Regulation of Ultraviolet B-induced Phosphorylation of Histone H3 at Serine 10 by Fyn Kinase*

Ultraviolet B (UVB) induces phosphorylation of histone H3 at serine 10, and mitogen-activated protein kinases are involved in this signal transduction pathway. Here we provide evidence that Fyn kinase, a member of the Src kinase family, is involved in the UVB-induced phosphorylation of histone H3 at serine 10. UVB distinctly increased Fyn kinase activity and phosphorylation. Fyn kinase inhibitors 4-amino-5-(4-chlorophenyl)-7( t -butyl)pyrazol(3,4-d)pyramide and leflunomide, an Src kinase inhibitor, suppressed both UVB-induced phosphorylation of histone H3 at serine 10 and Fyn kinase activity and phosphorylation. UVB-induced phosphorylation of histone H3 at serine 10 was blocked by either a dominant-negative mutant of Fyn (DNM-Fyn) kinase or small interfering RNA of Fyn kinase. UVB-induced phosphorylation and activities of ERKs and protein kinase B/Akt were markedly inhibited by DNM-Fyn kinase. However, DNM-Fyn kinase did not inhibit UVB-induced phosphorylation of p38 MAPK or c-Jun N-terminal kinases. Active Fyn kinase phosphorylated histone H3 at serine 10 in vitro , and the phosphorylated Fyn kinase could translocate into the nucleus of HaCaT cells. These results indicate that Fyn kinase plays Cell study the role of in the regulation of H3 at serine 10 through regulation of ERKs and Akt. MAP kinases, including ERKs and p38 MAP kinases, are involved in UVB-induced phosphorylation of histone H3 at serine 10. Here we provide evidence showing that Fyn kinase, a member of the Src kinase family, is located upstream of the MAP kinases cascade and plays a key role in UVB-induced phosphorylation of histone H3 at serine 10. Inhi- bition of Fyn kinase by dominant-negative mutant Fyn kinase (DNM-Fyn) or the Fyn kinase inhibitors PP2 or leflunomide or siRNA Fyn kinase causes inhibition of phosphorylation of histone H3 (serine 10). DNM-Fyn kinase also inhibits UVB-induced phosphorylation of Akt and ERKs but not p38 MAP kinase or JNKs. Fyn kinase is involved in UVB-induced phosphorylation of histone H3 at serine 10 mediated through the Akt and ERKs/MSK1 pathways.

Experimental evidence suggests that solar ultraviolet irradiation is the most important environmental carcinogen leading to the development of malignant skin melanoma (1)(2)(3). UV irradiation results in suppression of the immune system (4) and chronic skin damage including premature aging (5). According to wavelength, UV is divided into UVA 1 (UVA I, 340 -400 nm; UVA II (320 -340 nm), UVB (280 -320 nm), and UVC (180 -280 nm). UVA and UVB are recognized as the chief carcinogenic components of sunlight with relevance to human skin cancer (1,2). UVB irradiation has the ability to stimulate oncogene c-fos promoter expression (6), to accumulate c-fos mRNA and c-Fos protein, and to increase activator protein-1 activation (7). In addition, it also induces tumor suppressor p53 (8 -10) and p16 (INK4A-ARF) expression (8). By using oligonucleotide microarray analysis, researchers found that several biological processes, including basal transcription, splicing, and translation, as well as proteasome-mediated degradation, are globally affected by UVB irradiation in human keratinocytes (11). These results demonstrate the complexity of the UVB response. Various signal transduction pathways, including extracellular signal-regulated kinases (ERKs), p38 kinase, and c-Jun N-terminal kinases (JNKs), are involved in UVBinduced cell proliferation or apoptosis (1,7,(12)(13)(14). We recently found that UVB induces phosphorylation of histone H3 at serine 10 or serine 28 through MAP kinases or mitogen-and stress-activated protein kinase 1 (MSK1) (15,16). However, much less is known about the UVB-induced phosphorylation of histone H3, especially the upstream mediators in the signal transduction pathway of UVB irradiation.
Modification of histones is an important element in the regulation of gene expression, and histone H3 phosphorylation at serine 10 has traditionally been regarded as a marker for mitosis (17,18). Increased phosphorylation of histone H3 at serine 10 was found in mitogen-stimulated and oncogenetransformed mouse fibroblasts (19). Phosphorylation of H3 at serine 10 corresponds with chromatin relaxation and gene expression in interphase, whereas in mitosis it is associated with chromosome condensation (20). Thus to identify the responsible kinases and the circumstances under which histone H3 at serine 10 becomes phosphorylated is important.
Fyn kinase is a member of the Src family of nonreceptor protein-tyrosine kinases and is associated with outgrowth during development and regeneration of the central and peripheral nervous systems (21,22). Fyn kinase can interact with numerous other signal molecules, and it has diverse biological functions including signaling via the T cell receptor, regulation of brain function, and adhesion-mediated signaling (23). Recent research indicates that Fyn kinase is expressed in T cells, and it is the first signaling molecule to be activated downstream of the T cell receptor (24). Fyn kinase is also involved in epidermal growth factor receptor signaling and disrupts the function of the integrin ␣ 6 ␤ 4 at hemidesmosomes and regulates normal keratinocyte migration and squamous carcinoma invasion (25). In fact, more and more evidence shows that Src kinase family members interact with MAP kinases in response to various stimuli. For example, activation of MAP kinase by cAMP requires an Src family kinase (SRC, YES, and FYN) that lies downstream of protein kinase A in the murine fibroblast cell line SYF (26). Reactive oxygen species-induced JNKs activation is mediated by Src kinase (27), and reactive oxygen species activate p90 ribosomal S6 kinase via Fyn kinase (28). Overall, Src kinases interact widely with serine/threonine kinases when cells are exposed to a variety of stimuli.
To explore further the mechanism of UVB-induced carcinogenesis, we used UVB to induce phosphorylation of histone H3 at serine 10. We found that UVB induced phosphorylation of histone H3 at serine 10 in a time-and dose-dependent manner and dramatically increased Fyn kinase activity. Fyn kinase inhibitors, PP2 and leflunomide, an Src kinase inhibitor, inhibited UVB-induced Fyn kinase activity and phosphorylation of histone H3 at serine 10 in a dose-dependent manner. Dominant-negative (DNM) Fyn kinase and siRNA Fyn kinase interrupted UVB-induced phosphorylation of histone H3 at serine 10. DNM-Fyn kinase inhibited UVB-induced ERKs and Akt1 activities and phosphorylation. This study indicates that Fyn kinase plays a key role in UVB-induced phosphorylation of histone H3 at serine 10 through regulation of Akt1 and ERKs.

EXPERIMENTAL PROCEDURES
Materials-Eagle's minimal essential medium (MEM), L-glutamine, and Lipofectamine TM 2000 reagent were from Invitrogen. Plasmids of pCMV5-wild type-huFyn and pCMV5-huFyn-K299M mutant vector were kindly provided by Dr. Marilyn D. Resh (Sloan-Kettering Institute for Cancer Research, New York). Antibodies to detect phosphorylation of histone 3 at serine 10 and total histone H3 protein, phosphorylation of ERKs, p38 MAP kinase, and JNKs antibodies and nonphospho-Fyn kinase antibody were from Upstate Biotechnology, Inc. (Lake Placid, NY). Phospho-Fyn (Thr-12) was from Santa Cruz Biotechnology (Santa Cruz, CA). Fetal bovine serum (FBS) was from Gemini Bio-Product (Calabasas, CA). Gentamicin sulfate was from BioWhittaker, Inc. (Walkersville, MD). The Akt kinase assay kit and ERKs kinase assay kit were from Cell Signaling Technology Inc. Polyvinylidene difluoride (PVDF) membrane was from Millipore (Bedford, MA). The Folin and Ciocalteus phenol reagent and modified Lowry protein assay kit were from Pierce. The sense and antisense oligonucleotides for siRNA of Fyn kinase were synthesized by Sigma.
Extraction of Acid-soluble Proteins-After UVB treatment, cells were harvested and washed with ice-cold phosphate-buffered saline (PBS). Extraction of the acid-soluble proteins was performed according to the protocol described by Upstate Biotechnology, Inc. Briefly, cells were scraped from the plates after treatment and centrifuged at 1000 rpm at 4°C for 10 min. Cells were washed once with ice-cold PBS and resuspended with 10 volumes of lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 1.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol), and sulfuric acid was then added to a final concentration of 0.2 N, and extractions were left on ice for 45 min. Extraction solutions were centrifuged at 12,000 rpm for 10 min at 4°C in a microcentrifuge, and the acid-insoluble pellets were discarded. Supernatant fractions were transferred to fresh tubes and precipitated on ice for 45 min at a final concentration of 20% trichloroacetic acid. Samples were then centrifuged at 12,000 rpm for 10 min at 4°C, and the pellets were washed once with 0.1% acidic acetone and once with pure acetone. Acid-soluble proteins were dissolved in 0.1 N NaOH solution and stored at ؊20°C. Protein quantity was determined according to the instructions provided in the modified Lowry protein assay kit.
Assay of Phosphorylated H3 at Serine 10 -Phosphorylation of histone H3 at serine 10 was detected as described previously (29). In brief, a specific antibody against phosphorylation of histone H3 at serine 10 or total histone H3 was employed to detect the UVB-induced phosphorylation of histone H3 at serine 10 using a Western blot assay. Membranebound proteins were detected with enzyme-catalyzed fluorescence (Amersham Biosciences) and analyzed using the Storm 840 Scanner (Amersham Biosciences).
Establishing the Stably Transfected DNM-Fyn-JB6 Cells-By using plasmids pCMV5-wild type-huFyn and pCMV5-huFyn-K299M mutant vector, we established these stable transfections according to the protocol from Invitrogen. All of these cells were selected in media containing 500 g/ml G418 for 2 weeks, and the G418 concentration was then decreased to 250 g/ml and maintained. G418-selected cells were tested for phosphorylation and activity of Fyn kinase.
Akt Kinase Assay-The ability of DNM-Fyn kinase to inhibit the UVB-induced phosphorylation of Akt was tested by using an Akt kinase assay kit (Cell Signaling Technology Inc.). In brief, cells were treated with UVB at various doses for the desired times. Cells were washed once with ice-cold PBS after removing the media, and then 0.5 ml of 1ϫ ice-cold cell lysis buffer plus 1 mM phenylmethylsulfonyl fluoride was added to each plate, and the cells were scraped and transferred to fresh tubes. Cells were sonicated on ice four times for 5 s each and centrifuged for 10 min at 4°C, and the supernatant fractions were transferred to other fresh tubes. Cell lysate protein (200 g) and beads (20 l) with immobilized Akt1 1G1 monoclonal antibody were added together by gently rocking for 3 h at 4°C. These tubes were centrifuged for 1 min at 4°C and washed twice with 500 l of cell lysis buffer and then washed with 500 l of 1ϫ kinase buffer. The pellets were suspended in 20 l of 1ϫ kinase buffer supplemented with 200 M ATP and 2 g of GSK-3 fusion protein and incubated for 45 min at 30°C. The reaction was terminated with 10 l of 3ϫ SDS sample buffer. The samples were denatured at 95-100°C for 5 min before they were separated by 8% SDS-PAGE. The proteins were transferred to PVDF membranes, and Akt1 kinase activity was analyzed by Western blotting using a phospho-GSK-3␣/␤ (serine 21/9) antibody.
ERKs MAP Kinase Assay-The ability of DNM-Fyn kinase to inhibit UVB-induced phosphorylation of ERKs was tested using a p44/42 MAP kinase assay kit (Cell Signaling Technology Inc.). The treatment and lysis of cells were performed as described above. Cell lysate protein (200 g) and immobilized phospho-p44/42 MAP kinase (Thr-202/Tyr-204) antibody beads (20 l) were added together by gently rocking for 3 h at 4°C. These tubes were centrifuged and washed twice. The pellets were suspended in 20 l of 1ϫ kinase buffer supplemented with 200 M ATP and 2 g Elk-1 fusion protein and incubated for 45 min at 30°C. The reaction was terminated with 10 l of 3ϫ SDS sample buffer. The samples were denatured at 95-100°C for 5 min before they were separated by 10% SDS-PAGE. The proteins were transferred to PVDF membranes, and ERK kinases activity was analyzed by Western blotting using a phospho-Elk-1 (serine 383) antibody.
Fyn Kinase Assay-UVB-induced activation of Fyn kinase was detected by a 32 P-labeled radioactive method. In brief, 200 g of cell lysate protein were mixed with protein-A/G beads (20 l) in advance for 1 h at 4°C. The mixture was centrifuged at 12,000 rpm for 5 min at 4°C, and the supernatant fraction was added to phospho-Fyn (Thr-12) antibody (20 l) and gently rocked for 3 h at 4°C. These tubes were centrifuged and washed twice. The pellets were suspended in 20 l of 1ϫ kinase buffer supplemented with 10 l of diluted [␥-32 P]ATP solution and 2.5 l of Src substrate peptide (250 M) and incubated for 30 min at 30°C. A 20-l aliquot was transferred onto P81 paper and washed tree times with 0.75% phosphoric acid for 5 min per wash and one time with acetone for 2 min. Radioactive incorporation was determined by scintillation counter. The same experiments were repeated three times. Cells were then incubated in fresh 0.1% FBS/DMEM for another 2 h before being treated with UVB and harvested at the indicated time (A) or treated with UVB at the indicated dose and harvested after treatment for 30 min (C). Acidic proteins were extracted as described under "Experimental Procedures." By using Western blot analysis, phosphorylation of histone H3 at serine 10 was detected by using the Storm PhosphorImager analysis system (Amersham Biosciences). The level of total histone H3 protein (B and D) was determined as described under "Experimental Procedures." Small Interfering RNA (siRNA) Against Fyn Kinase and Establishing the Stable Expression Cell Line-To study further the role of Fyn kinase in the regulation of phosphorylation of histone H3 at serine 10, we successfully "knocked down" Fyn kinase expression levels by the siRNA method. The sense oligonucleotide of Fyn kinase used for siRNA was 5Ј-TTTGCAGCTCGGAAGGAGATTGGTTCAAGAGACAATCTCCTTC-CGAGCTGTTT TT-3Ј, and the antisense is 5Ј-CTAGAAAAACAGCTC-GGAAGGAGATTGGTCTCTTGAACCAATCTCCTTCCGAGCTG-3Ј. In the sense sequence, the boldface letters indicate a Bbs1 endonuclease site, and italic underlined letters show the RNA loop. In the antisense sequence, the boldface letters show an XbaI endonuclease site. The ligated pair of oligonucleotides was inserted into the mU6pro vector. Annealing, ligation, and colony screening were done as described previously (30). The oligonucleotide synthesis and sequencing of the inserted sequences in the mU6pro vector were performed by Sigma. The reconstructed plasmid with mU6pro vector contained the siRNA Fyn kinase sequence and is named siRNA-Fyn-mU6pro. The plasmid siR-NA-Fyn-mU6pro and pcDNA3.1 were stably transfected into JB6 Cl41 cells using the Lipofectamine TM 2000 reagent. The transfected cells were selected with G418 as described previously, and Fyn kinase expression level was confirmed by a Western blot assay using an anti-Fyn kinase antibody (Upstate Biotechnology, Inc., Lake Placid, NY).
Active Fyn Kinase Phosphorylated Histone H3 at Serine 10 in Vitro-Phosphorylation of pure histone H3 by active Fyn kinase was carried out according to the manufacturer's instructions (Upstate Biotechnology, Inc.). In brief, 8 g of pure histone H3 was incubated for 1 h at 30°C with the active Fyn kinase and 200 M ATP in 30 l of kinase reaction buffer (25 mM Tris, pH 7.5, 5 mM ␤-glycerol phosphate, 2 mM dithiothreitol, 0.1 mM Na 3 VO 4 , 10 mM MgCl 2 ). The samples were added to 10 l of 4ϫ SDS sample buffer and separated by 15% SDS-PAGE. The phosphorylation of histone H3 at serine 10 and total histone H3 protein were detected by Western blotting with specific antibodies from Upstate Biotechnology, Inc.
Phosphorylated Fyn Kinase Translocation Assay-HaCaT cells were seeded and cultured in 100-mm dishes for 24 h. Cells were starved in serum-free DMEM for 48 h after cells reached 85-90% confluence. At various times (15, 30, or 60 min) after UVB irradiation (4 kJ/m 2 ), nuclear and cytoplasmic proteins were extracted according to manufacturer's instructions (NE-PER Nuclear and Cytoplasmic Extraction Reagents; Pierce). The protein concentration was determined by the modified Lowry protein assay kit (Pierce). Phosphorylated Fyn kinase was detected by Western blot using a phospho-specific Fyn (Thr-12) antibody. To show that the nuclear proteins did not mix with cytoplasmic proteins, the specific cytoplasmic protein, ␣-tubulin, was also detected by Western blot using an anti-␣-tubulin antibody. Equal protein loading was monitored by the level of ␤-actin.

RESULTS
UVB Induces Phosphorylation of Histone H3 at Serine 10 in a Time-and Dose-dependent Manner-HaCaT keratinocytes were derived from adult human skin, and although spontaneously immortalized, they remain highly related to their normal cell counterparts, and it is an ideal cell line for studying the HaCaT cells were treated with UVB and harvested at the indicated times (A) or treated with UVB at the indicated dose and harvested after treatment for 30 min (B). Cells were starved for 48 h. 200 g of cell lysate protein were mixed with protein-A/G beads (20 l) in advance for 1 h at 4°C. The mixture was centrifuged at 12,000 rpm for 5 min at 4°C. The supernatant fraction was incubated with a phospho-Fyn kinase antibody and rocked for 3 h at 4°C. Samples were washed twice with kinase buffer, and Fyn kinase activity was evaluated by determining the phosphorylation of the 32 P-labeled amount of phosphorylation of Src substrate peptide as described under "Experimental Procedures." Compared with control, * indicates p Ͻ 0.05 and ** indicates p Ͻ 0.01. effects of UVB on human skin (31,32). In this study, HaCaT cells were used to analyze the UVB-induced phosphorylation of histone H3 at serine 10. The time response study indicated that phosphorylation of histone H3 at serine 10 gradually increases from 15 to 30 min following treatment with UVB (4 kJ/m 2 ) (Fig.  1A). The level of total histone H3 protein did not change (Fig.  1B). The dose-response study indicated that phosphorylation of histone H3 gradually increases after treatment with increasing doses of UVB (1, 2, or 4 kJ/m 2 ) (Fig. 1C) with no effect on total histone H3 protein levels (Fig. 1D). These results indicate that phosphorylation of histone H3 at serine 10 is induced by UVB in a dose-and time-dependent manner.
UVB Strongly Increases Fyn Kinase Activity and Phosphorylation of Fyn Kinase at Thr-12-Our results also showed that UVB induced Fyn kinase activity in a time- (Fig. 2A) and dose-dependent (Fig. 2B) manner. We found that a 21-fold increase in Fyn kinase activity occurred in UVB-treated cells compared with untreated control cells ( Fig. 2A). Fyn kinase activity reached a maximum level when cells were treated with 1 kJ/m 2 of UVB, and doses up to 4 kJ/m 2 produced a similar induction of activity. Because we already reported that 4 kJ/m 2 of UVB distinctly induced phosphorylation of histone H3 at serine 28 or serine 10 in JB6 cells (15,16), a dose of 4 kJ/m 2 of UVB was used to further study the stress function of UVB. UVB induced phosphorylation of Fyn kinase (Thr-12) also in a time-and dose-dependent manner (Fig. 3, A and C), and nonphospho-Fyn kinase was unchanged (Fig. 3, B and D).
Fyn Kinase Inhibitors Greatly Suppress UVB-induced Phosphorylation of Histone H3 at Serine 10 -The above data indicate that UVB induces phosphorylation of histone H3 at serine 10 and simultaneously induces Fyn kinase activity. Therefore, we hypothesized that Fyn kinase would play a key role in the UVB-induced phosphorylation of histone H3 at serine 10, and H3 phosphorylation would be changed by inhibition of Fyn kinase. In this study, we found that PP2, a Fyn kinase inhibitor (33), and leflunomide, an Src kinase inhibitor (34), markedly inhibited the UVB-induced phosphorylation of histone H3 at serine 10 in a dose-dependent manner (Fig. 4, A and C, respectively) but did not change the total histone H3 protein level (Fig. 4, B and D). This result not only confirms our hypothesis but also indicates that Fyn kinase plays an important role in UVB-induced phosphorylation of histone H3 at serine 10.
Fyn Kinase Inhibitors Suppress Fyn Kinase Activity and Phosphorylation-Because Fyn kinase inhibitors markedly suppressed UVB-induced phosphorylation of histone H3 at serine 10, we determined whether these inhibitors can affect Fyn kinase activity and phosphorylation under the same conditions. We found that either PP2 or lefunomide decisively inhibited UVB-induced Fyn kinase activity (Fig. 5A) and phosphorylation (Fig. 5B) in a dose-dependent manner.

Dominant-negative (DNM) Mutant Fyn Kinase Inhibits UVB-induced Phosphorylation of Histone H3 at Serine 10 -To
explore further the role of Fyn kinase in UVB-induced phosphorylation of histone H3 at serine 10, we used DNM-Fyn and WT-Fyn plasmids to establish stably expressed DNM-Fyn and WT-Fyn transfected JB6 cells. Our data show that phosphorylation of histone H3 at serine 10 induced by UVB was obviously inhibited in DNM-Fyn-transfected cells (Fig. 6C), but the total histone H3 protein level did not change (Fig. 6D). Meanwhile, DNM-Fyn also distinctly inhibited UVB-induced phosphorylation of Fyn kinase (Fig. 6A), but the total Fyn kinase protein level was also unchanged (Fig. 6B). These results indicate that Fyn kinase plays a very important role in mediating the phosphorylation of histone H3 at serine 10 induced by UVB.
Small Interfering RNA (siRNA) Fyn Kinase Inhibits UVBinduced Phosphorylation of Histone H3 at Serine 10 -We also used the siRNA method to knock down the Fyn kinase expression level, and we then determined the effects on UVB-induced phosphorylation of histone H3 at serine 10. First, we confirmed the efficiency of siRNA for knocking down Fyn kinase level using Western blotting and an anti-Fyn antibody. Our results indicated that the Fyn kinase protein level decreased to 5% of that observed in control cells (Fig. 7A). ␤-Actin protein level was used to monitor equal protein loading in each well (Fig. 7B). These results indicate that the siRNA-Fyn kinase works well in vivo. Further results indicated that UVB increased phosphorylation of histone H3 at serine 10 in the "mock" mU6pro vector plus pcDNA3.1 vector-transfected cells but not in the siRNA-Fyn (mU6pro plus pcDNA3.1 vector) stably transfected cells in a time-and dose-dependent manner (Fig. 7, C and E). Total histone H3 levels were unchanged (Fig. 7, D and F).

DNM Fyn Kinase Inhibits UVB-induced Phosphorylation of Akt (Serine 473) and ERKs (Thr-203/Tyr-204)-Fyn kinase is
involved in UVB-induced phosphorylation of histone H3 at serine 10; however, the signal transduction components mediated by Fyn kinase leading to that phosphorylation are unknown. We hypothesized that Fyn kinase is located upstream of the known UVB-stimulated signal transduction pathway,  and Fyn kinase can regulate several downstream kinases, which are then translocated into the nucleus where they phosphorylate histone H3 in vivo. We assessed the status of several MAP kinases and Akt phosphorylation in WT-Fyn cells and DNM-Fyn cells. We found that UVB only stimulated ERKs (Thr-202/Tyr-204) and Akt (serine 473) phosphorylation (Fig. 8, E and G) but not p38 MAP kinase or JNKs (Fig. 8, A and C) in WT-Fyn cells. ERKs and Akt were not affected by UVB in DNM-Fyn cells. The total protein levels of p38 MAP kinase, ERKs, JNKs and Akt were unchanged in either cell type (Fig.  8, B, D, F, and H).

DNM-Fyn Kinase Inhibited Akt and ERKs
Activity-To confirm the results described above (Fig. 8), we further examined UVB-stimulated activity of ERKs and Akt in WT-Fyn and DNM-Fyn cells. A nonradioactive Akt and p44/p42 MAP kinase assay kit was used to test for Akt activity and ERKs activity, respectively. The Akt kinase substrate was GSK3, and the ERKs substrate was Elk1. Our results indicated that phosphorylation of GSK3-␣ and -␤ decreased in UVB-treated DNM-Fyn-JB6 cells (Fig. 9A). The nonphosphorylated protein level of GSK3 was unchanged (Fig. 9B). Phosphorylation of Elk1 dramatically decreased in UVB-treated DNM-Fyn-JB6 cells com-  (5, 15, or 30 min) after UVB treatment, cell lysates were used to determine the phosphorylated or nonphosphorylated level of Fyn kinase (Thr-12) using a phospho-Fyn (Thr-12) or Fyn kinase antibody, respectively (A and B). Acidic proteins were extracted as described under "Experimental Procedures." Phosphorylation of histone H3 at serine 10 was detected with a phosphohistone H3 (serine 10) antibody (C). The level of total histone H3 protein was detected with a nonphospho-histone H3 antibody (D). pared with WT-Fyn-JB6 cells (Fig. 9C), and total Elk1 protein level was unchanged (Fig. 9D). These data showed that DNM-Fyn kinase also inhibited UVB-induced Akt and ERKs activity.
Active Fyn Kinase Phosphorylated Histone H3 at Serine 10 in Vitro-Furthermore, we tested whether Fyn kinase could directly phosphorylate histone H3 at serine 10 in vitro. We used pure histone H3 protein as the Fyn kinase substrate, and we incubated it with active Fyn kinase and then analyzed phosphorylation of histone H3 at serine 10 by Western blot. Our results showed that active Fyn kinase strongly phosphorylated histone H3 at serine 10 in vitro (Fig. 10A) in a dose-dependent manner. Totallab software was used to analyze the density value (Fig. 10B). The nonphosphorylated level of total histone H3 protein was unchanged (Fig. 10C). These data indicate that Fyn kinase can phosphorylate the histone H3 protein at serine 10 in vitro.
Phosphorylated Fyn Kinase Is Translocated into the Nucleus-To determine whether Fyn kinase can phosphorylate histone H3 in vivo, we first confirmed that the phosphorylated Fyn kinase (Thr-12) can be translocated to the nucleus from the cytosol in HaCaT cells. Thirty minutes after UVB treatment, phosphorylated Fyn kinase (Thr-12) increased in both the cytosol and nucleus (Fig. 11A). To confirm that the nuclear proteins were not mixed with cytosolic proteins, the samples were analyzed by Western blot using an ␣-tubulin antibody. ␣-Tubulin is known to be found only in the cytoplasm and not in the nucleus. The results indicated that no ␣-tubulin was present in the nucleus, thus confirming that the nuclear fraction was not contaminated with cytosolic proteins (Fig. 11B). Equal loading of samples was monitored by ␤-actin expression level (Fig. 11C). These results indicated that phosphorylated Fyn kinase also can be translocated to the nucleus similar to other kinases. DISCUSSION Fyn kinase is a member of the Src family of tyrosine kinases, and it has diverse biological functions, including signaling via the T cell receptor, regulation of brain function, and adhesionmediated signaling (21)(22)(23)(24). In the present study, we found that Fyn kinase plays a key role in the regulation of UVBinduced phosphorylation of histone H3 at serine 10. Early   FIG. 7. siRNA knocked down the Fyn kinase protein level and markedly inhibited UVB-induced phosphorylation of histone H3 at serine 10. Stably expressed siRNA-Fyn or mock (mU6pro plus pcDNA3.1) JB6 cells were cultured in 5% FBS/MEM at 37°C in a 5% CO 2 atmosphere. These cells were starved in 0.1% FBS/MEM for 48 h. Cell lysates were used to analyze Fyn kinase protein levels by Western blot with an anti-Fyn antibody, and the acidic proteins were extracted as described under "Experimental Procedures." A indicates the total Fyn kinase protein from mock and siRNA-Fyn JB6 cells. B indicates the ␤-actin protein level indicating equal loading of samples. Cells were treated with UVB as described, and phosphorylation of histone H3 at serine 10 was detected by Western blot using a specific antibody (C and E). Total histone H3 protein level (D and F) was determined as described under "Experimental Procedures."

FIG. 8. Dominant-negative Fyn kinase inhibited UVB-induced phosphorylation of Akt and ERKs but not p38 MAP kinase or JNKs.
Stably expressed DNM-Fyn or WT-Fyn JB6 cells were starved in 0.1% FBS/MEM for 48 h and then treated with UVB (4 kJ/m 2 ). At various times (5, 15, or 30 min) after UVB treatment, cells were harvested with cell lysis buffer. The cell lysate was used to analyze phosphorylation of p38 MAP kinase, JNKs, Akt, and ERKs by Western blot assay using specific antibodies against phosphorylation of p38 kinase (A), JNKs (C), Akt (E), and ERKs (G), respectively. Total p38 kinase, JNKs, Akt1, and ERKs protein levels were determined by Western blot using antibodies against p38 kinase (B), JNKs (D), Akt (F) and ERKs (H), respectively. studies show that UVB induces phosphorylation of histone H3 at serine 10 or serine 28 in JB6 cells, which is mediated through MAP kinases or mitogen-and stress-activated protein kinase (MSK1) (15,16). However, little is known about the upstream mediators in UVB-induced phosphorylation of histone H3. To characterize further the carcinogenic effect of UVB and explore the mechanism of UVB-induced phosphorylation of histone H3 at serine 10, we used the HaCaT cell line. The HaCaT cell line is a spontaneously transformed human epithelial cell line derived from human adult skin, and it maintains full epidermal differentiation capacity (35). This cell line is obviously immortal and has a transformed phenotype in vitro but remains a nontumorigenic human keratinocyte (35). An early study indicates that sublethal doses of UVB produce a strong induction of c-jun and c-fos transcripts in HaCaT cells (36). Here we found that UVB strongly induced phosphorylation of histone H3 at serine 10 in human HaCaT cells in a doseand time-dependent manner (Fig. 1). UVB also markedly increased Fyn kinase activity (Fig. 2) and phosphorylation (Fig.  3) in a time-and dose-dependent manner. Another study showed that UVB irradiation results in the up-regulation of the transcription factor activator protein-1 in HaCaT cells and specifically increased c-fos and JunD expression (37). Moreover, active immediate-early gene expression including that of the proto-oncogene c-fos is associated with phosphorylation of histone H3 at serine 10 (37, 38). Our results clearly indicate that UVB induces phosphorylation of histone H3 at serine 10 in the human keratinocyte HaCaT cell line, and Fyn kinase activity plays a key role in the UVB-induced phosphorylation of histone H3 at serine 10.
To confirm further that Fyn kinase is involved in UVBinduced phosphorylation of histone H3 at serine 10, PP2, a Fyn kinase inhibitor (33), and leflunomide, an Src family kinase inhibitor (34), were employed to suppress Fyn kinase activity. Our data indicate that PP2 and leflunomide indeed inhibited UVB-induced phosphorylation of histone H3 at serine 10 in a dose-dependent manner (Fig. 4). Under the same conditions, PP2 and leflunomide also inhibited UVB-induced Fyn kinase activity (Fig. 5A) and phosphorylation at Thr-12 (Fig. 5B). These results strongly illustrate that Fyn kinase is involved in UVB-stimulated phosphorylation of histone H3 at serine 10.
To exclude nonspecific characteristics of chemical inhibitors, we transfected WT-Fyn and DNM-Fyn plasmids into JB6 Cl41 cells, which are derived from mouse skin and are a well developed cell culture model for studying tumor promotion (39,40). In the present study, we found that Fyn kinase-deficient cells inhibited UVB-induced phosphorylation of Fyn kinase at Thr-12 (Fig. 6A) and phosphorylation of histone H3 at serine 10 FIG. 9. Dominant-negative Fyn kinase inhibited UVB-induced Akt and ERKs activity. DNM-Fyn JB6 and WT-Fyn JB6 cells were cultured and treated with UVB as described. The GSK3 fusion protein was used as a substrate for Akt and the Elk1 fusion protein for ERKs. Akt kinase activity was assessed by determining phosphorylation of GSK3, which was detected by a specific phospho-GSK3␣/␤ antibody. A, indicates the level of phosphorylation of GSK␣/␤, and B, indicates total GSK3 protein level. ERKs kinase activity was evaluated by determining phosphorylation of Elk1 (serine 383), which was detected by a specific phospho-Elk1 (serine 383) antibody (C). D, indicates the total Elk1 protein level.
FIG. 10. Active Fyn kinase phosphorylates histone H3 at serine 10 in vitro. An active Fyn kinase assay was carried out according to the manufacturer's instructions. Pure histone H3 (8 g) was used as the Fyn kinase substrate. Defined units of Fyn kinase (1 unit is defined as the amount of Fyn required to catalyze the transfer of 1 pmol of phosphate to protein substrate in 1 min at 30°C in kinase buffer in a 30-l reaction volume) were used to analyze the doseresponse for phosphorylation of histone H3 at serine 10 by Fyn kinase (A and B). The density value of A was measured and shown in B. Total histone H3 was detected with a nonphospho-histone H3 antibody (C). induced by UVB (Fig. 6C). Moreover, we further confirmed the importance of Fyn kinase function in UVB-induced phosphorylation of histone H3 at serine 10 by the siRNA method. Our results indicated that UVB-induced phosphorylation of histone H3 was also dramatically inhibited by siRNA Fyn kinase compared with control cells transfected with only the vector plasmid (Fig. 7, C and E). Fyn kinase has many functions. For example, Fyn is one of the signals involved in regulating myelination during development (41), and morphological differentiation of oligodendrocytes requires activation of Fyn tyrosine kinase (42). Here our results clearly indicate that Fyn kinase is a key mediator in regulating the UVB-induced phosphorylation of histone H3 at serine 10.
To explore the mechanism of Fyn kinase in the regulation of phosphorylation of histone H3 at serine 10, we determined the phosphorylation level of other kinases. All of these kinases are associated with phosphorylation of histone H3 at serine 10 (15,16). Our results showed that DNM Fyn kinase inhibited UVBinduced ERKs and Akt1 phosphorylation (serine 473) (Fig. 8, E and G), but phosphorylation levels of p38 MAP kinase and JNKs were unchanged (Fig. 8, A and C) compared with wild type Fyn plasmid-transfected cells. Moreover, UVB-induced Akt1 (Fig. 9A) and ERKs activity (Fig. 9C) were both distinctly restrained in DNM-Fyn kinase cells. These data indicate that Fyn kinase regulates UVB-induced phosphorylation of histone H3 at serine 10 through Akt1 and ERKs. A previous study showed that Akt1 is involved in arsenite-induced phosphorylation of histone H3 at serine 10 (29). In the present study, we provide evidence indicating that Akt1 is also involved in UVBinduced phosphorylation of histone H3 at serine 10, and its upstream kinase is Fyn kinase. Recent studies indicate that Akt1 associates with and activates IB kinase (IKK) (43)(44)(45). IKK-␣ kinase directly phosphorylates histone H3 in vivo, and histone H3 phosphorylation by IKK-␣ is critical for cytokineinduced gene expression (46). Moreover, our results indicate that Fyn kinase can also phosphorylate histone H3 protein at serine 10 in vitro (Fig. 10), and the phosphorylated Fyn kinase can be translocated to the nucleus from the cytosol (Fig. 11). These results strongly indicate that Fyn kinase probably phosphorylates histone H3 at serine 10 in vivo and also suggest that Fyn kinase may regulate UVB-induced phosphorylation of histone H3 at serine 10 by itself and through the Akt1/IKK-␣ pathway. On the other hand, DNM-Fyn kinase also inhibited UVB-induced phosphorylation of MSK1 (data not shown), which is involved in mediation of histone H3 phosphorylation and is downstream of ERKs (47). A recent study indicated that oxidase-dependent reactive oxygen species signaling and ERK1/2 phosphorylation are both controlled by Fyn kinase activation (48). Fyn kinase is still required for activation and phosphorylation of ERK1/2 stimulated by insulin-like growth factor-I (49) or thrombin (50). Fyn kinase regulates UVB-induced phosphorylation of histone H3 at serine 10 also through the Fyn/ERKs/MSK1 pathway.
The present study shows that Fyn kinase is involved in UVB-induced phosphorylation of histone H3 at serine 10 and that Fyn kinase is another important regulator that mediates the UVB-induced phosphorylation of histone H3 at serine 10. UVB also induces phosphorylation of histone H3 at serine 10 through a p38 MAP kinase pathway (15) (Fig. 12). This study provides powerful evidence that illustrates a probable mechanism of UVB in carcinogenesis in cells and animal models. However, many questions, including how UVB light enters into cells, still need to be answered. FIG. 11. Phosphorylated Fyn kinase is translocated to the nucleus. HaCaT cells were starved in serum-free DMEM for 36 h at 37°C in a 5% CO 2 atmosphere after cells reached 90% confluence. At various times (15, 30, or 60 min) after UVB treatment, cells were harvested according to the instructions included with the NE-PER nuclear (N) and cytoplasmic (C) extraction reagents kit. Cytoplasmic proteins and nuclear proteins were separately loaded into 10% SDS-polyacrylamide gels, and a Western blot assay was employed to detect protein expression level using anti-phospho-Fyn (Thr-12) (A), anti-␣-tubulin (B), and ␤-actin (C) antibodies.

FIG. 12. Fyn kinase is involved in UVB-induced phosphorylation of histone H3 at serine 10 through regulation of ERKs and
Akt. MAP kinases, including ERKs and p38 MAP kinases, are involved in UVB-induced phosphorylation of histone H3 at serine 10. Here we provide evidence showing that Fyn kinase, a member of the Src kinase family, is located upstream of the MAP kinases cascade and plays a key role in UVB-induced phosphorylation of histone H3 at serine 10. Inhibition of Fyn kinase by dominant-negative mutant Fyn kinase (DNM-Fyn) or the Fyn kinase inhibitors PP2 or leflunomide or siRNA Fyn kinase causes inhibition of phosphorylation of histone H3 (serine 10). DNM-Fyn kinase also inhibits UVB-induced phosphorylation of Akt and ERKs but not p38 MAP kinase or JNKs. Fyn kinase is involved in UVB-induced phosphorylation of histone H3 at serine 10 mediated through the Akt and ERKs/MSK1 pathways.