LysRS Serves as a Key Signaling
Molecule in the Immune Response
by Regulating Gene Expression
Nurit Yannay-Cohen,1,5Irit Carmi-Levy,1,5Gillian Kay,1Christopher Maolin Yang,2Jung Min Han,3D. Michael Kemeny,2
Sunghoon Kim,3Hovav Nechushtan,4,* and Ehud Razin1,*
1Department of Biochemistry, The Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School,
Jerusalem 91120, Israel
2Department of Microbiology and Immunology Program, Life Science Institute, National University of Singapore 117456,
Republic of Singapore
3Center for Medicinal Protein Network and Systems Biology, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul
National University, Seoul 151-742, Korea
4Oncology Department, Hadassah Hebrew University Medical Center, P.O. Box 12272, Jerusalem 91120, Israel
5These authors contributed equally to this work
*Correspondence: firstname.lastname@example.org (H.N.), email@example.com (E.R.)
two decades ago. Here, we used LysRS silencing in
mast cells in combination with transfected normal
role played by LysRS in the production of Ap4A in
response to immunological challenge. Upon such
challenge, LysRS was phosphorylated on serine 207
in a MAPK-dependent manner, released from the
multisynthetase complex, and translocated into the
nucleus. We previously demonstrated that LysRS
forms a complex with MITF and its repressor Hint-1,
which is released from the complex by its binding to
Ap4A, enabling MITF to transcribe its target genes.
Here, silencing LysRS led to reduced Ap4A produc-
tion in immunologically activated cells, which re-
sulted in a lower level of MITF inducible genes. Our
data demonstrate that specific LysRS serine 207
phosphorylation regulates Ap4A production in immu-
nologically stimulated mast cells, thus implying that
LysRS is a key mediator in gene regulation.
Aminoacyl-tRNA synthetases (aaRSs) are extremely conserved
during evolution. AaRSs play a central role in the translation of
the language of nucleotides into the amino acid sequence of
protein. This reaction occurs in the cytoplasm of all living cells
(Ibba and Soll, 2000). Several studies have revealed that in addi-
tion to the well-known role in the catalysis of amino acid to the
cognate tRNA, many of the aaRS have noncanonical roles (Szy-
manski et al., 2000). For instance, glutamyl-prolyl-tRNA synthe-
tase (GluProRS) is involved in gene-specific silencing of transla-
tion (Sampath et al., 2004). Another example was observed with
tyrosyl-tRNA synthetase, which was secreted by the cells during
apoptosis and acquired cytokine activities (Wakasugi and
Schimmel, 1999a, 1999b).
Lysyl-tRNA synthetase (LysRS) was previously shown to be
localized in the cytoplasmic department (Gunasekera et al.,
2004) and can be found in mammalian cells mostly in multisyn-
thetase complex (MSC) (Bandyopadhyay and Deutscher,
1971), which contains three nonenzymatic proteins, p43, p18,
and p38, and nine different aaRSs. Many of these aaRSs have
been found to be involved in different signaling pathways; there-
fore, the MSC was termed ‘‘signalosome.’’ While several aaRSs
have been demonstrated to be able to produce diadenosine tet-
raphosphate (Ap4A), which is composed of two adenosine moie-
ties joined in 50-50linkage by a chain of four phosphates, it has
been reported that LysRS is the major contributor to the produc-
tion of this nucleotide (Wahab and Yang, 1985). Moreover, the
free form of LysRS from rat liver was reported to synthesize
higher levels of Ap4A than the form associated to the MSC (Wa-
hab and Yang, 1985). Several studies have demonstrated that
the concentrations of Ap4A increase after exposure of cells to
various forms of metabolic stress (heat, oxidative, nutritional,
and DNA damage). For that reason they have been described
as ‘‘alarmones’’ in cellular and metabolic stress both in prokary-
otes (Lee et al., 1983) and in eukaryotes (Varshavsky, 1983).
These studies implicated a crucial role for Ap4A in the regula-
tion of the cellular response to various stresses (reviewed by
Kisselev et al., 1998). Additionally, in vitro studies show that
phosphorylation of aaRS does not affect the aminoacylation
reaction but increases Ap4A production by up to 6-fold (Dang
and Traugh, 1989). None of these studies, however, was fol-
lowed by any systematic attempts to reveal the regulatory
mechanisms of Ap4A production.
We have been studying the regulation of MITF and USF2 for
over a decade (Levy et al., 2002; Nechushtan and Razin, 1998,
2002; Sonnenblick et al., 2004). During our studies, we discov-
ered that LysRS forms a complex with each of these
Molecular Cell 34, 603–611, June 12, 2009 ª2009 Elsevier Inc. 603
transcription factors and hypothesized that this association is
not related to the well-known role of LysRS as a tRNA synthe-
tase, but to one of its ‘‘moonlight’’ functions as a producer of
Ap4A (Lee et al., 2004; Lee and Razin, 2005). Furthermore, we
demonstrated by utilizing either external introduction of Ap4A
or silencing of the endogenous Ap4A hydrolase (Carmi-Levy
et al., 2008) that increased levels of Ap4A are associated with
increased MITF and USF2 transcriptional activity.
Here, we propose a signaling pathway in which phosphoryla-
tion of LysRS on a specific serine residue affects its dissociation
from the MSC and enhances its capability to synthesize Ap4A
and, therefore, to regulate MITF gene expression.
The Induction of Ap4A Synthesis in IgE-Ag-Activated
RBL Cells Is Mediated by LysRS
To determine whether, indeed, LysRS is responsible for the level
of Ap4A in mast cells, LysRS was knocked down in a rat baso-
philic cell line (RBL) using the short interfering RNA (siRNA)
approach. This siRNA was designed to be complementary to
evant nucleotide sequence was used as the control (NR siRNA).
Downregulation of the LysRS protein was observed 24 hr after
transfection of the RBL cells with siRNA, and the levels of the
protein remained low for up to 48 hr (Figure 1A). There was
almost no Ap4A accumulation in LysRS siRNA-transfected IgE-
Ag activated RBL cells, whereas its level in the NR siRNA treated
cells was similar to that in activated cells without transfection
(Figure 1B). These results are complementary to our previous
findings that Ap4A levels were increased in cells overexpressing
LysRS (Lee and Razin, 2005).
The effect of LysRS siRNA on total protein synthesis was
determined by introducing35[S]-methionine into cells 24 hr after
the administration of siRNA against LysRS. No significant
change in the total cellular protein synthesis was observed in
the LysRS knockdown cells compared to controls (Figure S1
Fc3RI Aggregation Induces the Release of LysRS
from the MSC
As mentioned above, LysRS is usually found in mammalian cells
as a part of the MSC; however, in eukaryotic cells, it is several-
fold more efficient at producing Ap4A when found dissociated
from the MSC. Thus, we examined whether LysRS molecules
are released from the MSC upon immunological activation of
RBL cells. In order to assess this dissociation, gel filtration chro-
matography of protein extracts derived from activated or nonac-
tivated RBL cells was performed. As shown in Figure 2A, in
nonactivated RBL cells, LysRS was mostly associated with the
MSC. However, following activation, a shift of the LysRS mole-
cules into the low molecular weight (MW) fractions was
observed. Thus, LysRS molecules dissociated from the MSC
upon mast-cell activation by Fc3RI aggregation.
The dissociation of other proteins from the MSC (p43, ArgRS,
and MetRS) was examined after cells were cultured with IgE-Ag
reduced molecular size after the stimulus.
We subsequently determined whether the dissociation from
the MSC was a result of a posttranslational modification, such
as a rapid phosphorylation. We found an indication for this
hypothesis, as in vitro studies have shown that phosphorylation
of aaRSs does not affect the aminoacylation reaction but
increases Ap4A production by up to 6-fold (Dang and Traugh,
with phosphospecific antibodies and immunoblot analysis with
anti-LysRS demonstrated phosphorylation of LysRS on serine
(Figure 2B), but not on threonine residues after cell activation
The MAPK pathway is one of the most important mast-cell
pathways involved in signaling via Fc3RI aggregation stimuli
(Furuno et al., 2001; Santini and Beaven, 1993; Tsai et al.,
1993). A key enzyme in this pathway is MAPK/ERK kinase
(MEK). To determine whether LysRS phosphorylation is medi-
ated by MEK, cells were treated with IgE followed by 10 mM
U0126, a known, specific inhibitor of MEK (Bain et al., 2007; De-
Silva et al., 1998), and then activated with antigen for 30 min. The
results clearly show that this inhibitor blocked the serine phos-
phorylation of LysRS (Figure 2C).
In addition to the immunoprecipitation experiment, we
used 2D gel electrophoresis in order to further confirm the
Figure 1. Silencing of LysRS Results in Abrogation of Immune
Induction of Ap4A
(A) RBL cells were transfected with siRNA, and whole-cell proteins were
extracted 24 hr and 48 hr after the transfection. The level of LysRS was deter-
mined by western blotting analysis using antibody against LysRS. One repre-
sentative experiment out of three is shown.
(B) Ap4A levels were determined in RBL cells transfected with siRNA against
LysRS and with NR siRNA. After 24 hr, the cells were activated with 100 ng/
ml IgE anti-DNP and 100 ng/ml DNP for 15 min. The mean and standard error
of the mean (SEM) of three experiments is shown.
LysRS Is a Key Mediator in Gene Regulation
604 Molecular Cell 34, 603–611, June 12, 2009 ª2009 Elsevier Inc.
phosphorylation of LysRS upon IgE-Ag activation. It should be
noted that the antibodies anti-LysRS and anti-Myc reacted at
the expected molecular weight of either rat LysRS or Myc-
human LysRS with calculated pIs of 6.16 and 5.77, respectively.
Phosphorylation of LysRS was not detected in unstimulated
cells, whereas in cells activated with IgE-Ag, spots shifted to
the left side of the gel, indicating a more acidic pI compatible
with phosphorylation. Moreover, this phosphorylation was
specifically found in the low MW fractions, that is, the free form
of LysRS (Figure 2D). This phosphorylation was totally blocked
by the presence of alkaline phosphatase (AP) and U0126, which,
in accordance to the immunoprecipitation results, indicates the
involvement of MAPK in LysRS phosphorylation.
Next, proteins were extracted from cells triggered by Fc3RI
aggregation due to 30 min antigen activation, and these extracts
were subjected to gel filtration chromatography. As shown in
Figure 2E, the use of MEK inhibitor (U0126) completely pre-
vented the release of LysRS from the MSC (second panel),
whereas the p38 MAPK inhibitor, SB203580 (Bain et al., 2007),
did not effect the dissociation of LysRS from the MSC (lower
panel). Thus, the release of LysRS from the MSC was dependent
on phosphorylation of its serine residues via MEK.
Serine 207 Phosphorylated LysRS Is Required
for Ap4A Synthesis
The involvement of the MAPK pathway in LysRS activity was
further investigated by searching for ERK consensus motifs
(X-S/T-P) within the LysRS sequence using multiple sequence
alignment (Figure S4). Two serine residues within an ERK
consensus motif in LysRS were detected (S207 and S470).
Based on this, four fused Myc tag constructs of wild-type and
mutated human LysRS (hLysRS) were constructed (WT,
S207A, S470A, and S207A/S470A). Each one of the constructs
was administered to the cells after the endogenous LysRS was
knocked down by siRNA. Under these conditions, no gene
silencing of the exogenous hLysRS occurred (Figure 3A). The
Ap4A assay was carried out on extracts derived from immuno-
logically activated RBL cells that were transfected with each of
the constructs. As can be seen in Figure 3B, mutation S207A
of hLysRS significantly reduced Ap4A production in these trans-
fected RBL cells, similar to cells transfected with rat LysRS
siRNA. Identical results were obtained for the cells transfected
with the double mutation (S207A/S470A). Ap4A accumulation
was not affected as a result of the transfection of RBL cells
with the S470A mutation. These observations clearly demon-
strated that phosphorylation of serine 207, which is within the
ERK consensus motif, is required for the synthesis of Ap4A by
Since phosphorylation of LysRS at Ser207 was found to have
a major effect on Ap4A synthesis, we next analyzed the
Figure 2. Mast Cell IgE-Ag Stimulation Induces LysRS MAPK-
Dependent Serine Phosphorylation and Release from the Multisyn-
(A) Lysates from RBL cells that were either immunologically activated for
30 min or nonactivated were subjected to size-exclusion chromatography.
Two milliliter fractions were collected. The eluted proteins in each fraction
were analyzed by immunoblotting with anti-LysRS. One representative exper-
iment out of three is shown.
(B) Serine phosphorylation of LysRS in mast cells is mediated by Fc3RI activa-
tion. Lysates from cells treated with IgE and antigen for 15 or 30 min were
immunoprecipitated (IP) with anti-LysRS antibody and subjected to western
blot analysis (WB) with antibody against anti-phosphorylated-serine (P-Ser).
One representative experiment out of three is shown.
(C) Lysates from cells treated with IgE-Ag alone (control) or with IgE followed
by 10 mM U0126 for 10 min and then activated with antigen for 30 min, were
immunoprecipitated (IP) with anti-P Ser and subjected to western blot analysis
(WB) with antibodies against LysRS. One representative experiment out of
three is shown.
(D) Lysates from RBL cells treated with IgE-Ag alone, or with IgE followed by
10 mM U0126 for 10 min and then activated with antigen for 30 min, were sub-
jected to 2D electrophoresis on a pH 4?7 gradient and a 8% polyacrilammide
gel. The gel was blotted with anti-LysRS antibody.
panel), with U0126 (10 mM) for 10 min (second panel), or with SB203580 (5 mM)
for 2 hr (lower panel) were subjected to size-exclusion chromatography. The
eluted proteins in each fraction were analyzed by immunoblotting with anti-
LysRS. One representative experiment out of three is shown.
LysRS Is a Key Mediator in Gene Regulation
Molecular Cell 34, 603–611, June 12, 2009 ª2009 Elsevier Inc. 605
phosphorylation status of LysRS WT and S207A variants upon
immunological activation of mast cells. Strikingly, while the WT
variant, similarly to the endogenous LysRS in Figure 2D, was
shown to shift to the acidic side of the gel following IgE-Ag stim-
ulation, the S207A variant was not affected by the trigger
When recombinant LysRS proteins (WT/S207A) were incu-
bated with [g-32P]ATP in the presence of recombinant ERK1
and analyzed via SDS-PAGE, LysRS WT was clearly phosphor-
significantly reduced (Figure 3D). Using densitometry analysis,
the observed reduction in the phosphorylation was found to
In order to further establish phosphorylation of Ser207 by
MAPK as a crucial step toward Ap4A production, we transfected
cells with either LysRS WT or LysRS S207D variants. Ap4A
synthesis in cells transfected with the LysRS S207D variant
was found to be insensitive to the MEK inhibitor U0126
(Figure 3E). Moreover, the S207D variant was shown to have
constitutive Ap4Aproduction activityeven in quiescent RBLcells
when compared to WT (Figure 3F).
IgE-Ag Activation, via MAPK Pathway in RBL Cells,
Induces Translocation of LysRS from the Cytosol
into the Nucleus
LysRS has been identified as an MITF-interacting protein, using
a construct containing the MITF bHLH-Zip domain as bait in
yeast two-hybrid library screening (Razin et al., 1999). This
protein-protein interaction within the complex was later verified
by GST pull-down assays and coimmunoprecipitations (Lee
et al., 2004; Nechushtan and Razin, 2002). Since MITF transcrip-
tional activity is compartmentalized to the nucleus, we examined
whether aggregation of Fc3RI by IgE-Ag that induced the release
of LysRS from the MSC (as we described above) also causes
translocation of LysRS from the cytosol to the nuclear compart-
nuclear translocation occurred 30 min after cell activation by
IgE-Ag (Figure 4A). Confirmation of these findings was carried
Figure 3. Serine 207 Phosphorylated LysRS Is Required for Ap4A Synthesis
(A) RBL cells were transfected with rat LysRS siRNA (as described in the Experimental Procedures). Twenty-four hours later, cells were transfected with human
LysRS variants (WT, S207A, S470A, or S207A/S470A). Next, the cells were incubated with IgE and challenged with DNP for 30 min. The cell extracts were
analyzed by western blot with anti-Myc or anti-LysRS antibodies. One representative experiment out of three is shown.
(B) RBL cells were transfected and activated as described above. The Ap4A assay was performed on the cells extracts as described in the Experimental Proce-
dures. The mean and standard error of the mean (SEM) of three experiments is shown.
(C) Lysates from IgE-Ag-activated and -nonactivated RBL cells transfected either with WT or withS207A LysRS variants were subjected to2D electrophoresis on
a pH 4?7 gradient and a 8% polyacrilammide gel. The gel was blotted with anti-myc antibody.
(D) Recombinant LysRS proteins (WT/S207A) were expressed as His fusion proteins then phosphorylated in vitro in the presence of recombinant, active ERK1
and g32P-ATP. Samples were resolved on SDS-polyacrylamide gel and blotted, and the radioactive bands were detected by autoradiography.
(E) Ap4A levels were determined in RBL cells transfected with either WT or S207D LysRS variants, cultured with or without U0126 (10 mM) for 10 min, and then
activated with antigen for 15 min. The mean and standard error of the mean (SEM) of three experiments is shown.
(F) Ap4A levels were determined in quiescent RBL cells transfected with either WT or S207D LysRS variants. The mean and standard error of the mean (SEM) of
three experiments is shown.
LysRS Is a Key Mediator in Gene Regulation
606 Molecular Cell 34, 603–611, June 12, 2009 ª2009 Elsevier Inc.
with anti-LysRS (Figure 4B).
We then examined the effect of U0126 and PD 098059,
another specific inhibitor of MEK (Alessi et al., 1995), on LysRS
nuclear translocation in order to determine whether this translo-
cation is affected by MAPK activity. As shown in Figure 4C, both
of these inhibitors prevented the nuclear translocation of LysRS
in immunologically activated mast cells. Thus, LysRS is translo-
cated to the nucleus in a MAPK-dependent fashion following IgE
and antigen stimulation.
In order to determine whether the production of Ap4A is
dependent on the MAPK pathway, RBL cells were treated with
U0126, and Ap4A production was examined. As seen in
Figure 4D, MEK inhibition resulted in a significant decrease in
Ap4A levels in RBL cells. Thus, we concluded that the MAPK
pathway is involved in the synthesis of Ap4A by LysRS.
Subsequent to our observations of MAPK-dependent nuclear
presence of LysRS and MAPK-dependent Ap4A production, we
wanted to determine if this nuclear localization is a prerequisite,
exclusive condition for Ap4A synthesis. To address this, we
transfected cells with a LysRS S207A variant that was linked to
a strong NLS. This variant was expressed in the nucleus despite
icant difference in Ap4A production between the cytosolic and
nuclear LysRS S207A expression (Figure S5B). Hence, nuclear
localization by itself of the LysRS S207A variant cannot lead to
induction of Ap4A synthesis.
The Involvement of the MAPK Pathway in MITF
To determine the direct effect of LysRS on the transcriptional
activity of MITF, the transcript levels of two of its responsive
genes, tryptophan hydroxylase (TPH) and mast-cell protease 5
(MCP5) (Ito et al., 1998; Morii et al., 1997), were measured in
immunologically activated RBL cells in which LysRS had been
knocked down using the corresponding siRNA.These two target
genes showed a marked decrease in their transcript levels in
LysRS knockdown cells (Figures 5A and 5B), indicating that
Culturing RBLcells with MAPKinhibitor prior to immunological
activation prevented the process of LysRS dissociation from the
MSC, phosphorylation, and translocation into the nucleus. The
downstream effect of LysRS being retained in the cytoplasm
was further examined by measuring Ap4A levels and assessing
MITF transcriptional activity by quantifying transcription of its
target genes. The immunological induction of TPH and MCP5
5C and 5D). Furthermore, the LysRS S207D variant was shown
to enhance transcription of TPH compared to WT (Figure 5E).
These results strongly indicate that in activated mast cells,
via Ser207 phosphorylation in the MAPK pathway.
Among the aaRSs family, LysRS is the major contributor to the
production of Ap4A (Wahab and Yang, 1985). LysRS is usually
found in mammalian cells as a part of the MSC (Han et al.,
2003; Robinson et al., 2000); however, in eukaryotic cells, it is
several-fold more efficient at producing Ap4A when found disso-
ciated from the MSC (Wahab and Yang, 1985). Additionally,
Figure 4. Identification of LysRS Nuclear Translocation in RBL Cells
following Fc3RI Aggregation
microscopy.Datashown are representativeof threeindependent experiments.
(B) Nuclear and cytoplasmic fractions were isolated from RBL cells activated
with IgE and antigen. The subcellular extracts were analyzed by western blot
with anti-LysRS antibodiy. Antitubulin was used as a cytoplasmic marker while
(C) RBL cells were pretreated with IgE and with either MAPK inhibitor U0126
anti-LysRS using Cy5-labeled secondary antibody. The cells were analyzed by
confocal laser scanning microscopy. One of three independent experiments is
(D) RBL cells were activated for 15 min by IgE-Ag with or without U0126 treat-
error of the mean (SEM) of three experiments is shown.
LysRS Is a Key Mediator in Gene Regulation
Molecular Cell 34, 603–611, June 12, 2009 ª2009 Elsevier Inc. 607
in vitro studies have shown that phosphorylation of aaRS
enhances Ap4A production (Dang and Traugh, 1989).
Using the siRNA technique, we have demonstrated directly
that in mast cells, inhibition of LysRS expression in vivo caused
a substantial decrease in the induction of Ap4A in response to
Fc3RI aggregation. Our previous studies suggested that an
increase in Ap4A can induce the release of Hint-1 from MITF
(Lee et al., 2004). Since the level of Ap4A needed for this release
was relatively high, and given that we have demonstrated the
binding of LysRS to this transcription factor, we investigated
by which pathway(s) LysRS could produce high amounts of
Ap4A leading to the release of Hint-1 from its bound transcription
factor. Our assumption was that LysRS plays a pivotal role in
Ap4A production, and thus, we expected that this activity of
LysRS would be regulated by an inducible process and would
involve dissociation from the MSC.
The MSC, which contains three scaffold proteins and nine
aaRSs, has been referred to recently as a ‘‘signalosome’’ (Han
et al., 2006), since many of its aaRSs elements, aside from their
catalytic roles in protein synthesis, are also involved in signaling
pathways. Another aaRS with a ‘‘moonlighting,’’ nonconven-
tional role, GluProRS, has been shown to be regulated through
its release from the MSC following interferon g induction (Sam-
path et al., 2004). Similarly, the small form of arginine tRNA
synthetase, which is located in the cytoplasm outside the
MSC, was shown to play a role in posttranslational modification,
whereas the longer form, which is responsible for translation,
was found in the MSC (Ferber and Ciechanover, 1987).
Figure 5. Determination of the Expression Levels of
MITF-Responsive Genes in Cells Administered siRNA
(A and B) The mRNA quantitation of MCP5 and TPH was deter-
Expression levels were normalized to b-actin housekeeping gene.
The mean and standard error of the mean (SEM) of three experi-
ments is shown.
(C and D) RBL cells were activated for 4 hr by IgE-Ag with or
without U0126. The mRNA quantitation of MCP-5 and TPH was
determined by real-time PCR as described above. One of two
independent experiments is shown.
(E) RBL cells were transfected with either WT or S207D hLysRS
variants and activated for 4 hr with IgE-Ag. TPH mRNA level was
determined by real-time PCR as described above. The mean and
standard error of the mean (SEM) of five experiments is shown.
Our results demonstrated that LysRS is released
from the MSC following Fc3RI aggregation of mast
serineresidue andisdependent ontheinductionof the
The MAPK pathway is one of the most important
mast-cell pathways involved in signaling via the stim-
ulus of Fc3RI aggregation (Furuno et al., 2001; Santini
and Beaven, 1993; Tsai et al., 1993). Moreover, MEK
activation is required for antigen-stimulated secretion
in mast cells (Hirasawa et al., 1995).
Our immunohistochemical studies and cellular frac-
tionation assays revealed that a much greater propor-
tion of LysRS can be found within the nucleus following Fc3RI
aggregation. Using MEK inhibitors, we have clearly demon-
strated that this nuclear translocation was dependent upon acti-
vation of the MAPK pathway. Interestingly, both reduction of
cellular levels of LysRS by specific siRNA and the use of the
MEK inhibitor U0126 led to reduced mRNA levels of several
target genes of MITF in mast cells.
It has previously been reported that the MAPK cascade is crit-
ical in the activation of various transcription factors, such as
ATF3 and AP-1, and it is also involved in cytokine production
in mast cells (Garrington et al., 2000). Here we have revealed
for the first time a new MAPK pathway branch in activated
mast cells that leads to gene expression via the phosphorylation
of LysRS and the network of MITF and Hint-1.
Combining all of our data, wepropose amodel in which LysRS
is phosphorylated on the serine 207 residue through the MAPK
pathway following cellular activation. This phosphorylation is fol-
lowed by the release from the MSC of LysRS, which then trans-
locates into the nucleus. The released serine 207 phosphory-
lated LysRS can then produce higher levels of Ap4A, with
profound cellular effects via binding to Ap4A binding proteins.
One such effect is the removal of the repressor Hint-1 from
MITF, enabling it to transcribe its target genes.
Direct binding of LysRS to molecules such as transcription
factors should allow very high levels of Ap4A in the vicinity of
thesemolecules (Figure 6). Thus, based on our data, wepropose
that in immunologically activated cells, LysRS has a signal trans-
duction role besides its other well-defined roles.
LysRS Is a Key Mediator in Gene Regulation
608 Molecular Cell 34, 603–611, June 12, 2009 ª2009 Elsevier Inc.
The antibody anti-LysRS was custom made against a specially designed
determinant KEVLLFPAMKPE (Hy Laboratories Ltd., Israel). Anti-phosphoser-
ine antibody was purchased from Zymed Laboratories (San Francisco, CA).
Anti-phosphothreonine and anti-Myc were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-MetRS and anti-ArgRS were purchased
from Abcam (Abcam Ltd., Cambridge, UK). Anti-p43/AIMP1 was produced as
previously described (Han et al., 2006).
This assay detects the relative amount of Ap4A present in extracts of mamma-
lian cells. For each determination, cells were grown to about 80% confluence.
The cell layer was lysed with trichloroacetic acid. Extraction and measurement
by luminometry of the nucleotides were performed as described previously
(Murphy et al., 2000). Results were normalized by Bradford protein assay.
(Razin et al., 1999). RBL cells were sensitized first with anti-DNP IgE mono-
clonal antibody (SPE-7, Sigma-Aldrich Corp., St. Louis, MO) and then chal-
lenged with DNP (Sigma-Aldrich Corp.). IgE antibody was centrifuged
(18,000 g, 5 min) before use to remove aggregates.
Chemical Inhibitor Treatment
U0126, PD098059, and SB203580 were purchased from Sigma-Aldrich Corp.
(St. Louis, MO).
Human LysRS was subcloned into the EcoRI and XbaI sites of the pSC2+MT
vector (Invitrogen). This vector was used for the production LysRS mutant by
site-directed mutagenesis in which serine was replaced by alanine at both 207
and 470 positions (LysRS S207A/S470A). Human LysRS S207A variant was
subcloned into pCMV/myc/cyto and pCMV/myc/nuc vectors. The fidelity of
all constructs was verified by direct sequencing.
Gel Filtration Chromatography of Cell Lysates
Cell extracts were applied to a Superdex 200 column (30 3 1 cm from
Amersham Biosciences) using AKTA Explorer (Amersham Biosciences) and
eluted at a flow rate of 0.8 ml/min in buffer containing 20 mM Tris-HCL
(pH 7.4), 150 mM NaCl, 10% glycerol, and 0.5% Triton X-100. Thyroglobulin
(669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (163 kDa), BSA
(67 kDa), and OvaAlb (44 kDa) were used as molecular weight standards.
The eluted proteins in each fraction were analyzed by immunoblotting with
Gel Electrophoresis and Western Blots
Proteins were resolved by 10% SDS-PAGE under reducing conditions and
were transferred to nitrocellulose membranes. Visualization of reactive
proteins was performed by enhanced chemiluminescence.
The immunoprecipitation of the specific proteins from RBL cells was carried
out as previously described (Levy et al., 2002).
2D electrophoresis was performed as previously described (Han et al., 2008).
Cells were solubilized in 2-D-lysis buffer (7 M urea, 2 M thiourea, 4% w/v,
CHAPS, 100 mM DTT). Cell lysates were loaded to immobilized pH gradient
strip gels (linear pH gradient 4–7, 7 cm). Isoelectric focusing was performed
at 4,000 V until the total volt-hours reached 10 kV hours using PROTEAN
IEF cell (Bio-Rad). Following two-step equilibration with 375 mM Tris-HCl
(pH 8.8), 6 M urea, 2% SDS, 20% glycerol, 2% DTT, and 2.5% iodoacetamide,
the IPG strips were embedded on top of 8% SDS-PAGE gels and sealed with
2% agarose. Proteins were separated based on their molecular weight.
Expression and Purification of LysRS Proteins
cDNA of human LysRS WT or S207A were subcloned into pET28a (Novagen).
Recombinant LysRS proteins were expressed as His fusion proteins and puri-
fied by Ni2+-bound His-Bind resin. Cells were lysed and sonicated in 30 ml of
lysis buffer (20 mM KH2PO4, 500 mM NaCl, 2 mM b-mercaptoethanol [pH 7.8]
containing 0.5 mM PMSF, 1 mg/ml leupeptin, and 5 mg/ml aprotinin). The
lysates were centrifuged at 20,000 g for 1 hr at 4?C. The supernatant was incu-
bated with 1 ml of Ni2+-bound His-Bind resin at 4?C overnight with constant
agitation. The resin was washed with 10 column volumes of washing buffer
A (20 mM KH2PO4, 500 mM NaCl, 2 mM b-mercaptoethanol, and 10% glyc-
erol [pH 6.0]), washing buffer B (20 mM KH2PO4, 500 mM NaCl, 2 mM b-mer-
captoethanol, and 10% glycerol [pH 5.2]), and washing buffer C (20 mM
KH2PO4, 500 mM NaCl, 2 mM b-mercaptoethanol [pH 7.8], and 50 mM imid-
azole). Bound protein was eluted with elution buffer (20 mM KH2PO4, 500 mM
NaCl, and 2 mM b-mercaptoethanol [pH 7.8]) containing 300 mM imidazole.
The fractions eluted with 300 mM imidazole buffer were pooled and injected
onto PD-10 gel-filtration columns equilibrated with phosphate-buffered saline.
The fractions were then analyzed by SDS-PAGE and western blotting.
In Vitro Phosphorylation of LysRS by ERK1
LysRS proteins (200 ng) were incubated with 20 ng of recombinant ERK1 in
phosphorylation buffer (20 mM Tris/HCl [pH 7.5], 25 mM b-glycerophosphate,
5 mM EGTA, 1 mM Na3VO4, 1 mM DTT, 0.12 mM ATP, and 2 mCi [g-32P] ATP
[3000 Ci/mmol]) for 15 min. The reaction mixture was then electrophoresed
through an 8% SDS-polyacrylamide gel. The dried gel was exposed to autora-
Real-Time Quantitative Polymerase Chain Reaction
Candidate MITF responsive gene transcription was measured using real-time
quantitative PCR. mRNAs of MITF target genes were quantified by SYBR-
green incorporation (ABgene SYBR green ROX Mix, ABgene). Real-time
PCR was performed on Rotor-Gene sequence detection system (Corbett,
Australia). The genes whose mRNA levels were quantified by real-time PCR
were rat TPH, MCP5, and b-actin.
Figure 6. Proposed Model for LysRS as a Signaling Molecule
Following specific stimuli, LysRS is serine phosphorylated in a MAPK-depen-
dent fashion, dissociates from the MSC, and translocates from the cytoplasm
to the nucleus. The phosphorylation on serine residue 207 elevates Ap4A
levels, leads to the dissociation of Hint-1 from MITF, and allows this transcrip-
tion factor to activate its responsive genes.
LysRS Is a Key Mediator in Gene Regulation
Molecular Cell 34, 603–611, June 12, 2009 ª2009 Elsevier Inc. 609
Cells were transfected with a siRNA duplex consisting of two complementary
21 nucleotide RNA strands with 30dTdT overhangs (QIAGEN Inc., CA) in order
otide sequence found in rat mRNA of LysRS, and a nonrelevant nucleotide
sequence was used as the control (NR siRNA). The target sequence of the
specific siRNAs for LysRS was TTCGTTCACATCAATAACAAA. The nonrele-
vant control sequence was AATTCTCCGAACGTGTCACGT.
Amaxa Nucleofector technology (Amaxa, Cologne, Germany) was used for
transfecting cells. 2 3 106cells were transfected with 3 mg of the selected
siRNA oligonucleotide according to the manufacturer’s protocol. Briefly, the
cells were resuspended in 100 ml nucleofector solution. RNA was added,
and the mixture transferred into an electroporation cuvette. Nucleofector solu-
tion was used to stabilize the cells during electroporation, which was per-
formed using the T-20 program. The cells were suspended in 2 ml of cell
culture medium immediately after electroporation.
Indirect Fluorescent Immunocytochemistry
RBL cells were grown on glass coverslips in 6-well plates. After extensive
washing with PBS, the cells were fixed with 1.5 ml 4% formaldehyde in PBS
for 10 min. The fixed cells were then washed with PBS and permeabilized
with 1.5 ml Triton 1003 diluted 1:2 with PBS containing 7.5 mg bovine serum
albumin. After 45 min blocking with normal donkey serum, the cells were
stained either with rabbit anti-LysRS followed by the addition of Cy5-conju-
gated goat anti-rabbit IgG (Jackson ImmunoResearch Inc., West Grove, PA).
Fluorescence analysis was performed using Zeiss LSM 410 confocal laser
scanning system connected to Zeiss Axiovert 135M microscope (Zeiss,
Germany) as previously described (Sonnenblick et al., 2004).
Supplemental Data include five figures and can be found with thisarticleonline
This work was supported by the United States Binational Science Foundation
(E.R., 2003-009); the Israeli Academy of Science (E.R., 144/04); the German-
Israel Foundation for Scientific Research and Development (E.R., I-726-
10.2); the Morasha Foundation Fund (H.N.); the Acceleration Research of
KOSEF (S.K., 2009-0063498); and the 21st Frontier Functional Proteomics
of Hebrew University. We would like to thank Dr. Mario Lebendiker from The
Protein Purification Facility, Wolfson Centre for Applied Structural Biology,
Hebrew University of Jerusalem, for his help in gel filtration chromatography.
Received: September 11, 2008
Revised: December 31, 2008
Accepted: May 26, 2009
Published: June 11, 2009
Alessi, D.R., Cuenda, A., Cohen, P., Dudley, D.T., and Saltiel, A.R. (1995). PD
098059 is a specific inhibitor of the activation of mitogen-activated protein
kinase kinase in vitro and in vivo. J. Biol. Chem. 270, 27489–27494.
Bain, J., Plater, L., Elliott, M., Shpiro, N., Hastie, C.J., McLauchlan, H., Kle-
vernic, I., Arthur, J.S., Alessi, D.R., and Cohen, P. (2007). The selectivity of
protein kinase inhibitors: a further update. Biochem. J. 408, 297–315.
Bandyopadhyay, A.K., and Deutscher, M.P. (1971). Complex of aminoacyl-
transfer RNA synthetases. J. Mol. Biol. 60, 113–122.
Carmi-Levy, I., Yannay-Cohen, N., Kay, G., Razin, E., and Nechushtan, H.
(2008). Diadenosine tetraphosphate hydrolase is part of the transcriptional
regulation network in immunologically activated mast cells. Mol. Cell. Biol.
Dang, C.V., and Traugh, J.A. (1989). Phosphorylation of threonyl- and seryl-
tRNA synthetase by cAMP-dependent protein kinase. A possible role in the
regulation of P1, P4-bis(50-adenosyl)-tetraphosphate (Ap4A) synthesis.
J. Biol. Chem. 264, 5861–5865.
J.M., and Scherle, P.A. (1998). Inhibition of mitogen-activated protein kinase
kinase blocks T cell proliferation but does not induce or prevent anergy.
J. Immunol. 160, 4175–4181.
Ferber, S., and Ciechanover, A. (1987). Role of arginine-tRNA in protein degra-
dation by the ubiquitin pathway. Nature 326, 808–811.
Furuno, T., Hirashima, N., Onizawa, S., Sagiya, N., and Nakanishi, M. (2001).
Nuclear shuttling of mitogen-activated protein (MAP) kinase (extracellular
signal-regulated kinase (ERK) 2) was dynamically controlled by MAP/ERK
Garrington, T.P., Ishizuka, T., Papst, P.J., Chayama, K., Webb, S., Yujiri, T.,
Sun, W., Sather, S., Russell, D.M., Gibson, S.B., et al. (2000). MEKK2 gene
disruption causes loss of cytokine production in response to IgE and c-Kit
ligand stimulation of ES cell-derived mast cells. EMBO J. 19, 5387–5395.
Gunasekera, N., Lee, S.W., Kim, S., Musier-Forsyth, K., and Arriaga, E. (2004).
Nuclear localization of aminoacyl-tRNA synthetases using single-cell capillary
electrophoresis laser-induced fluorescence analysis. Anal. Chem. 76, 4741–
Han, J.M., Kim, J.Y., and Kim, S. (2003). Molecular network and functional
Res. Commun. 303, 985–993.
Han, J.M., Lee, M.J., Park, S.G., Lee, S.H., Razin, E., Choi, E.C., and Kim, S.
(2006). Hierarchical network between the components of the multi-tRNA
synthetase complex: implications for complex formation. J. Biol. Chem. 281,
Han, J.M., Park, B.J., Park, S.G., Oh, Y.S., Choi, S.J., Lee, S.W., Hwang, S.K.,
Chang, S.H., Cho, M.H., and Kim, S. (2008). AIMP2/p38, the scaffold for the
multi-tRNA synthetase complex, responds to genotoxic stresses via p53.
Proc. Natl. Acad. Sci. USA 105, 11206–11211.
Hirasawa, N., Santini, F., and Beaven, M.A. (1995). Activation of the mitogen-
activated protein kinase/cytosolic phospholipase A2 pathway in a rat mast
cell line. Indications of different pathways for release of arachidonic acid and
secretory granules. J. Immunol. 154, 5391–5402.
Ibba, M., and Soll, D. (2000). Aminoacyl-tRNA synthesis. Annu. Rev. Biochem.
Ito, A., Morii, E., Maeyama, K., Jippo, T., Kim, D.K., Lee, Y.M., Ogihara, H.,
Hashimoto, K., Kitamura, Y., and Nojima, H. (1998). Systematic method to
obtain novel genes that are regulated by mi transcription factor: impaired
expression of granzyme B and tryptophan hydroxylase in mi/mi cultured
mast cells. Blood 91, 3210–3221.
Kisselev, L.L., Justesen, J., Wolfson, A.D., and Frolova, L.Y. (1998). Diadeno-
sine oligophosphates (Ap(n)A), a novel class of signalling molecules? FEBS
Lett. 427, 157–163.
Lee, P.C., Bochner, B.R., and Ames, B.N. (1983). AppppA, heat-shock stress,
and cell oxidation. Proc. Natl. Acad. Sci. USA 80, 7496–7500.
Lee, Y.N., and Razin, E. (2005). Nonconventional involvement of LysRS in the
mast cells. Mol. Cell. Biol. 25, 8904–8912.
tRNA synthetase and Ap4A as signaling regulators of MITF activity in Fc3RI-
activated mast cells. Immunity 20, 145–151.
Levy, C., Nechushtan, H., and Razin, E. (2002). A new role for the STAT3 inhib-
itor, PIAS3: a repressor of microphthalmia transcription factor. J. Biol. Chem.
Morii, E., Jippo, T., Hashimoto, K., Kim, D.-K., Lee, Y.-M., Ogihara, H., Tsujino,
K., Kim, H.-M., and Kitamura, Y. (1997). Abnormal expression of mouse mast
LysRS Is a Key Mediator in Gene Regulation
610 Molecular Cell 34, 603–611, June 12, 2009 ª2009 Elsevier Inc.
cell protease 5 gene in cultured mast cells derived from mutant mi/mi mice. Download full-text
Blood 90, 3057–3066.
Murphy, G.A., Halliday, D., and McLennan, A.G. (2000). The Fhit tumor
suppressor protein regulates the intracellular concentration of diadenosine
triphosphate but not diadenosine tetraphosphate. Cancer Res. 60, 2342–
Nechushtan, H., and Razin, E. (1998). Deciphering the early-response tran-
scription factor networks in mast cells. Immunol. Today 19, 441–444.
Nechushtan, H., and Razin, E. (2002). The function of MITF and associated
proteins in mast cells. Mol. Immunol. 38, 1177–1180.
Razin, E., Zhang, Z.C., Nechushtan, H., Frenkel, S., Lee, Y.N., Arudchandran,
R., and Rivera, J. (1999). Suppression of microphthalmia transcriptional
activity by its association with protein kinase C-interacting protein 1 in mast
cells. J. Biol. Chem. 274, 34272–34276.
Robinson, J.C., Kerjan, P., and Mirande, M. (2000). Macromolecular assem-
blage of aminoacyl-tRNA synthetases: quantitative analysis of protein-protein
interactions and mechanism of complex assembly. J. Mol. Biol. 304, 983–994.
Sampath, P., Mazumder, B., Seshadri, V., Gerber, C.A., Chavatte, L., Kinter,
M., Ting, S.M., Dignam, J.D., Kim, S., Driscoll, D.M., and Fox, P.L. (2004).
Noncanonical function of glutamyl-prolyl-tRNA synthetase: gene-specific
silencing of translation. Cell 119, 195–208.
Santini, F., and Beaven, M.A. (1993). Tyrosine phosphorylation of a mitogen-
activated protein kinase-like protein occurs at a late step in exocytosis.
Studies with tyrosine phosphatase inhibitors and various secretagogues in
rat RBL-2H3 cells. J. Biol. Chem. 268, 22716–22722.
Sonnenblick, A., Levy, C., and Razin, E. (2004). Interplay between MITF,
PIAS3, and STAT3 in mast cells and melanocytes. Mol. Cell. Biol. 24,
Szymanski, M., Deniziak, M., and Barciszewski, J. (2000). The new aspects of
aminoacyl-tRNA synthetases. Acta Biochim. Pol. 47, 821–834.
Tsai, M., Chen, R.H., Tam, S.Y., Blenis, J., and Galli, S.J. (1993). Activation of
MAP kinases, pp90rsk and pp70–S6 kinases in mouse mast cells by signaling
through the c-kit receptor tyrosine kinase or Fc epsilon RI: rapamycin inhibits
activation of pp70–S6 kinase and proliferation in mouse mast cells. Eur. J.
Immunol. 23, 3286–3291.
cally acting alarmone? Cell 34, 711–712.
Wahab, S.Z., and Yang, D.C. (1985). Synthesis of diadenosine 50,5000-P1,P4-
tetraphosphate by lysyl-tRNA synthetase and a multienzyme complex of ami-
noacyl-tRNA synthetases from rat liver. J. Biol. Chem. 260, 5286–5289.
Wakasugi, K., and Schimmel, P. (1999a). Highly differentiated motifs respon-
sible for two cytokine activities of a split human tRNA synthetase. J. Biol.
Chem. 274, 23155–23159.
Wakasugi, K., and Schimmel, P. (1999b). Two distinct cytokines released from
a human aminoacyl-tRNA synthetase. Science 284, 147–151.
LysRS Is a Key Mediator in Gene Regulation
Molecular Cell 34, 603–611, June 12, 2009 ª2009 Elsevier Inc. 611