Involvement of ERKs, RSK2 and PKR in UVA ... - Carcinogenesis

Carcinogenesis vol.28 no.7 pp.1543–1551, 2007 doi:10.1093/carcin/bgm070 Advance Access publication April 2, 2007

Involvement of ERKs, RSK2 and PKR in UVA-induced signal transduction toward phosphorylation of eIF2a (Ser51) Tatyana A.Zykova, Feng Zhu, Yiguo Zhang, Ann M.Bode and Zigang Dong
Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA
To whom correspondence should be addressed. Tel: þ507 437 9600; Fax: þ507 437 9606; Email:[email protected]

Double-stranded RNA-dependent protein kinase R (PKR) has been implicated in anti-viral (antitumor) and apoptotic responses. PKR is activated by extracellular stresses and phosphorylates the a subunit of protein synthesis initiation factor eIF2, thereby inhibiting protein synthesis and impeding virus multiplication. Phosphorylation of eIF2a in mammalian cells has been shown to be increased after ultraviolet (UV) stress and to be required for UV-induced repression of protein translation. UVA is an important etiological factor in skin carcinogenesis and we observed that UVA induced phosphorylation of PKR (Thr451) and eIF2a (Ser51) in mouse skin epidermal JB6 Cl41 cells. The induction was suppressed by the MEK1 inhibitor, PD 98059. UVA stimulation of PKR and eIF2a phosphorylation was also inhibited by a dominant-negative mutant (DNM) of ERK2- or RSK2-deficient cells (RSK2). An inhibitor of p38, SB 202190 or a DNM of p38a kinase (DNM-p38a) suppressed UVA-induced phosphorylation of eIF2a (Ser51) but had no effect on phosphorylation of PKR (Thr451). Our data indicated that phosphorylation of PKR at Thr451 is mediated through ERK2 and RSK2, but not through p38 kinase, and is involved in the regulation of Ser51 phosphorylation of eIF2a in UVA-irradiated JB6 cells. In vitro and in vivo kinase assays indicated that phosphorylation of eIF2a at Ser51 occurred indirectly through ERK2, RSK2 or p38 kinase in the cellular response to UVA. These data may lead to the use of these signaling molecules as targets to develop more effective chemopreventive agents with fewer side effects to control UV-induced skin cancer.

Introduction The double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) is a ubiquitously expressed serine/threonine protein kinase, which is activated by interferon, dsRNA, growth factors and stress signals. PKR has been shown to play a broad role in the control of cell proliferation and differentiation, apoptosis and transcriptional regulation (1–5). Human PKR consists of two functionally distinct domains: an N-terminal dsRNA-binding regulatory domain and a C-terminal kinase catalytic domain (4,5). Thr446 and Thr451 in the activation loop of human PKR were found to be critical for kinase activity (6). Replacement of Thr451 with alanine in the activation loop of PKR abolishes the ability of PKR to phosphorylate eIF2a (7). The a subunit of the eukaryotic translation initiation factor 2 (eIF2a) is a major substrate of PKR (8–10). Phosphorylation of eIF2a at Ser51 by PKR leads to inhibition of protein synthesis (11,12). PKR is thought to exhibit anti-proliferative and tumor suppressor activities (13), but also has been suggested to be a positive regulator of cancer cell growth (14). As a result of the phosphorylation of eIF2a (Ser51), eIF2 forms a highaffinity sequestering interaction with its own guanine nucleotide exAbbreviations: DNM, dominant-negative mutant; dsRNA, double-stranded RNA; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; IP, immunoprecipitation; JNK, c-Jun N-terminal kinase; MAPK, mitogenactivated protein kinase; MEM, minimum essential medium; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PKR, protein kinase R; SDS, sodium dodecyl sulfate; UV, ultraviolet.

change factor, eIF2B. This complex inhibits the recycling of GTP for GDP on eIF2 prior to assembly of the 43S initiation complex, thus causing a general reduction in protein synthesis and control of cell growth and differentiation (15,16). Ultraviolet (UV) irradiation has been shown to inhibit protein synthesis in rat fibroblasts (17). UVA constitutes .95% of the solar UV that reaches the Earth’s surface and activates various signaling pathways that are either oncogenic or protective or both. Many of these pathways are mediated primarily through signaling cascades involving mitogen-activated protein kinases (MAPKs), including the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs), and the p38 kinases, which control the activities of various transcription factors (18,19). The suggestion has been made that although higher doses of UVA, compared with UVC, are necessary to induce gene transcription, the possibility of reaching these doses of UVA is more likely to occur physiologically than is attaining the lower doses of UVC required to produce a similar effect (20). UVC-induced eIF2a phosphorylation and translational inhibition are mediated by pancreatic eIF2a kinase/RNA-dependent protein kinaselike endoplasmic reticulum, which is important for resolving protein misfolding in the endoplasmic reticulum (21), and general control non-derepressible-2, which is activated by amino acid limitation (22,23). General control non-derepressible-2 is central to recognition of UV stress and eIF2a phosphorylation provides resistance to stressinduced apoptosis (23). The central importance of the PKR/eIF2a pathway in the cellular response to stress is indicated by the number of eukaryotic viruses that must disable PKR or reverse the phosphorylation of eIF2a to effect productive infection (24). Whether UVA stimulates phosphorylation of PKR or eIF2a is as yet unknown. Data regarding the role of PKR and the mechanism by which MAPKs may mediate PKR phosphorylation are also lacking. Here, we report that PKR and eIF2a phosphorylation in JB6 Cl41 cells is significantly increased in response to UVA irradiation. In this study, the role of the upstream MAPK pathways, specifically ERK2 and RSK2, in UVA-induced phosphorylation of PKR and eIF2a was examined. Our data indicated that in the cellular response to UVA, phosphorylation of PKR at Thr451 is mediated through ERK2 and RSK2 and is involved in the regulation of Ser51 phosphorylation of eIF2a in UVAirradiated JB6 cells. These data may lead to the use of these signaling molecules as targets to develop more effective chemopreventive agents with fewer side effects to control UV-induced skin cancer. Materials and methods Materials Eagle’s minimum essential medium (MEM), fetal bovine serum (FBS) and gentamicin were purchased from BioWhittaker (Walkersville, MD). MEM-a was obtained from Invitrogen Corporation (Grand Island, NY) and L-glutamine and G418 sulfate were from Life Science Technologies (Grand Island, NY). GST antibody kits and antibodies to detect phosphorylated RSK2 (Thr573), ÔnonÕ-phosphorylated (Np)-RSK, phosphorylated eIF2a and phosphorylated or non-phosphorylated ERKs were from Cell Signaling Technology (Beverly, MA). The antibody used to detect PKR [pT451] was from Biosource International (Camarillo, CA) and the antibody used for detection of PKR (D-20) was from Santa Cruz Biotechnology (Santa Cruz, CA). The p38 kinase inhibitor SB 202190, the MEK1 inhibitor PD 98059 and b-actin were purchased from Sigma St. Louis, MO and the antibody to detect Np-eIF2a was a gift from Dr S.Kimball (Department of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, Hershey, PA). Cell lines and plasmids Mouse epidermal tumor promotion-sensitive (Pþ) JB6 Cl41 cells were cultured in MEM containing 5% FBS, 2 mM L-glutamine and 25 lg/ml gentamicin at 37°C in humidified air with 5% CO2. JB6 Cl41 cell lines stably transfected with empty CMV-neo vector (CMV-neo) or a dominant-negative mutant

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(DNM) of ERK2 (DNM-ERK2) or p38a (DNM-p38a) were prepared and identified as described previously (25,26). Before each experiment, transfectants were selected with G418 and tested with their respective phospho-specific antibodies. GM09621 is a human lymphoblast cell line containing wild-type RSK2 (Wt-RSK2) genes and GM03317 is a RSK2-deficient (RSK2) lymphoblast line from a patient with Coffin-Lowry syndrome. These two cell lines were acquired from the NIGMS Human Genetic Cell Repository, Coriell Institute for Medical Research (Camden, NJ) and were cultured as described previously (27). PKRþ/þ and PKR/ murine embryonic fibroblasts were a gift from Dr C.Bell (Ottawa Regional Cancer Centre, Ottawa, Ontario, Canada) (28). The plasmid pEGST-PKR used for expression of GST–PKR and the pGEX-eIF2a plasmid for expression of GST–eIF2a were gifts from Dr Takayasu Date (Department of Biochemistry, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan) (29). UVA irradiation of cells The UVA source used was a Philips TL100w/10R system from Ultraviolet Resources International (Lakewood, OH). It consisted of a Magnetek transformer number 799-XLH-TC-P, 120 V 60 Hz and six bulbs, each 6 ft long. UVA irradiation filtered through 6 mm of plate glass, eliminating UVB and UVC light at all wavelengths below 320 nm, was performed on cultured cells in a box with two ventilation fans installed to eliminate thermal stimulation. These adjustments were necessary because the normal UVA lamps also produce a small amount of UVB and UVC. Analysis of PKR (Thr451) and eIF2a (Ser51) phosphorylation with phosphospecific antibodies Equal numbers (7  105) of JB6 Cl41, the CMV-neo Cl41 or DNM-ERK2 cell lines were cultured in 5% FBS/MEM for 12–15 h in 10 cm diameter dishes until they reached 70–80% confluence. The Wt-RSK2 and RSK2 cell lines were cultured in RPMI 1640 medium supplemented with 20% FBS, 2 mM Lglutamine, 100 U/ml penicillin and 100 lg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C. The PKRþ/þ and PKR/ cell lines were cultured in MEM-a (Invitrogen) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 lg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C. Cells were treated with UVA irradiation and harvested at different times after exposure to UVA. The cells were washed once with ice-cold phosphate-buffered saline (PBS) and disrupted in 200 ll of RIPA buffer (1 PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM Na3VO4 and 1 mM aprotinin and 1 mM phenylmethylsulfonyl fluoride). The samples were sonicated and centrifuged at 14 000 r.p.m. for 15 min in a microcentrifuge. The quantity of protein was determined by the Bradford method (30). The samples (30–50 lg of protein) with 5 SDS were heated at 95°C for 5 min, cooled on ice and centrifuged at 14 000 r.p.m. for 5 min. Then the samples were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and subsequently transferred onto an Immobilon-P transfer membrane (Millipore, Chelmsford, MA). The phosphorylation of PKR (Thr451) and eIF2a (Ser51) was selectively detected by western blotting using the respective phospho-specific antibodies. Some transfer membranes were washed with stripping buffer [7 M guanidine hydrochloride, 50 mM glycine (pH 10.8), 0.05 mM EDTA, 0.1 M KCl and 20 mM b-mercaptoethanol] and used with other primary phospho- or non-phospho-specific antibodies or anti-b-actin. Antibody-bound proteins were detected by a chemiluminescence (enhanced chemifluorescence, Amersham Pharmacia Biotech, Piscataway, NJ) and analyzed using the Storm 840 Scanner (Molecular Dynamics, Sunnyvale, CA). Non-irradiated cell samples were used as negative controls. Effect of MEK1 or p38 kinase inhibitors on phosphorylation of PKR (Thr451) or eIF2a (Ser51) JB6 Cl41 cells were cultured to 80% confluence. Before the cells were irradiated with UVA, they were incubated for 1 h with different concentrations of the MEK1 inhibitor, PD 98059, or p38 kinase inhibitor, SB 202190. Then the cells were exposed to UVA (80 kJ/m2) and incubated for an additional 30 min at 37°C. The cells were disrupted with RIPA buffer. Western blotting analysis was performed with antibodies to detect phosphorylated PKR (Thr451), non-phosphorylated PKR, phosphorylated eIF2a (Ser51) or non-phosphorylated eIF2a. GST fusion protein expression and pull-down assays (Escherichia coli) Rosettaä (DE3) (Novagen, Madison, WI) freshly transformed with the pEGST-PKR plasmid and E.coli BL21 (DE3) (Novagen) freshly transformed with the pGEX-eIF2a plasmid were grown with 100 lg/ ml ampicillin at 37oC to an OD600 of 0.8 and induced by isopropyl-b-D-thiogalactopyranoside at a final concentration of 1 mM. After induction, the cells were cultured at 26°C for another 4 h. The bacterial cells were harvested by centrifugation, sonicated and re-suspended in 1 PBS buffer. Cellular debris was removed by centrifugation for 30 min at 15 000 r.p.m. at 4°C. The super-

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natant fraction was added to GST–agarose beads (Pierce, Rockford, IL), and gently rocked at 4°C for 1 h before centrifugation at 2500 r.p.m. for 5 min. Resin-bound GST fusion proteins were washed three times with PBS. The GST fusion products were then eluted by addition of 1 ml of elution buffer containing 10 mM glutathione in 50 mM Tris–HCl (pH 8.0). The eluted affinitypurified GST fusion proteins were resolved by 10% SDS-PAGE and stained with Coomassie Brilliant Blue R-250 (Bio-Rad, Richmond, CA) for identification of GST–PKR and GST–eIF2a. Kinase assays for in vitro PKR and eIF2a phosphorylation Samples containing the GST–PKR or GST–eIF2a proteins were incubated at 30°C for 30 min with active MAP kinase 2/ERK2 or RSK2/MAPKAP kinase 1b or p38a/SAPK2a kinase (50 mU each) (Upstate Biotechnology, Charlottesville, VA) in 1 kinase buffer A [50 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 1 mM DTT and 0.01% Brij 35] (Cell Signaling Technology) containing 5 mM ATP. The reactions were stopped by adding 5 SDS sample buffer. Then phosphorylation of PKR (Thr451) and eIF2a (Ser51) was analyzed by western blot using a phospho-PKR (Thr451) or phospho-eIF2a antibody, respectively. GST–PKR or GST–eIF2a was utilized as an internal control to verify equal protein loading. Immunoprecipitation assay and kinase activity assays for PKR and eIF2a After culturing for 12–24 h, the JB6 cells were harvested at 30 min following UVA irradiation (80 kJ/m2) and disrupted in 250 ll of immunoprecipitation (IP) buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (vol/vol) Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerol phosphate, 1 mM Na3VO4, 10 lg/ml leupeptin, 10 lg/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride]. The clarified supernatant fractions containing equal amounts of protein were subjected to IP followed by western blot analysis. An antibody against total ERK2, RSK2 or p38a was used for IP and the immune complex beads were washed twice with PBS. The immunoprecipitated ERK2, RSK2 or p38a kinase was incubated for 1 h at 30°C with 5 ll of 1 kinase buffer A, 1 ll (20 lM) of unlabeled ATP, 2 ll (1 lg) of GST–PKR or GST–eIF2a as substrates and 42 ll H2O (total volume of 50 ll). The reaction was stopped by the addition of 5 SDS sample buffer and then proteins were separated by 8% SDS-PAGE followed by western blot analysis with a phosphor-PKR (Thr451) or phospho-eIF2a (Ser51) antibody.

Results UVA induces a strong phosphorylation of PKR at Thr451 and eIF2a at Ser51 The question of whether UVA stimulates PKR (Thr451) or eIF2a (Ser51) phosphorylation was investigated herein by using western blot analysis with specific antibodies to detect PKR phosphorylation at Thr451 and eIF2a phosphorylation at Ser51. A strong phosphorylation of PKR (Thr451) was induced in a dose- (Figure 1A) and timedependent (Figure 1B) manner following UVA exposure of mouse epidermal tumor promotion-sensitive JB6 Cl41 cells. We also observed a marked UVA-induced phosphorylation of eIF2a (Ser51) that was dose- (Figure 1C) and time-dependent (Figure 1D). Maximal phosphorylation of PKR or eIF2a was induced by 80 kJ/m2 of UVA (Figure 1A and C, respectively), which is a physiologically relevant dose. For example, on a typical summer day (90°F) in August at 1:30 p.m. in Austin, MN, UVA exposure was measured to be 1.46 mW/ cm2, which corresponds to 14.6 J/m2. Therefore, a dose of 450 J/m2 would be equivalent to 31 s of UVA exposure under these specific conditions. Thus, under these same conditions, a dose of 80 000 J/m2 would be equivalent to 1.5 h of exposure, which is clearly physiologically achievable and therefore relevant. Phosphorylation of PKR (Thr451) occurred as early as 15 min after irradiation with UVA (80 kJ/ m2) and increased to a maximal level at 30 min after UVA (Figure 1B). The eIF2a phosphorylation at Ser51 also began 15 min after irradiation with UVA (80 kJ/m2) and increased to a maximal level by 60 min after UVA (Figure 1D). For further experiments, we used irradiation with UVA at a dose of 80 kJ/m2 and harvested cells 30 min after irradiation. The correct size of p-eIF2a (Ser51) was verified using PC12 cells treated with thapsigargin as a positive control (Figure 1E). PKR is required for UVA-induced eIF2a phosphorylation We used PKR wild-type (PKRþ/þ) and PKR knockout (PKR/) cells to assess whether PKR plays a role in UVA-induced phosphorylation

UVA-specific induction of eIF2a signaling

Fig. 1. UVA induces phosphorylation of PKR and eIF2a and PKR is required for UVA-induced eIF2a phosphorylation. JB6 Cl41 cells were or were not irradiated with UVA at the indicated doses (A and C), or with UVA at 80 kJ/m2 (B and D). The cells were then harvested at 30 min (A and C) or at the indicated times (B and D) after UVA stimulation. PKR and eIF2a proteins in the cell lysates were separated by 10% SDS-PAGE followed by western blot analysis with specific antibodies to detect phosphorylation of PKR (p-PKR) at Thr451, eIF2a at Ser51 or total non-phosphorylated (Np)-PKR or Np-eIF2a. Non-irradiated samples served as negative controls. (E) A sample of PC12 cells treated with thapsigargin (Cell Signaling Technology) was used for correct sizing of p-eIF2a (Ser51). (F) PKRþ/þ and PKR/ cells were used to compare UVA-induced (80 kJ/m2) phosphorylation of eIF2a. Np-eIF2a and b-actin were used as loading controls for p-elF2a. NpPKR was verified to be absent in PKR/ cells (third panel). These data are representative of at least three independent experiments.

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of eIF2a (Ser51). Phosphorylation of PKR in PKR/ cells was confirmed to be absent, and importantly, UVA-induced phosphorylation of eIF2a at Ser51 was strongly attenuated in PKR/ knockout cells compared with PKRþ/þ cells (Figure 1F). No change in the nonphosphorylated level of eIF2a or b-actin expression was observed in these cell lines. These results support our hypothesis that UVAinduced eIF2a phosphorylation is mediated in part by PKR. UVA-stimulated phosphorylation of PKR (Thr451) and eIF2a (Ser51) is blocked by PD 98059 Although ERKs have been shown to be involved in a variety of cellular biological processes (18), their involvement in the phosphorylation of PKR and eIF2a is not fully elucidated. PD 98059, an inhibitor of MEK1, strongly suppresses ERKs phosphorylation (31,32). In our present experiments, PD 98059 was used to examine UVA-induced phosphorylation of PKR (Thr451) and eIF2a (Ser51). This inhibitor markedly suppressed the phosphorylation of both kinases (Figure 2A and B). The phosphorylation of ERKs (p-ERKs) was used to verify the effectiveness of the MEK inhibitor, PD 98059. Secondly, a DNM of ERK2 showed a marked decrease in UVA-induced PKR (Thr451) and eIF2a (Ser51) phosphorylation (Figure 2C). The DNMERK2 cell line also displayed a markedly reduced phosphorylation of ERKs (p-ERKs) compared with the CMV-neo cell line (Figure 2C). These experiments suggested that ERK2 is involved as an upstream kinase in the UVA-induced phosphorylation of PKR (Thr451) and eIF2a (Ser51). An important downstream substrate for ERKs is the RSK2 protein (33). UVA-stimulated phosphorylation of RSK at Thr573 was also substantially inhibited in DNM-ERK2 cells compared with the control CMV-neo cells (Figure 2C, seventh panel). The total levels of Np-ERKs, Np-PKR, Np-eIF2a and b-actin were unaffected by DNM-ERK2 expression. RSK2 is required for mediating UVA-stimulated phosphorylation of PKR and eIF2a We further assessed whether RSK2, a downstream target of ERKs, is involved in mediating the UVA-induced PKR (Thr451) and eIF2a (Ser51) phosphorylation response. The RSK2 cell line has a markedly reduced UVA-induced expression of RSK2 compared with the normal wild-type cell line (Figure 2D, third panel). Our results also showed that RSK2 cells had a strong inhibitory effect on UVA-induced eIF2a (Ser51) phosphorylation as well as phosphorylation of PKR (Thr451) (Figure 2D, first and fourth panels, respectively). However, expression of Np-PKR, Np-eIF2a or b-actin was not different between the two cell lines. Thus, these data indicated that RSK2 is required for mediating UVA-stimulated phosphorylation of PKR at Thr451 and eIF2a at Ser51. The p38 kinase is not required for mediating UVA-stimulated PKR phosphorylation, but is required for mediating UVA-stimulated phosphorylation of eIF2a We treated JB6 Cl41 cells with SB 202190, a specific inhibitor of p38 kinase activity. Results from this experiment showed that UVAinduced Thr451 phosphorylation of PKR was not suppressed by pretreatment with SB 202190, whereas UVA-induced phosphorylation of eIF2a at Ser51 was inhibited after pre-treatment with SB 202190 (Figure 3A). In addition, data showed that stimulation of Thr451 phosphorylation of PKR with UVA was not prevented. On the other hand, stimulation of Ser51 phosphorylation of eIF2a with UVA was suppressed by expression of DNM-p38a compared with control JB6 cells expressing the empty CMV-neo vector (Figure 3B). However, DNMp38a blocked p38 phosphorylation (p-p38) following UVA irradiation (Figure 3B, bottom). These results suggested that p38 kinase is most probably not involved in the regulation of phosphorylation of PKR (Thr451) but is involved in the regulation of phosphorylation of eIF2a (Ser51) in UVA-irradiated JB6 Cl41 cells. ERK2 and RSK2 phosphorylate PKR at Thr451 For identification of the protein kinases directly responsible for phosphorylating PKR (Thr451) and eIF2a (Ser51), we expressed and puri-

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fied GST–PKR and GST–eIF2a from (E.coli). Gels stained with Coomassie Brilliant Blue R250 show bands containing GST–PKR (Figure 4A) and GST–eIF2a (Figure 5A). We then examined whether certain MAPKs are responsible for direct phosphorylation of PKR (Thr451) or eIF2a (Ser51) using GST–PKR or GST–eIF2a as the kinase substrates, respectively. Results of western blot analysis of in vitro kinase reactions (Figure 4B) and pull-down assays with GST– PKR (Figure 4C) showed that active ERK2 or RSK2, but not p38a kinase, phosphorylated PKR at Thr451. Thus, the ERK2/RSK2 signaling pathway and not p38a-mediated signaling plays a positive regulatory role in the UVA induction of PKR phosphorylation. The results of the in vitro kinase reaction for eIF2a (Ser51) indicated that active ERK2, RSK2 or p38a did not directly phosphorylate eIF2a at Ser51 (Figure 5B). However, the pull-down assay using GST–eIF2a indicated that ERK2, RSK2 or p38a was associated with a strongly phosphorylated eIF2a (Ser51) (Figure 5C). Thus, the ERK2, RSK2 or p38a kinase signaling pathways indirectly plays a regulatory role in UVA induction of eIF2a phosphorylation. PKR phosphorylates eIF2a in vitro and in vivo Samples containing the GST–eIF2a proteins were incubated at 30°C for 30 min with PKR (1 lg) (Upstate Biotechnology). Then phosphorylation of eIF2a (Ser51) was analyzed by western blot using a phospho-eIF2a (Ser51) antibody. The result of this in vitro kinase reaction indicated that active PKR phosphorylates eIF2a at Ser51 (Figure 6A). JB6 cells were harvested at 30 min following UVA irradiation (80 kJ/ m2). The clarified supernatant fractions containing equal amounts of protein were subjected to IP with PKR followed by western blot analysis. The IP complex was incubated for 1 h at 30°C with ATP and 1 lg of GST–eIF2a as substrate. Proteins were separated by 8% SDS-PAGE followed by western blot analysis with a phospho-eIF2a antibody. The results of the kinase assay indicated that immunoprecipitated PKR phosphorylated eIF2a at Ser51 in vivo (Figure 6B). GST–eIF2a was utilized as an internal control to verify equal protein loading (Figure 6A and B, bottom). Discussion Experimental evidence supported by epidemiological findings suggests that solar UV irradiation is the most important environmental carcinogen leading to the development of skin cancers (18). The MAPK signaling cascades are targets for UV and are important in the regulation of the multitude of UV-induced cellular responses. Progress in understanding the mechanisms of UV-induced signal transduction could lead to the use of these protein kinases as specific targets for the prevention and control of skin cancer. Experimental studies involving cell culture have utilized a range of UVA exposures from a low dose of 450 J/m2 to a maximal dose of 300 kJ/m2, and the effect on activation and phosphorylation of the MAPKs is variable (18,19). The phosphorylation and activation of ERKs, JNKs and p38 kinases in response to UVA are not universal phenomena, and the response may depend on the cells studied and the doses and duration of UVA applied. In human lens epithelial cells, UVA (450–2000 J/m2) had no effect on any of the three MAPK pathways, whereas in mouse epidermal JB6 cells, UVA (2000–160 000 J/m2) activated all three MAPK pathways (18,19). Research data (18) indicates that UVA exposures from a dose of 40 kJ/m2 to a dose of 160 kJ/m2 (from 15 to 720 min) induced phosphorylation of RSK2, ERKs, JNKs or p38 kinase in mouse epidermal JB6 promotion-sensitive Cl41 cells. In human keratinocytes, UVA (3000–6000 J/m2) has been shown to stimulate the activation of all three MAPK pathways (34). In human NCTC 2544 keratinocytes, a dose-dependent stimulation of ERK activation was observed in response to UVA irradiation ranging from 60 000 to 240 000 J/m2 (35). In the present study, we used a range of UVA exposures from a dose 20 to 80 kJ/m2 and demonstrated that UVA irradiation (80 kJ/m2) induced maximal phosphorylation of PKR (Thr451) and eIF2a (Ser51). Using PKRþ/þ and PKR/ cells, we found that PKR played a critical role in the phosphorylation of

UVA-specific induction of eIF2a signaling

Fig. 2. ERK2 and RSK2 are required for UVA-induced phosphorylation of PKR and are involved in modulating phosphorylation of PKR (Thr451) and eIF2a (Ser51). (A and B) JB6 cells were or were not pre-incubated for 1 h with the indicated doses of the MEK1 inhibitor, PD 98059. Then cells were or were not irradiated with UVA (80 kJ/m2) and harvested 30 min later. Subsequently, the cell lysates were assayed for phosphorylation of PKR (Thr451) (A) or eIF2a (Ser51) (B). The phosphorylation of ERKs (p-ERKs) served as a control for PD 98059. Np-PKR, Np-eIF2a or NP-ERKs served as loading controls. (C) Cell lines stably expressing an empty vector (CMV-neo) or a DNM of ERK2 (DNM-ERK2) were harvested at the indicated times after irradiation with UVA (80 kJ/m2). The cell lysates were used to determine the phosphorylated levels of PKR (Thr451), eIF2a (Ser51), ERKs or RSK (Thr573) (C). Total levels of Np-PKR, Np-eIF2a, Np-ERKs or b-actin, respectively, served as loading controls. (D) Wild-type RSK2 (Wt-RSK) and RSK2-deficient (RSK2) cells were harvested at the indicated times following irradiation with UVA (80 kJ/m2) and phosphorylated levels of eIF2a (Ser51) or PKR (Thr451) were determined. Total levels of Np-eIF2a, Np-PKR and b-actin served as loading controls. RSK (Np-RSK) was verified to be absent in RSK2 cells (third panel). These data are representative of at least three independent experiments.

eIF2a (Ser51) following UVA stress. MAPKs are important for the regulation of a multitude of physiological cellular functions, such as development, growth, proliferation, differentiation, apoptosis, inflammation and carcinogenesis (18). PKR also contributes to p38 MAPK pathway activation (36) and interacts with and activates MAPK kinase 6, providing a mechanism for regulating p38 kinase activation in response to dsRNA stimulation (37). Our in vitro and in vivo results

demonstrated that p38 is not required for PKR (Thr451) phosphorylation in JB6 cells after UVA irradiation, but is indirectly required for eIF2a (Ser51) phosphorylation. Further, PKR is involved in serine phosphorylation of STAT3 through the activation of ERKs (38). Using peritoneal macrophages isolated from PKRþ/þ and PKR/ mice, Maggi et al. (39) reported that the genetic absence of PKR does not modulate the ability of dsRNA to induce IL-1 expression and release

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Fig. 3. DNM-p38a did not prevent UVA-stimulated PKR (Thr451), but inhibited UVA-induced phosphorylation of eIF2a (Ser51). (A) JB6 cells were or not preincubated for 1 h with SB 202190 at the indicated doses and were or not irradiated with UVA (80 kJ/m2) and then harvested 30 min after irradiation. (B) JB6 cells were stably transfected with an empty vector (CMV-neo) or a DNM of p38a kinase (DNM-p38a). The two cell lines were harvested at the indicated times following irradiation with UVA (80 kJ/m2). (A and B) Subsequently, the cell lysates were assayed for phosphorylation of eIF2a (Ser51) or PKR (Thr451). Total levels of Np-eIF2a or Np-PKR served as loading controls. Phosphorylation of p38 was verified to be absent in DNM-p38a cells (B, fifth panel). These data are representative of at least three independent experiments.

Fig. 4. Kinase assay to determine PKR (Thr451) phosphorylation in vitro and in vivo. (A) Purified GST–PKR proteins were separated by 10% SDS-PAGE and visualized with Coomassie Brilliant Blue R-250. (Lane Mr 5 molecular weight.) (B) Phosphorylation and total levels of GST–PKR fusion proteins by active ERK2, RSK2 or p38a kinase were assayed by western blotting using a specific phosphor-PKR (Thr451) or GST antibody. (C) JB6 Cl41 cells were or were not exposed to UVA (80 kJ/m2) and harvested after 30 min. ERK2, RSK2 or p38a kinase was immunoprecipitated using the appropriate antibody. The immunoprecipitated ERK2, RSK2 or p38a kinase was incubated with GST–PKR as substrate with ATP. Proteins were separated by 8% SDS-PAGE followed by western blot analysis to detect phosphorylation of PKR (Thr451) or total GST–PKR protein. These data are representative of at least three independent experiments.

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UVA-specific induction of eIF2a signaling

Fig. 5. Kinase assay to determine eIF2a (Ser51) phosphorylation in vitro and in vivo. (A) Purified GST–eIF2a proteins were separated by 10% SDS-PAGE and visualized by Coomassie Brilliant Blue R-250 staining (Lane Mr 5 molecular weight). (B) GST–eIF2a was used as a substrate for active ERK2, RSK2 or p38a kinase. Phosphorylation of eIF2a protein was performed by western blotting using a specific p-eIF2a (Ser51) or GST antibody. (C) JB6 Cl41 cells were or were not exposed to UVA (80 kJ/m2) and harvested after 30 min. ERK2, RSK2 or p38a kinase was immunoprecipitated using the appropriate antibody. The immunoprecipitated ERK2, RSK2 or p38a kinase was incubated with GST–eIF2a as substrate with ATP. Proteins were separated by 8% SDS-PAGE followed by western blot analysis to detect phosphorylation of eIF2a (Ser51) or total GST–eIF2a protein. These data are representative of at least three independent experiments.

or stimulate ERKs phosphorylation. Others have tested whether PKR is an upstream mediator of MAP kinase activation and the results suggested that PKR mediates the activation of ERKs when induced by chemical agents including deoxynivalenol, anisomycin and emetine (40). On the other hand, Hsu et al. (41) showed that PKR had no affect on ERKs activation in response to lipopolysaccharide. In the present study, the MEK1 inhibitor, PD 98059, and DNM-ERK2 cells were used to examine the role of the MEK/ERK2 in the UVA-induced phosphorylation of PKR (Thr451) and eIF2a (Ser51). In contrast to the results discussed above, PD 98059 markedly suppressed UVA-induced phosphorylation of PKR (Thr451) and eIF2a (Ser51), and experiments with DNM-ERK2 cells strongly suggested that ERK2 is an upstream kinase involved in the UVA-induced phosphorylation of PKR and eIF2a. Other substrates of ERKs also include RSK2 (32), and UV-induced phosphorylation and activation of RSK2 have been shown to be mediated by ERKs (42). ERKs activation of RSK2 results in diverse cellular responses including proliferation, differentiation and apoptosis (43). Our experiments showed that UVA-induced phosphorylation of PKR (Thr451) and eIF2a (Ser51) was markedly decreased in the RSK2 cell line, indicating that RSK2 is required for mediating UVA-stimulated phosphorylation of PKR (Thr451) and eIF2a (Ser51). In vitro and in vivo kinase assays showed that ERK2 or RSK2 can directly phosphorylate PKR (Thr451) and are indirectly required for eIF2a (Ser51) phosphorylation in JB6 cells after UVA irradiation. The status of eIF2a phosphorylation is controlled by PKR, which utilizes eIF2a as its primary substrate. Phosphorylation of eIF2a in mammalian cells is significantly increased after UVC irradiation and this conserved mechanism is required for the trans-

lation repression in response to UVC stress (21–23). Wu et al. (21) reported that UVC-induced eIF2a phosphorylation is specifically mediated by pancreatic eIF2a kinase/RNA-dependent protein kinase-like endoplasmic reticulum. In contrast, Deng et al. (22) demonstrated that pancreatic eIF2a kinase/RNA-dependent protein kinase-like endoplasmic reticulum is not required for UVC-induced eIF2a phosphorylation and attributed the discrepancy to different doses of UVC and cell line (i.e. MCF-7, HIT, COS-1) specificity. The cellular signaling response is UV wavelength dependent (18). In vivo and in vitro experimental studies have used a range of UVA, UVB or UVC exposures in multiple doses, and the observed effects on activation and phosphorylation of the MAPKs are varied. The biological effects produced by UVA, UVB or UVC involve different targets, indicating that the mechanisms by which UVA, UVB or UVC induce transcriptional activation differ substantially and the activation of MAPKs by UV displays dependencies on dose, time and wavelength (18). We used PKRþ/þ and PKR/ cells to demonstrate the critical role of PKR in the phosphorylation for eIF2a (Ser51) following UVA stress. Together with data from in vitro and in vivo kinase assays, our results demonstrated that PKR is a direct downstream serine/threonine protein kinase of ERK2 and RSK2. After UVA stress, PKR integrates multiple signals from these kinases, directly phosphorylates eIF2a (Ser51) and functions to link the regulation of translation (Figure 7). Meric et al. (44) described the basic principles of translational control, the alterations encountered in cancer and selected therapies targeting translation initiation to help elucidate new therapeutic avenues. EPA, an n-3 polyunsaturated fatty acid, blocks cell division by inhibiting translation initiation (45). EPA releases Ca2þ from

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Fig. 6. UVA-activated PKR phosphorylates eIF2a in vitro. (A) GST–eIF2a was used as a substrate for active PKR. The indicated GST–eIF2a fusion proteins were assayed by western blotting with a specific p-eIF2a (Ser51) or GST antibody. (B) JB6 Cl 41 cells were or were not treated with UVA (80 kJ/m2) and harvested after 30 min. PKR was immunoprecipitated with anti-PKR antibody. The immune complex of PKR was incubated with GST–eIF2a as substrate and proteins were separated by 8% SDS-PAGE followed by western blot analysis to detect phosphorylation of eIF2a (Ser51) or total GST–eIF2a protein. These data are representative of at least three independent experiments.

Fig. 7. Schematic diagram shows the involvement of ERK2 and RSK2 as the upstream kinases in the UVA-induced phosphorylation of PKR (Thr451) and eIF2a (Ser51). The signaling pathways of UVA-induced PKR phosphorylation at Thr451 transduce through ERK2 and RSK2. PKR is required for UVA-induced eIF2a (Ser51) phosphorylation, which leads to an inhibition of protein synthesis. [ indicates activation.

intracellular stores while inhibiting their refilling, thereby activating PKR. PKR, in turn, phosphorylates and inhibits eIF2a, resulting in the inhibition of protein synthesis at the level of translation initiation. Similarly, clotrimazole inhibits cell growth through activation of PKR and phosphorylation of eIF2a (46). A novel tumor suppressor gene mda-7 is being developed as a gene therapy agent and also

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induces and activates PKR, which leads to phosphorylation of eIF2a and induction of apoptosis (47). PKR is activated by flavonoids, such as genistein and quercetin, which suppress tumor cell growth, with phosphorylation of eIF2a and inhibition of protein synthesis (48). Neoplastic progression in melanoma and colon cancer is associated with increased expression and activity of the interferon-inducible protein kinase, PKR (49). PKR phosphorylation and the phosphorylation of eIF2a are 7- to 40-fold higher in breast carcinoma cell lines compared with non-transformed epithelial cell lines and, correspondingly, a larger proportion of eIF2a is present in a phosphorylated state in carcinoma cell lines than in non-transformed cell lines (14). These data do not support the concept of PKR as a classic tumor suppressor but instead suggest a positive regulatory role of PKR in growth control in tumor development. PKR and eIF2a phosphorylation play a significant role in apoptosis of neuroblastoma cells and could be involved in the molecular signaling events leading to neuronal apoptosis and death and might be a new target in neuroprotection (50). Progress in understanding the mechanisms of UV-induced signal transduction could lead to the use of these protein kinases as specific targets for the prevention and control of skin cancer. Because the ozone layer blocks UVC exposure, UVA and UVB are probably the chief carcinogenic components of sunlight with relevance for human skin cancer (51– 53). Significant contributions to the elucidation of the specific signal transduction pathways involved in UV-induced skin carcinogenesis have been made over the past few years, and most evidence suggests that the cellular signaling response is UV wavelength dependent. Identifying ÔnonÕ-toxic strong antioxidants (tea polyphenols, curcumin, silymarin, genistein, garlic compounds, apigenin, resveratrol and others)—capable of preventing ultraviolet radiation-induced skin cancer—has become an important area of research. A wide range of such agents have been shown to prevent skin cancer in cell and animal model systems (54). Acknowledgements We thank Dr Takayasu Date for the generous gift of plasmids pEGST-PKR and pGEX-eIF2a, Dr John C.Bell for the generous gift of PKRþ/þ and PKR/ cell lines and identification of these cells and Dr Scot Kimball for the gift of nonphospho-eIF2a antibody. We thank Ms Andria Hansen for her secretarial assistance. This work was supported in part by The Hormel Foundation and National Institutes of Health grants CA77646, CA111356 and CA111536. The University of Minnesota is an equal opportunity educator and employer. Conflict of Interest Statement: None declared.

UVA-specific induction of eIF2a signaling

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