Originally published In Press as doi:10.1074/jbc.M002573200 on May 8, 2000
J. Biol. Chem., Vol. 275, Issue 29, 22520-22525, July 21, 2000
Ultraviolet Light-induced Stimulation of the JNK
Mitogen-activated Protein Kinase in the Absence of Src Family Tyrosine
Kinase Activation*
Kambiz
Amdjadi
§¶ and
Bartholomew M.
Sefton
From the Molecular Biology and Virology Laboratory,
The Salk Institute for Biological Studies,
La Jolla, California 92037 and the § Department of
Molecular Pathology, University of California, San Diego,
La Jolla, California 92093
Received for publication, March 27, 2000, and in revised form, April 27, 2000
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ABSTRACT |
In T cells, the JNK mitogen-activated protein
kinase is activated by simultaneous stimulation of the T-cell receptor
and CD28 or by a number of stress stimuli including ultraviolet light, hydrogen peroxide, and anisomycin. Lck, a Src family kinase, is essential for T-cell receptor-mediated activation of JNK. We asked whether Lck was also involved in stress-mediated activation of JNK. JNK
was activated by ultraviolet light irradiation in all of the four
T-cell lines we examined, but Lck was not. Additionally, JNK activation
by ultraviolet light, hydrogen peroxide, and anisomycin was completely
normal in T cells lacking Lck. These data suggest that Lck is not
activated by ultraviolet light irradiation, nor is it required for JNK
activation in T cells by any of the stress stimuli we tested. We also
examined JNK activation by ultraviolet light in mouse fibroblasts
expressing no known Src kinases. The activation of JNK by ultraviolet
light was completely normal in these cells. Finally, treatment of
lymphoid and epithelial cells with a Src kinase family inhibitor
PP2-reduced tyrosine phosphorylation of cellular proteins markedly
without affecting ultraviolet light-induced activation of JNK. These
results suggest that Src kinases are not essential for ultraviolet
light-induced activation of JNK in a diverse variety of cell types.
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INTRODUCTION |
The c-jun N-terminal kinases
(JNK),1 also known as the
stress-activated protein kinases, are members of the mitogen-activated protein kinase family (1-3). They are stimulated by exposure of cells
to a diversity of stimuli including protein synthesis inhibitors such
as anisomycin, oxidative agents such as hydrogen peroxide, genotoxins
such as ultraviolet light (UV), and cytokines such as interleukin-1
(2-6). The c-jun N-terminal kinases are also stimulated
during lymphocyte activation following stimulation of the T-cell
receptor and CD28 (7). The JNK family
kinases2 have a number of
identified substrates including activated transcription factor-2 and
c-jun (8, 9). Phosphorylation of serine 63 and serine 73 on
c-jun by JNK is necessary for maximal c-jun
transcriptional activity (1, 10). JNK1 and JNK2 are widely expressed in
diverse cell types including lymphocytes, whereas JNK3 expression is
restricted to brain neurons (8, 9, 11). JNK1 null mice or JNK2 null mice are viable. Both strains, however, exhibit decreased
activation-induced T-cell death and an imbalance in TH1- and
TH2-mediated immune responses (12-14). These data suggest that JNK1
and JNK2 are both important for regulation of cell viability and
differentiation in T lymphocytes. Deletion of both JNK1 and JNK2 causes
embryonic lethality due to severe disregulation of apoptosis during
brain development (15).
In HeLa cells, UV irradiation has been reported to activate the c-Src
tyrosine-protein kinase and based on the inhibitory effect of
expressing catalytically inactive v-src, it has been concluded that Src activity is essential for UV-induced activation of
JNK in these cells (16). However, neither the mechanism of Src
activation by UV light nor how this leads to JNK stimulation is clear.
It is likely that Lck, a Src kinase expressed predominantly in T
lymphocytes (17), is involved in activation of JNK following ligation
of the T-cell receptor and CD28 (18). Lck is required for T cell
maturation (19, 20) and signaling from the T-cell antigen receptor in
mature T lymphocytes. A derivative of the Jurkat T-cell line that
has lost expression of full-length Lck, JCaM.1, is unresponsive to
stimulation through the T-cell receptor (20). These JCaM.1 cells have
greatly reduced calcium flux into the cytoplasm and JNK activation
under conditions that fully stimulate their parental Jurkat T cells
(18, 20). Because reintroduction of Lck restores signaling, calcium
flux, and JNK activation, the absence of Lck is responsible for the
signaling defect from the T cell receptor in these cells.
The mechanism for activation of JNK after UV exposure is not
understood. UV-induced activation of JNK is not necessarily because of
chromosomal DNA damage, because JNK stimulation is observed in
enucleated cells (21). This suggests that there is at least one UV
target other than DNA responsible for activation of JNK. A role for
reactive oxygen intermediates in the propagation of the UV signal has
been suggested. N-acetylcysteine, a glutathione precursor
that elevates the reducing potential in the cytoplasm, inhibits
UV-induced JNK activation and c-jun phosphorylation (16, 22-24). This suggests that UV irradiation may lead to elevated levels
of oxidants that either directly or indirectly lead to JNK stimulation.
Lck is activated by hydrogen peroxide (25-27), a progenitor of
reactive oxygen intermediates. Because UV apparently generates reactive
oxygen intermediates and has been reported to activate the c-Src
kinase, it was reasonable to suspect that ultraviolet light might also
activate Lck in T cells. Furthermore, the finding that Lck is essential
for stimulation of JNK and extracellular signal-regulated kinase
mitogen-activated protein kinases after T-cell activation (18)
suggested that Lck is essential for propagation of some upstream
signals to these mitogen-activated protein kinases. We therefore asked
whether and how UV irradiation activated Lck in T cells and whether Src
family kinases were essential for UV-induced activation of JNK in a
variety of cells including T lymphocytes.
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EXPERIMENTAL PROCEDURES |
Construction of Lck Mutants and Generation of Stable Cell
Lines--
Wild type and mutant Lck constructs were derived from
murine lck cDNA (28). The construction of A2F505 Lck has
been described (25, 29). 208F rat fibroblasts were infected with the
retroviral expression vector LXSN containing either wild type or A2F505
Lck as described (29).
Cell Lines--
208F rat fibroblasts expressing wild type or
A2F505 Lck were maintained in Dulbecco-Vogt modified Eagle's medium
(Mediatech) supplemented with 10% calf serum (Gemini), and 600 µg/ml G418 (Geneticin). Jurkat (30), JCaM.1 (20), and HPB-MLT (31) cells, all human leukemia T-cell lines, were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. D011.10 cells (32), a
murine CD4+ T-cell hybridoma, were maintained in
Dulbecco-Vogt modified Eagle's medium supplemented with 10% fetal
bovine serum, nonessential amino acids, 1 mM sodium
pyruvate, and 50 µM 
-mercaptoethanol. CTLL-2 T cells
were grown in the same medium but supplemented with 2 mM
glutamine, 20 units/ml of human interleukin-2, and 10% supernatant of
concanavalin A-stimulated rat splenocytes.
S
YF mouse embryo fibroblasts
were grown in Dulbecco-Vogt modified Eagle's medium supplemented with
10% fetal bovine serum (33). The
SYF cell line, using the SV40
large T antigen for immortalization, was derived from embryos produced
by breeding the mice homozygous mutant for c-yes and
heterozygous mutant for c-src and c-fyn.
Preparation of Cell Lysates for Isolation of Lck and Fyn by
Immunoprecipitation--
For immunoprecipitation of Lck and Fyn, cells
were lysed in radioimmune precipitation buffer (150 mM
NaCl, 10 mM sodium phosphate, pH 7.2, 1% (w/v) sodium
deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 0.2 mM sodium vanadate, 50 mM NaF, 2 mM
EDTA, 100 kallikrein inhibitor units/ml aprotinin) for 20 min at
4 °C at a concentration of 1 × 107 cells/ml.
Lysates were clarified by centrifugation at 35,000 × g
for 45 min at 4 °C. Lck and Fyn were immunoprecipitated with rabbit
polyclonal antisera (34). After 45 min at 4 °C, 30 µl of a 10%
solution of Staphylococcus aureus cells (Pansorbin cells, Calbiochem)
were added to each lysate, and incubation was continued at 4 °C for
an additional 30 min. The immune complexes were collected by
centrifugation, washed three times in radioimmune precipitation buffer
buffer, and twice in buffer (50 mM Tris, pH 7.5, 150 mM NaCl).
Immunoblotting--
Immunoprecipitates or whole cell extracts
were subjected to electrophoresis on 15% SDS-polyacrylamide gels and
transferred to a polyvinylidene difluoride membrane (Immobilon-P,
Millipore). Western blotting was performed with polyclonal rabbit
anti-Lck (34) or anti-Fyn sera. The antibody-antigen complexes were
visualized by either enhanced chemiluminescence detection system
(Amersham Pharmacia Biotech) or 125I-protein A (ICN) as
described previously (35, 36).
In Vitro Kinase Assay--
Lck and Fyn immunoprecipitates were
incubated with 5 µCi of [
-32P]ATP (3000 Ci/mmol;
Amersham Pharmacia Biotech) in PIPES kinase buffer (40 mM
PIPES, pH 7.0, 10 mM MnCl2) for 10 min at
30 °C. For assays using Val5-angiotensin II (Star
Biochemicals) as an exogenous substrate, immunoprecipitates were
incubated with 2 mM angiotensin II in kinase buffer and 15 µCi of [-32P]ATP for 1, 3, and 5 min at room
temperature. The reactions were stopped by the addition of 5%
trichloroacetic acid and centrifuged, and the angiotensin-containing
supernatant was absorbed onto Whatman P81 phosphocellulose paper
(Whatman). The paper was washed with 0.5% phosphoric acid to remove
unincorporated [-32P]ATP. The -32P
incorporated into angiotensin was measured using liquid scintillation counting.
Isolation of JNK with GST-Jun Precipitation and JNK Kinase
Assay--
Cells were lysed in modified whole cell extract buffer (25 mM HEPES, pH 7.7, 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton
X-100, 0.5 mM dithiothreitol, 20 mM
-glycerophosphate, 0.1 mM sodium vanadate, 0.5 mM phenylmethylsulfonyl fluoride, 100 kallikrein inhibitor
units/ml aprotinin) for 20 min at 4 °C as described (3). The lysates
were clarified by centrifugation at 35,000 × g for 45 min at 4 °C. Clarified extracts were diluted such that the final
concentration of the whole cell extract buffer was 20 mM
HEPES, pH 7.7, 75 mM NaCl, 2.5 mM
MgCl2, 0.1 mM EDTA, 0.05% Triton X-100, 0.5 mM dithiothreitol, 20 mM -glycerophosphate, 0.1 mM sodium vanadate, 0.5 mM
phenylmethylsulfonyl fluoride, and 100 kallikrein inhibitor units/ml
aprotinin. The extracts were mixed with 10 µg of GST-Jun bound to 10 µl of glutathione-agarose (Amersham Pharmacia Biotech). The mixture
was tumbled at 4 °C for 3 h in a microcentrifuge tube and
subsequently spun at 10,000 × g for 1 min. The pellet
was washed four times in 1 ml of HEPES binding buffer (20 mM HEPES, pH 7.7, 50 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05%
Triton X-100). The collected beads were resuspended in 30 µl of
kinase buffer (20 mM HEPES, pH 7.6, 20 mM
MgCl2, 20 mM -glycerophosphate, 20 mM p-nitrophenyl phosphate, 0.1 mM
sodium vanadate, 2 mM dithiothreitol) containing 5 µCi of
[-32P]ATP (3000 Ci/mmol; Amersham Pharmacia Biotech).
After 20 min at 30 °C, the reaction was terminated by the addition
of SDS-polyacrylamide gel loading sample buffer.
Treatment of Cells With Hydrogen Peroxide, Pervanadate,
Anisomycin, Ultraviolet Light, and PP2--
Adherent cells were seeded
at 1 × 106 cells on 10-cm plates and allowed to
recover for 24 h. They were subsequently starved in medium
containing 0.25% calf serum for 16 h. Hydrogen peroxide was
diluted from a 30% stock solution immediately before use (Mallinkrodt) and added at the final concentration of 500 µM.
Pervanadate was prepared by oxidizing sodium vanadate (Fisher) with a
100-fold molar excess of hydrogen peroxide at room temperature for 20 min. The residual hydrogen peroxide was degraded in a 20-min incubation with catalase (Sigma) at room temperature, and pervanadate was used at
a final concentration of 200 µM. Anisomycin (Calbiochem) was administered at a final concentration of 50 µg/ml. UV irradiation was performed as described previously (3). Tissue culture medium was
removed to abrogate absorption of UV light by the phenol red in the
medium. Adherent cells were exposed directly on the plate to a
calibrated UV light source (General Electric, two G15T8 15 W bulbs)
that emitted 8.5 J/m2/s. The dose of UV irradiation was
controlled by varying the length of exposure to the lamp. Unless
indicated otherwise, cells were exposed to 40 J/m2 UV
irradiation. After treatment with one of the aforementioned agents, the
cells were incubated for 15 and 40 min at 37 °C for Lck and JNK
assays, respectively. Cells were washed with ice-cold isotonic
Tris-buffered saline and subsequently lysed directly on the tissue
culture plates. Suspension cells were counted and resuspended at 1 × 107 cells/ml in tissue culture medium containing 0.25%
calf serum. Suspension cells were exposed to the same doses of each
stimulus used for adherent cells. UV irradiation was performed in
medium lacking phenol red. After treatment, suspension cells were
collected by centrifugation at 400 × g in a Beckman
TJ-6 centrifuge for 5 min and washed once with chilled isotonic
Tris-buffered saline before lysis. PP2 (Calbiochem) was used to
specifically inhibit Src family tyrosine-protein kinases (37). Cells
were pretreated with either 10 or 20 µM PP2 for 2 h
before exposure to UV light. The cells were incubated for an additional
40 min after UV irradiation in the presence of PP2 before lysis.
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RESULTS |
Lck Activity Is Unaffected by Irradiation with Ultraviolet Light in
Jurkat T Cells--
We asked whether Lck activity was altered by UV
irradiation in human Jurkat T cells. To confirm that these cells
responded to UV irradiation, we assayed Lck and JNK activity in
parallel. A GST-jun chimera was used to bind kinases that
interact with the N terminus of c-jun, and the bound kinase
was subsequently assayed in vitro for its ability to
phosphorylate the GST-jun protein. We confirmed that this
method of measuring JNK activity gave similar results to
immunoprecipitation of epitope-tagged JNK1 and JNK2. Because both JNK1
and JNK2 are expressed in T cells and our assay does not distinguish
between them, here the term JNK refers to both JNK1 and JNK2.
Irradiation of Jurkat T cells with 40 J/m2 UV light led to
a 10-fold increase in the activity of JNK (Fig.
1A). Lck was unaffected by
this dose of UV irradiation (Fig. 1B). In contrast,
pervanadate (200 µM) stimulated both Lck and JNK 10-fold,
and hydrogen peroxide (500 µM) activated Lck and JNK 5- 10-fold, respectively (Fig. 1, A and B).
UV-induced activation of JNK was detectable in Jurkat cells after
irradiation with as little as 20 J/m2 ultraviolet light.
JNK activity increased steadily in response to increasing doses of UV
irradiation up to 1000 J/m2, where 25-fold activation was
seen, and declined gradually at higher doses (data not shown). In
contrast, Lck activity was not changed detectably at any dosage of UV
irradiation up to 1200 J/m2, a level of irradiation 60-fold
higher than that required to activate JNK (Fig. 1E). Optimum
activation of JNK occurs 40 min after UV irradiation. Lck activation
due to a stress stimulus such as hydrogen peroxide peaks 15 min after
stimulation (25). We considered the possibility that Lck was in fact
activated by UV irradiation for a brief period and that its activity
had returned to basal levels by the time we measured it. Therefore, we
assayed Lck activity at 0, 2, 5, 15, and 30 min after 200 J/m2 UV irradiation. We found no detectable change in Lck
activity at any time following UV stimulation (Fig. 1F).
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Fig. 1.
Effect of UV, H2O2,
and pervanadate on activity of Lck and JNK in Jurkat T cells. For
A-D, Jurkat T cells were exposed to either 40 J/m2 ultraviolet light (UV), 500 µM hydrogen
peroxide (H2O2), 200 µM
pervanadate (PV), or mock irradiation. A, lysates
for the analysis of JNK activity were prepared 40 min after stimulation
and assayed in vitro for their ability to phosphorylate
GST-Jun. The samples were fractionated by SDS-PAGE. Incorporation of
32P into GST-Jun was quantified on a PhosphorImager and
normalized to the amount of GST-Jun substrate in each sample.
B, Lck immunoprecipitates were prepared 15 min after
stimulation and assayed in vitro for their ability to
phosphorylate an exogenous substrate, Val5-angiotensin II.
Assay results are expressed as the rate of labeled phosphate
incorporation into the substrate/arbitrary unit of Lck. C,
for normalization, Lck protein levels in the samples were measured by
Western blotting of a fraction of the immunoprecipitates with antibody
to Lck and 125I-protein A. D, GST-Jun quantities in each sample were determined by
staining with Coomassie Brilliant Blue. E, Lck
immunoprecipitates were prepared 15 min after irradiation of Jurkat T
cells with the indicated dose of ultraviolet light, 200 µM pervanadate (PV), or mock irradiation.
F, lysates for the analysis of Lck activity were prepared at
the indicated time points (min) after 200 J/m2 UV
irradiation. Mock-irradiated cells were lysed 30 min after sham
irradiation, and cells stimulated with 500 µM hydrogen
peroxide (H2O2) were lysed after 15 min of
exposure.
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To determine whether the lack of UV-induced activation of Lck activity
in T cells was peculiar to Jurkat cells, we asked whether UV activated
Lck in mouse D011.10 hybridoma T cells, human HPB-MLT leukemic T cells,
and mouse CTLL-2 T cells. UV irradiation activated JNK 8-10-fold in
all three cell lines but had no detectable effect on Lck activity in
any of the cell lines (data not shown). In contrast, pervanadate
activated both JNK and Lck in all three cell lines 8-10- and
3-5-fold, respectively.
Lck Activity in 208F Rat Fibroblasts Is Insensitive to Ultraviolet
Light--
Activation of c-Src by UV irradiation has been observed in
adherent cells (16). Although we could not detect UV-induced Lck
activation in T cells, we asked whether more robust UV-induced activation of Lck might occur when Lck was expressed in adherent cells.
We therefore examined the effect of UV irradiation on the activity of
Lck expressed stably in rat 208F fibroblasts. 40 J/m2
ultraviolet light induced an 8-10-fold increase in JNK activity in
these cells (Fig. 2A). Lck
activity was unaffected under the same conditions (Fig. 2C).
To confirm that we had not missed a brief period of Lck activation, we
examined Lck activity at 0, 2, 5, 15, and 30 min after 200 J/m2 UV irradiation. Lck activity in adherent cells
remained unchanged at all time points after stimulation as we had
observed in Jurkat T cells (data not shown).
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Fig. 2.
Effect of UV and H2O2
on activity of wild type Lck, A2F505 Lck, and JNK in 208F
fibroblasts. Cells expressing wild type (A and
C) or A2F505 Lck (B and D) were
exposed to either 40 J/m2 ultraviolet light (UV), 500 µM hydrogen peroxide (H2O2), or
mock irradiation. Lysates for the analysis of JNK activity were
prepared 40 min after stimulation and assayed in vitro for
their ability to phosphorylate GST-Jun. The samples were fractionated
by SDS-PAGE. The amount of GST-Jun in each sample was determined by
staining with Coomassie Blue. Incorporation of 32P into
GST-Jun was quantified on a PhosphorImager and normalized to the
amount of GST-Jun substrate in each sample (A and
B). Lck immunoprecipitates were prepared 15 min after
stimulation and assayed in vitro for their ability to
phosphorylate Val5-angiotensin II. Assay results are
expressed as the rate of labeled phosphate incorporation by the
substrate/arbitrary unit of Lck (C and
D).
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It has been postulated that the UV signal is propagated by a reactive
oxygen species (16, 22). We have shown previously that the activity of
A2F505 Lck, a cytosolic Lck mutant, is noticeably more sensitive to
oxidants than wild type Lck. Hydrogen peroxide, which stimulates wild
type Lck 3-5-fold, activates A2F505 Lck as much as 20-fold (29).
Therefore, we speculated that A2F505 Lck might be more sensitive to
UV-induced free radicals than wild type Lck. UV irradiation (40 J/m2) increased JNK activity 25-40-fold in 208F
fibroblasts expressing A2F505 Lck but did not alter the activity of
A2F505 Lck detectably (Fig. 2, B and D). In
contrast, hydrogen peroxide activated both A2F505 Lck and JNK over
10-fold. These results indicate that neither wild type nor A2F505 Lck
are activated by UV light irradiation in 208F fibroblasts under
conditions that stimulate JNK dramatically.
Ultraviolet Light-induced Activation of JNK in T Cells Is Not
Dependent on Lck--
JNK is stimulated in T cells after activation
induced by cross-linking with anti-CD3 and anti-CD28 antibodies (7). It
has been shown that this CD3/CD28-dependent activation of
JNK in T cells is Lck-dependent (18). Although Lck was not
activated detectably by UV light in T cells, we asked whether Lck was
required for UV-induced activation of JNK. To this end we utilized
JCaM.1 cells, a derivative of Jurkat T cells lacking functional Lck
(20). JCaM.1 cells are largely unresponsive to T-cell receptor
stimulation. JNK activity in JCaM.1 cells was increased over 25-fold by
UV irradiation (Fig. 3A).
Treatment of both JCaM.1 and Jurkat cells with anisomycin or
pervanadate induced a 15-20-fold increase in JNK activity, and
exposure to hydrogen peroxide yielded a 5-7-fold increase in JNK
activity (Fig. 3, A and B). In each case, the extent of JNK activation was indistinguishable from that observed with
parental Jurkat cells. We therefore concluded that Lck is not required
for activation of JNK by these stress stimuli in T cells.
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Fig. 3.
JNK activity in JCaM.1 and Jurkat cells.
JCaM.1 (A) and Jurkat (B) cells were treated with
either 40 J/m2 ultraviolet light (UV), 50 µg/ml of
anisomycin (Aniso), 200 µM pervanadate
(PV), 500 µM hydrogen peroxide
(H2O2), or mock irradiation. Lysates for the
analysis of JNK activity were prepared, normalized, and presented as
described in the legend to Fig. 1.
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To examine the possibility that Fyn, another Src family kinase
expressed in Jurkat cells, played a role in the propagation of the
UV-induced signal, we measured Fyn activity after UV irradiation in
both Jurkat and JCaM.1 cells. Exposure to UV light had no detectable effect on Fyn activity (data not shown).
UV-induced Activation of JNK Is Undiminished in Mouse Embryonic
Fibroblasts Lacking Src Family Kinase Function--
It has been shown
that UV activation of JNK is normal in cells derived from mouse embryos
lacking the c-Src gene (33). This demonstrated that c-Src itself was
not absolutely essential for UV-mediated activation of JNK, but left
open the possibility that another Src kinase expressed in these cells,
c-yes, c-fyn, or c-lyn, compensated
for the absence of c-src. We therefore asked whether the
UV-induced stimulation of JNK was intact in mouse embryonic fibroblasts
that lack detectable levels of any known Src kinases (33). We compared
a cell line derived from a mouse embryo lacking the c-src,
c-yes, and c-fyn genes
(SYF) to a cell line derived
from littermates with intact c-src alleles (S+YF). Lyn, the only other Src
kinase that has been reported to be expressed in fibroblasts, was not
detectable in SYF cells
(33).3 We additionally
examined UV-induced activation of JNK in triple knockout cells
(SYF) that express c-Src from
a stably introduced c-Src expression vector. UV irradiation stimulated
JNK 4-6-fold in all three cell lines (Fig.
4).
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Fig. 4.
JNK activity in mouse embryonic fibroblasts
lacking Src kinases. Triple knock out
SYF cells, double knock out
S+YF cells derived from a
littermate embryo, and SYF
cells expressing c-Src from a stably introduced c-Src expression vector
were treated with either 40 J/m2 ultraviolet light (UV),
200 µM pervanadate (PV), or mock irradiation.
Lysates were prepared 40 min after stimulation and were assayed
in vitro for their ability to phosphorylate GST-jun. The
samples were fractionated by SDS-PAGE, stained with Coomassie Blue, and
quantified on a PhosphorImager.
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JNK Activity Is Unaffected by Treatment with the Src Kinase
Inhibitor PP2--
The pyrazolopyrimidine PP2 is a fairly specific
inhibitor of Src family kinases. It inhibits the in vitro
activity of Lck and Fyn kinases with an IC50 of 5 nM (37). In contrast, the IC50 of PP2 for
ZAP-70 and JAK2, two tyrosine kinases expressed in hematopoetic cells
that are not Src family members, is greater than 100 and 50 µM PP2, respectively (37). As another approach for
evaluating a role for Src kinases in UV-induced activation of JNK, we
asked whether PP2 suppressed UV activation of JNK in 293T, Jurkat, and
HeLa cells. A 2-h PP2 (20 µM) treatment of 293T cells
ectopically expressing a genetically activated Lck (F505) reduced
tyrosine phosphorylation of cellular proteins over 90% (Fig.
5A). JNK activation in these
cells was largely unaffected by treatment with PP2 (Fig.
5B). In three experiments, we detected no more than 15%
reduction in JNK activation after treatment with PP2 (20 µM) for 2 h. PP2 was equally effective in reducing
cellular tyrosine phosphorylation in Jurkat and HeLa cells, whereas JNK activation by UV irradiation remained unaffected in these cells (data
not shown).
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Fig. 5.
JNK activity in 293T cells treated with the
Src kinase inhibitor PP2. 293T cells expressing activated F505 Lck
were pretreated with either 10 or 20 µM PP2 for 2 h
and then exposed to either 40 J/m2 ultraviolet light (UV)
or mock irradiation. Lysates were prepared 40 min after stimulation
with continuous PP2 treatment. A, clarified lysates were
fractionated by SDS-PAGE stained with antiphosphotyrosine rabbit
polyclonal antibody and 125I-protein A. B,
lysates for the analysis of JNK activity were assayed in
vitro for their ability to phosphorylate GST-jun. The samples were
fractionated by SDS-PAGE, stained with Coomassie Blue, and quantified
on a PhosphorImager.
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DISCUSSION |
Exposure of bacteria to UV light and other DNA damaging agents
induces the SOS response (38, 39). Either DNA damage or by-products of
DNA damage are thought to initiate this response. The mammalian stress
response was also originally attributed to DNA damage. However, recent
work has shown that stress responses can occur in the absence of DNA
damage (40-43) and that not all DNA damaging agents yield identical
responses or produce the same outcome (40, 44). The stress response to
reactive oxygen intermediates and UV light is likely to be triggered in
the cytoplasm or the plasma membrane, because both nuclear factor-
B
and activator protein-1 are activated by UV light in enucleated cells
(21). It has been reported that UV irradiation activates the c-Src
protein kinase in HeLa cells and UV-induced activation of JNK is
inhibited by expression of a catalytically inactive v-Src protein (16). This suggested that c-Src was an essential component of the cellular response to ultraviolet light. Here, we asked whether UV-induced activation of Src kinases occurred in lymphocytes, and if so, was it
important. Because Lck is an abundant member of the Src family of
tyrosine-protein kinases in T lymphocytes, is activated readily by
hydrogen peroxide, and is known to be essential for CD3/CD28-dependent activation of JNK, we examined the role
of Lck in UV-induced activation of JNK in T cells (18, 25, 28, 45).
Exposure of either human or murine T cells to UV light did not
stimulate Lck under conditions that activated JNK robustly (Fig. 1). We
then asked whether UV-induced activation of Src kinases was unique to
adherent cells. However, neither wild type Lck nor the hypersensitive
A2F505 mutant of Lck were stimulated after UV irradiation of
fibroblasts in which these Lck proteins were expressed ectopically
(Fig. 2). Although Lck was not activated by UV irradiation, the
possibility remained that Lck was required for the stimulation of JNK
in an activation-independent manner. We therefore asked whether UV
could activate JNK in JCaM.1 cells that lack Lck. We found that JNK was
activated to the same extent in the Lck-deficient JCaM.1 cells as in
the parental Jurkat T cells (Fig. 3). Fyn, another Src kinase, was also
unaffected by exposure to UV light in JCaM.1 and Jurkat cells,
indicating that another abundant Src kinase was not compensating for
the loss of Lck (data not shown).
We also examined the ability of UV to activate JNK in fibroblasts that
express no known Src kinases (33). We found that UV-induced activation
of JNK was intact in SYF
cells that express no Src kinases and indistinguishable from that
observed in cells from littermates with functional c-src gene (Fig. 4). We finally evaluated the role of the Src family kinases
in the UV response using the Src kinase inhibitor PP2. Under conditions
where PP2 treatment inhibited more than 90% of total tyrosine
phosphorylation, UV-induced activation of JNK in 293T cells ectopically
expressing activated Lck remained largely intact (Fig. 5). PP2
treatment similarly reduced tyrosine phosphorylation in Jurkat T cells
and human HeLa cells without noticeably affecting UV-induced activation
of JNK (data not shown).
We conclude that the activity of Src kinases is not required for
stimulation of JNK by UV irradiation in any of the cells we tested. Our
results do not, however, suggest that Src kinases or tyrosine-protein
kinases cannot activate JNK. Sodium vanadate, which induces elevated
levels of tyrosine protein phosphorylation, activates JNK in a variety
of cell types, and overexpression of Lck stimulates JNK in Jurkat T
cells (18), rather our data suggest that in most cells Src kinases are
not necessarily involved in, or required for, UV-induced activation of
JNK. HeLa cells, in which c-Src has been reported to play an important
role in UV signaling, appear to be an exceptional cell line in this
respect (16). We however observed that treatment of HeLa cells with the
Src kinase inhibitor PP2 did not affect UV-induced activation of JNK
(data not shown). It is possible that the observed inhibitory effect of
inactive v-Src was due to general toxicity from overexpression of a
catalytically inactive Src molecule that is unburdened by intramolecular regulation. Interestingly, NIH3T3 cells lacking the
c-Src gene exhibit reduced JNK activation by the alkylating agent
methyl methanesulfonate but have normal UV-induced activation of JNK
(46).
The apparent dispensability for Src kinases in activation of JNK is
certainly not unprecedented. Stimulation of the Fas/APO-1/CD95 receptor, a member of the tumor necrosis factor (TNF) receptor family
(47), results in JNK activation and apoptosis in a wide variety of cell
types (48). JNK activation by Fas stimulation is, however, normal in
Lck-deficient cells, suggesting that Fas-induced activation of JNK is
not Lck-dependent (49). TNF-
treatment results in
clustering of the ubiquitously expressed p55 TNF- receptor and
subsequent stimulation of JNK in a wide variety of cell types (7, 48,
50, 51). Irradiation of cells with UV is also reported to lead to the
clustering of TNF- receptors (52). This clustering may be in fact
critical in UV-induced activation of JNK because irradiation with UV at
10 °C, below the transition temperature of cellular membranes, fails
to induce both TNF- receptor clustering and JNK activation (52). The TNF- receptor-associated death domain protein and TNF-
receptor-associated factor bind to the clustered TNF- receptors
(53-55) and mediate signaling to JNK (56-59). UV-induced activation
of JNK may be dependent on triggering of the TNF- receptor and
signaling through TNF- receptor-associated death domain protein and
TNF- receptor-associated factor proteins, whereas Lck and other Src
kinases may be involved in activation of JNK only through the pathway
downstream of the T-cell receptor and CD28.
| |
ACKNOWLEDGEMENTS |
We thank Estella Jacinto for providing
numerous JNK reagents, and we are indebted to Richard Klinghoffer and
Phillipe Soriano for the very generous gift of the
SYF cells. We thank Gary
Chiang and Roberta Schulte for critical reading of the manuscript.
| |
FOOTNOTES |
*
This work was supported by Grants CA14195, CA17289, and
CA42350 from the NCI, National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Supported by Training Grant DK07202 from the National
Institutes of Health. To whom correspondence should be addressed:
Molecular Biology and Virology Laboratory, The Salk Institute for
Biological Studies, 10010 North Torrey Pines Rd., La Jolla, California
92037. Tel.: 858-453-4100 (ext. 1477); Fax: 858-457-4765; E-mail:
kamdjadi@aim.salk.edu.
Published, JBC Papers in Press, May 8, 2000, DOI 10.1074/jbc.M002573200
2
Because both JNK1 and JNK2 are comparably
expressed in T cells and our assay measures c-jun N-terminal
kinase activity, in this report JNK refers to both JNK1 and JNK2.
3
K. Amdjadi and B. M. Sefton, unpublished results.
| |
ABBREVIATIONS |
The abbreviations used are:
JNK, c-jun N-terminal kinase;
PIPES, 1,4-piperazinediethanesulfonic acid;
GST, glutathione
S-transferase;
TNF, tumor necrosis factor;
PAGE, polyacrylamide gel electrophoresis.
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