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Mitogen- and Stress-activated Protein Kinase 1 Mediates Activation of Akt by Ultraviolet B Irradiation

2001; Elsevier BV; Volume: 276; Issue: 27 Linguagem: Inglês

10.1074/jbc.m101164200

ISSN

1083-351X

Autores

Masaaki Nomura, Akira Kaji, Weiya Ma, Shuping Zhong, Guangming Liu, G. Tim Bowden, K Miyamoto, Zigang Dong,

Tópico(s)

CRISPR and Genetic Engineering

Resumo

In this study, we investigated the mechanism by which UVB irradiation activates Akt (also known as protein kinase B (PKB)) in mouse epidermal JB6 cells. Treatment with a phosphatidylinositol 3-kinase inhibitor, LY 294002, or expression of a dominant negative mutant of p85 (regulatory component of phosphatidylinositol 3-kinase) inhibited UVB-induced Akt activation. Interestingly, Akt activation by UVB was attenuated by treatment with PD 98059, a specific mitogen-activated protein kinase/extracellular signal-regulated protein kinase (Erk) kinase 1 inhibitor, or SB 202190, a specific p38 kinase inhibitor. Furthermore, the expression of a dominant negative mutant of Erk2 or p38 kinase, but not that of c-Jun N-terminal kinase 1 (JNK1), blocked UVB-induced Akt activation. The expression of a dominant negative mutant of p85 or treatment with LY 294002 also inhibited UVB-induced Erk phosphorylation. The UVB-activated mitogen-activated protein kinase members, which were immunoprecipitated from cells exposed to UVB, did not phosphorylate Akt. Instead, Akt was phosphorylated at both threonine 308 and serine 473 and activated by UVB-activated mitogen- and stress-activated protein kinase 1 (Msk1). The expression of a Msk1 C-terminal kinase-dead mutant inhibited UVB-induced phosphorylation and activation of Akt. These data thus suggested that UVB-induced Akt activation was mediated through Msk1, which is a downstream kinase of the Erk and p38 kinase signaling pathways. In this study, we investigated the mechanism by which UVB irradiation activates Akt (also known as protein kinase B (PKB)) in mouse epidermal JB6 cells. Treatment with a phosphatidylinositol 3-kinase inhibitor, LY 294002, or expression of a dominant negative mutant of p85 (regulatory component of phosphatidylinositol 3-kinase) inhibited UVB-induced Akt activation. Interestingly, Akt activation by UVB was attenuated by treatment with PD 98059, a specific mitogen-activated protein kinase/extracellular signal-regulated protein kinase (Erk) kinase 1 inhibitor, or SB 202190, a specific p38 kinase inhibitor. Furthermore, the expression of a dominant negative mutant of Erk2 or p38 kinase, but not that of c-Jun N-terminal kinase 1 (JNK1), blocked UVB-induced Akt activation. The expression of a dominant negative mutant of p85 or treatment with LY 294002 also inhibited UVB-induced Erk phosphorylation. The UVB-activated mitogen-activated protein kinase members, which were immunoprecipitated from cells exposed to UVB, did not phosphorylate Akt. Instead, Akt was phosphorylated at both threonine 308 and serine 473 and activated by UVB-activated mitogen- and stress-activated protein kinase 1 (Msk1). The expression of a Msk1 C-terminal kinase-dead mutant inhibited UVB-induced phosphorylation and activation of Akt. These data thus suggested that UVB-induced Akt activation was mediated through Msk1, which is a downstream kinase of the Erk and p38 kinase signaling pathways. protein kinase B cytomegalovirus dominant negative mutant dithiothreitol extracellular signal-regulated protein kinase fetal bovine serum c-Jun N-terminal kinase mitogen-activated protein MAP kinase-activated protein kinase minimal essential medium 3-[N-morpholino]propanesulfonic acid mitogen- and stress-activated protein kinase phosphatidylinositol 3,4, 5-triphosphate-dependent protein kinase phosphatidylinositol 3-kinase Phosphatase andTensin ribosomal S6 protein kinase kinase Ultraviolet irradiation, especially in the UVB range (290–320 nm), accounts for most of the harmful biological effects associated with sunlight, including cancer in mammals and malformations in amphibians (1Elder D.E. J. Invest. Dermatol. 1989; 92: 297S-303SAbstract Full Text PDF PubMed Scopus (56) Google Scholar, 2Nomura T. Nakajima H. Hongyo T. 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These findings suggest that UV irradiation induces the activation of Akt. In a wide range of cellular systems, Akt has been shown to control intracellular pathways responsible for preventing cell death in response to a variety of extracellular stimuli (15Marte B.M. Downward J. Trends. Biochem. Sci. 1997; 22: 355-358Abstract Full Text PDF PubMed Scopus (649) Google Scholar, 16Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2222) Google Scholar, 17Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (966) Google Scholar). Furthermore, recent work has shown that Akt is not only a “cell survival” kinase but it may play an important role in protein synthesis, glycogenesis, and regulation of cell cycle progression (18Medema R.H. Kops G.J. Bos J.L. Burgering B.M. 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Chem. 2001; 276: 3517-3523Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar) recently showed that the p38 kinase pathway regulates Akt activation in human neutrophils. The Erk signaling pathway also may be associated with anti-apoptotic effects (47Bonni A. Brunet A. West A.E. Datta S.R. Takasu M.A. Greenberg M.E. Science. 1999; 286: 1358-1362Crossref PubMed Scopus (1682) Google Scholar, 48Meier P. Evan G. Cell. 1998; 95: 295-298Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Therefore, we investigated the involvement of the MAP kinase signaling pathways in UVB-induced Akt activation. In this study, we demonstrated that activation of Akt is induced by UVB irradiation and that the activation of Akt is mediated by Erks and p38 kinase through their downstream kinase, mitogen- and stress-activated protein kinase 1 (Msk1), in addition to the PI3-K/PDK pathway. Fetal bovine serum (FBS) was from Gemini Bio-Product (Calabasas, CA); gentamicin was from BioWhittaker, Inc. (Walkersville, MD); Eagle's minimal essential medium (MEM) andl-glutamine were from Life Technologies, Inc.; the MAP kinase/Erk kinase 1 specific inhibitor PD 98059 and the PI3-K inhibitor LY 29402 were from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA); the p38 kinase inhibitor SB 202190 was from Calbiochem (La Jolla, CA); Akt fusion protein, radioactive and nonradioactive Akt immunoprecipitation kinase assay kit, anti-Msk1 antibody, anti-MAP kinase-activated protein kinase (MAPKAP-K) 2 antibody, and anti-ribosomal S6 protein kinase kinase (Rsk) 1 antibody were from Upstate Biotechnology, Inc. (Lake Placid, NY); Elk-1, c-Jun, and ATF-2 fusion protein, Akt antibody, phospho-specific Akt (threonine 308 or serine 473), Bad (serine 136), Elk-1 (serine 383), c-Jun (serine 63), and ATF-2 (threonine 71), PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from New England BioLabs, Inc. (Beverly, MA); phospho-JNK antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz Biotechnology (Santa Cruz, CA); and phosphatidylinositol was from Sigma. Dominant negative mutants of Erk2, p38 kinase, JNK1, and PI3-K subunit p85 were generous gifts from Drs. Melanie H. Cobb (49Frost J.A. Geppert T.D. Cobb M.H. Feramisco J.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3844-3848Crossref PubMed Scopus (203) Google Scholar), Mercedes Rincon (50Rincon M. Enslen H. Raingeaud J. Recht M. Zapton T. Su M.S. Penix L.A. Davis R.J. Flavell R.A. EMBO J. 1998; 17: 2817-2829Crossref PubMed Scopus (359) Google Scholar, 51Chen Y.R. 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Ding M. Bowden G.T. Dong Z. J. Biol. Chem. 1997; 272: 27753-27757Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The source of UVB was a bank of four Westinghouse F520 Lamps (National Biological, Twinsburg, OH) at 6 J/S/m light in the UVB range. Approximately 10% of the remaining radiation from the F520 lamp is in the UVA region (320 nm). Although almost no UVC leakage occurred, the UVB irradiation was carried out in a UVB exposure chamber fitted with a Kodak Kodacel K6808 filter that eliminates all wavelengths below 290 nm. This lamp is one of the most frequently used UVB sources for the study of carcinogenesis. The International Agency for Research on Cancer refers to this lamp as a source emitting mainly UVB irradiation for the study of cancer induction in animals. UVB irradiation was measured using the UVX radiometer from UVP (UVX-31). The JB6 mouse epidermal cell line Cl 41 (JB6 Cl 41) and its stable transfectants, Cl 41 CMV-neo, Cl 41 DN-Erk2, Cl 41 DN-p38, Cl 41 DN-JNK1, Cl 41 DN-p85, Cl 41 CMV5, and Cl 41 Msk1 C-dead, were grown at 37 °C in MEM supplemented with 5% heat-inactivated FBS, 2 mml-glutamine, and 25 μg/ml gentamicin. The dominant negative mutants of Erk2, p38 kinase, or JNK1 were subcloned into a mammalian expression vector plasmid, CMV-neo. A dominant negative mutant plasmid of PI3-K p85 subunit or Msk1 C-terminal kinase-dead mutant plasmid or its control vector CMV5 plasmid was transfected or co-transfected with CMV-neo in JB6 Cl 41 cells by using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instructors. The stable transfectants were obtained by selection for G418 resistance (300 μg/ml) and further confirmed by assay of respective activities (44Huang C. Ma W.Y. Dong Z. Oncogene. 1999; 18: 2828-2835Crossref PubMed Scopus (64) Google Scholar,55Huang C. Li J. Ma W. Dong Z. J. Biol. Chem. 1999; 274: 29672-29676Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 56Huang C. Ma W. Maxiner A. Sun Y. Dong Z. J. Biol. Chem. 1999; 274: 12229-12235Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 57Huang C. Schmid P.C. Ma W. Schmid H.O. Dong Z. J. Biol. Chem. 1997; 272: 4187-4194Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Immunoblotting was carried out as described previously (44Huang C. Ma W.Y. Dong Z. Oncogene. 1999; 18: 2828-2835Crossref PubMed Scopus (64) Google Scholar, 55Huang C. Li J. Ma W. Dong Z. J. Biol. Chem. 1999; 274: 29672-29676Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 56Huang C. Ma W. Maxiner A. Sun Y. Dong Z. J. Biol. Chem. 1999; 274: 12229-12235Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). In brief, JB6 Cl 41 cells and their stable transfectants were cultured to 80% confluence. The cells were starved in 0.1% FBS MEM for 48 h at 37 °C. Then, medium was changed to fresh 0.1% FBS MEM, and cells were incubated for another 2–4 h at 37 °C. Before the cells were treated with UVB or insulin, they were treated with or without LY 294002, PD 98059, or SB 202190 for 1 h. Then, the cells were exposed to UVB (4 kJ/m2) and subsequently incubated for the indicated periods or for 30 min or treated with insulin (2.5 μg/ml) for the indicated periods or for 5 min at 37 °C in the presence of inhibitors. The cells were then lysed, and immunoblot analysis performed by using the antibodies against Akt, Erks, JNKs, or p38 kinase or the phospho-specific antibodies against their phosphorylated proteins. Antibody-bound proteins were detected by chemiluminescence (ECF from Amersham Pharmacia Biotech) and analyzed using the Storm 840 scanner (Molecular Dynamics). Cells were treated with UVB (4 kJ/m2) as described above. The cells were lysed in 400 μl of lysis buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 1 mm Na2EDTA, 1 mm EGTA, 1 mm Na3VO4, 1 mm ν-glycerophosphate, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μm microcystin). The lysates were sonicated and centrifuged, and the supernatant fraction was incubated with the phospho-specific Erks, p38 kinase, or JNKs antibody with gentle rocking for 6–10 h at 4 °C. Then, the protein A/G plus agarose was added, and the incubation was continued for another 4 h. The beads were washed twice with 500 μl of lysis buffer and twice with 500 μl of kinase buffer (25 mm Tris, pH 7.5, 5 mm ν-glycerophosphate, 2 mmdithiothreitol (DTT), 0.1 mmNa3VO4, 10 mm MgCl2). The kinase reactions were carried out in the presence of 200 μm ATP at 30 °C for 30 min using 2 μg of Elk-1, ATF-2, or c-Jun as substrate for Erks, p38 kinase, or JNKs, respectively. The phosphorylated proteins were detected by immunoblotting using phospho-specific antibodies. PI3-K activity was carried out as described previously (57Huang C. Schmid P.C. Ma W. Schmid H.O. Dong Z. J. Biol. Chem. 1997; 272: 4187-4194Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In brief, cells were treated with UVB (4 kJ/m2) as described above. The cells were lysed in 400 μl of lysis buffer (20 mm Tris-HCl, pH 7.4, 137 mmNaCl, 1 mm MgCl2, 10% glycerol, 1% Nonidet P-40, 1 mm DTT, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 10 μmaprotinin, 10 μm leupeptin). The lysate was sonicated and centrifuged, and the supernatant fraction was incubated with 20 μl of agarose conjugated with monoclonal anti-phosphotyrosine antibody PY20 with gentle rocking overnight at 4 °C. The agarose beads were washed twice with each of the following buffers: 1) PBS with 1% Nonidet P-40, 1 mm DTT, 0.1 mm sodium orthovanadate; 2) 100 mm Tris-HCl, pH 7.6, 0.5m LiCl, 1 mm DTT, 0.1 mm sodium orthovanadate; and 3) 10 mm Tris-HCl, pH 7.6, 0.1m NaCl, 1 mm DTT, 0.1 mm sodium orthovanadate. The beads were incubated for 5 min on ice in 20 μl of Buffer 3, and then 20 μl of 0.5 mg/ml phosphatidylinositol (previously sonicated in 50 mm Hepes, pH 7.6, 1 mm EGTA, 1 mm NaH2PO4) was added. After 5 min at room temperature, 10 μl of the reaction buffer was added (50 mm MgCl2, 100 mm Hepes, pH 7.6, 250 μm ATP containing 10 μCi of [γ-32P]ATP), and the beads were incubated for an additional 15 min. The reaction was stopped by the addition of 15 μl of 4 n HCl and 130 μl of chloroform:methanol (1:1). After vortexing for 30 s, 30 μl of the chloroform phase was spotted onto 1% potassium oxalate-coated silica gel H plates (previously baked at 110 °C for 1 h). The plates were developed in choloroform/methanol/NH4OH/H2O (60:47:2:11.3) and dried at room temperature. Radiolabeled spots were quantified using the Storm 840 scanner (Molecular Dynamics). Cells were treated with UVB (4 kJ/m2), lysates were prepared from the cells, and the immunoprecipitation was carried out using the phospho-specific Erk, p38 kinase, or JNKs antibody or the specific Msk1, MAPKAP-K2, or Rsk1 antibody as described above (under “Assay for Erk, p38 Kinase, and JNK Activities”). The kinase reactions were carried out at 30 °C for 30 min in the presence of kinase buffer with 200 μmATP and 250 ng of inactive Akt fusion protein as substrate. The phosphorylated protein was detected by immunoblotting using phospho-specific Akt antibodies. Cells were treated with UVB (4 kJ/m2) or insulin (2.5 μg/ml), lysates were prepared from the cells, and the immunoprecipitation was carried out using 4 μg of anti-Akt1/PKBα PH domain or anti-Msk1 antibody as described above. The enzyme immune complex was washed three times with 0.5 ml of lysis buffer and once with 100 μl of assay dilution buffer (20 mm MOPS, pH 7.2, 25 mm ν-glycerophosphate, pH 7.0, 1 mmsodium orthovanadate, 1 mm DTT). For the Akt kinase assay, the enzyme immune complex was added to 10 μl of assay dilution buffer, 40 μm protein kinase A inhibitor peptide, 0.4 mm Akt substrate peptide, and 1 μCi/μl [γ-32P]ATP, and for the Msk kinase assay, it was added to 20 μl of assay dilution buffer, 10 μl of 0.4 mm Akt substrate peptide, and 1 μCi/μl [γ-32P]ATP. The reaction was incubated for 10 min at 30 °C and centrifuged, and then 30 μl of the supernatant fraction was transferred onto P81 phosphocellulose paper and allowed to bind for 30 s. The P81 papers were washed three times in 0.75% phosphoric acid and then washed once in acetone, and γ-32P incorporation was measured by scintillation counting. Cells were treated with UVB (4 kJ/m2), lysates were prepared from the cells, and the immunoprecipitation was carried out using 4 μg of anti-Akt1/PKBα PH domain antibody as described above. The enzyme immune complex was added to 12.5 μl of assay dilution buffer, 10 μl (3 μg) of Bad protein, and 50 μl of magnesium/ATP mixture (75 mm MgCl2 and 500 μm ATP in assay dilution buffer). The reaction was incubated for 10 min at 30 °C and centrifuged, and then 20 μl of the supernatant fraction was used as a sample for immunoblotting. Immunoblot analysis was performed by using the phospho-specific Bad (serine 136) antibody, and antibody-bound proteins were detected by chemiluminescence (ECF from Amersham Pharmacia Biotech) and analyzed using the Storm 840 scanner (Molecular Dynamics). Cells were treated with UVB (4 kJ/m2), lysates were prepared from the cells, and the immunoprecipitation was carried out using 4 μg of anti-Msk1 or anti-MAPKAP-K2 antibodies as described above. The enzyme immune complex was added to 12.5 μl (250 ng) of Akt in assay dilution buffer, 10 μl (3 μg) of Bad protein, and 50 μl of magnesium/ATP mixture (75 mm MgCl2 and 500 μmATP in assay dilution buffer). The reaction was incubated for 30 min at 30 °C and centrifuged, and then 20 μl of the supernatant fraction was used as a sample of immunoblotting. Immunoblot analysis was performed by using the phospho-specific Bad (serine 136) antibody. Antibody-bound proteins were detected by chemiluminescence (ECF fromAmersham Pharmacia Biotech) and analyzed using the Storm 840 scanner (Molecular Dynamics). Because the phosphorylation of threonine 308 and serine 473 is a prerequisite for the catalytic activity of Akt (19Muise-Helmericks R.C. Grimes H.L. Bellacosa A. Malstrom S.E. Tsichlis P.N. Rosen N. J. Biol. Chem. 1998; 273: 29864-29872Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 33Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. 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