Hyperphosphorylation of Retinoblastoma Protein and P53 by Okadaic Acid, a Tumor Promoter

Home , P53, Retinoblastoma protein, Tumor promotion

]CANCER RESEARCH53,239-241, January 15, 19931 Advances in Brief

Hyperphosphorylation of Retinoblastoma Protein and p53 by Okadaic Acid, a Tumor Promoter

Jun Yatsunami, Atsumasa Komori, Tetsuya Ohta, Masami Suganuma, and Hirota Fujiki 2 Cancer Prevention Division, National Cancer Center Research Institute, Tokyo 104, Japan

Abstract a hyperphosphorylated Rb position in one-dimensional SDS-PAGE for primary human fibroblasts labeled by [35S]methionine. The hy- A potent tumor promoter, okadaic acid, induced hyperphosphorylation perphosphorylation of p53 was autoradiographically determined for of tumor suppressor proteins, retinoblastoma protein and p53, by in vitro both cases. These findings are the first to describe the hyperphospho- incubation with nuclei isolated from rat regenerating liver as well as by incubation with primary human fibroblasts. Most of the retinoblastoma rylation of tumor suppressor gene products induced by a tumor pro- protein migrated to a hyperphosphorylated position in electrophoresis. moter, okadaic acid. The phosphorylation of p53 was increased at a rate 8 times that in non- Materials and Methods treated primary human fibroblasts. Hyperphosphorylation of tumor suppressor proteins, mediated through inhibition of protein phosphatases 1 Okadaic acid was isolated from the black sponge, Halichondria okadai (7). and 2A, is involved in tumor promotion by okadaic acid. The significance [3~-32p]ATP, [35S]methionine, and 32pi were obtained from Amersham, Buck- of hyperphosphorylation of the retinoblastoma protein and p53 is dis- inghamshire, United Kingdom. Male Fischer 344 rats, 7 weeks old, were cussed in relation to the regulation of the cell cycle. purchased from Charles River Japan, Inc., Kanagawa, Japan. The nuclei (200 lag), which were isolated from the rat liver 2 days after partial hepatectomy and Introduction from the liver without hepatectomy (8), were incubated with 2.5 ]aM ['y-32p]ATP Okadaic acid class tumor promoters, which are potent inhibitors of (3.7 MBq/ml) and various concentrations of okadaic acid in a buffer containing protein phosphatases 1 and 2A, induce tumors from initiated cells in 50 rnM Tris-HC1 (pH 7.6), 10 n~ MgC12, 1 mM dithiothreitol, and 1 mM various organs, mediated through common biochemical and molecular [ethylenebis(oxyethylenenitrilo)]tetraacetic acid for 10 min at 30~ After re- processes (1). Their mechanisms of action are named the okadaic acid action, the nuclei were immediately solubilized in 1 ml of the lysis buffer containing 25 mM Tris-HCI (pH 7.4), 50 rnM NaCI, 0.5% sodium deoxycholate, pathway; i.e., okadaic acid binds to the okadaic acid receptors, cata- 2% Nonidet P-40, 0.2% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl lytic subunits of protein phosphatases 1 and 2A; inhibits their activ- fluoride, and 50 pg/ml aprotinin and then centrifuged at 12,000 • g for 10 min. ities, resulting in increased phosphorylation of cellular and nuclear The supernatants were treated with either an anti-Rb protein antibody (G3-245; proteins; and induces expression of genes responsible for cell prolif- PharMingen) or an anti-p53 antibody (PAb421; Oncogene Science) in ice- eration. water for 2 h. Each immunocomplex was absorbed on protein A-Sepharose 4B The tumor suppressor gene products, Rb 3 protein and p53, which (Pharmacia) in ice-water for 1 h and washed 3 times with the lysis buffer. are nuclear phosphoproteins, are proposed to regulate cell growth in a Immunoprecipitates were subjected to SDS-PAGE, and the radioactivity was negative manner (2). Although the loss or inactivation of tumor sup- determined by BAS-2000 Image Analyzer (Fuji Film Co., Tokyo, Japan). Their pressor genes in human and rodent tumors has been extensively an- radioactivities in nuclei isolated from regenerating and normal livers were alyzed (3, 4), alteration of the tumor suppressor genes in mouse skin determined by five and three different experiments, respectively. Primary human fibroblasts (5 • 105 cells) were placed in a culture dish with two-stage carcinogenesis was not often found (5). If the alteration in 4 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal the tumor suppressor genes is a later event in the process of tumor calf serum (9). For the study of Rb protein phosphorylation in the cells, Rb promotion and tumor progression, how can tumor suppressor gene protein was labeled with [35S]methionine, rather than 32p~. Since hyperphos- products be involved in a process of tumor promotion? On the basis phorylated Rb protein has a unique feature which causes it to migrate differ- of evidence that hyperphosphorylated forms of Rb protein and p53 are ently than the hypophosphorylated Rb protein in SDS-PAGE, this was taken as inactive forms, we recently hypothesized that sustained hyperphos- a parameter to estimate the degree of Rb protein phosphorylation. The medium phorylation of these proteins is an alternative explanation for the loss was replaced by a methionine-deficient medium and incubated for 14 h. [35S]- of function of both tumor suppressor gene products in the okadaic acid Methionine, at a concentration of 3.7 MBq/ml, was added to the medium and pathway (6). it was further incubated for 3 h. The cells were treated with okadaic acid at various concentrations for length of times indicated in the text. The cells were To identify hyperphosphorylation of the Rb protein and p53, nuclei solubilized in the lysis buffer. The supematants were immunoprecipitated with isolated from rat regenerating liver and primary human fibroblasts the anti-Rb protein antibody, and the immunoprecipitates were subjected to 7% were treated with okadaic acid. The tumor suppressor proteins were SDS-PAGE. Radioactivity was determined by BAS-2000 Image Analyzer. As precipitated with specific monoclonal antibodies. Hyperphosphoryla- for p53 phosphorylation, the cells were incubated with phosphate-deficient tion of the Rb protein was estimated by incorporation of 32Pi into Rb Dulbecco's modified Eagle's medium for 14 h and labeled with 32Pi at a protein for in vitro incubation with the nuclei and by the migration to concentration of 7.4 MBq/ml for 3 h. The cells were treated with okadaic acid and solubilized, as described above. Cell lysates were immunoprecipitated with Received 11/2/92; accepted 11/30/92. the anti-p53 antibody, the immunoprecipitates were subjected to 10% SDS- The costs of publication of this article were defrayed in part by the payment of page PAGE, and radioactivity was determined by BAS-2000 Image Analyzer. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Results and Discussion 1This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Science, and Culture; a grant from the Ministry of Health and Welfare for a Comprehensive10-Year Strategy for Cancer Control, Japan; and grants from Phosphorylation of Rb Protein and p53 in Nuclei in Vitro. In- the Foundation for Promotion of Cancer Research, the Uehara Memorial Life Science cubation of the nuclei of the regenerating and normal liver with Foundation, and the Princess Takamatsu Cancer Research Fund. okadaic acid resulted in phosphorylation of numerous proteins, deter- 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: Rb, retinoblastoma; TPA, 12-O-tetradecanoylphorbol- mined by autoradiography of SDS-PAGE of the nuclear lysates (data 13-acetate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamidegel electrophoresis. not shown). Immunoprecipitation analysis with an anti-Rb protein 239

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1993 American Association for Cancer Research. TUMOR SUPPRESSOR PROTEINS AND OKADAIC ACID antibody revealed that the hypophosphorylated Rb protein has a molecular weight of 105,000 in SDS-PAGE and the hyperphosphorylated 300 Rb protein decreases its electrophoretic mobility, as reported previ- ously (10, 11). Okadaic acid induced phosphorylation of Rb protein ._c O linearly, at concentrations of 1 nM to 1 laM, by incubation with the o nuclei of the regenerating liver in vitro, whereas okadaic acid did not t). o3 increase the phosphorylation in the nuclei of normal liver. It is of to interest to note that the basal level of the phosphorylation in the nuclei o of the regenerating liver was twice as much as that in the nuclei of normal liver (Fig. 1). The nuclei isolated from regenerating rat liver ,i.a are thought to contain activated protein kinases and protein phosphatases, whereas the nuclei isolated from normal liver are less active, "oom 100, k due to their remaining in Go. In regenerating liver, various protein O kinases, such as p34 cdc2 or cyclin-dependent kinase are activated, .g. because the Rb protein is phosphorylated by p34 cdc2 or its related tr kinases (12). Carlberg et al. (13) reported that the highest protein kinase activity was found in cells from the early S phase, whereas 0 I 1 I I 101 10 3 protein phosphatase activity was most pronounced during G2 plus M. These results are in agreement with our evidence that okadaic acid Concentration (nM ) was effective only on the nuclei of the regenerating liver, but not on Fig. 2. Phosphorylation of p53 protein in nuclei isolated from regenerating (0) and those of normal liver. normal (Q)) rat liver by incubation with okadaic acid. Nuclei were isolated as described in Fig. 1. Nuclear lysates were immunoprecipitatedwith PAb421 and subjected to SDS- Phosphorylation of p53 in the nuclei isolated from regenerating PAGE. Phosphorylationof p53 in nuclei of the normal liver was expressed as 100% and liver increased up to 100 nM okadaic acid and saturated at concentra- was the same as that of the regeneratingliver. Phosphorylationof p53 was determinedby the same procedure as that of Rb protein. Bars, SD. tions of okadaic acid from 100 nM to 1 ~ (Fig. 2). However, phosphorylation of p53 in the nuclei isolated from normal liver with okadaic acid was not increased significantly. In this case, the basal 1 2 3 4 5 levels of phosphorylation of p53 were the same with both kinds of nuclei. The difference between the dose-response curves of Rb protein and p53 may be explained by various factors: (a) the half-life of Rb 105 kDa .--~ protein is more than 10 h (11), whereas that of p53 is short, 5-20 min (14); (b) Rb protein shows more than 20 tryptic phosphopeptides in 2-dimensional peptide mapping (15), whereas p53 shows 6 major Fig. 3. Phosphorylation of Rb protein in primary human fibroblasts induced by okadaic peptides (16); and (c) Rb protein has a molecular weight twice as large acid. Cells labeled by [353]methionine were treated with okadaic acid at concentrations of 0, 1, 10, 100 and 1000 nM for 2 h (Lanes 1-5, respectively),kDa, molecular weight in thousands.

600 as p53. In addition, the experimental results indicated that the Rb v protein is a more stable phosphoprotein with more phosphorylation e'- sites than p53. Phosphorylation of Rb Protein and p53 in Primary Human Fibroblasts. The hypophosphorylated and hyperphosphorylated Rb -Qn- 400 proteins, labeled by [35S]methionine, were differentiated by electro- ~6 phoretic mobility. Both Rb proteins were well immunoprecipitated by an anti-Rb protein antibody, however. As Fig. 3, Lane 1, shows, primary human fibroblasts not treated with okadaic acid contained

o large amounts of hypophosphorylated Rb protein and small amounts ='5 200 of hyperphosphorylated Rb protein. Treatment with various concentrations of okadaic acid for 2 h (Fig. 3, Lanes 2 to 5) decreased > electrophoretic mobility of the Rb protein, depending on the concentrations of okadaic acid, indicating that the Rb protein was hyper- nt- phosphorylated in various degrees, and with 100 nM okadaic acid, 0 "-// ' I I I i most of the Rb protein was hyperphosphorylated. The time course of 101 10 3 Rb protein phosphorylation in the cells treated with 100 nM okadaic Concentration (nM ) acid was recorded for up to 240 min (data not shown). The Rb protein, Fig, 1. Phosphorylation of Rb protein in nuclei isolated from regenerating (0) and which had started being phosphorylated within 30 min, shifted to normal (O) rat liver by incubationwith okadaicacid. Nuclei isolated from the liver 2 days hyperphosphorylated positions, depending on the incubation time after partial 'hepatectomy and from normal liver were incubated with various concentra- (data not shown). These results were well correlated with the increase tions of okadaic acid. Nuclear lysates were immunoprecipitatedwith G3-245 and subjected to SDS-PAGE. Radioactivity was analyzed by BAS-2000 Image Analyzer. Phos- of phosphorylation of the Rb protein in the nuclei of the regenerating phorylation of Rb protein in nuclei of the normal liver was expressed as 100% and that in liver treated with okadaic acid in vitro. nuclei of the regeneratingliver was twice as much as that in the normal liver. Each point represents the mean _-_ SD (bars) of various experiments, as reported in "Materials and The p53 was immunoprecipitated with an anti-p53 antibody from Methods." the lysates of the cells. Without okadaic acid treatment, p53 did not 240

show any strong phosphorylation. The phosphorylation of p53 was that p53 causes a tetramer to become active (21), the molecules of increased by treatment with okadaic acid, at concentrations of 100 nM hyperphosphorylated p53, which are unable to associate with each and 1 pM, at a rate 8 and 9 times of that in nontreated primary human other, disturb the expression of downstream genes that negatively fibroblasts, respectively (Fig. 4A). Like the Rb protein, okadaic acid control growth (22). Our results first show that a tumor promoter, induced time-dependent phosphorylation of p53 in the cells that okadaic acid, induced hyperphosphorylation of two products of tumor strongly increased at 60 rain (Fig. 4B). These results clearly indicated suppressor genes at the same time. It has not yet been clarified that phosphorylation of the Rb protein and p53 was induced through whether phosphorylation of both proteins is involved in the process of inhibition of protein phosphatases 1 and 2A by okadaic acid, i.e., the tumor promotion or whether only one protein is actively involved. We okadaic acid pathway. simply assume that these hyperphosphorylated proteins might be sig- This experiment clearly showed that a tumor promoter, okadaic nificant factors regulating the proliferation of initiated cells. acid, at concentrations of 1 nM to 1 iuM, induced phosphorylation of tumor suppressor gene products, probably resulting in inactivation of References their function. We think that posttranslational modification is involved in the alteration of tumor suppressor function during tumor promotion. 1. Fujiki, H., Suganuma, M., Nishiwaki, S., Yoshizawa, S., Yatsunami, J., Matsushima, R., Furuya, H., Okabe, S., Matsunaga, S., and Sugimura, T. Specific mechanistic These concentrations were compared with those of okadaic acid, aspects of animal tumor promoters: the okadaic acid pathway. In: R. D'Amato, T. J. which was used for the 3-methylcholanthrene-induced transformation Slaga, W. Farland, and C. Henry (eds.), Relevance of Animal Studies to the Evaluation in BALB/3T3 cells (17). Two concentrations of okadaic acid, 12.5 and of Human Cancer Risk, pp. 337-350. New York: John Wiley & Sons, Inc., 1992. 2. Weinberg, R. A. Tumor suppresser genes. Science (Washington DC), 254:1138-1146, 25 nM, significantly induced cell transformation during 2-week treat- 1991. ment. Therefore, hyperphosphorylation of Rb protein and p53 is 3. Benedict, W. E, Xu, H-J., Hu, S-X., and Takahashi, R. Role of the retinoblastoma thought to be induced by okadaic acid during the process of cell gene in the initiation and progression of human cancer. J. Clin. Invest., 85: 988-993, 1990. transformation. 4. Hollstein, M., Sidransky, D., Vogelstein, B., and Harris, C. C. p53 Mutations in human How are the hyperphosphorylated tumor suppressor proteins inac- cancers. Science (Washington DC), 253: 49-53, 1991. tivated and how do they induce proliferation? It is reported that 5. Ruggeri, B., Caamano, J., Goodrow, T., DiRado, M., Bianch, A., Trono, D., Conti, C. J., and Klein-Szanto, A. J. P. Alteration of the p53 tumor suppressor gene during phosphorylated Rb protein loses contact with the cellular oncopro- mouse skin tumor progression. Cancer Res., 51: 6615~621, 1991. teins, such as EIA, resulting in the removal of E2F from its complex 6. Fujiki, H. Is the inhibition of protein phosphatase 1 and 2A activities a general (18). This allows the transcription factor to be activated. The conver- mechanism of tumor promotion in human cancer development? Mol. Carcinog., 5: 91-94, 1992. sion from hyperphosphorylated Rb protein to a hypophosporylated 7. Tachibana, K., Scheuer, E J., Tsukitani, Y., Kikuchi, H., Van Engen, D., Clardy, J., one during the M phase is induced by the presence of protein phos- Gopichand, Y., and Schmitz, E J. Okadaic acid, a cytotoxic polyether from two phatase 1 (19), which is also inhibited by okadaic acid. Thus, okadaic marine sponges of the genus Halichondria. J. Am. Chem. Soc., 103: 2469-2471, 1981. acid might induce a cyclin-independent state and sustain the hyper- 8. Hosoya, T., Nagai, Y., Inagaki, T., and Hayashi, M. In vivo effect of androgen and phosphorylated state, both of which cause deregulation of the cell cycloheximide on the RNA synthesis in isolated nuclei of rat ventral prostates. J. cycle. It is also reported that p53 is phosphorylated at amino- and Biochem. (Tokyo), 84: 151%1528, 1978. 9. Yatsunami, J., Fujiki, H., Suganuma, M., Yoshizawa, S., Eriksson, J. E., Olson, M. O. carboxyl-terminal sites and the amino-terminal sites are dephospho- J., and Goldman, R. D. Vimentin is hyperphosphorylated in primary human fibro- rylated by protein phosphatase 2A (20). Therefore, the amino-terminal blasts treated with okadaic acid. Biochem. Biophys. Res. Commun., 107:1165-1170, sites are hyperphosphorylated by okadaic acid. Although it is known 1991. 10. DeCaprio, J. A., Ludlow, J. W., Lynch, D., Furukawa, Y., Griffin, J., Piwnica-Worms, H., Huang, C-M., and Livingston, D. M. The product of the retinoblastoma suscep- A 1 2 3 4 5 tibility gene has properties of a cell cycle regulatory element. Cell, 58: 1085-1095, 1989. 11. Mihara, K., Cao, X-R., Yen, A., Chandler, S., Driscoll, B., Murphree, A. L., T'Ang, A., and Fung, Y. K-I. Cell cycle dependent regulation of phosphorylation of the human retinoblastoma gene product. Science (Washington DC), 246: 1300-1303, 1989. 12. Lin, B. T-Y., Gruenwald, S., Morla, A. O., Lee, W-H., and Wang, J. Y. J. Retinoblas- 53 kDa,,,-~ toma cancer suppressor gene product is substrate of the cell cycle regulator cdc2 kinase. EMBO J., 10: 858-864, 1991. 13. Carlberg, U., Nilsson, A., Skog, S., Palmquist, K., and Nygard, O. Increased activity of the eEF-2 specific, Ca 2+ and calmodulin dependent protein kinase III during the S-phase in Ehrlich ascites cells. Biochem. Biophys. Res. Commun., 180: 1372-1376, 1991. 14. Reich, N. C., Oren, M., and Levine, A. J. Two distinct mechanisms regulate the level of a cellular tumor antigen, p53. Mol. Cell. Biol. 3: 2143-2150, 1983. 15. Templeton, D. J., Park, S. H., Lanier, L., and Weinberg, R. A. Nonfunctional mutants of the retinoblastoma protein are characterized by defects in phosphorylation, viral oncoprotein association, and nuclear tethering. Proc. Natl. Acad. Sci. USA, 88: 3033-3037, 1991. 16. Meek, D. W., and Eckhart, W. Phosphorylation of p53 in normal and simian virus B 1 2 3 4 5 40-transformed NIH 3T3 cells. Mol. Cell. Biol., 8: 461-465, 1988. 17. Sakai, A., and Fujiki, H. Promotion of BALB/3T3 cell transformation by the okadaic acid class of tumor promoters, okadaic acid and dinophysistoxin-1. Jpn. J. Cancer i ,84 Res., 82: 518-523, 1991. 18. Bagchi, S., Raychandhuri, E, and Nevins, J. R. Adenovirus E1A proteins can disas- sociate heteromeric complexes involve the E2F transcription factor: a novel mecha- 53 kDa,--~ nism for E1A trans-activation. Cell, 62: 659--669, 1990. 19. DeCaprio, J. A., Furukawa, Y., Ajchenbaum, E, Griffin, J. D., and Livingston, D. M. The retinoblastoma-susceptibility gene product becomes phosphorylated in multiple stages during cell cycle entry and progression. Proc. Natl. Acad. Sci. USA, 89: 1795-1798, 1992. Fig. 4. Phosphorylation of p53 in primary human fibroblasts induced by okadaic acid. 20. Wang, Y., and Eckhart, W. Phosphorylation sites in the amino-terminal region of (A) Cells labeled by 32p i for 3 h were treated with okadaic acid at concentrations of 0, 1, mouse p53. Proc. Natl. Acad. Sci. USA, 89: 4231-4235, 1992. 10, 100 and 1000 nM, for 2 h (Lanes 1-5, respectively). (B) Cells were treated with 100 21. Stenger, J. E., Mayr, G. A., Mann, K., and Tegtmeyer, E Formation of stable p53 nM okadaic acid for 0, 15, 30, 60, and 120 min, respectively, p53 was immunoprecipitated homotetramers and multiples of tetramers. Mol. Carcinog., 5: 102-106, 1992. with PAb421 and subjected to 10% SDS-PAGE. Radioactivity was analyzed by BAS-2000 22. Vogelstein, B., and Kinzler, K. W. p53 Function and Dysfunction. Cell, 70: 523-526, Image Analyzer. kDa, molecular weight in thousands. 1992. 241

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1993 American Association for Cancer Research. Hyperphosphorylation of Retinoblastoma Protein and p53 by Okadaic Acid, a Tumor Promoter

Jun Yatsunami, Atsumasa Komori, Tetsuya Ohta, et al.

Cancer Res 1993;53:239-241.

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