Earlysignalingeventsinducedby280-nmUV irradiation
Yukihito Kabuyama
1
, Miwako K. Homma
1
, Tomohiro Kurosaki
2
and Yoshimi Homma
1
1
Department of Biomolecular Science, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine,
Fukushima, Japan;
2
Department of Molecular Genetics, Institute for Hepatic Research, Kansai Medical University, Moriguchi, Japan
The d epletion of stratospheric o zone results in increased UV
(ultraviolet) light below 300 nm, and has signi®cant eects
on biological systems. To better understand the eects of UV
in this range, earlysignalingeventsinducedby monochro-
matic UV light were investigated using the ch icken B cell line
DT40 and mutants lacking protein tyrosine kinases ( PTKs).
Among MAP kinase family proteins, P38 MAP kinase (P38)
was selectively and immediately activated by 280 nm UV
light in cultured DT40 cells. Activation of P38 was com-
pletely inhibited in cells de®cient in Lyn and Btk. Introduc-
tion of wild-type Btk, but not kinase-inactive Btk, restored
the P 38 activation. In contrast, P38 activation was not
aected in Syk-de®cient cells. Tyrosine phosphorylation of
Lyn was inducedby 280 n m UV light, and pretreatment of
cells with orthovanadate, an inhibitor of protein tyrosine
phosphatase (PTP), enhanced both L yn phosphorylation
and P38 activation. These r esults show that Lyn and Btk are
upstream regulators of the P38 s ignaling pathway activated
by 280 nm UV light and that t he triggering event likely
involves inactivation of PTP. Furthermore, cell death
induced by 280 nm UV i rradiation were a ugmented by Btk
depletion or a speci®c inhibitor for P38, and partially
blocked in Lyn-de®cient cells, suggesting that the Lyn±Btk±
P38 pathway promotes cell s urvival while other Lyn path-
ways stimulate cell death.
Keywords: ultraviolet; signal transduction; protein tyrosine
kinase; protein tyrosine phosphatase.
Ultraviolet (UV) sunlight is an important environmental
factor in the etiology of skin cancer, aging and immuno-
suppression [1,2]. T he harmful effects of UV light are mainly
attributed to the UVB ( 280±320 nm) r ange, and it is
excessive exposure to these wavelengths that accounts for
the risk of stratospheric ozon e depletion in biological and
ecological systems [3]. Studies have shown that irradiating
mammalian cells with UVB light leads to transcriptional
activation of immediate early genes such as c-fos and c-jun
[4,5]. This UV response d epends on several primary target
molecules, including chromosomal DNA. I n DNA, U V
light induces pyrimidine dimers and 6-4 photoproducts,
resulting in mutations and carcinogenesis [6,7]. Mammalian
cells also respond to DNA damage by transcribing the genes
encoding cellular proteins that control DNA repair, DNA
synthesis, transcription, and c ell cycle regulation. However,
nuclear events triggered by DNA damage are not the only
response to UV irradiation. Recent studies have revealed
that UVirradiation activates several cytoplasmic s ignal
transduction pathways [8±10], including pathways regulated
by extracellular signal regulated kinases (ERKs), c-Jun
N-terminal kinases (JNKs), and P38 MAP kinases (P38).
Although much is known about the regulation and
function of MAP k inase pathways, t he mechanism by w hich
UV light triggers the activation is poorly understood. It has
been suggested that reactive oxygen intermediates (ROI),
such as singlet oxygen, superoxide radicals, hydroxyl
radicals, and H
2
O
2
, are increased in response to UV and
may be key regulators of UV-induced signaling pathways
[11±14]. More recent studies [15,16] have shown that UV
irradiation also causes oxidative damage to catalytic
sulfhydryl groups of protein tyrosine phosphatases (PTPs)
that dephosphorylate transmembrane receptor tyrosine
kinases, such as epidermal growth factor receptor.
Decreased phosphatase activity, combined with high intrin-
sic kinase activity of t he receptor tyrosine kinase, results in
the activation of signal transduction pathways such as
ERK, which correlates with t he UV-induced inhibition of
EGFR dephosphorylation [15,16]. T hese results are the ®rst
direct evidence of the regulation o f PTKs and PTPs by
UV-induced oxidative damage, and the function of these
enzymes as regulators of signaling pathways responsive to
irradiation. Previously, we r eported that MAP kinases [17]
and PtdIns 3-kinases [18] are regulated separately and
independently in a s trict wavelength-speci®c manner. In
particular, P 38 was activated byUV light at around
280 nm. In the present study, we further invest igated early
signaling eventsinducedby 280 nm UV i rradiation. We also
present evidence that Lyn, B tk and P38 are involved i n the
cell death response to UV-irradiation at 280 nm.
MATERIALS AND METHODS
Cell culture and UV irradiation
Wild-type and mutant DT40 cells were cultured in RPMI
1640 medium (Sigma) supplemented with 10% fetal bovine
serum and 1% chicken serum. The cell density was
Correspondence to Y. Homma, Fukushima Medical University School
of Medicine, Fukushima 960-1295, Japan. Fax: + 8 1 24 548 3041,
Tel.: + 81 24 548 2111 ext. 281 0, E-mail: yoshihom@fmu.ac.jp
Abbreviations:UVB,ultravioletB;ROI,reactiveoxygenintermedi-
ates; JNK, c-Jun N-terminal kinase; MAP kinase, mitogen activated
protein kinase; ERK, extracellular signal-related kinase; P38, P38
MAP kinase; PtdIns 3-kinase, p hosphatidylinositol 3-kinase; EGF,
epidermal growth factor; TNF, tumor necrosis factor; PTK, protein
tyrosine kinase; PTP, protein tyrosine phosphatase; MTT,
[3-(4,5-dimethylthiazol-s-yl)-2,5-diphenyl] tetrazolium bromide.
(Received 14 August 2001, revised 6 November 2001, accepted 23
November 2001)
Eur. J. Biochem. 269, 664±670 (2002) Ó FEBS 2002
maintained at 1±5 ´ 10
5
cellsámL
)1
. T he culture medium
was replaced with NaCl/P
i
, and the cell concentration w as
adjusted to 1 ´ 10
6
cellsámL
)1
. Irradiation was carried out
in quartz cuvettes using a spectro¯uorophotometer
(RF5300PC, Shimazu, Tokyo, Japan) as a source of
monochromatic UV. The UV energy was controlled by
the irradiation time and monitored with a broadband
energy meter ( 13PEM001, Melles Griot, Boulder, C O,
USA). Wild-type and kinase-inactive Btk cDNAs were
cloned into p Apuro expression vector [25]. For DNA
transfection into DT40 cells, DNA were line arized and
electroporated as described p reviously [25]. C ell clones
expressing Btk were selected in the presence of puromycin
(0.5 lgámL
)1
). The expression of Btk was analysed by
immunoblotting, and clones, in which the expression level of
Btk was almost same to that in wild-typ e cells, were used in
this study.
P38 kinase assay
The activity of P38 was measured by in vitro kinase assays
using PHAS-1 as substrate [17]. Lysates were prepared by
solubilizing cells in buffer containing 20 m
M
Tris/HCl
(pH 7 .4), 1% (w/v) NP-40, 0.27
M
sucrose, 1 m
M
EDTA,
1m
M
EGTA, 10 m
M
b-glycerophosphate, 1 m
M
benzami-
dine, 50 m
M
NaF, 10 lgámL
)1
pepstatin A, 10 lgámL
)1
aprotinin, and 10 lgámL
)1
leupeptin. P38 was immunopre-
cipitated using anti-P38 Ig (N-17, Santa Cruz Biotechnol-
ogy, Inc., Santa Cruz, CA, USA), resuspended in reaction
buffer (25 m
M
Hepes/NaOH, pH 7.5, 10 m
M
magnesium
acetate, 50 l
M
ATP), and incubated for 15 min at 37 °C
with [c-
32
P] ATP ( 50 lCiámL
)1
) and substrate PHAS-1
(250 lgámL
)1
). The substrates were resolved by SDS/PAGE
(14% acrylamide) and visualized by autoradiography. The
incorporation of phosphate was quanti®ed using a Fuji BAS
1000 bioimaging analyz er.
Immunoblotting
Immunodetection of tyrosine-phosphorylated proteins
was carried out using anti-phosphotyrosine Ig (Promega).
Total P38, Lyn and Btk w ere probed with anti-P38 (N-
17, Santa Cruz Biotechnology, Inc), anti-Lyn or anti-Btk
Ig [19], respectively. Incubatio n with secondary antibody
conjugated to horseradish peroxidase was f ollowed by
chemiluminescence detection (Amersham Pharmacia
Biotech).
Dephosphorylation of Lyn
in vitro
Wild-type DT40 cells were pretreated with 1 m
M
iodoace-
tamide for 15 min to inactivate PTPs. Cells were then
exposed to UV light ( 280 nm) for 5 min to enhance the
phosphorylation of Lyn. C ell lysates prepared from irradi-
ated cells were referred to as the Ôphosphorylated LynÕ
fraction. On the other hand, Lyn-de®cient cells were either
treatedwithNa
3
VO
4
for 10 min, or irradiated with 280 nm
UV light for 10 min. The cell lysates prepared from these
cells were used as ÔphosphataseÕ fractions. In the in vitro Lyn
dephosphorylation assay, the Ôphosphorylated LynÕ fraction
and the ÔphosphataseÕ fractions were mixed and incubated at
37 °C for speci®c periods. The reaction was stopped by
addition of an equal volume of 2 ´ SDS sample buffer, and
tyrosine phosphorylation of Lyn was analyzed by Western
blotting with anti-Lyn and anti-phosphotyrosine Ig.
Cell viability
Cell viability was measured by an [3-(4,5-dimethylthiazol-
s-yl)-2,5-diphenyl] tetrazolium bromide (MTT) assay at 24 h
post-UV irradiation a s d escribed [18]. B rie¯y, cells were
treated with MTT (®nal concentration, 0.5 mgámL
)1
)and
incubated for 30 min, prior to r emoval of the medium and
addition of dimethylsulfoxide (500 lL) to solubilize the
MTT formazon product. Absorbances were measured at
595 nm, and plotted as a m easure of the relative number of
cells, normalized to nonirradiated cells.
RESULTS
Wavelength-speci®c activation of P38
by monochromatic UV irradiation
Chicken DT40 cells were exposed to monochromatic UV
light ranging from 260 to 360 nm a t increments of 20 nm,
and lysates of irradiated cells were analyzed for P38 kinase
activity. As shown in Fig. 1A, P38 was activated at 260
and 280 nm, with the most effective wavelength being
280 nm. Next, activation of P38 by 280 nm UV light was
determined as a function of time (Fig. 1B) and energy
dosage (Fig. 1C). UV led to a rapid activation of P38,
which p eaked within seconds after irradiation, and
declined to basal l evels by 2 min. The k inase activity
increased in a energy dependent manner up to 80 Jám
)2
,
and then decreased at higher energy levels. No cytotoxic
effect was observed a t the higher energy levels. P38 was
not activated by UVA light or long-wavelength UVB light.
Activation of JNK or ERK was not observed within
10 min in cells irradiated with monochromatic UV light at
any wavelength or energy dose examined (data not
shown). These results clearly indicate a selective activation
of P38 by U V irradiation at 280 nm in chicken B cells and
was c onsistent with the r esults of our previous study in
human T cell lines [17].
280 nm UVinduced P38 activation requires
Btk and Lyn, but not Syk
To identify regulatory factors involved in the activation of
P38 kinase, we examined the effect of the tyrosine kinase
inhibitor, genistein, on the activation. Pretreatment of cells
with genistein completely inhibited the UV-induced activa-
tion of P38 (Fig. 2A), suggesting that genistein-sensitive
protein t yrosine kinases (PTKs) are involved in stimulation
of P38 activity. To examine the PTKs regulating UV-
induced P38 activation, we compared the P38 response in
wild-type ce lls and mutant cells de®cient in nonreceptor
PTKs, Lyn, Syk, and Btk. No differences in endogenous
P38 expression levels were observed between the wild-type
and these PTK-de®cient cells. As shown in Fig. 2B, UV-
induced P38 activation was completely inhibited in Lyn- or
Btk-de®cient cells, whereas the kinase activity was main-
tained in Syk-de®cient cells. Introduction of wild-type Btk
restored the P38 activation inducedby 280 nm UV irradi-
ation (Fig. 2C), but expression of the kinase-inactive Btk
did not. Taken together, these results clearly indicate that
Ó FEBS 2002 PTP inhibition by 280 nm UV (Eur. J. Biochem. 269) 665
Lyn and Btk, but not Syk, are essential for the activation of
P38 and that Btk is required for UVsignaling to P38 kinase.
It has been reported that tyrosine phosphorylation of Lyn
is required f or activation in B cell receptor signaling systems
[20]. Therefore, the regulation of Lyn activity byUV was
monitored indirectly, by analyzing its reactivity with anti-
phosphotyrosine Ig. Figure 2D shows the pro®les of
280 nm UV-induced protein tyrosine phosphorylation in
wild-type vs. PTK-de®cient cells. Several proteins showed
enhanced tyrosine phosphorylation after UVirradiation in
wild-type cells. The major tyrosine-phosphorylated protein,
appearing w ith a molecular mass of 57 kDa, was identi®ed
as Lyn by immunoblotting with anti-Lyn Ig (Fig. 2D).
Enhancement of tyrosine phosphorylation inducedby UV
light was abolished in Lyn-de®cient ce lls. Therefore, Lyn is
an upstream kinase in the 280 nm UV signaling. On the
other hand, Lyn tyrosine phosphorylation was slightly
affected in Btk-de®cient cells, suggesting that Btk is partially
involved in the phosphorylation o f Lyn.
Involvement of protein tyrosine phosphatase
in 280 nm UVinduced P38 activation
A recent study [16] showing that protein tyrosine phospha-
tases (PTPs) are inhibited by ROI suggests the involvement
of tyrosine phosphatases in the regulation of Lyn induced
by 280 nm UV. The observation that the activation of P38
by 280 nm UV light was completely inhibited by antioxi-
dants s uch a s reduced glutathione, vitamin E and mannitol
(Fig. 2 A), suggests a critical role for ROI and possibly PTPs
in the activation. Therefore, the involvement of PTPs was
examined by pretreating D T40 cells with vanadate, a
nonspeci®c inhibitor of phosphatases, and then irradiating
them with 280 nm of UV. If tyrosine phosphatases function
upstream in this pathway, treatment with phosphatase
inhibitors should synergize in signaling. Vanadate by itself
caused a slight increase of Lyn phosphorylation and P38
activation even in the absence o f UV stimulation (Fig. 3). A
signi®cant enhancement in Lyn phosphorylation and P38
activation was observed in cells pretreated with vanadate
before the UV stimulation (Fig. 3A,B). These results
indicate that PTPs likely function upstream of Lyn.
To further examine the involvement of PTPs in the
280 nm UV signaling, we prepared a Ôphosphorylated Lyn Õ
fraction and two different Ôphosphatase Õ fractions and
monitored d ephosphorylation of the Ôphosphorylated LynÕ
induced by the ÔphosphataseÕ.TheÔphosphorylated LynÕ
fraction was prepared from UV-treated DT40 cells which
were previously incubated with iodoacetamide to inactivate
PTPs. The ÔphosphataseÕ fractions were independently
Fig. 1. Activation of P38 by 280 nm UV irradiation. (A) Wavelength-
dependent activation of P38. Wild-type DT40 cells were exposed for
5 min (from )5 min to 0 m in) to monochromatic UV irradiation
ranging from 260 nm to 360 nm in increments of 20 nm. Irradiated
energy varied with wavelength (30 Jám
)2
at 260 nm UV; 60 Jám
)2
at
280 nm UV; 300 Jám
)2
at 350 and 360 nm UV). Immediately following
the irradiation, cell lysates were prepared and P38 activity was ana-
lyzed using PHAS-1 as substrate. The total amount of P38 MAP
kinase was also analyzed by immunoblotting using anti-P38 Ig. (B )
Time dependence of P38 activation by monochromatic UV. DT40 cells
were exposed to UV at 280 nm. At the indicated times after irradiation,
P38 activity was analyzed as in (A). (C) Dose-dependence of P38
activation. DT40 cells we re exposed to the indicated doses of UV ligh t
(280 n m, irradiation for 50 s to 1 0 min). Cell extracts were prepared
and kinase activities o f P38 were analyzed as in (A). Autoradiograms
A±C a re each representative of ®ve independent expe riments. Sum-
maries of results (means SE, n 5) are shown.
666 Y. Kabuyama et al. (Eur. J. Biochem. 269) Ó FEBS 2002
obtained from nonstimulated, 280 nm UV-irradiated, and
vanadate-treated Lyn-de®cient DT40 cells. The Ôphospho-
rylated L ynÕ fraction was mixed with the ÔphosphataseÕ
fraction and the resulting dephosphorylation of Lyn was
detected by anti-phosphotyrosine immunoblotting (Fig. 4).
As expected, mixing with the ÔphosphataseÕ derived from
nonstimulated cells led to the immediate dephosphorylation
of Lyn in the Ôphosphorylated LynÕ fraction. In contrast, this
dephosphorylation was completely inhibited when the
ÔphosphataseÕ fraction derived from the UV-irradiated and
vanadate-treated cells was used ( Fig. 4). These results
indicate that 280 nm UV inhibits Lyn-directed PTPs, which
might be a triggering event to activate the P38 signaling
pathway inducedby the irradiation.
Role of Lyn, Btk, and P38 in UV- induced cell death
We examined the involvement of PTKs and P38 in cell
death p rocess following UV irradiation. As shown in
Fig. 5, irradiation of cells with 280 nm UV light le d t o
Fig. 2. 280 nm UV-induced P38 activation requires Lyn and Btk, but not Syk. (A) Eect of tyrosine kinase inhibitor and antioxidants on P38
activation. DT40 cells were preincubated for 20 min either with genistein (100 l
M
), vitamin E (100 l
M
), GSH (250 l
M
), or mannitol (100 m
M
), and
then irradiated with 280 nm UV light. P38 activities were analyzed as described in Fig. 1. (B) P38 activation in PTK-de®cient cells. Wild-type (WT),
Lyn-de®cient (Lyn
±
), Syk-de®cient (Syk
±
), and B tk-de® cient (Btk
±
) cells w ere irradiated with 280 nm UV light and P38 act ivity was analyzed a s
described in Fig. 1. (C) Expression of Btk restores 280 nm UV-induced P38 activation in Btk-de®cient cells. Wild-type cells (WT), Btk-de®cient cells
(Btk
±
), Btk-de®cient cells transfected with wild-type btk cDNA (Btk
±
/Btk), and Btk-de®cient cells transfected with kinase-inactive btk cDNA [Btk
±
/
Btk(K
±
)] were irradiated with 280 nm UV light, and P38 activity was analyzed. (D) Tyrosine phosphorylation of whole cell proteins in Lyn-, Syk- or
Btk-de®cient DT40 cells. At 1 min after UVirradiation at 280 nm, w hole cell lysates were prepared and analyzed by immunoblotting with anti-
phosphotyrosine Ig. Tyrosin e-phosphorylated Lyn was immunoprecipitated with anti-phosphotyrosine Ig and visualized by immuno blotting with
anti-Lyn Ig (25).
Fig. 3. Involvement of protein tyrosine phos-
phatases in Lyn phosphorylation and P38 acti-
vation. Wild-type DT40 cells were
preincubated f or 30 min with Na
3
VO
4
(0, 0.1,
and 1 m
M
), and then exposed to UV light at
280 nm. P38 activity (A) and tyrosine phos-
phorylation of Lyn (B) w ere analyzed as
described in Fig. 2. The total amount of P38
(A) and Lyn (B) was analyzed by imunoblot-
ting. Autoradiograms A and B are each rep-
resentative of ®ve independent experiments.
Summaries of results (means SE, n 5) are
shown.
Ó FEBS 2002 PTP inhibition by 280 nm UV (Eur. J. Biochem. 269) 667
signi®cant drop in the viability o f wild-type DT40 cells in a
dose-dependent fashion. DNA fragmentation was d etected
in these irradiated cells (data not shown). Cells de®cient in
Btk showed nearly a twofold enhancement of toxicity to
280 nm UV irradiation, as compared to wild-type cells
(Fig. 5A). Expression of wild-type B tk in the Btk-de®cien t
cells restored the cell viability to wild-type levels (Fig. 5B),
whereas kinase-inactive Btk failed to rescue the cell
viability. The involvement of P38 activation in the
UV-induced cell death was assessed using SB203580, a
speci®c inhibitor of P38 kinase. Treatment with SB203580
signi®cantly enhanced the response inducing cell death, and
completely inhibited P38 activity in the se cells (Fig. 5A).
These results indicate an important role for t he P38
signaling pathway in protecting cell death following UV
irradiation. On the o ther hand, a de®ciency of Lyn had the
opposite effect, rendering cells resistant to 280 nm
UV-induced cell death. This suggests that Lyn induces cell
death through a precise mechanism d istinct from t he
Btk±P38 pathway (Fig. 6).
DISCUSSION
We have reported that MAP kinases and PtdIns 3-kinases
are r egulated sep arately and independently in a strict
wavelength-speci®c manner [17,18]. In particular, P38 was
selectively activated byUV light at around 280 n m. In the
present s tudy, we investigated earlysignalingevents induced
by UVirradiation at 280 nm using D T40 and PTK-
defective mutants thereof. The r esults demonstrated that
activation of P 38 was completely inhibited in cells de®cient
in Lyn and Btk, but not in Syk-de®cient cells. Tyrosine
phosphorylation of Lyn was inducedby 280 nm UV, and
pretreatment of cells with orthovanadate, an inhibitor of
PTPs, enhanced both Lyn phosphorylation and P38 acti-
vation. The tyrosine phosphorylation of Lyn was signi®-
cantly diminished in the Lyn-de®cient mutant. In contrast,
the phosphorylation of Lyn was clearly unaffected in the
Btk-defective mutant. These results show that Lyn and Btk
are upstream regulators o f the P38 signaling pathway
activated by 280 nm UV, and that Lyn s eems to be an
upstream regulator of Btk.
Using the same DT40 cell lines, it has been found that
PTK controls activation of MAP kinases; ERK is activated
by Syk and JNK i s activated by both Syk and Btk in B cell
receptor signaling systems [22]. Moreover, B cell receptor-
mediated P38 activation requires both S yk and Lyn, but n ot
Btk. On the o ther hand, Syk i s required for JNK activation
in cells treated with high doses of H
2
O
2
, whereas in cells
treated with low doses of H
2
O
2
, the activation of JNK is n ot
dependent on Syk [21]. Osmotic stress induces the activation
of Lyn and Syk, but does not lead to activation of JNK [21].
Thus, different stimulatory signals activate different sets of
PTKs, resulting in different patterns of activation of MAP
kinase proteins. Our ®nding that Lyn a nd Btk r egulate
280 nm UV-induced P38 signaling reveals a novel mech-
anism, distinct from ®ndings made w ith the B cell r eceptor
systems [26].
The initial cellular signals that f ollow UVirradiation and
trigger the activation of downstream MAP kinase signaling
pathways are still controversial, but in large part, appear to
be independent of chromosomal DNA damage [23]. High
doses of UVC have been shown to provoke ligand-
independent activation of EGFR and PDGFR, resulting
in activation of ERK [15,16]. This process is mediated by the
inactivation of receptor-directed phosphatases via ROI
generated by U V irradiation. I n addition, the f unctional
down-modulation of receptors for EGF, TNF, and IL-1 is
suf®cient to block UVB-induced activation of JNK, imply-
ing important roles for these receptors in t he JNK response
to UV. Recently, Mihail et al. have also reported a novel
signaling pathway to JNK, initiated by rRNA damage to
functionally active ribosomes [24]. However, t he early
signaling events that induce the activation of P38 are not
well understood. The evidence presented here supports that
the activation of Lyn inducedby suppression o f PTPs is an
important t riggering event in the activation of P38 kinase by
280 nm UV. This is consistent with the experimental ®nding
that this process is blocked by antioxidants (Fig. 2A). These
results establish that the UV-induced activation of MAP
kinase proteins is triggered by similar mechanisms, involv-
ing inactivation of PTPs potentially through t he generation
of ROI. We speculate that Lyn-directed P TPs are sensitive
to ROI speci®cally generated at 280 nm UV. The nature of
Fig. 4. UVirradiation inhibits the activity of Lyn-directed protein
tyrosine phosphatases. Cell extracts were prepared as follows. Wild-
type DT40 cells pretreated with iodoacetamide to irreversibly inacti-
vate p rotein tyrosine phosphatases w e re e xp osed to UV light at
280 nm to induce tyrosine phosphorylation of Lyn and cell extracts
were p repared (referred to as the Ôphosphorylated LynÕ fractio n). Lyn-
de®cient cells were incubated in the absence or presence of Na
3
VO
4
for
30 min, or irradiated with 280 nm UV for 10 min, and cell lysates were
preparedandusedastheÔphosphataseÕ fraction. The Ôphosphorylated
LynÕ fraction was m ixed with the ÔphosphataseÕ fraction or the buer
used fo r lysate preparation, a nd then incubated at 37 °C for 0, 1, or
5 m in. Reactions were stopped by adding an equal volume of 2 ´ SDS
sample buer, and tyrosine phosphorylation of Lyn was analyzed by
immunoblotting. The autoradiogram (top) is representative of three
independent experiments. A summary of the results (means SE,
n 3) is shown in the bottom panel.
668 Y. Kabuyama et al. (Eur. J. Biochem. 269) Ó FEBS 2002
ROI generated byUV at various wavelengths, and the
identi®cation of speci®c PTPs that are inactivated by UV
irradiation need to be studied further.
The result that a speci®c inhibitor for P38, SB203580,
abolished the cell d eath response (Fig. 5) demonstrates that
the P38 signaling pathway controls cell fate i n 280 nm UV-
irradiated cells. Namely, P38 has an important function in
the cell survival process. Because 280 nm UV-dependent
activation of P38 is observed in a number of mammalian
cells [17], P38 activation may be a signal against UV-
induced cell death commonly conserved among cell types.
Although Lyn and Btk function as upstream regulators of
P38, their effects o n cell viability are quite different. The cell
death response inducedby 280 nm UVirradiation was
augmented by Btk depletion as by a speci®c inhibitor for
P38, and partially blocked in L yn-de®cient cells, suggesting
that P38 promotes cell survival whereas Lyn bifurcates
towards cell survival and cell death pathways.
Based on the ®ndings of this study, we p ropose the
following model for the regulation of P38 by 280 nm UV in
DT40 cells (Fig. 6). UVirradiation selectively regulates Lyn
and Btk tyrosine kinases via mechanisms involving inhibi-
tion of PTPs. It is likely that Btk is activated downstream of
Lyn, although both tyrosine kinases are necessary for the
initial UV-triggered events to induce P38 activation. Lyn
generates a t least two signaling p athways; a Lyn ±Btk
pathway activates P38 to produce signals promoting
survival, while other Lyn pathway provokes cell death. In
this context, Lyn controls the divergence of two pathways,
which regulate the balance between cell death and survival
processes.
ACKNOWLEDGEMENT
This study was supported by g rants from the Ministry of Educ ation,
Science, Sports and Culture.
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Fig. 6. A model for 280 nm UV-induced signal transduction. UV
induces t he generation of ROI i n irradiated c ells, which inhibits
Lyn-directed PTPs, resulting in the apparent activation of Lyn. Lyn
regulates two distinct signaling pathways; one induces cell death, and
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signaling pathway.
Fig. 5. Lyn, Btk, and P38 regulate cell viability
after UV irradiation. (A) UV-induced cell
death in PTK-de®cient DT 40 cells. Wild-type
(open circle), Lyn-de®cient (open d iamond),
Syk-de®cie nt (ope n square ), and B tk-d e®cient
DT40 (close triangle) cells were irradiated with
280 nm UV light at the indicated doses. The
cell viability was examined by MTT a ssay
after 24 h. Wild-type DT 40 cells were pre-
treated with 10 l
M
SB203580 for 10 min and
their viability was also analyzed (closed circle).
Data represent the me ans SE of three
independent experiments. P38 activity in
SB203580-treated c ells was analyzed as
described in Fig. 1. (B) Cell viability of Btk-
de®cient cells (Btk
±
), and wild-type or kinase-
inactive btk transfected cells [Btk
±
/Btk, and
Btk
±
/Btk(K
±
), respectively]. Cells were irradi-
ated with 280 nm UV light (60 Jám
)2
), and cell
viability was m easured as in (A). The
expression levels of Btk were shown in
Fig. 2C.
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. was
selectively activated by UV light at around 280 n m. In the
present s tudy, we investigated early signaling events induced
by UV irradiation at 280 nm. 38 was activated by UV light at around
280 nm. In the present study, we further invest igated early
signaling events induced by 280 nm UV i rradiation.