNA breaks , and analyzed the mRNA
levels of the zDHHC genes. Only zDHHC11, 17, 20 and
24 mRNA showed increase of about twofold, while the rest
exhibited no significant change. The expression of a wide
spectrum of PATs in primary MEFs suggests that protein
palmitoylation may play important roles in these cells.
Impaired DNA damage‑induced p53 activation in the presence of 2BP
We then wanted to study the possible roles of protein
palmitoylation in DNA damage response. Since it was
unfeasible to simultaneously silence most of the PATs
with interference RNA, we used 2BP, a substrate analog
inhibitor that had been widely used to block PAT
activity [30–32]. Although it may have activities other than
palmitoylation inhibition , we have shown that 2BP
at the concentrations of 50 μM and above was able to
inhibit total PAT activities in cultured cells [12, 33].
Indeed, reduced protein palmitoylation was observed in
MEFs in the presence of 50 μM 2BP (Additional file 1:
Figure S1). Additionally, 2BP showed very modest effects
on the expression of a few zDHHC genes (Additional
file 1: Figure S2).
Western blot analysis showed that Dox-induced
increase in the protein levels of p53, phosphorylation
of p53 at Ser15, and the protein levels of p21 and Bax,
targets of p53, were inhibited in the presence of 2BP
(Fig. 2a). While Dox induced a near fivefold increase of
phosphorylated p53 in normal MEFs by 24 h, the increase
was only 2.5-fold or none in cells with 50 and 100 μM
2BP pre-treatment, respectively.
To further confirm these findings, we analyzed
mRNA levels of p21 (Cdkn1a), Bax, and Puma (Bbc3)
using relative quantitative PCR. We found that Dox
treatment increased the mRNA levels of p21 and Bax
but not Puma in MEFs (Fig. 2b). 2BP significantly
inhibited the induction of p21 and Bax expression
at the mRNA levels in response to Dox treatment
(Fig. 2b). Since one of the main functions of 2BP was
to inhibit PATs, these results suggest that protein
palmitoylation is required for the optimal induction of
p53 target genes.
Impaired DNA damage‑induced Atm activation in the presence of 2BP
The induction and activation of p53 is dependent on Atm
activation in response to double-stranded DNA breaks.
We found decreased activation of Atm in primary MEFs,
justified by decreased Atm phosphorylation on Ser1981
(Fig. 3a, b) in the presence of 2BP. Although Ser1981
phosphorylation is not required for Atm activation
in vivo, it is an autophosphorylation commonly used as
an indication of Atm activation . Moreover, we found
that 2BP also impeded DNA damage-induced Smad1
activation (Fig. 3c), another cellular event that requires
Atm activation .
Impaired DNA damage foci formation in the presence of 2BP
We also looked at the formation of DNA damage foci
in the presence of 2BP. In normal MEFs, Dox treatment
induced DNA damage foci positive for γH2AX, TopBP1,
and Brca1, which are believed to be signal propagation
centers as well as DNA repair centers (Fig. 4a, b). In
2BPtreated cells, the number of foci positive for γH2AX was
higher than those in controls, especially at 8 and 16 h
after Dox treatment. These results suggest that 2BP
interferes with the assembly/disassembly of the DNA damage
foci or the DNA repair process.
Impaired DNA damage‑induced cell cycle arrest in the presence of 2BP
DNA damage eventually induces apoptosis or cell cycle
arrest. Since 2BP compromised Atm activation and p53
induction and activation, we suspected that 2BP might
affect Dox-induced cell cycle arrest and apoptosis. Cell
cycle arrest was analyzed by flow cytometry (Fig. 5a).
Dox treatment led to an accumulation of cells in the
G2/M phase, a decrease in G1 phase cells, and a
modest decrease in S phase cells in wild type MEFs. 2BP
pre-treatment impeded Dox-induced increase in the
percentage of cells arrested in the G2/M phase compared to
control cells, without altering the percentage of S phase
cells (Fig. 5b). These results suggest that protein
palmitoylation is required for proper G2/M cell cycle arrest
in DNA damage response. However, we found that 2BP
showed some toxicity to MEFs at the doses used to inhibit
protein palmitoylation, which may reflect a requirement
for protein palmitoylation for cell survival, and the
combination of 2BP and Dox caused more cell death
(Additional file 1: Figure S3). This prevented us from further
testing the role of 2BP-suppressed p53 activation in DNA
compromised the activation of Atm and the induction
of p53 and its target protein p21 (Fig. 6a–c).
DHHC16 deficiency impaired the activation of Atm‑p53
We have previously reported the identification of one
of the PATs, encoded by zDHHC16 gene. This
protein, Aph2, was originally identified as a c-Abl
interacting protein (Abl-philin2) . c-Abl is involved in
DNA damage response. In particular, it is required for
Atm-p53 activation [35–38]. Biochemical and genetic
studies have shown that at least one protein,
phospholamban, a cardiac muscle specific protein, was
palmitoylated by Aph2 . In addition, MEFs
overexpress zDHHC16 also showed increased total
protein palmitoylation (Additional file 1: Figure S4a).
Although Dox did not affect the expression of DHHC16
at the mRNA level (Additional file 1: Figure S2), Dox
induced nuclear translocation of ectopically-expressed
DHHC16 (Additional file 1: Figure S4b). We have tried
to raise anti-DHHC16 antibodies but those antibodies
could not recognize endogenous DHHC16. This could
be due to the fact that DHHC16 is a membrane protein.
The lack of anti-DHHC16 antibodies prevents us to
study the expression, localization, and modification of
endogenous DHHC16 at the moment. To test whether
DHHC16 also plays a role in DDR, we treated primary
MEFs deficient for zDHHC16 gene with Dox for
different periods of time. Western blot analysis revealed that
zDHHC16 deficiency, like inhibition of PATs with 2BP,
DHHC16 deficiency impaired DNA damage foci formation and cell death
We next examined DNA damage foci formation in
zDHHC16 deficient MEFs. Dox-treated zDHHC16−/−
MEFs showed similar patterns of foci formation as the
2BP-treated wild type MEFs. The number of TopBP1
or Brca1 positive foci in zDHHC16−/− MEFs was
significantly higher than that in wild type cells (Fig. 7a–d).
We also tested DNA damage-induced cell cycle arrest
and apoptosis in zDHHC16−/− and wild type MEFs.
We found that Dox-induced G2/M cell cycle arrest in
Aph2−/− MEFs were not significantly different from
those in wild type MEFs (data not shown). However,
zDHHC16−/− MEFs showed a modest increase in cell
survival compared to wild type MEFs (Fig. 7e). These
results suggest that zDHHC16 plays a role in DNA
damage-induced cell death.
In this study, we showed that 2BP impaired Dox-induced
DNA damage response, in particular the activation of
the Atm-p53 pathway, and led to disrupted activation of
the cell cycle checkpoint and DNA damage foci
dynamics in primary MEFs. Since 2BP is a general PAT
inhibitor which also binds palmitoylated proteins, we further
showed that the defective DNA damage responses
observed in 2BP-treated cells were largely replicated in
MEFs deficient for zDHHC16, one of the 23
palmitoyltransferases. Collectively our data suggested that
protein palmitoylation carried out by PATs, in particular
by zDHHC16, plays an important role in DNA damage
response. Since DDR, in particular the Atm-p53
pathway, is the major tumor suppression scheme, our findings
also provide a possible explanation on how some zDHHC
proteins exert their tumor suppression activities .
Palmitoylation is a common protein post-translational
modification [1, 3, 14]. This is also evident by the
expression of a variety of zDHHC genes in primary MEFs.
However, little is known about the specific in vivo
functions of each PAT. zDHHC16 knockout mice showed
neonatal lethality with severe cardiac and ocular defects.
It appears that one of the substrates of zDHHC16 protein
in heart is phospholamban, which is largely responsible
for cardiac defects observed in zDHHC16 knockout mice
. Here we found that cells deficient of zDHHC16 also
had defective DNA damage response, a function that has
not been previously ascribed to zDHHC16 or any other
How does zDHHC protein facilitate Atm activation?
Since Atm is not known to be modified by palmitoylation,
zDHHC16 and other PATs likely regulate the protein(s)
that affect Atm activation upon DNA damage. Atm
activation requires MRN complex (a protein complex
consisting of Mre11, Rad50 and Nbs1), DNA
conformational change, and/or DNA breaks. The generally agreed
major function of palmitoylation is to increase the
hydrophobicity of the targeted protein, thus facilitate protein
anchoring to membrane and subsequent interaction
with other proteins [1, 2, 13]. Since most of the DNA
damage foci proteins and signaling molecules are
localized in the nucleus, it is more likely that palmitoylation
affects the stability and/or complex assembly of the
proteins that are involved in DNA damage response rather
than their membrane association. One such candidate
may be histone protein, which has been reported to be
palmitoylated [39, 40]. Palmitoylation of histone proteins
may affect the remodeling of chromatin structures, which
may in turn affect DNA damage foci formation and/or
DNA conformation, eventually lead to alteration of Atm
activation and DNA repair [21, 41]. Whether histone
proteins are substrates of zDHHC16 warrants further
Alternatively, zDHHC16 protein may regulate DNA
damage response through c-Abl. zDHHC16 was originally
identified as a c-Abl interacting protein, which was also
named Aph2 . c-Abl is activated in Atm-dependent
manner in response to DNA damage. Activated c-Abl
helps to up-regulate p53 and p73 expression and also
plays a positive role in maximal Atm/Atr activation [35–
37]. zDHHC16 seems to have a similar role as c-Abl in
Atm activation in DNA damage response, yet c-Abl is not
a substrate of zDHHC16 . It is possible that zDHHC16
may regulate DDR via c-Abl in a
palmitoylation-independent manner. Further exploration on the nature of
interaction between zDHHC16 and c-Abl will help
understand how zDHHC16 affects DNA damage response.
In summary, this study, for the first time, uncovered a
critical role for protein palmitoylation and more
specifically zDHHC16 in DNA damage response. These
findings advance our understanding of the regulation of
DNA damage response and provide a possible
explanation on how protein palmitoylation is involved in cancer
development. Moreover, our results expand the role of
palmitoylation on cellular activities and calls for further
research on palmitoylated nuclear proteins.
Additional file 1: Figure S1. 2BP inhibited protein palmitoylation in
MEFs. Figure S2. The effect of 2BP on zDHHC gene expression in MEFs.
Figure S3. The effect of 2BP and Dox on MEF cell survival. Figure S4.
Overexpressed zDHHC16 increased protein palmitoylation in MEFs and
was translocated into the nucleus in response to Dox.
zDHHC: zinc‑finger and Asp ‑His‑His‑ Cys domains; PAT: protein acyltransferase;
DDR: DNA damage response; PIKKs: phosphatidylinositol‑3 kinase ‑related
kinases; MEF: mouse embryonic fibroblast; 2BP: 2‑bromopalmitate; DOX: doxo ‑
rubicin; PI: propidium iodide; GAPDH: glyceraldehyde 3‑phosphate; RT: reverse
transcription; PCR: polymerase chain reaction.
JL, LZ and BL designed the research, analyzed the data and wrote the paper. NC
and LZ did the western blot, immunofluorescent staining and FACS analysis; JKL
and YQR performed the RT, realt‑ime PCR and cytotoxicity analysis, HL isolated
primary mouse embryonic fibroblasts; PZ and JW provided critical review on the
whole project. All authors have read and approved the final manuscript.
We would like to thank Lina Gao and Haiyang Xie for their technical support.
Availability of data and material
The original data of the real‑time PCR experiments and data images for the
microscopy work and western blot analysis will be available upon request.
The authors declare that they have no competing interests.
1. Resh MD . Palmitoylation of ligands, receptors, and intracellular signaling molecules . Sci STKE . 2006 ; 359 : re14 .
2. Linder ME , Deschenes RJ . Palmitoylation: policing protein stability and traffic . Nat Rev Mol Cell Biol . 2007 ; 8 : 74 - 84 .
3. Blaskovic S , Adibekian A , Blanc M , van der Goot GF. Mechanistic effects of protein palmitoylation and the cellular consequences thereof . Chem Phys Lipids . 2014 ; 180 : 44 - 52 .
4. Korycka J , Lach A , Heger E , Boguslawska DM , Wolny M , Toporkiewicz M , Augoff K , Korzeniewski J , Sikorski AF . Human DHHC proteins: a spotlight on the hidden player of palmitoylation . Eur J Cell Biol . 2012 ; 91 : 107 - 17 .
5. Roth AF , Wan J , Bailey AO , Sun B , Kuchar JA , Green WN , Phinney BS , Yates JR 3rd, Davis NG . Global analysis of protein palmitoylation in yeast . Cell . 2006 ; 125 : 1003 - 13 .
6. Martin BR , Wang C , Adibekian A , Tully SE , Cravatt BF . Global profiling of dynamic protein palmitoylation . Nat Methods . 2012 ; 9 : 84 - 9 .
7. Adams MN , Christensen ME , He Y , Waterhouse NJ , Hooper JD . The role of palmitoylation in signalling, cellular trafficking and plasma membrane localization of protease‑activated receptor ‑2 . PLoS ONE . 2011 ; 6 : e28018 .
8. Yang W , Di Vizio D , Kirchner M , Steen H , Freeman MR . Proteome scale characterization of human S‑acylated proteins in lipid raft ‑ enriched and non‑raft membranes . Mol Cell Proteom . 2010 ; 9 : 54 - 70 .
9. Mukai J , Liu H , Burt RA , Swor DE , Lai WS , Karayiorgou M , Gogos JA . Evidence that the gene encoding ZDHHC8 contributes to the risk of schizophrenia . Nat Genet . 2004 ; 36 : 725 - 31 .
10. Saleem AN , Chen YH , Baek HJ , Hsiao YW , Huang HW , Kao HJ , Liu KM , Shen LF , Song IW , Tu CP , et al. Mice with alopecia, osteoporosis, and systemic amyloidosis due to mutation in Zdhhc13, a gene coding for palmitoyl acyltransferase . PLoS Genet . 2010 ; 6 : e1000985 .
11. Mill P , Lee AW , Fukata Y , Tsutsumi R , Fukata M , Keighren M , Porter RM , McKie L , Smyth I , Jackson IJ . Palmitoylation regulates epidermal homeostasis and hair follicle differentiation . PLoS Genet . 2009 ; 5 : e1000748 .
12. Zhou T , Li J , Zhao P , Liu H , Jia D , Jia H , He L , Cang Y , Boast S , Chen YH , et al. Palmitoyl acyltransferase Aph2 in cardiac function and the development of cardiomyopathy . Proc Natl Acad Sci USA . 2015 ; 112 : 15666 - 71 .
13. Yeste‑ Velasco M , Linder ME , Lu YJ . Protein S‑palmitoylation and cancer . Biochim Biophys Acta . 2015 ; 1856 : 107 - 20 .
14. Greaves J , Chamberlain LH . DHHC palmitoyl transferases: substrate interactions and (patho) physiology . Trends Biochem Sci . 2011 ; 36 : 245 - 53 .
15. Hanahan D , Weinberg RA . Hallmarks of cancer: the next generation . Cell . 2011 ; 144 : 646 - 74 .
16. Elledge SJ . The DNA damage response-self‑awareness for DNA: the 2015 Albert Lasker Basic Medical Research Award . JAMA. 2015 ; 314 : 1111 - 2 .
17. Haber JE . Deciphering the DNA damage response . Cell . 2015 ; 162 : 1183 - 5 .
18. Jackson SP , Bartek J . The DNA‑ damage response in human biology and disease . Nature . 2009 ; 461 : 1071 - 8 .
19. Rothkamm K , Barnard S , Moquet J , Ellender M , Rana Z , Burdak‑Rothkamm S. DNA damage foci: meaning and significance . Environ Mol Mutagen . 2015 ; 56 : 491 - 504 .
20. Rogakou EP , Boon C , Redon C , Bonner WM . Megabase chromatin domains involved in DNA double‑strand breaks in vivo . J Cell Biol . 1999 ; 146 : 905 - 16 .
21. Wu J , Clingen PH , Spanswick VJ , Mellinas‑ Gomez M , Meyer T , Puzanov I , Jodrell D , Hochhauser D , Hartley JA . Gamma‑H2AX foci formation as a pharmacodynamic marker of DNA damage produced by DNA crosslinking agents: results from 2 phase I clinical trials of SJG‑ 136 ( SG2000 ). Clin Cancer Res . 2013 ; 19 : 721 - 30 .
22. Paull TT . Mechanisms of ATM Activation . Annu Rev Biochem . 2015 ; 84 : 711 - 38 .
23. Shiloh Y , Ziv Y. The ATM protein kinase: regulating the cellular response to genotoxic stress, and more . Nat Rev Mol Cell Biol . 2013 ; 14 : 197 - 210 .
24. Chau JF , Jia D , Wang Z , Liu Z , Hu Y , Zhang X , Jia H , Lai KP , Leong WF , Au BJ , et al. A crucial role for bone morphogenetic protein‑Smad1 signalling in the DNA damage response . Nat Commun . 2012 ; 3 : 836 .
25. Ruan X , Zuo Q , Jia H , Chau J , Lin J , Ao J , Xia X , Liu H , Habib SL , Fu C , et al. P53 deficiency‑induced Smad1 upregulation suppresses tumorigenesis and causes chemoresistance in colorectal cancers . J Mol Cell Biol . 2015 ; 7 : 105 - 18 .
26. Davda D , El Azzouny MA , Tom CT , Hernandez JL , Majmudar JD , Kennedy RT , Martin BR . Profiling targets of the irreversible palmitoylation inhibitor 2‑bromopalmitate . ACS Chem Biol . 2013 ;8: 1912 - 7 .
27. Jennings BC , Nadolski MJ , Ling Y , Baker MB , Harrison ML , Deschenes RJ , Linder ME . 2 ‑Bromopalmitate and 2‑(2‑hydroxy‑5‑nitro ‑benzylidene)‑ benzo[b]thiophen‑3‑ one inhibit DHHC‑mediated palmitoylation in vitro . J Lipid Res . 2009 ; 50 : 233 - 42 .
28. Resh MD . Use of analogs and inhibitors to study the functional significance of protein palmitoylation . Methods . 2006 ; 40 : 191 - 7 .
29. Carvalho C , Santos RX , Cardoso S , Correia S , Oliveira PJ , Santos MS , Moreira PI . Doxorubicin: the good, the bad and the ugly effect . Curr Med Chem . 2009 ; 16 : 3267 - 85 .
30. Garant KA , Shmulevitz M , Pan L , Daigle RM , Ahn DG , Gujar SA , Lee PW . Oncolytic reovirus induces intracellular redistribution of Ras to promote apoptosis and progeny virus release . Oncogene . 2015 ; 35 ( 6 ): 771 - 82 .
31. Fukata Y , Dimitrov A , Boncompain G , Vielemeyer O , Perez F , Fukata M. Local palmitoylation cycles define activity‑regulated postsynaptic subdo ‑ mains . J Cell Biol . 2013 ; 202 : 145 - 61 .
32. Zheng B , DeRan M , Li X , Liao X , Fukata M , Wu X. 2 ‑bromopalmitate analogues as activity‑based probes to explore palmitoyl acyltransferases . J Am Chem Soc . 2013 ; 135 : 7082 - 5 .
33. Leong WF , Zhou T , Lim GL , Li B . Protein palmitoylation regulates osteoblast differentiation through BMP‑induced osterix expression . PLoS ONE . 2009 ; 4 : e4135 .
34. Li B , Cong F , Tan CP , Wang SX , Goff SP . Aph2, a protein with a zf‑DHHC motif, interacts with c‑Abl and has pro ‑apoptotic activity . J Biol Chem . 2002 ; 277 : 28870 - 6 .
35. Maiani E , Diederich M , Gonfloni S. DNA damage response: the emerging role of c‑Abl as a regulatory switch? Biochem Pharmacol . 2011 ; 82 : 1269 - 76 .
36. Meltser V , Ben‑ Yehoyada M , Shaul Y. c ‑Abl tyrosine kinase in the DNA damage response: cell death and more . Cell Death Diff . 2011 ; 18 : 2 - 4 .
37. Shaul Y , Ben‑ Yehoyada M. Role of c‑Abl in the DNA damage stress response . Cell Res . 2005 ; 15 : 33 - 5 .
38. Wang X , Zeng L , Wang J , Chau JF , Lai KP , Jia D , Poonepalli A , Hande MP , Liu H , He G , et al. A positive role for c‑Abl in Atm and Atr activation in DNA damage response . Cell Death Diff . 2011 ; 18 : 5 - 15 .
39. Chen X , Du Z , Shi W , Wang C , Yang Y , Wang F , Yao Y , He K , Hao A. 2 ‑Bromo ‑ palmitate modulates neuronal differentiation through the regulation of histone acetylation . Stem Cell Res . 2014 ; 12 : 481 - 91 .
40. Wilson JP , Raghavan AS , Yang YY , Charron G , Hang HC . Proteomic analysis of fatty‑acylated proteins in mammalian cells with chemical reporters reveals S‑acylation of histone H3 variants . Mol Cell Proteom . 2011 ; 10 ( M110 ): 001198 .
41. Hsiao KY , Mizzen CA. Histone H4 deacetylation facilitates 53BP1 DNA damage signaling and double‑strand break repair . J Mol Cell Biol . 2013 ; 5 : 157 - 65 .