Báo cáo khoa học: Early signaling events induced by 280-nm UV irradiation pot

To better understand the effects of UV in this range, early signaling events induced by monochro- matic UV light were investigated using the chicken B cell line DT40 and mutants lacking

Early signaling events induced by 280-nm UV irradiation

Yukihito Kabuyama', Miwako K Homma’, Tomohiro Kurosaki? and Yoshimi Homma’

‘Department of Biomolecular Science, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima, Japan; *Department of Molecular Genetics, Institute for Hepatic Research, Kansai Medical University, Moriguchi, Japan

The depletion of stratospheric ozone results in increased UV

(ultraviolet) light below 300 nm, and has significant effects

on biological systems To better understand the effects of UV

in this range, early signaling events induced by monochro-

matic UV light were investigated using the chicken 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 deficient in Lyn and Btk Introduc-

tion of wild-type Btk, but not kinase-inactive Btk, restored

the P38 activation In contrast, P38 activation was not

affected in Syk-deficient cells Tyrosine phosphorylation of

Lyn was induced by 280 nm UV light, and pretreatment of

cells with orthovanadate, an inhibitor of protein tyrosine phosphatase (PTP), enhanced both Lyn phosphorylation and P38 activation These results show that Lyn and Btk are upstream regulators of the P38 signaling pathway activated

by 280 nm UV light and that the triggering event likely

involves inactivation of PTP Furthermore, cell death

induced by 280 nm UV irradiation were augmented by Btk depletion or a specific inhibitor for P38, and partially blocked in Lyn-deficient cells, suggesting that the Lyn—Btk— P38 pathway promotes cell survival 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] The harmful effects of UV light are mainly

attributed to the UVB (280-320 nm) range, and it is

excessive exposure to these wavelengths that accounts for

the risk of stratospheric ozone 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 depends on several primary target

molecules, including chromosomal DNA In DNA, UV

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 cell cycle regulation However,

nuclear events triggered by DNA damage are not the only

response to UV irradiation Recent studies have revealed

that UV irradiation activates several cytoplasmic signal

transduction pathways [8-10], including pathways regulated

by extracellular signal regulated kinases (ERKs), c-Jun

N-terminal kinases (JNKs), and P38 MAP kinases (P38)

Correspondence to Y Homma, Fukushima Medical University School

of Medicine, Fukushima 960-1295, Japan Fax: + 81 24 548 3041,

Tel.: + 81 24 548 2111 ext 2810, E-mail: yoshihom@fmu.ac.jp

Abbreviations: UVB, ultraviolet B; ROI, reactive oxygen intermedi-

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, phosphatidylinositol 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)

Although much is known about the regulation and function of MAP kinase pathways, the mechanism by which

UV light triggers the activation is poorly understood It has been suggested that reactive oxygen intermediates (ROT), such as singlet oxygen, superoxide radicals, hydroxyl radicals, and Hs, 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 the receptor tyrosine kinase, results in the activation of signal transduction pathways such as

ERK, which correlates with the UV-induced inhibition of

EGER dephosphorylation [15,16] These results are the first direct evidence of the regulation of PTKs and PTPs by UV-induced oxidative damage, and the function of these enzymes as regulators of signaling pathways responsive to

irradiation Previously, we reported that MAP kinases [17]

and PtdIns 3-kinases [18] are regulated separately and independently in a strict wavelength-specific manner In particular, P38 was activated by UV light at around

280 nm In the present study, we further investigated early signaling events induced by 280 nm UV irradiation We also present evidence that Lyn, Btk and P38 are involved in 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

maintained at 1-5 x 10° cellsmL7! The culture medium

was replaced with NaCl/P;, and the cell concentration was

adjusted to 1 x 10° cellsmL™' Irradiation was carried out

in quartz cuvettes using a_ spectrofluorophotometer

(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 (13PEMO001, Melles Griot, Boulder, CO,

USA) Wild-type and kinase-inactive Btk cDNAs were

cloned into pApuro expression vector [25] For DNA

transfection into DT40 cells, DNA were linearized and

electroporated as described previously [25] Cell clones

expressing Btk were selected in the presence of puromycin

(0.5 pgmL7') The expression of Btk was analysed by

immunoblotting, and clones, in which the expression level of

Btk was almost same to that in wild-type 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 mm _ Tris/HCl

(pH 7.4), 1% (w/v) NP-40, 0.27 mM sucrose, 1 mm EDTA,

1 mm EGTA, 10 mm ÿ-glycerophosphate, | mm benzami-

dine, 50 mm NaF, 10 ugmL~” pepstain A, 10 ugmL""

aprotinin, and 10 ug-mL"' 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 mm Hepes/NaOH, pH 7.5, 10 mm magnesium

acetate, 50 um ATP), and incubated for 15 min at 37 °C

with [y-°P] ATP (50 wCimL™') and substrate PHAS-1

(250 gmL~') The substrates were resolved by SDS/PAGE

(14% acrylamide) and visualized by autoradiography The

incorporation of phosphate was quantified using a Fuji BAS

1000 bioimaging analyzer

Immunoblotting

Immunodetection of tyrosine-phosphorylated proteins

was carried out using anti-phosphotyrosine Ig (Promega)

Total P38, Lyn and Btk were probed with anti-P38 (N-

17, Santa Cruz Biotechnology, Inc), anti-Lyn or anti-Btk

Ig [19], respectively Incubation with secondary antibody

conjugated to horseradish peroxidase was followed by

chemiluminescence detection (Amersham Pharmacia

Biotech)

Dephosphorylation of Lyn in vitro

Wild-type DT40 cells were pretreated with 1 mm 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 Cell lysates prepared from irradi-

ated cells were referred to as the ‘phosphorylated Lyn’

fraction On the other hand, Lyn-deficient cells were either

treated with Na3VOg, for 10 min, or irradiated with 280 nm

UV light for 10 min The cell lysates prepared from these

cells were used as ‘phosphatase’ fractions In the in vitro Lyn

dephosphorylation assay, the ‘phosphorylated Lyn’ fraction

and the ‘phosphatase’ fractions were mixed and incubated at

37 °C for specific periods The reaction was stopped by

addition of an equal volume of 2 x 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 24h post-UV irradiation as described [18] Briefly, cells were treated with MTT (final concentration, 0.5 mgmL™') and incubated for 30 min, prior to removal of the medium and addition of dimethylsulfoxide (500 uL) to solubilize the MTT formazon product Absorbances were measured at

595 nm, and plotted as a measure of the relative number of

cells, normalized to nonirradiated cells

RESULTS Wavelength-specific activation of P38

by monochromatic UV irradiation Chicken DT40 cells were exposed to monochromatic UV light ranging from 260 to 360 nm at 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 peaked within seconds after irradiation, and declined to basal levels by 2 min The kinase activity increased in a energy dependent manner up to 80 Jm?, and then decreased at higher energy levels No cytotoxic effect was observed at 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 UV irradiation at 280 nm in chicken B cells and was consistent with the results of our previous study in human T cell lines [17]

280 nm UV induced 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 tyrosine 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 cells and mutant cells deficient in nonreceptor

PTKs, Lyn, Syk, and Btk No differences in endogenous

P38 expression levels were observed between the wild-type and these PTK-deficient cells As shown in Fig 2B, UV- induced P38 activation was completely inhibited in Lyn- or Btk-deficient cells, whereas the kinase activity was main- tained in Syk-deficient cells Introduction of wild-type Btk restored the P38 activation induced by 280 nm UV irradi- ation (Fig 2C), but expression of the kinase-inactive Btk did not Taken together, these results clearly indicate that

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Lyn and Btk, but not Syk, are essential for the activation of

P38 and that Btk is required for UV signaling to P38 kinase

It has been reported that tyrosine phosphorylation of Lyn

is required for activation in B cell receptor signaling systems

[20] Therefore, the regulation of Lyn activity by UV was

monitored indirectly, by analyzing its reactivity with anti-

phosphotyrosine Ig Figure 2D shows the profiles of

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 min) to monochromatic UV irradiation ranging from 260 nm to 360 nm in increments of 20 nm Irradiated energy varied with wavelength (30 Jm™~ at 260 nm UV; 60 Jim”? at

280 nm UV; 300 J'm Ÿ 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 were exposed to the indicated doses of UV light (280 nm, irradiation for 50 s to 10 min) Cell extracts were prepared and kinase activities of P38 were analyzed as in (A) Autoradiograms A-C are each representative of five independent experiments Sum- maries of results (means + SE, n = 5) are shown

280 nm UV-induced protein tyrosine phosphorylation in wild-type vs PTK-deficient cells Several proteins showed enhanced tyrosine phosphorylation after UV irradiation in wild-type cells The major tyrosine-phosphorylated protein, appearing with a molecular mass of 57 kDa, was identified

as Lyn by immunoblotting with anti-Lyn Ig (Fig 2D) Enhancement of tyrosine phosphorylation induced by UV light was abolished in Lyn-deficient cells Therefore, Lyn is

an upstream kinase in the 280 nm UV signaling On the other hand, Lyn tyrosine phosphorylation was slightly affected in Btk-deficient cells, suggesting that Btk is partially involved in the phosphorylation of Lyn

Involvement of protein tyrosine phosphatase

in 280 nm UV induced 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 such as reduced glutathione, vitamin E and mannitol (Fig 2A), suggests a critical role for ROI and possibly PTPs

in the activation Therefore, the involvement of PTPs was

examined by pretreating DT40 cells with vanadate, a nonspecific 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 of UV stimulation (Fig 3) A significant 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 ‘phosphorylated Lyn’ fraction and two different ‘phosphatase’ fractions and monitored dephosphorylation of the ‘phosphorylated Lyn’ induced by the ‘phosphatase’ The ‘phosphorylated Lyn’ fraction was prepared from UV-treated DT40 cells which were previously incubated with iodoacetamide to inactivate PTPs The ‘phosphatase’ fractions were independently

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WT BC B/Btk BtC/Bk(K”) Fig 2 280 nm UV-induced P38 activation requires Lyn and Btk, but not Syk (A) Effect of tyrosine kinase inhibitor and antioxidants on P38 activation DT40 cells were preincubated for 20 min either with genistein (100 pm), vitamin E (100 um), GSH (250 um), or mannitol (100 mm), and then irradiated with 280 nm UV light P38 activities were analyzed as described in Fig 1 (B) P38 activation in PT K-deficient cells Wild-type (WT), Lyn-deficient (Lyn), Syk-deficient (Syk ), and Btk-deficient (Btk ) cells were irradiated with 280 nm UV light and P38 activity was analyzed as described in Fig 1 (C) Expression of Btk restores 280 nm UV-induced P38 activation in Btk-deficient cells Wild-type cells (WT), Btk-deficient cells (Btk ), Btk-deficient cells transfected with wild-type btk cDNA (Btk /Btk), and Btk-deficient 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-deficient DT40 cells At 1 min after UV irradiation at 280 nm, whole cell lysates were prepared and analyzed by immunoblotting with anti- phosphotyrosine Ig Tyrosine-phosphorylated Lyn was immunoprecipitated with anti-phosphotyrosine Ig and visualized by immunoblotting with anti-Lyn Ig (25)

obtained from nonstimulated, 280 nm UV-irradiated, and

vanadate-treated Lyn-deficient DT40 cells The ‘phospho-

rylated Lyn’ fraction was mixed with the ‘phosphatase’

fraction and the resulting dephosphorylation of Lyn was

detected by anti-phosphotyrosine immunoblotting (Fig 4)

As expected, mixing with the ‘phosphatase’ derived from

nonstimulated cells led to the immediate dephosphorylation

of Lyn in the ‘phosphorylated Lyn’ fraction In contrast, this

dephosphorylation was completely inhibited when the

‘phosphatase’ fraction derived from the UV-irradiated and

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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 induced by the irradiation

Role of Lyn, Btk, and P38 in UV- induced cell death

We examined the involvement of PTKs and P38 in cell death process following UV irradiation As shown in Fig 5, irradiation of cells with 280 nm UV light led to

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Fig 4 UV irradiation 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 protein tyrosine phosphatases were exposed to UV light at

280 nm to induce tyrosine phosphorylation of Lyn and cell extracts

were prepared (referred to as the ‘phosphorylated Lyn’ fraction) Lyn-

deficient cells were incubated in the absence or presence of Na3VO4 for

30 min, or irradiated with 280 nm UV for 10 min, and cell lysates were

prepared and used as the ‘phosphatase’ fraction The ‘phosphorylated

Lyn’ fraction was mixed with the ‘phosphatase’ fraction or the buffer

used for lysate preparation, and then incubated at 37 °C for 0, 1, or

5 min Reactions were stopped by adding an equal volume of 2 x SDS

sample buffer, 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

significant drop in the viability of wild-type DT40 cells in a

dose-dependent fashion DNA fragmentation was detected

in these irradiated cells (data not shown) Cells deficient 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 Btk in the Btk-deficient

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

specific inhibitor of P38 kinase Treatment with SB203580

significantly enhanced the response inducing cell death, and

completely inhibited P38 activity in these cells (Fig 5A)

These results indicate an important role for the P38

signaling pathway in protecting cell death following UV

irradiation On the other hand, a deficiency 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 distinct from the

Btk—P38 pathway (Fig 6)

DISCUSSION

We have reported that MAP kinases and PtdIns 3-kinases are regulated separately and independently in a strict

wavelength-specific manner [17,18] In particular, P38 was

selectively activated by UV light at around 280 nm In the present study, we investigated early signaling events induced

by UV irradiation at 280 nm using DT40 and PTK- defective mutants thereof The results demonstrated that activation of P38 was completely inhibited in cells deficient

in Lyn and Btk, but not in Syk-deficient cells Tyrosine phosphorylation of Lyn was induced by 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 signifi- cantly diminished in the Lyn-deficient 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 of the P38 signaling pathway activated by 280 nm UV, and that Lyn seems 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 is activated by both Syk and Btk in B cell

receptor signaling systems [22] Moreover, B cell receptor-

mediated P38 activation requires both Syk and Lyn, but not Btk On the other hand, Syk is required for JNK activation

in cells treated with high doses of H,O>, whereas in cells

treated with low doses of H,Os>, the activation of JNK is not 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 finding that Lyn and Btk regulate

280 nm UV-induced P38 signaling reveals a novel mech- anism, distinct from findings made with the B cell receptor

systems [26]

The initial cellular signals that follow UV irradiation 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 PDGER, resulting

in activation of ERK [15,16] This process is mediated by the inactivation of receptor-directed phosphatases via ROI generated by UV irradiation In addition, the functional down-modulation of receptors for EGF, TNF, and IL-1 is sufficient to block UVB-induced activation of JNK, imply- ing important roles for these receptors in the JNK response

to UV Recently, Mihail e¢ al have also reported a novel signaling pathway to JNK, initiated by rRNA damage to functionally active ribosomes [24] However, the early signaling events that induce the activation of P38 are not well understood The evidence presented here supports that the activation of Lyn induced by suppression of PTPs is an important triggering event in the activation of P38 kinase by

280 nm UV This is consistent with the experimental finding 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 the generation

of ROI We speculate that Lyn-directed PTPs are sensitive

to ROI specifically generated at 280 nm UV The nature of

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Fig 5 Lyn, Btk, and P38 regulate cell viability

after UV irradiation (A) UV-induced cell

death in PTK-deficient DT40 cells Wild-type

(open circle), Lyn-deficient (open diamond),

Syk-deficient (open square), and Btk-deficient

DT40 (close triangle) cells were irradiated with

280 nm UV light at the indicated doses The

cell viability was examined by MTT assay

after 24 h Wild-type DT40 cells were pre-

treated with 10 um SB203580 for 10 min and

their viability was also analyzed (closed circle)

Data represent the means + SE of three

independent experiments P38 activity in

0.8 -

0.6 -

0.4 -

0.5 ¬

SB203580-treated cells was analyzed as 0.2

described in Fig 1 (B) Cell viability of Btk-

deficient cells (Btk ), and wild-type or kinase-

inactive btk transfected cells [Btk /Btk, and

0 20 |

UV dose (J/m*)

- SB203580

WT Btk Btk /Btk Btk /Btk(K )

"

Btk /Btk(K ), respectively] Cells were Irradi- r ua + SB203580 280 nm UV

ated with 280 nm UV light (60 J-m~’), and cell

viability was measured as in (A) The

expression levels of Btk were shown in

Fig 2C

ROI generated by UV at various wavelengths, and the

identification of specific PTPs that are inactivated by UV

irradiation need to be studied further

The result that a specific inhibitor for P38, SB203580,

abolished the cell death response (Fig 5) demonstrates that

the P38 signaling pathway controls cell fate in 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

UV

|

ROI! production

+?

protein tyrosine phosphataso

inhibition

Lyn activation

Btk activation

v

v

P38 activation

Cell death

Cell survival

Fig 6 A model for 280 nm UV-induced signal transduction UV

induces the generation of ROI in irradiated cells, which inhibits

Lyn-directed PTPs, resulting in the apparent activation of Lyn Lyn

regulates two distinct signaling pathways; one induces cell death, and

the other promotes cell survival through the activation of a Btk—P38

signaling pathway

0 15 30 45 60 0 15 30 45 60

UV dose (J/m2)

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 on cell viability are quite different The cell death response induced by 280 nm UV irradiation was augmented by Btk depletion as by a specific inhibitor for P38, and partially blocked in Lyn-deficient cells, suggesting that P38 promotes cell survival whereas Lyn bifurcates towards cell survival and cell death pathways

Based on the findings of this study, we propose the following model for the regulation of P38 by 280 nm UV in DT40 cells (Fig 6) UV irradiation selectively regulates Lyn and Btk tyrosine kinases via mechanisms involving inhibi- tion of PTPs It is likely that Btk 1s activated downstream of Lyn, although both tyrosine kinases are necessary for the initial UV-triggered events to induce P38 activation Lyn generates at least two signaling pathways; 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 grants from the Ministry of Education, Science, Sports and Culture

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