FRAP DNA-Dependent Protein Kinase Mediates a Late Signal Transduced from Ultraviolet-Induced DNA Damage  Daniel B. Yarosh, Nicholas Bizios, Jeannie Kibitel, Karina Goodtzova, Dawn Both, Shari Goldfarb, Bryan Green, David Brown  Journal of Investigative Dermatology  Volume 114, Issue 5, Pages 1005-1010 (May 2000) DOI: 10.1046/j.1523-1747.2000.00964.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Fluorescence micrograph showing nuclear localization of FRAP protein. NHEK were double-stained with DAPI to identify nuclei (A) and with FITC-tagged antibodies against FRAP (B) and the same field was examined with two filter sets. The arrow indicates a typical cell showing nuclear localization of FRAP. Journal of Investigative Dermatology 2000 114, 1005-1010DOI: (10.1046/j.1523-1747.2000.00964.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 FRAP induction following UV irradiation is dependent on UV dose and time. (A) HaCaT cells were irradiated with increasing doses of UVB and after 24 h the intensity of FRAP antibody staining (in arbitrary units) in the nucleus was measured using computerized image analysis. (B) NHEK cells were irradiated with 500 J UVB per m2 and at time points after irradiation the intensity of FRAP antibody staining in the nucleus was measured using computerized image analysis. Pyknotic NHEK cells, which showed little or no FRAP staining, were excluded from the analysis. For each data point in the panels between 32 and 123 cells were analyzed. Error bars represent the standard deviation of each measurement. Journal of Investigative Dermatology 2000 114, 1005-1010DOI: (10.1046/j.1523-1747.2000.00964.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 FRAP phosphorylates p53 in vitro and is stimulated by UV-irradiated DNA. FRAP and ATM were immunoprecipitated from cell extracts of p53-deficient HaCaT cells and then mixed with ATP and p53 protein in vitro. Phosphorylation of p53 serines and threonines was detected by ELISA. Lanes 1–4, immunoprecipitated FRAP + FKBP12 + p53; lane 2, with added λ-DNA; lane 3, with added UV-irradiated λ-DNA instead; lane 4, including UV-irradiated λ-DNA and 2 nM rapamycin. Positive control for the immunoprecipitation and kinase activity were in lanes 5 and 6: immunoprecipitated ATM + p53. Positive controls for the ELISA detection were in lane 7 (phosphoserine-bovine albumin) and lane 8 (phosphothreonine-bovine serum albumin). Negative controls for the ELISA detection were: lane 9, p53 protein alone; lane 10, FKBP12 protein alone; lane 11, bovine serum albumin alone. The gray and black bars denote two different experiments. Each measurement was in duplicate and the values were averaged. Journal of Investigative Dermatology 2000 114, 1005-1010DOI: (10.1046/j.1523-1747.2000.00964.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 UV stimulation of p53 phosphorylation in vivo is inhibited by rapamycin. Western blot of (A) total p53 and (B) serine 15 phosphorylated p53 in cell extracts prepared 24 h after irradiation. Lane 1, p53 control protein; lane 2, unirradiated cell extract; lane 3, unirradiated cells treated with 2 nM rapamycin; lane 4, cells irradiated with 200 J UVB per m2; lane 5, cells irradiated with 500 J UVB per m2; lane 6, cells irradiated with 1000 J UVB per m2; lane 7, cells irradiated with 500 J per m2 and treated with rapamycin. Journal of Investigative Dermatology 2000 114, 1005-1010DOI: (10.1046/j.1523-1747.2000.00964.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Rapamycin inhibits phosphorylation of p70S6K. (A) Western blot of phosphorylated p70S6K in HaCaT cells. Lane 1, cells irradiated with 300 J UVB per m2 at 37°C and incubated for 24 h; lane 2, cells preincubated for 30 min at 10°C, irradiated with UVB, and incubated for 10 min at 10°C before transfer to 37°C for 24 h; lane 3, cells treated with 2 nm rapamycin and irradiated with UVB. (B) Low molecular weight proteins from the same gel stained with Coomassie showing equivalent protein loading among the lanes. Journal of Investigative Dermatology 2000 114, 1005-1010DOI: (10.1046/j.1523-1747.2000.00964.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 UVB induction of TNF-α protein and transcript is inhibited by rapamycin. (A) Western blot of TNF-α extracts of NHEK. Lane 1, irradiated with 200 J UVB per m2; lane 2, 30 min pretreated with 2 nM rapamycin, irradiated with 200 J UVB per m2, and 24 h post-treated with rapamycin; lane 3, treated with 1 μg LPS per ml; lane 4, treated with LPS and rapamycin. (B) CAT activity assay in repair-deficient XP12BE cells carrying the TNF-α CAT transgene, as described byKibitel et al. (1998). The black arrows indicate the chloramphenicol substrate and the brackets indicate the acetylated forms. Lane 1, substrate alone; lane 2, untreated extract; lane 3, cells irradiated with 100 J UVB per m2 and incubated for 18 h; lane 4, cells treated with 2 nM rapamycin; lane 5, cells irradiated with UVB and treated with rapamycin; lane 6, untreated cells; lane 7, cells irradiated with UVB; lane 8, cells irradiated with UVB and treated with 500 nM wortmannin; lane 9, untreated cells; lane 10, cells irradiated with UVB; lane 11, cells irradiated with UVB and treated with 200 nM staurosporine; lane 12, cells treated with 1 μg LPS per ml; lane 13, cells treated with LPS and rapamycin. Journal of Investigative Dermatology 2000 114, 1005-1010DOI: (10.1046/j.1523-1747.2000.00964.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Early versus late gene activation by UV irradiation and inhibition by rapamycin. (A) TNF-α CAT activity assay as in Figure 5b(B) and measured as described inKibitel et al. (1998). Baseline CAT activity has been subtracted. Cells were held at 10°C (@10c) or 37°C for 30 min prior to and 10 min after irradiation, or treated with 2 nM rapamycin (+rap) for 30 min prior to irradiation with 100 J UVB per m2 and then incubated for 3 or 24 h before assay. (B) PGE2 assay of HaCaT cells irradiated with UVB at 10°C (as noted) or 37°C or treated with rapamycin (+rap). After 3 h the PGE2 in the serum-free supernatant was assayed by ELISA. Each measurement was in duplicate and the values were averaged. Journal of Investigative Dermatology 2000 114, 1005-1010DOI: (10.1046/j.1523-1747.2000.00964.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Inhibition of systemic contact hypersensitivity by UV irradiation and protection by rapamycin. Ear swelling was measured 24 and 48 h after challenge by DNFB. Mice were either untreated (None, n = 2); or DNFB was applied after four consecutive days of either no treatment (DNFB alone, n = 3); treatment with 0.3% rapamycin (+rapamycin, n = 5); irradiation with 200 J UVB per m2 (+UVB, n = 4); or rapamycin followed by UVB (+Rapamycin + UVB, n = 5). Black bars represent the measurements at 24 h and gray bars represent the measurements at 48 h. The asterisks denote that the differences between the values at 24 and 48 h for +UVB and +Rapamycin + UVB were both statistically significant (p < 0.001). Journal of Investigative Dermatology 2000 114, 1005-1010DOI: (10.1046/j.1523-1747.2000.00964.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 A model of DNA damage induction of TNF-α mediated by FRAP. UV irradiation induces DNA damage in the form of cyclobutane pyrimidine dimers, and this structural alteration in DNA is recognized by the FRAP protein. The FRAP kinase activity then phosphorylates target proteins, including the p53, nuclear p70S6K, and RNA Pol II proteins. Rapamycin can bind FKBP12 to the FRAP protein and prevent this phosphorylation, thereby blocking later events. The phosphorylated target proteins then modulate gene transcription and translation. In particular, p70S6K translocates to the cytoplasm, where it phosphorylates ribosomal proteins and alters their patterns of translation. The result is induction of TNF-α protein and other late-gene proteins. One of the effects of elevated TNF-α is to suppress the contact hypersensitivity immune response. Journal of Investigative Dermatology 2000 114, 1005-1010DOI: (10.1046/j.1523-1747.2000.00964.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions