Early signaling events induced by 280-nm UV 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 eects on biological systems. To better understand the eects of UV in this range, early signaling events induced by 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 aected in Syk-de®cient cells. Tyrosine phosphorylation of Lyn was induced by 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 UV irradiation 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 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. 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 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 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 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 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 UV signaling 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 by UV 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 UV irradiation 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 induced by 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 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 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 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 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) Eect 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 UV irradiation 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 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 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 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 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 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 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 induced by 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. 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 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 buer 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 buer, 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 by UV 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 induced by 280 nm UV irradiation 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). UV irradiation 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. REFERENCES 1. Nomura, T., Nakajima, M., Hongyo, T., Taniguchi, E., Fukuda, K., Li, L., Kurooka, M., Sutoh , M., Hande, P ., K awaguchi, T., Ueda, M. & Takatera, H. (1997) Induction of c ancer, acidic keratosis, and speci®c p53 mutations b y UVB light in human s kin maintained in severe combined immunode®cient mice. Cancer Res. 57, 2081±2084. 2. Gange, W.R. & Rosen, C.F. (1986) UVA eects on mammalian skin and cells. Photochem. Photobiol. 43, 701±705. 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 the other promotes cell survival through the activation o f a Btk±P38 signaling pathway. Fig. 5. Lyn, Btk, and P38 regulate cell viability after UV irradiation. 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