Signal Transduction Pathways Involved in Phosphorylation and Activation of p70S6K Following Exposure to UVA Irradiation
2001; Elsevier BV; Volume: 276; Issue: 24 Linguagem: Inglês
10.1074/jbc.m009047200
ISSN1083-351X
AutoresYiguo Zhang, Ziming Dong, Masaaki Nomura, Shuping Zhong, Nanyue Chen, Ann M. Bode, Zigang Dong,
Tópico(s)Viral Infectious Diseases and Gene Expression in Insects
ResumoUltraviolet light A (UVA) plays an important role in the etiology of human skin cancer, and UVA-induced signal transduction has a critical role in UVA-induced skin carcinogenesis. The upstream signaling pathways leading to p70S6Kphosphorylation and activation are not well understood. Here, we observed that UVA induces phosphorylation and activation of p70S6K. Further, UVA-stimulated p70S6K activity and phosphorylation at Thr389 were blocked by wortmannin, rapamycin, PD98059, SB202190, and dominant negative mutants of phosphatidylinositol (PI) 3-kinase p85 subunit (DNM-Δp85), ERK2 (DNM-ERK2), p38 kinase (DNM-p38), and JNK1 (DNM-JNK1) and were absent in Jnk1−/− or Jnk2−/− knockout cells. The p70S6K phosphorylation at Ser411 and Thr421/Ser424 was inhibited by rapamycin, PD98059, or DNM-ERK2 but not by wortmannin, SB202190, DNM-Δp85, or DNM-p38. However, Ser411, but not Thr421/Ser424 phosphorylation, was suppressed in DNM-JNK1 and abrogated in Jnk1−/− orJnk2−/− cells. In vitro assays indicated that Ser411 on immunoprecipitated p70S6K proteins is phosphorylated by active JNKs and ERKs, but not p38 kinase, and Thr421/Ser424 is phosphorylated by ERK1, but not ERK2, JNKs, or p38 kinase. Moreover, p70S6Kco-immunoprecipitated with PI 3-kinase and possibly PDK1. The complex possibly possessed a partial basal level of phosphorylation, but not at MAPK sites, which was available for its activation by MAPKs in vitro. Thus, these results suggest that activation of MAPKs, like PI 3-kinase/mTOR, may be involved in UVA-induced phosphorylation and activation of p70S6K. Ultraviolet light A (UVA) plays an important role in the etiology of human skin cancer, and UVA-induced signal transduction has a critical role in UVA-induced skin carcinogenesis. The upstream signaling pathways leading to p70S6Kphosphorylation and activation are not well understood. Here, we observed that UVA induces phosphorylation and activation of p70S6K. Further, UVA-stimulated p70S6K activity and phosphorylation at Thr389 were blocked by wortmannin, rapamycin, PD98059, SB202190, and dominant negative mutants of phosphatidylinositol (PI) 3-kinase p85 subunit (DNM-Δp85), ERK2 (DNM-ERK2), p38 kinase (DNM-p38), and JNK1 (DNM-JNK1) and were absent in Jnk1−/− or Jnk2−/− knockout cells. The p70S6K phosphorylation at Ser411 and Thr421/Ser424 was inhibited by rapamycin, PD98059, or DNM-ERK2 but not by wortmannin, SB202190, DNM-Δp85, or DNM-p38. However, Ser411, but not Thr421/Ser424 phosphorylation, was suppressed in DNM-JNK1 and abrogated in Jnk1−/− orJnk2−/− cells. In vitro assays indicated that Ser411 on immunoprecipitated p70S6K proteins is phosphorylated by active JNKs and ERKs, but not p38 kinase, and Thr421/Ser424 is phosphorylated by ERK1, but not ERK2, JNKs, or p38 kinase. Moreover, p70S6Kco-immunoprecipitated with PI 3-kinase and possibly PDK1. The complex possibly possessed a partial basal level of phosphorylation, but not at MAPK sites, which was available for its activation by MAPKs in vitro. Thus, these results suggest that activation of MAPKs, like PI 3-kinase/mTOR, may be involved in UVA-induced phosphorylation and activation of p70S6K. UVB, and UVC, ultraviolet light A, B, and C, respectively p70/p85 ribosomal S6 kinases proline-directed Ser/Thr 12-O-tetradecanoylphorbol-13-acetate epidermal growth factor phosphatidylinositol 3-phosphoinositide-dependent protein kinase 1 mammalian target of rapamycin mitogen-activated protein kinase extracellular signal-regulated kinase c-Jun N-terminal kinase p38 MAPK or p38 kinase protein phosphatase 1 p90 ribosomal S6 kinase(s) Eagle's minimum essential medium fetal bovine serum phosphate-buffered saline dithiothreitol polyacrylamide gel electrophoresis dominant negative mutant 4-morpholinepropanesulfonic acid phospho- nonphospho- Ultraviolet light A (UVA)1 (320–400 nm) comprises ∼95% of the total solar UV reached the earth (1Grether-Beck S. Buettner R. Krutmann J. Biol. Chem. 1997; 378: 1231-1236PubMed Google Scholar), because all the ultraviolet C (UVC) (200–290 nm) and most of the ultraviolet B (UVB) (290–320 nm) radiation are absorbed by the earth's stratospheric ozone layer (2Tyrrell R.M. Feige U. Morimoto R.I. Yahara I. Polla B. Stress-inducible Cellular Responses. Birkhäuser Verlag, Basel, Switzerland1996: 255-271Crossref Google Scholar). Currently, UVA, like UVB, is considered to be a complete carcinogen (3Scharffetter-Kochanek K. Wlaschek M. Brenneisen P. Schauen M. Blaudschun R. Wenk J. Biol. Chem. 1997; 378: 1247-1257PubMed Google Scholar) and to play an important role in the etiology of human skin cancer (4De Laat J.M. de Gruijl F.R. Cancer Surv. 1996; 26: 173-191PubMed Google Scholar). But the activation of signaling molecules and their pathways implicated in the process following UVA irradiation (5Bae G.U. Seo D.W. Kwon H.K. Lee H.Y. Hong S. Lee Z.W. Ha K.S. Lee H.W. Han J.W. J. Biol. Chem. 1999; 274: 32596-32602Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 6Bender K. Blattner C. Knebel A. Iordanov M. Herrlich P. Rahmsdorf H.J. J. Photochem. Photobiol. B Biol. 1997; 37: 1-17Crossref PubMed Scopus (233) Google Scholar) are not well understood. Therefore, the study of UVA-induced signal transduction will help in understanding the molecular mechanisms underlying UVA-induced carcinogenesis. Activation of tumor cell proliferation requires an accelerated rate of protein synthesis, which is regulated in part by intracellular activation of several signaling protein kinase cascades that interact with the translational machinery of the ribosome (7Kimball S.R. Vary T.C. Jefferson L.S. Annu. Rev. Physiol. 1994; 56: 321-348Crossref PubMed Scopus (182) Google Scholar). Among them, S6 is a component of ribosomal proteins and is located at the interface between 40 and 60 S ribosomal proteins (8Stewart M.J. Thomas G. Bioessays. 1994; 16: 809-815Crossref PubMed Scopus (69) Google Scholar). Phosphorylation of S6 at multiple serine sites on its C terminus was shown to be correlated with increased translation, especially of mRNAs containing a polypyrimidine tract in their 5′-untranslated regions (9Peterson R.T. Schreiber S.L. Curr. Biol. 1998; 8: R248-R250Abstract Full Text Full Text PDF PubMed Google Scholar). This family of mRNAs constitutes as few as 100–200 genes but makes up 20–30% of the total cellular mRNA, indicating that they are important for cell cycle progression. The family of serine/threonine kinases that mediate S6 phosphorylation are known as ribosomal S6 kinases, one of which is a 70-kDa S6 kinase (p70S6K) (8Stewart M.J. Thomas G. Bioessays. 1994; 16: 809-815Crossref PubMed Scopus (69) Google Scholar). Accumulating evidence suggests that the prominent role of p70S6Kactivation in mitogenesis may be to promote translation of mRNAs necessary for cell growth and division and to generate many of the molecules necessary for driving the cell cycle from G0/G1 to S phase (9Peterson R.T. Schreiber S.L. Curr. Biol. 1998; 8: R248-R250Abstract Full Text Full Text PDF PubMed Google Scholar). Initially, p70S6K was isolated from mitogen-stimulated Swiss mouse 3T3 cells (10Jenö P. Ballou L.M. Novak-Hofer I. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 406-410Crossref PubMed Scopus (144) Google Scholar). Subsequently, two isoforms of p70S6K (p70S6K/p85S6K, collectively termed p70S6K or S6K1) were found in purification, cloning, and expression studies (11Reinhard C. Thomas G. Kozma S.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4052-4056Crossref PubMed Scopus (98) Google Scholar). Both isoforms are encoded by the same transcript with alternative translational start sites (12Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (608) Google Scholar). Based on evidence that an additional 23-amino acid extension at the N terminus of p85S6K was shown to function as a nuclear localization signal, p85S6K appears to be exclusively nuclear, whereas p70S6K is largely cytoplasmic (11Reinhard C. Thomas G. Kozma S.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4052-4056Crossref PubMed Scopus (98) Google Scholar, 12Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (608) Google Scholar). The p85S6K may be responsible for phosphorylation of the free chromatin-bound nuclear form of S6 (13Franco R. Rosenfeld M.G. J. Biol. Chem. 1990; 265: 4321-4325Abstract Full Text PDF PubMed Google Scholar, 14Reinhard C. Fernande A. Lamb N.J. Thomas G. EMBO J. 1994; 13: 1557-1565Crossref PubMed Scopus (179) Google Scholar). Recently, deletion of the p70S6K gene was shown to have no effect on S6 phosphorylation, 5′-untranslated region mRNA translation, or the rate of cell growth, but it resulted in a small mouse phenotype (15Shima H. Pende M. Chen Y. Fumagalli S. Thomas G Kozma S.C. EMBO J. 1998; 17: 6649-6659Crossref PubMed Google Scholar). In p70 S6K −/− mice, another S6 kinase (S6K2) with a 70% overall amino acid homology with p70S6K and a potential nuclear localization signal at the C terminus was found to partially compensate for loss of p70S6K function (15Shima H. Pende M. Chen Y. Fumagalli S. Thomas G Kozma S.C. EMBO J. 1998; 17: 6649-6659Crossref PubMed Google Scholar). Recently, another nuclear S6 kinase-related kinase was cloned and identified as a novel nuclear target of Akt (16Koh H. Jee K Lee B. Kim J. Kim D. Yun Y.H. Kim J.W. Choi H.S. Chung J. Oncogene. 1999; 18: 5115-5119Crossref PubMed Scopus (67) Google Scholar). Generally, the p70S6K family plays a key role in the control of cell size, growth, and proliferation. Consistent with this concept, inhibition of p70S6Kactivation by microinjection of neutralizing antibodies (17Lane H.A. Fernandez A. Lamb N.J. Thomas G. Nature. 1993; 363: 170-172Crossref PubMed Scopus (318) Google Scholar) or treatment of cells with rapamycin, an inhibitor of mammalian target of rapamycin (mTOR)-p70S6K (18Chung J. Kuo C.J. Crabtree G.R. Blenis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1031) Google Scholar, 19Kuo C.J. Chung J. Fiorentino D.F. Flanagan W.M. Blenis J. Crabtree G.R. Nature. 1992; 358: 70-73Crossref PubMed Scopus (568) Google Scholar, 20Price D.J. Grove J.R. Clavo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (590) Google Scholar), severely impeded cell cycle progression. Although p70S6K is known to be activated by various stimuli including growth factors, cytokines, 12-O-tetradecanoylphorbol-13-acetate (TPA), oncogenic products, Ca2+, and inhibitors of protein synthesis (5Bae G.U. Seo D.W. Kwon H.K. Lee H.Y. Hong S. Lee Z.W. Ha K.S. Lee H.W. Han J.W. J. Biol. Chem. 1999; 274: 32596-32602Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar,21Chou M.M. Blenis J. Curr. Opin. Cell Biol. 1995; 7: 806-814Crossref PubMed Scopus (245) Google Scholar), the signal transduction pathway mediating p70S6K is poorly understood. An array of independently regulated protein kinases (12Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (608) Google Scholar, 22Weng Q.-P. Kozlowski M. Belham C. Zhang A. Comb M.J. Avruch J. J. Biol. Chem. 1998; 273: 16621-16629Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 23Eguchi S. Iwasaki H. Ueno H. Frank G.D. Motley E.D. Eguchi K. Marumo F. Hirata Y. Inagami T. J. Biol. Chem. 1999; 274: 36843-36851Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) are known to activate 70S6K via phosphorylation of at least eight Ser/Thr sites in its three separate domains. Thr229 in the p70S6K activation loop within the catalytic domain has been shown to be phosphorylatedin vivo through the phosphatidylinositol (PI)-3 kinase pathway (24Avruch J. Mol. Cell. Biochem. 1998; 182: 31-48Crossref PubMed Scopus (324) Google Scholar) and in vitro selectively by 3-phosphoinositide-dependent protein kinase 1 (PDK1) (25Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (731) Google Scholar). Thr229 phosphorylation has been shown to enable p70S6K activity and be repressed by wortmannin, an inhibitor of PI 3-kinase (26Weng Q.-P. Andrabi K. Klippel A. Kozlowski M.T. Williams L.T. Avruch J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5744-5748Crossref PubMed Scopus (202) Google Scholar). Additionally, similar to Thr229, Ser371 located in the kinase extension domain, has been shown to influence p70S6K activity, and its phosphorylation is also regulated by the PI 3-kinase-dependent pathway (27Moser B.A. Dennis P.B. Pullen N. Pearson R.B. Williamson N.A. Wettenhall R.E. Kozma S.C. Thomas G. Mol. Cell. Biochem. 1997; 17: 5648-5655Crossref Scopus (88) Google Scholar). Thr389 is another site for mitogen-stimulated phosphorylation and is situated in a conserved 65-amino acid segment located immediately C-terminal to the catalytic domain (12Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (608) Google Scholar, 22Weng Q.-P. Kozlowski M. Belham C. Zhang A. Comb M.J. Avruch J. J. Biol. Chem. 1998; 273: 16621-16629Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). It plays an especially important role in p70S6K activation, because it influences both the phosphorylation of Thr229 in vitro by PDK1 and p70S6K activity (22Weng Q.-P. Kozlowski M. Belham C. Zhang A. Comb M.J. Avruch J. J. Biol. Chem. 1998; 273: 16621-16629Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). Although p70S6K was shown to be phosphorylated by mTOR in vitro (28Burnett P.E. Barrow R.K. Cohen N.A. Snyder S.H. Sabatini D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1432-1437Crossref PubMed Scopus (947) Google Scholar, 71Isotani S. Hara K. Tokunaga C. Inoue H. Avruch J. Yonezawa K. J. Biol. Chem. 1999; 274: 34493-34498Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), a p70Δ2–46/ΔCT104 construct was shown to still be activated and phosphorylated at Thr389 in vivo in the presence of rapamycin, an mTOR inhibitor (12Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (608) Google Scholar, 22Weng Q.-P. Kozlowski M. Belham C. Zhang A. Comb M.J. Avruch J. J. Biol. Chem. 1998; 273: 16621-16629Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 29Dennis P.B. Pullen N. Kozma S.C. Thoma G. Mol. Cell. Biol. 1996; 16: 6242-6251Crossref PubMed Scopus (224) Google Scholar). Other studies indicated that mTOR activity probably suppresses protein phosphatase 2A-mediated dephosphorylation of p70S6K (30Parrott L.A. Templeton D.J. J. Biol. Chem. 1999; 274: 24731-24736Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 31Peterson R.T. Desai B.N. Hardwick J.S. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4438-4442Crossref PubMed Scopus (429) Google Scholar, 32Westphal R.S. Cffee Jr., R.L. Marotta A. Pelech S.L. Wadzinski B.E. J. Biol. Chem. 1999; 274: 687-692Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). At the same time that p70S6K is activated by mitogens, the extracellular signal-regulated kinases (ERKs) pathway is also stimulated (23Eguchi S. Iwasaki H. Ueno H. Frank G.D. Motley E.D. Eguchi K. Marumo F. Hirata Y. Inagami T. J. Biol. Chem. 1999; 274: 36843-36851Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), indicating that the activation of p70S6Kappears to coincide with that of ERKs. The proline-directed Ser/Thr (S/T-P) sites (Ser411, Ser418, Thr421, and Ser424) within the autoinhibitory domain of p70S6K are in a consensus motif similar to those known to serve as recognition determinants for mitogen-activated protein kinases (MAPKs). Indeed, ERKs were shown to phosphorylate p70S6K in vitro, suggesting that the Ras/ERK pathway controlled p70S6K activation (33Mukhopadhyay N.K. Price D.J. Kryriakis J.M. Pelech S. Sanghera J. Avruch J. J. Biol. Chem. 1992; 267: 3325-3335Abstract Full Text PDF PubMed Google Scholar); however, other studies (34Chung J. Grammer T.C. Lemon K.P. Kazlauskas A. Blenis J. Nature. 1994; 370: 71-75Crossref PubMed Scopus (656) Google Scholar, 35Ming X.F. Burgering B.M. Wennström S. Claesson-Welsh L. Heldin C.H. Bos J.L. Kozma S.C. Thomas G. Nature. 1994; 371: 426-429Crossref PubMed Scopus (204) Google Scholar) showed that ERKs were neither necessary nor sufficient for p70S6K activation. But more recently, the ERK cascade, like PI 3-kinase/Akt cascades, has been demonstrated to be a prerequisite for p70S6K activation (23Eguchi S. Iwasaki H. Ueno H. Frank G.D. Motley E.D. Eguchi K. Marumo F. Hirata Y. Inagami T. J. Biol. Chem. 1999; 274: 36843-36851Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 36Papst P.J. Sugiyama H. Nagasawa M. Lucas J.J. Maller J.L. Terada N. J. Biol. Chem. 1998; 273: 15077-15084Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Taken together, p70S6K activation appears to require a complex array of separate, concurrent phosphorylations at multiple sites catalyzed by various protein kinases, but its precise mechanisms of activation are as yet unclear. In our work, we provide evidence that MAPK pathways, like the PI 3-kinase/mTOR pathways, are implicated in phosphorylation and activation of p70S6K in response to UVA irradiation. Chemicals were of the best grades available commercially. Eagle's minimum essential medium (MEM) and fetal bovine serum (FBS) were from Whittaker Biosciences, Inc. (Walkersville, MD); Dulbecco's modified Eagle's medium,l-glutamine, gentamicin, and G418 sulfate were from Life Technologies, Inc.; aprotinin, leupeptin, 12-O-tetradecanoylphorbol-13-acetate (TPA), PD98059, SB202190, and rapamycin were purchased from Sigma; wortmannin was from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA); PD169316 was from Alexis® Biochemicals, Inc. (San Diego, CA); and epidermal growth factor (EGF) was from Collaborative Research (Madison, WI). The phosphospecific antibodies against phosphorylated sites of ERKs (Tyr204 of p44 and p42), c-Jun N-terminal kinases (JNKs) (Thr183/Tyr185), p38 kinase (Thr180/Tyr182), and antibodies to nonphospho-ERKs, -JNKs, and -p38 kinase were from New England Biolabs, Inc. (Beverly, MA). The polyclonal antibodies against phospho-p70S6K at Ser411, Thr421/Ser424, or Thr389 and anti-nonphospho-p70S6K antibodies were also from New England Biolabs. Mouse anti-phosphospecific p70S6K(Ser411) mouse monoclonal antibody (A-6) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Active ERK1, ERK2, JNK1, JNK2, and p38 kinase and p70S6K S6 activity assay kits were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The CMV-neo vector plasmid was constructed as previously reported (37Huang C. Ma W-Y. Dong Z. Mol. Cell. Biol. 1996; 16: 6427-6435Crossref PubMed Scopus (153) Google Scholar). Mouse epidermal JB6 promotion-sensitive Cl 41 and its stable transfectants with CMV-neo mass1 (Cl 41) (38Huang C. Ma W-Y. Li J. Goranson A. Dong Z. J. Biol. Chem. 1999; 274: 14595-14601Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) or with dominant negative mutants of ERK2 (DNM-ERK2) (39Watts R.G. Huang C. Young M.R. Li J.J. Dong Z. Pennie W.D. Colburn N.H. Oncogene. 1998; 17: 3493-3498Crossref PubMed Scopus (107) Google Scholar), JNK1 (DNM-JNK1) (38Huang C. Ma W-Y. Li J. Goranson A. Dong Z. J. Biol. Chem. 1999; 274: 14595-14601Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), p38 kinase (DNM-p38) (40Huang C. Ma W-Y. Maxiner A. Sun Y. Dong Z. J. Biol. Chem. 1999; 274: 12229-12235Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar), or PI 3-kinase p85 subunit (DNM-Δp85) (41Huang C. Schmid P.C. Ma W.-Y. Schmid H.H. Dong Z. J. Biol. Chem. 1997; 272: 4187-4194Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) were established as previously reported and cultured in monolayers using Eagle's MEM supplemented with 5% heat-inactivated FBS, 2 mml-glutamine, and 25 μg/ml gentamicin at 37 °C in humidified air with 5% CO2. Before each experiment, the transfectants were selected with G418 and tested with their phosphospecific MAPK antibodies. The UVA source used was a Philips TL100w/10R system from Ultraviolet Resources International (Lakewood, OH). It consists of a Magnetek transformer number 799-XLH-TC-P, 120 V, 60 Hz, and six bulbs each 6 feet long. UVA irradiation filtered through about 6 mm of plate glass, eliminating most of UVB and UVC light at all wavelengths below 320 nm, was performed on cultured cells in the UVA box with two ventilation fans installed to eliminate thermal stimulation. These adjustments were necessary, because the normal UVA lamps can also produce a small amount of UVB and UVC. The UVB irradiation was carried out in a UVB chamber with a transluminator emitting UVB light protons and was fitted with an Eastman Kodak Co. Kodacel K6808 filter that eliminates all wavelengths below 290 nm. This was also necessary, because a normal UVB lamp can generate a small number of UVC light protons. UVC radiation performed was from germicidal lamps. To assess the roles of different signaling pathways in p70S6Kphosphorylation, we pretreated JB6 Cl 41 cells for 1–2 h before UVA irradiation with Me2SO or kinase inhibitors including PD98059, SB202190, PD169316, wortmannin, or rapamycin dissolved in Me2SO. Immunoblot analysis for detection of phosphorylated proteins for ERKs, JNKs, and p38 kinase was carried out using the phosphospecific MAPK antibodies as reported previously (38Huang C. Ma W-Y. Li J. Goranson A. Dong Z. J. Biol. Chem. 1999; 274: 14595-14601Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 39Watts R.G. Huang C. Young M.R. Li J.J. Dong Z. Pennie W.D. Colburn N.H. Oncogene. 1998; 17: 3493-3498Crossref PubMed Scopus (107) Google Scholar, 40Huang C. Ma W-Y. Maxiner A. Sun Y. Dong Z. J. Biol. Chem. 1999; 274: 12229-12235Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 41Huang C. Schmid P.C. Ma W.-Y. Schmid H.H. Dong Z. J. Biol. Chem. 1997; 272: 4187-4194Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The antibody-bound protein complexes were detected by Western immunoblotting using a chemiluminescent detection system (ECL, New England Biolabs). Some transfer membranes were washed with stripping buffer (7 m guanidine hydrochloride, 50 mm glycine, pH 10.8, 0.05 mm EDTA, 0.1m KCl, and 20 mm β-mercaptoethanol) and reprobed with other primary phosphospecific or nonphosphospecific antibodies. Cells (40 × 104 to 80 × 104) were seeded into 100-mm dishes and cultured for 24–48 h until the cells reached 80–90% confluence. The Cl 41, DNM-ERK2, DNM-JNK1, DNM-p38, or DNM-Δp85 cells were starved for 24–48 h in MEM containing 0.1% FBS, 2 mml-glutamine, and 25 μg/ml gentamicin. After treatment with UVA or kinase inhibitors as indicated (prior to irradiation), the cells were washed once with ice-cold phosphate-buffered saline (PBS) and lysed in 200 μl of SDS sample lysis buffer containing 62.5 mm Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol, 50 mm dithiothreitol (DTT), and 0.1% bromphenol blue. The lysed samples were scraped into 1.5-ml tubes and sonicated for 5–10 s. Samples containing equal amounts of protein (Bio-Rad protein assay) were loaded into each lane of an 8% SDS-polyacrylamide gel for electrophoresis (SDS-PAGE) and subsequently transferred onto an Immobilon P transfer membrane. The phosphorylated p70S6Kprotein was selectively detected by Western immunoblotting using a chemiluminescent detection system and phosphospecific antibodies against p70S6K phosphorylation at Ser411, Thr421/Ser424, or Thr389. Nonphosphorylated p70S6K was used as a control to verify equal protein loading. p70S6Kactivity was measured by an immune complex kinase assay using an S6 peptide, AKRRRLSSLRA, as a substrate according to the procedure recommended in the S6 kinase assay kit (Upstate Biotechnology) (5Bae G.U. Seo D.W. Kwon H.K. Lee H.Y. Hong S. Lee Z.W. Ha K.S. Lee H.W. Han J.W. J. Biol. Chem. 1999; 274: 32596-32602Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Briefly, cell lysates were prepared from JB6 Cl 41 cells or Cl 41 cell lines expressing DNM-ERK2, DNM-JNK1, DNM-p38, or DNM-Δp85 grown in 100-mm dishes. After starving by replacing medium with 0.1% FBS-MEM, the cells were or were not pretreated with inhibitors as described above and then irradiated with UVA (160 kJ/m2). The cells were harvested at the times indicated and lysed in 300 μl of buffer A containing 20 mm Tris (pH 7.5), 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% (v/v) Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerol phosphate, 1 mmNa3VO4, 1 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mm phenylmethylsulfonyl fluoride. The cell lysates were clarified by centrifugation at 17,000 × gfor 5 min at 4 °C. The supernatant fractions containing equal amounts of proteins were incubated with p70S6K antibody at 4 °C overnight and then for an additional 4 h with protein A/G-Sepharose beads (Santa Cruz Biotechnology). After washing four times with PBS, the immunoprecipitates were incubated at 30 °C for 10 min in a mixture of the following: 20 μl of assay dilution buffer (20 mm MOPS, pH 7.2, 25 mm β-glycerol phosphate, 5 mm EGTA, 1 mmNa3VO4, and 1 mm DTT), 10 μl of substrate mixture (S6 peptide in assay dilution buffer), 10 μl of inhibitor mixture (20 μm PKC inhibitor peptide, 2 μm protein kinase A inhibitor peptide, and 20 μm compound R24571 in assay dilution buffer), and 10 μl of [γ-32P]ATP (1 μCi/μl; Amersham Pharmacia Biotech). To stop the reaction, each sample was spotted onto a numbered P81 paper square and washed three times (5 min each) with 0.75% phosphoric acid and once (5 min) with acetone. Each sample paper was transferred into a scintillation vial containing 5 ml of scintillation fluid and then counted in a β-scintillation counter. At the same time, immunoprecipitates isolated by nonimmune IgG serum instead of the p70S6K antibody were used as background controls. After subtraction of background from each of the samples, the UVA-stimulated p70S6K kinase activity was normalized to unstimulated controls and expressed as -fold change. Embryonic fibroblasts from normal,Jnk1−/−, and Jnk2−/− knockout mice were isolated and prepared according to the procedure of Loo and Cotman (42Loo D.T. Cotman C.W. Celis J.E. Cell Biology: A Laboratory Handbook. 2nd Ed. Academic Press, Inc., San Diego1998: 65-72Google Scholar). Cells were established in culture in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 2 mml-glutamine, 100 units/ml of penicillin, and 100 μg/ml of streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. For analysis of protein phosphorylation, the cells were starved by replacing growth medium with serum-free Dulbecco's modified Eagle's medium for 12 h, at which time cells were exposed to UVA. The cells were lysed with SDS sample buffer, and the protein concentration in the supernatant fraction of the cell lysates was determined (Bio-Rad assay). Equal amounts of protein were resolved by 8% SDS-PAGE, and phosphorylated and nonphosphorylated p70S6K proteins were determined by Western blotting analysis. Additionally, p70S6K kinase activity in these cell lines was measured following immunoprecipitation procedures as described above. JB6 Cl 41 cells were cultured in 100-mm dishes, starved for 24 h, and then lysed in buffer A alone. The cell lysates were centrifuged, and then the supernatant fractions were subjected to immunoprecipitation with rabbit anti-nonphosphorylated p70S6K polyclonal antibody as described above. Samples containing immunoprecipitated p70S6K were incubated with active ERK1, ERK2, JNK1, JNK2, or p38 kinases (Upstate Biotechnology) at doses as indicated in kinase buffer (50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 1 mm EGTA, 1 mm DTT, 5 mm ATP, and 0.01% Brij 35) (New England Biolabs) at 30 °C for 60 min. The reactions were stopped by adding SDS sample buffer, and phosphorylation of immunoprecipitated p70S6K protein was analyzed by using SDS-PAGE, Western blotting, and a chemiluminescent detection system. The first antibodies are mouse phosphospecific p70S6K (Ser411) monoclonal antibody (Santa Cruz Biotechnology) and rabbit phosphospecific p70S6K (Thr389, Thr421/Ser424) polyclonal antibodies (New England Biolabs). To further analyze whether p70S6K is activated by MAPKs in vitro, samples containing immunoprecipitated p70S6K were incubated at 30 °C for 30 min with S6 peptide plus active ERK1 (10 ng/μl), ERK2 (10 ng/μl), JNK1 (25 milliunits/μl), JNK2 (25 milliunits/μl), or p38 kinases (10 ng/μl) (Upstate Biotechnology), and p70S6K kinase activity was determined as described above. At the same time, incubation of immunoprecipitated p70S6K with S6 peptide only was used as a negative control, and incubations of S6 peptide with MAPKs were used as internal controls. The addition of bovine serum albumin instead of MAPKs or immunoprecipitated p70S6Kproteins was used as background control. After starvation for 48 h, JB6 Cl 41 cells were or were not irradiated with UVA at 160 kJ/m2 and then harvested at 15 or 30 min following irradiation. Immunoprecipitated p70S6K proteins were obtained by incubating the cell lysates with p70S6K antibody as described above. At the same time, immunoprecipitates with normal nonimmune IgG serum instead of p70S6K antibody were used as internal negative controls, and immunoprecipitates with antibodies against PI 3-kinase p85α (Z-8), p90 ribosomal S6 kinase (RSK) 3 (C-20), phosphotyrosine (PY99 from Santa Cruz Biotechnology), PDK1, Akt, RSK1, RSK2, and mitogen- and stress-activated protein kinase (Upstate Biotechnology) were used as positive controls. To examine whether p70S6K co-immunoprecipitates with PI 3-kinase, immunoprecipitated p70S6K proteins and corresponding PI 3-kinase controls were incubated with phosphatidylinositol 4,5-diphosphate (Sigma), a preferential substrate of PI 3-kinase, and then PI 3-kinase activity was determined by
Referência(s)