Hyperphosphorylation of Cytokeratins by Okadaic Acid Class 1\Imor Promoters in Primary Human Keratinocytes1

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(CANCER RESEARCH 53. 992-996. March 1. 1993] Hyperphosphorylation of Cytokeratins by Okadaic Acid Class 1\imor Promoters in Primary Human Keratinocytes1

Jun Yatsunami, Atsumasa Komori, Tetsuya Ohta, Masami Suganuma, Stuart H. Yuspa, and Hirota Fujiki2

Cancer Prevention Division, National Cancer Center Research Institute, Tokyo 104. Japan Â¡J.Yâ€žA.K., T. O., M. S., H. F.I and Laboratory of Cellular Carcinogenesis and Tumor Promotion. National Cancer Institute. Bethesda. Maryland 20892 /S. H. Y.Â¡

ABSTRACT present in epithelial cells. Expression of cytokeratins in the epidermis varies depending on cell types, stages of differentiation, the cellular Okadaic acid, dinophysistoxin-1 (35-methylokadaic acid), and calyculin growth environment, and skin diseases (6). Although the function of A are potent tumor promoters on mouse skin (H. Fujiki, M. Suganuma, S. Nishiwaki, S. Yoshizawa, J. Yatsunami, R. Matsushima, H. Furuya, individual cytokeratins in keratinocytes is not well understood, S. Okabe, S. Matsunaga, and T. Sugimura. In: R. D'Amato, T. J. Slaga, Epstein (7) recently reported that cytokeratin function is related to W. Farland, and C. Henry (eds.), Relevance of Animal Studies to the mechanical stability of the cells. In this experiment, we characterized Evaluation of Human Cancer Risk, pp. 337-350. New York: John Wiley the hyperphosphorylated cytokeratins in PHK 16-1 cells through im- and Sons, Inc., 1992). These tumor promoters, which are also inhibitors of munoprecipitation and Western blot analysis. The significance of the protein phosphatases 1 and 2A, induced hyperphosphorylation of ,17, hyperphosphorylation of cytokeratins was discussed in accordance 60,000, Mr 58,000, M, 56,000, A/r 52,000, MT 42,000, and Mr 27,000 pro with their functional alteration and with the changes in intermediate teins in PHK 16-1 cells, human keratinocytes immortalized by human papillomavirus type 16. Except for the .17, 27,000 protein, these hyper- filament networks that are associated with tumor promotion. phosphorylated proteins were identified to be cytokeratin peptides (CK) CK 5, CK 6, CK 7, CK 16, and CK 19, by anti-cytokeratin antibodies. CK MATERIALS AND METHODS 5 and CK 6 were more strongly phosphorylated than CK 16 and CK 19. The in vitro hyperphosphorylation of these cytokeratins was also found by Chemicals and Cells. Okadaic acid and dinophysistoxin-1 were isolated incubation with an enzyme fraction containing a mixture of protein phos- from the black sponge. Halichondria okadai (8). Calyculin A was isolated from phatase 2A and protein kinases isolated from mouse brain and various a marine sponge, Discodermia calyx (9). PHK 16-1 cells were kindly provided concentrations of dinophysistoxin-1. Indirect immunofluorescence micros by Dr. Shigeru Yasumoto at the Kanagawa Cancer Center Research Institute, copy with anti-cytokeratin antibodies revealed that the hyperphosphory- Yokohama, Japan (10). Fetal calf serum and MCDB 152 medium were lated cytokeratins had retracted to the perinuclear area. The hyperphos- purchased from Flow Laboratories, North Ryde, New South Wales, Australia, phorylated ,17, 27,000 protein was identified as a heat shock protein, and Kyokuto Pharmaceutical Co., Ltd., Tokyo, Japan, respectively. 32PÂ¡ HSP27. Hyperphosphorylation of HSP27 and intermediate filaments, such and [-/-"PJATP were obtained from Amersham. Buckinghamshire, United as cytokeratins, is one of the early biochemical changes, or pleiotropic Kingdom. effects, in cells induced by the okadaic acid class of tumor promoters. Antibodies. Antibody HK6 against cytokeratin peptide CK 6 was prepared by S. H. Y as described previously (11). Antibodies AE-1, AE-2, AE-3, AE-8, INTRODUCTION RCK 105, K8.13, and 19 Ks were purchased from ICN Biomedicals, Inc., Lisle, IL. Antibodies PKK-1 and PKK-2 were obtained from Labsystems, Okadaic acid, dinophysistoxin-1 (35-methylokadaic acid), and ca Helsinki, Finland. MAK-6 was purchased from Triton Bioscience, Inc., lyculin A are potent tumor promoters of the okadaic acid class on Alameda, CA. Anti-A/r 27,000 heat shock protein antibody SPA-800 was mouse skin initiated with DMBA3 (1). They bind to the catalytic purchased from Stressgen Biotechnologies Corp., Victoria, British Columbia, subunits of protein phosphatases 1 and 2A and inhibit their activities, Canada. resulting in the increase of phosphoproteins, i.e., the hyperphospho Hyperphosphorylation of Proteins in PHK 16-1 Cells. PHK 16-1 cells rylation of proteins (2, 3). This new mechanism of tumor promotion (1 x IO6) were cultured in a phosphate-deficient MCDB medium for 14 h. 32PÂ¡ was named the okadaic acid pathway (1). In primary human fibro- was added to the medium at a concentration of 1.85 MBq/ml and incubated for blasts treated with okadaic acid and dinophysistoxin-1, we have re 3 h. PHK 16-1 cells were further treated for 2 h with 100 nMof an okadaic acid cently demonstrated that vimentin, one of the intermediate filaments, class tumor promoter dissolved in dimethyl sulfoxide. Cells were solubilized in is a target protein for hyperphosphorylation (4). Therefore, we think lysis buffer consisting of 50 min 4-(2-hydroxyethyl)-l-piperazineethane- that various types of intermediate filament proteins are generally sulfonic acid (pH 7.4), 250 niM NaCl, 0.5% NP-40, 1 IHMphenylmethanesulfo- hyperphosphorylated in various cells by treatment with okadaic acid nyl fluoride, and 50 (Jg/ml of aprotinin, followed by centrifugation ( 12,000 rpm class tumor promoters. Okadaic acid, dinophysistoxin-1, and calyculin X 10 min). The soluble fraction was directly dissolved in electrophoresis buffer and applied to one-dimensional SDS-PAGE (12), followed by auto- A induced hyperphosphorylation of proteins other than vimentin sim ilarly in PHK 16-1 cells, human keratinocytes immortalized by human radiography. papillomavirus type 16 (5). This article reports that these hyperphos Immunoprecipitation of Hyperphosphorylated Proteins. Hyperphos phorylated proteins present in the soluble fraction of the cell lysates were phorylated proteins are cytokeratins. Â¡mmunoprecipitated with the anti-cytokeratin antibody K8.13 and anti-Afr Cytokeratin is the group name for the intermediate filaments 27,000 heat shock protein antibody SPA-800 as described previously (4). Both immunoprecipitates were subjected to one-dimensional SDS-PAGE and fol lowed by autoradiography. Received 6/17/92; accepted 12/22/92. Western Blot Analysis of Hyperphosphorylated Cytokeratins. PHK 16-1 The costs of publication of this article were defrayed in part by the payment of page cells (1 X IO6) labeled with 3-PÂ¡were treated with 100 nm dinophysistoxin-1 charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. for 2 h. The soluble fraction of the cell lysates was directly dissolved in 1This work was supported in part by Grants-in-Aid for Cancer Research from the electrophoresis buffer and subjected to one-dimensional SDS-PAGE. The pro Ministry of Education, Science and Culture, a grant from the Ministry of Health and Welfare for a Comprehensive 10-Year Strategy for Cancer Control, Japan, and grants from teins which were transferred to a nitrocellulose membrane were further reacted the Foundation for Promotion of Cancer Research, the Uehara Memorial Life Science with each of various anti-cytokeratin antibodies, as indicated in Table 1, and Foundation, and the Princess Takamatsu Cancer Research Fund. later stained with the immunoperoxidase method using the avidin-biotin com 2 To whom requests for reprints should be addressed. 1 The abbreviations used are: DMBA. 7,12-dimethylbenz(a)anthracene; CK, cytoker plex described previously (4). atin peptide; HSP27, M, 27,000 heat shock protein; SDS-PAGE, sodium dodecylsulfate- Two-dimensional Gel Electrophoresis of Cytokeratins. After labeling polyacrylamide gel electrophoresis; TPA, ^-O-tetradecanoylphorbol-lS-acetate. with 32PÂ¡,PHK 16-1 cells (1 x IO6) were treated with either 100 nui dinophy- 992 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1993 American Association for Cancer Research. HYPERPHOSPHORYLATED CYTOKERATINS BY OKADAIC ACID 1234 12

.60kDa 60 kDa '52kDa58 kDa 58 kDa 56 kDa 42 kDa 52 kDa 42 kDa

27 kDa Â«â€”27kDa

Fig. 1. Hyperphosphorylation of proteins in PHK 16-1 cells treated with okadaic acid class tumor promoters. After labeling with 32PÂ¡,cells(1 X IO6 cells) were incubated for 2 h with dimethyl sulfoxide as vehicle (Lane I), 100 nM okadaic acid (Lane 2), 100 nM dinophysistoxin-1 (Lane 3), and 100 nM calyculin A (Lane 4). kDa, kilodaltons. Fig. 2. Immunoprecipitation of hyperphosphorylated proteins in PHK I6-I cells. The cells were treated with 100 nM dinophysistoxin-1 for 2 h. Immunoprecipitates were separated by 10% SDS-PAGE followed by autoradiography. Immunoprecipitates of cell sistoxin-1 or vehicle for 2 h in a culture system. The total cell lysates were lysates were treated with normal mouse serum (iMiie /), anti-cytokeratin antibody K8.13 subjected to two-dimensional gel electrophoresis followed by autoradiography (Lane 2), and anti-Wr 27,000 heat shock protein antibody SPA-800 (Lane 3). kDa, kilodaltons. (13). Hyperphosphorylation of Cytokeratins in Vitro. The cytokeratin frac 52,000, 42,000, and 27,000 in PHK 16-1 cells (Fig. 1). The insoluble tion, which contains mainly CK 5, CK 6. CK 7, CK 16, and CK 19, along with trace amounts of CK 8 and CK 18, was isolated from PHK 16-1 cells, fraction of the lysis buffer also contained the hyperphosphorylated according to the method described by Achtstaetter (14). Fifty ug of cytoker- proteins, but the Mr 27,000 phosphoprotein was mainly found in the atins were incubated in vitro with an enzyme fraction (10 ug protein) contain soluble fraction. Moreover, the former four proteins are phosphory- ing a mixture of protein phosphatase 2A and protein kinases isolated from lated by only the okadaic acid class of tumor promoters and the Mr mouse brain, [7-12P]ATP (2.8 X IO4 Bq), and dinophysistoxin-1 at concentra 27,000 protein was phosphorylated by okadaic acid class and TPA- tions from I nM to 1000 nM for 10 min at 30Â°C(3). Trichloroacetic acid- type tumor promoters, suggesting a difference in their natures (data insoluble pellets were subjected to SDS-PAGE. Radioactivity of M, 42,000, M, not shown). 52,000, and M, 56,000-60,000 proteins was measured on a BAS-2000 Image Immunoprecipitation of Hyperphosphorylated Proteins. To test analyzer, Fuji Film Co. Indirect Immunofluorescence Microscopy. PHK 16-1 cells (5 X IO4/ whether the hyperphosphorylated proteins were cytokeratins, the cell chamber), plated on 4-chamber slides, were treated with 100 nM dinophysis lysates were immunoprecipitated by the monoclonal anti-cytokeratin toxin-1 for 15 min. After fixation of the cells with methanol, the slides were antibody K8.13, which mainly reacts with basic cytokeratins, and the treated with the anti-cytokeratin antibody AE-3 for 2 h and further incubated immunoprecipitates were separated by SDS-PAGE (Fig. 2). The an with secondary antibody conjugated with fluorescein for 1 h. The slides were tibody K8.13 significantly precipitated phosphoproteins that had the examined microscopically. same molecular weights as those in Fig. 1 (M, 60,000, Mr 58,000, Mr 52,000, and Mr 42,000) but it did not precipitate the Mr 27,000 protein. RESULTS In addition, the antibody K8.13 immunoprecipitated a Mr 56,000 phosphoprotein, which was later identified as cytokeratin 7 (Table 1), Hyperphosphorylation of Proteins in PHK 16-1 Cells. One-di although the Mr 56,000 phosphoprotein was difficult to distinguish in mensional SDS-PAGE of the soluble fraction of the lysates revealed Fig. 1. Normal mouse serum did not significantly precipitate any that okadaic acid, dinophysistoxin-1, and calyculin A induced hyper- phosphoproteins. The hyperphosphorylated M, 27,000 protein was phosphorylation of proteins with molecular weights of 60,000, 58,000, immunoprecipitated by anti-Mr 27,000, a heat shock protein, HSP27

Table 1 Western Util analysis of hyperphosphorylated cylokeratins with anti-cyltikeralin anlihiulies

AE-3Phosphoprotein 105(7)*AE-1 Ks(19)-IdentificationCK (1-8)"M, (9-19)+MAK-6 (8,14-16, 18,19)+PKK-2 (7, 16, 17,19)+19 60,000 + 5 Mr 58.000 + CK 6 Mr 56,000 + CK 7 Mr 52,000 CK 16 M, 42.000HK6(6)+RCK CK 19 ' Numbers in parentheses, specificity of cytokeratin peptide.

S S D 5 D S S 6 16 19

HSP27

Fig. 3. Two-dimensional gel electrophoresis of cytokeratins. The cells were treated with dimethyl sulfoxide as vehicle (A) or IOO RMdinophysistoxin-l (ÃŸ)for 2 h. The total cell lysates were subjected to two-dimensional gel electrophoresis followed by autoradiography. Numbers, cytokeratin peptides. IEF. isoelectric focusing.

(Fig. 2, Lane 3). These results showed that four of the phosphopro- specificities of the antibodies, phosphoproteins with molecular teins and the Mr 56.000 phosphoprotein are cytokeratins, but the Mr weights of 60,000, 58,000, 52,000, and 42,000 were identified to be 27,000 protein is not. CK 5, CK 6, CK 16, and CK 19, respectively (Table 1). In addition, Western Blot Analysis of Hyperphosphorylated Cytokeratins. the M, 56,000 phosphoprotein reacted with the antibody RCK 105 To characterize the cytokeratin peptides, the soluble fraction of cell against cytokeratin 7 (Table 1). CK 7 was not clearly visible in lysates was one-dimensionally separated and blotted onto nitrocellu one-dimensional SDS-PAGE, due to the smaller amount of protein lose membranes, then subjected to Western blot analysis using seven and a lesser amount of phosphorylation than those of CK 5 and CK 6. anti-cytokeratin antibodies, as indicated in Table I. According to Two-Dimensional Gel Electrophoresis of Cytokeratins. In this experiment, we used the total cell lysates because the hyperphosphorylated proteins had already been analyzed as described above. To confirm the cytokeratin peptide numbers, the gels were subjected to an autoradiography, not to Coomassie blue staining. The phosphoproteins of CK 5 and CK 6 were additionally separated into various spots, indicating different isoelectric points. Fig. 3 also showed that phosphorylation of CK 5 and CK 6 was much stronger than that of CK 16 and CK 19 in the cells treated with dinophysistoxin-l. Hyperphosphorylated CK 7 was clearly separated in two-dimensional SDS-PAGE (Fig. 3). 100 Hyperphosphorylation of Cytokeratins in Vitro. Fig. 4 illus trated the increase of in vitro phosphorylation of cytokeratins from their basal levels, depending on the concentrations of dinophysistox- o ro in-1, and expressed the increase of phosphorylation of the MT56,000- H- OJ 0 5 60,000 proteins, represented as cytokeratins CK 5, CK 6, and CK 7, oCC ~ 'sÂ° the A/r 52,000 protein as CK 16, and the M, 42,000 protein as CK 19. These results showed that in vitro hyperphosphorylated cytokeratins ot* â€”¿ Â£ are potential substrates for protein phosphatase 2A. 5, oVI Indirect Immunofluorescence Microscopy. The consequence of o.-C hyperphosphorylated cytokeratins was determined. An elaborated net work of cytokeratin filaments was seen in the cytoplasm of the non-

10' 102 103 treated PHK 16-1 cells (Fig. 5A). Treatment with dinophysistoxin-l for 15 min caused a significant retraction of cytokeratins from the Concentration ( nM ) cytoplasm into the perinuclear zone, a reaction probably associated Fig. 4. Hyperphosphorylation of cytokeratins iÂ«vitro.Cytokeratins obtained from PHK 16-1 cells were incubated with ("P]ATP. various concentration of dinophysistoxin-l (1 to with their hyperphosphorylation (Fig. 5ÃŸ).However, the cells did not 1000 nM), and an enzyme fraction containing protein pnosphatase 2A and protein kinases. show any morphological changes at this stage. Further treatment with Trichloroacetic acid-insoluble pellets were subjected to SDS-PAGE and autoradiography. dinophysistoxin-l induced morphological changes, such as bleb-like Increased radioactivity of CK 5, CK 6, and CK 7 (â€¢).CK 16 (â€¢).and CK 19 (A) were expressed as percentages. Points, mean of duplicate determinations. formations. 994 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1993 American Association for Cancer Research. HYPERPHOSPHORYLATED CYTOKERATINS BY OKADAIC ACID

ronmental factors (6, 7). PHK 16-1 cells used in the experiments contained mainly CK 5, CK 6, CK 7, CK 16, and CK 19. Basic cytokeratins CK 5 and CK 6 were phosphorylated much more than acidic cytokeratins CK 16 and CK 19 by dinophysistoxin-l. both in the cells and in cell-free systems. Various serine/threonine protein kinases are thought to be involved in hyperphosphorylation of basic as well as acidic cytokeratins be cause vimentin and desmin are phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, Ca2+/calmodulin-dependent protein kinase, and p34cdl:2 kinase (18-20). However, it is not well known what kinds of protein phosphatases are involved in dephos- phorylation of cytokeratins. Our results in Fig. 4 clearly showed that inhibition of protein phosphatase 2A by the okadaic acid class of tumor promoters induced in vitro hyperphosphorylation of cytoker atins. The results were well correlated with the evidence that in vitro hyperphosphorylation of vimentin was also obtained by inhibition of protein phosphatase 2A (4). Interestingly, three okadaic acid class tumor promoters (okadaic acid, dinophysistoxin-l, and calyculin A) have a similarly strong inhibitory activity toward protein phosphatase 2A, but not to protein phosphatase 1, and have similarly potent tumor promoting activity on mouse skin initiated with DMBA. Hyperphos phorylation of intermediate filaments induced by inhibition of protein phosphatase 2A seems to be well correlated with tumor promotion. How phosphorylation of intermediate filaments is related to tumor promotion has not been well investigated. TPA is reported to induce reorganization of intermediate filaments in mouse epidermal cells and chicken embryo fibroblasts (21, 22). The transient phosphorylation of intermediate filaments by TPA might cause only temporary subcellular morphological changes. Intermediate filaments are also associated with microfilaments, microtubles and nuclear membranes, and phos phorylation of intermediate filaments causes effects which might be a reflection of signal transduction by a tumor promoter. As for the nature of the Mr 27,000 protein, it was thought to be a ribosomal S6 protein, based on studies of the phosphoprotein in iso lated hepatocytes (23). However, using anti-Mr 27,000 heat shock protein antibody, we identified HSP27 as a new member of the target proteins for the okadaic acid pathway. It is of interest to note that HSP27 is also phosphorylated in vitro and in many cell lines treated with TPA (24). TPA and okadaic acid stimulate expression of various genes through the AP-1 complex binding site and various enhancer Fig. 5. Indirect immunofluorescence of hyperphosphorylaled cytokeratins. PHK 16-1 elements (25-27). We have recently demonstrated that okadaic acid cells treated with 0.1% dimethyl sulfoxide (A) and treated with dinophysistoxin-l (ÃŸ)for induced hyperphosphorylation of tumor suppressor gene products 15 min were fixed and stained for immunofluorescence. such as retinoblastoma protein and p53 in human fibroblasts (28). The functional interaction between phosphorylated intermediate filaments and phosphorylation of transcription factors, along with phosphory DISCUSSION lation of tumor suppressor gene products, should be further investi Tumor promoters of the okadaic acid class are potent inhibitors of gated. protein phosphatases 1 and 2A, whereas the TPA-type tumor promot ers are potent activators of protein kinase C (2, 15). Although their REFERENCES mechanisms of action are different, they induce tumor promotion on 1. Fujiki, H., Suganuma, M.. Nishiwaki, S.. Yoshizawa, S., Yatsunami. J., Mutsushima, R., Furuya, H., Okabe, S., Matsunaga, S., and Sugimura. T. 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Jun Yatsunami, Atsumasa Komori, Tetsuya Ohta, et al.

Cancer Res 1993;53:992-996.

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