Cardnogenesis vol.13 no. 12 pp.2443-2447, 1992
Hyperphosphorylation of cytokeratins 8 and 18 by microcystin-LR, a new liver tumor promoter, in primary cultured rat hepatocytes
Tetsuya Ohta, Rie Nishiwaki, Jun Yatsunami, Atsumasa Komori, Masami Suganuma and Hirota Fujiki1 Cancer Prevention EHvision, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104, Japan
Microcystin-LR (MC-LR), an inhibitor of protein phosphatases 1 and 2A, is a potent tumor promoter in rat liver initiated with diethylnltrosamine. To understand its biochemical process in hepatocytes, primary cultured rat hepatocytes were treated with MC-LR. MC-LR (1 /xM) induced phosphorylation of various proteins. Two 55 and 49 kDa proteins were phospborylated at a 3-fold higher rate than other proteins, and these proteins were identified to be cytokeratins 8 and 18 respectively, by immunoprecipitation and Western blot analysis using monoclonal anti-cytokeratin 8 and 18 antibodies. The basic cytokeratins 8 and 18 showed pi 6.4 and 5.4 respectively, in two-dimensional gel electrophoresis. MC-LR dose dependently increased phosphorylation of cytokeratins 8 and 18 in a cell-free system by incubation with a cytosolic fraction of rat liver containing both protein kinases and protein phosphatases 1 and 2A, and with [7-32P]ATP. Cytokeratins 8 and 18 were target proteins for phosphorylation induced by inhibition of protein phosphatases 1 and 2A, in vitro and in rat hepatocytes. Thus, the treatment of rat hepatocytes with MC-LR induced hyperphosphorylation of cytokeratins 8 and 18 associated with morphological changes, indicating that intermediate filament networks were rearranged in the cytoplasm. The hyperphosphorylation of cytokeratins is a significant biochemical process associated with liver tumor promotion. Introduction Microcystin-LR (MC-LR*), which contains leucine and arginine in two variable L-amino acids of cyclic heptapeptide is one of the microcystins isolated from blue-green algae, such as Microcystis, Anabena, Oscillatoria and Nostoc (1 —4). MC-LR had a potent tumor-promoting activity in rat liver initiated with diethylnitrosamine by repeated i.p. injections of a few ng per injection (5). Since MC-LR is a potent inhibitor of protein phosphatases 1 and 2A, the tumor-promoting activity in the liver is induced by the same mechanism of action as that of okadaic acid in mouse skin (5). We previously reported that the microcystins increased phosphorylation of various proteins in primary cultured rat hepatocytes, due to inhibition of protein phosphatases 1 and 2A (6). This paper reports the results of further experiments for characterizing the nature of proteins phosphorylated by MC-LR in rat hepatocytes. We recently reported that hyperphosphorylation of vimentin in primary human fibroblasts was induced by okadaic acid and dinophysistoxin-1 (35-methylokadaic acid, DTX-1) (7). In •Abbreviations: MC-LR, microcystin-LR; DTX-1, dinophysistoxin-1; PMSF, phenylmethanesulfonyl fluoride; HSP 27, heat shock protein 27. © Oxford University Press
Materials and methods Materials MC-LR was purified from lyophilized cells of laboratory cultured Microcystis aeruginosa pcc-7820 and natural bloom material dominated by M.aeruginosa, collected from a farm pond near Monroe, WI in 1985 (9,10). DTX-1 was purified from the black sponge, Halichondria okadai (11). Cytokeratins 8 and 18 were purchased from Boehringer Mannheim Yamanouchi Co., Japan. Monoclonal anticytokeratin antibodies were purchased: K8.13 from ICN Bkxnedicals Inc., Lisle, IL, LP3K from Cymbus Bioscience Ltd, Southampton, UK and PKK3 from Labsystems, Helsinki, Finland. Preparation of primary cultured rat hepatocytes Hepatocytes were isolated from male Fischer 344 rat liver digested with 0.05% collagenase, as described previously (12). Cells (2 x 106) were placed in a culture dish containing 2 nil of William's E medium enriched with 10% fetal calf serum, 1 nM dexamethasone, 600 ng/ml kanamycin and 250 ng/ml Fungizone, and were incubated for 30 h at 37°C in a 5% COj incubator. Hyperphosphorylation of proteins in primary cultured rat hepatocytes One day after preparation of primary cultured rat hepatocytes, the medium was replaced by phosphate-deficient Dulbecco's modified Eagle's medium and incubated for 14 h. Then [32P]orthophosphate was added to the medium at a concentration of 3.7 MBq/ml and incubated for 3 h. Cells were treated with either MC-LR or DTX-1 for 2 h, and further sonicated in lysis buffer [1 % deoxycholic acid, 1% NP-40, 7 mM NaCl, 1 mM M g d j , 7 mM Tris-HCl pH 7.4, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 jig/ml antipain]. After centrifugation, supematants were subjected to 10% SDS-PAGE (13). Incorporation of ^ P into the proteins was determined by autoradiography. Immunoprecipitation Aliquots (500/d) of cell h/sates were reacted with anti-cytokeratin armbody K8.13. The immunocomplex was absorbed to protein A sepharose 4B (Pharmacia) and washed with lysis buffer. The immunoprecipitates were separated on 10% SDS-PAGE, and further subjected to autoradiography. Western blot analysis After the treatment of rat hepatocytes with MC-LR as described above, the cell lysates were subjected to SDS-PAGE, and transferred to a nitrocellulose membrane. The membranes were reacted with a monoclonal anli-cytokeratin antibody (K8.13, LP3K or PKK3) and were stained by the immunoperoxidase method using the avidin—biotin complex (Vectastain ABC kit, Vector Laboratories, Burlingame).
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'To whom correspondence should be addressed
primary human keratinocytes, hyperphosphorylation of various cytokeratins was induced by the okadaic acid class tumor promoters, which are all potent inhibitors of protein phosphatases 1 and 2A (J.Yatsunami etal., manuscript in preparation). Vimentin and cytokeratins, which are types of intermediate filaments, are target proteins for phosphorylation induced by inhibition of protein phosphatases 1 and 2A. We assumed that the hvperphosphorylated 55 and 49 kDa proteins in rat hepatocytes treated with MC-LR were cytokeratins 8 and 18, by estimation of their mol wts (8). Immunoprecipitation with the monoclonal anti-cytokeratin 8 and 18 antibodies and two-dimensional gel electrophoresis revealed that cytokeratins 8 and 18 were hyperphosphorylated in primary cultured rat hepatocytes treated with either MC-LR or DTX-1 as a positive control. In addition, MC-LR dose dependently increased phosphorylation of cytokeratins 8 and 18 in vitro by incubation with a cytosolic fraction of rat liver. The role of hyperphosphorylation of cytokeratins in tumor promotion of rat liver is discussed.
T.OfaU et al.
1
kDa
2
3
S7-
68-
43 -
- 5 5 kDa - 4 9 kDa
31 -
55 kDa 49 kDa
Fig. 1. Hyperphosphorylation of 55 and 49 kDa proteins induced by MCLR and DTX-1. Control (lane 1), treated with 10 nM, 100 nM, 1 /»M and 10 yM MC-LR (lanes 2, 3, 4, 5) and 1 t>M DTX-1 (lane 6). The radioactivity of several proteins was measured by a BAS-2000 imageanalyzer. At a concentration of 10 pM MC-LR, phosphorylation of the 55 and 49 kDa proteins was —3-fold higher than that of the control. Two-dimensional gel electrophoresis After labeling with [32P]orthophosphate, rat hepatocytes were treated with MCLR for 1 h in a culture system. The cytoskeletal fraction was isolated according to the method described by Achtstactter el al. (14), and subjected to twodimensional gel electrophorcsis (15). Incorporation of 32 P into cytoskeletal proteins was determined by autoradiography. Phosphorylation of cytokeratins 8 and 18 in a cell-free system A cytosolic fraction of rat liver (50 /ig) containing both protein phosphatases 1 and 2A and protein kinases, [y-^PJATP (2.8 x 104 Bq) and either cytokerau'n 8 or 18 (15 /jg) as a substrate was incubated with various concentrations of MCLR for 10 min at 30°C (16). The reaction was stopped by addition of SDS sample buffer and heat treatment, and the reaction mixture was subjected to 10% SDS-PAGE. The radioactivity of cytokeratins 8 and 18 was measured by BAS-2000 image-analyzer (Fuji Film Co., Japan). The phosphorylation of cytokeratins 8 or 18 in the absence of MC-LR was expressed as 1.0.
Results Hyperphosphorylation of proteins in primary cultured rat hepatocytes Treatment of rat hepatocytes with MC-LR at various concentrations from 10 nM to 10 /tM induced phosphorylation of — 10 proteins with various mol wts (Figure 1). In particular, phosphorylation of two 55 and 49 kDa proteins was ~ 3-fold higher than that of other proteins, at a concentration of 10 /tM MC-LR (Figure 1, lane 5), demonstrating that these two proteins are hyperphosphorylated. The phosphorylation pattern by MCLR was almost the same as that by DTX-1 (Figure 1, lane 6), indicating that the phosphorylation of various proteins was induced by inhibition of protein phosphatases 1 and 2A in rat hepatocytes, because MC-LR and DTX-1 are both potent inhibitors of protein phosphatases 1 and 2A. The hyperphosphorylated proteins were found in both die soluble and insoluble fractions of lysis buffer (data not shown), indicating that these proteins become soluble in lysis buffer after phosphorylation, like phosphorylated cytokeratins in human keratinocytes treated with okadaic acid and DTX-1 (J.Yatsunami et al, manuscript in preparation). Before characterization of these two proteins, we further studied several effects on the cells, associated with hyperphosphorylation of proteins. Treatment with 1 /tM MC-LR or DTX-1 induced morphological changes in primary cultured rat hepatocytes from the normal round form to a bleb form, indicating that cytoskeletal 2444
Fig. 2. Autoradiogram of the lmmunoprecipitates of hyperphosphorylated 55 and 49 kDa proteins. Control (lane 1), treated with 10 /iM MC-LR (lane 2) and 1 ?M DTX-1 (lane 3).
networks were rearranged from the cell periphery to the cytoplasm, due to phosphorylation of cellular proteins (data not shown). Hyperphosphorylation of cytokeratins 8 and 18 in primary cultured rat hepatocytes Lysates of the cells treated with 10 /tM MC-LR or 1 /tM DTX-1 were immunoprecipitated with monoclonal anti-cytokeratin antibody K8.13 and resulted in precipitating hyperphosphorylated 55 and 49 kDa proteins (Figure 2). Since the anti-cytokeratin antibody used for the experiment cross-reacted with cytokeratins 1, 5, 6, 7, 8, 10, 11 and 18, the results indicated that hyperphosphorylated proteins are cytokeratins, probably cytokeratins 8 and 18, as estimated from their mol wts and from their prevalance in the hepatocytes. To identify the two cytokeratins, Western blot analysis of the cell lysates was performed with anti-cytokeratin 8 and 18 antibodies. As Figure 3, lanes 1 and 2, shows, the results with the monoclonal anti-cytokeratin antibody K8.13 confirmed the previous results that the hyperphosphorylated 55 and 49 kDa proteins were cytokeratins. Moreover, the monoclonal anticytokeratin 8 antibody, LP3K, specifically reacted with the 55 kDa protein in the cell lysates from the control cells and the cells treated with 1 /iM MG-LR as well (Figure 3, lanes 3 and 4). Similarly, the monoclonal anti-cytokeratin 18 antibody, PKK3, specifically cross-reacted with the 49 kDa protein in the cell lysates (Figure 3, lanes 5 and 6). Distinct from the immunoprecipitation of the cytokeratins, a cytoskeletal fraction was obtained from primary cultured rat hepatocytes treated with 1 /tM MC-LR or with a vehicle as a control. Since cytoskeletal fractions of hepatocytes contain mainly the two cytokeratins 8 and 18, two-dimensional gel electrophoresis clearly gave protein spots of 55 and 49 kDa associated with various pis (Figure 4). For example, a basic spot of 55 kDa protein showed pi 6.4 and that of 49 kDa protein
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22-
Pbosphorylatfon of cytokeratins
• 55 kDa • 49 kDa
55 kDa —
«-49kDa
1-b
1-a •IEF S D S
18
2-a
2-b
18 18
Fig. 4. Two-dimensional gel electrophorcsis of 32P-labeled cytoskeletal fraction of the primary cultured rat hepatocytes. Coomassie brilliant blue staining (1-a, 2-a), autoradiogram (1-b, 2-b). MC-LR (1 iiM) treated (1-a, 1-b), control (2-a, 2-b). The most basic non-phosphorylated 55 kDa cytokeratin showed a pi of 6.4 and that of the 49 kDa cytokeratin showed a pi of 5.4. The 55 and 49 kDa cytokeratin spots were determined by Western blot analysis (data not shown).
showed pi 5.4. All spots of the 55 and 49 kDa proteins reacted with anti-cytokeratin antibody K8.13 in Western blot analysis (data not shown), supporting the evidence that the 55 kDa protein is cytokeratin 8 and the 49 kDa protein is cytokeratin 18. Their autoradiograms showed these two cytokeratins were phosphoproteins. Interestingly, the cytoskeletal fraction of the cells treated with 1 /iM MC-LR contained cytokeratins 8 and 18 that were more strongly phosphorylated than that of the control cells (Figure 4, 1-b and 2-b). From these results, we concluded that the hyperphosphorylated 55 and 49 kDa proteins are cytokeratins 8 and 18 respectively. Phosphorylation of cytokeratins 8 and 18 in a cell-free system To test the possibility that cytokeratins 8 and 18 are substrates for phosphorylation mediated through inhibition of protein phosphatases 1 and 2 A by MC-LR or DTX-1, cytokeratins 8 and 18 were phosphorylated in a cell-free system. Figure 5 shows that cytokeratins 8 and 18 were phosphorylated by protein kinases in a cytosolic fraction of rat liver beyond a basal phosphorylation level, and their phosphorylation level was increased by MC-LR, due to inhibition of protein phosphatases 1 and 2A, also present in the cytosolic fraction. Interestingly, the phosphorylation of cytokeratin 8 was increased by MC-LR dose dependency, whereas that of cytokeratin 18 was saturated at concentrations from 10 nM to 1 /tM. At a concentration of 1 fiM MC-LR,
10°
10'
10'
10 3
Concentration ( nM ) Fig. S. Phosphorylation of cytokeratins 8 (O) and 18 ( • ) in a cell-free system. Concentrations of MC-LR were 0, 1, 10 and 100 nM and 1 jiM. The phosphorylation of cytokeratins 8 or 18 in the absence of MC-LR was expressed as 1.0.
cytokeratins 8 and 18 were phosphorylated at rates - 5 and 2.5 times greater than the basal phosphorylation level, indicating that cytokeratin 8 has many more serine/threonine phosphorylation sites than cytokeratin 18. 2445
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Fig. 3. Western blot analysis of the 55 and 49 kDa cytokeratins with monoclonal anti-cytokeratin antibodies. Immunoblotting was performed with monoclonal anti-cytokeratin antibodies K8.13 (lanes 1 and 2), LP3K (lanes 3 and 4) and PKK3 (lanes 5 and 6) according to the avidin-biotin complex method (29). Cytosolic fractions of cells were treated with a vehicle Canes 1, 3 and 5) and with 1 /M MC-LR (lanes 2, 4 and 6).
T.Ohta et al.
It has not been well investigated which protein kinases are involved in increased phosphorylation of cytokeratins in rat hepatocytes. The phosphorylation of cytokeratins 8 and 18 are induced in primary cultured rat hepatocytes treated with ethanol, vasopressin and 12-Otetradecanoylphorbol-13-acetate, indicating that protein kinase C is one of the responsible protein kinases (19). Epidermal growth factor treatment also results in hyperphosphorylation of the two cytokeratins in cultured rat hepatocytes (8). Baribault et al. (8) found that phosphorylation of cytokeratin 8 was much higher than that of cytokeratin 18 in non-stimulated hepatocytes. These results are in agreement with our in vitro phosphorylation by a cytosolic fraction. Intermediate filaments are significant target proteins for the okadaic acid pathway. We think that the turn-over rate of phosphorylation/dephosphorylation in intermediate filaments is more rapid than that of other proteins, such as HSP 27. Since hyperphosphorylation was found in fibroblasts and keratinocytes as well as in hepatocytes in this experiment, hyperphosphorylation of intermediate filaments induced by inhibition of protein phosphatases 1 and 2A is a general biochemical reaction that occurs in various cells (20). Hyperphosphorylation of cytokeratins is significant for tumor promotion in many respects. It was reported that intermediate filaments in the cell cortex formed a distinct sheet of matted filaments which enveloped entire hepatocytes (21). Therefore, it is easily explained why hyperphosphorylation of cytokeratins is associated with morphological changes in hepatocytes. The microcystins are potent hepatotoxic compounds, but their effects on cultured hepatocytes have not been fully investigated. Hooser et al. (22) reported that MC-LR induced plasma 2446
membrane blebbing and marked reorganization of actin microfilaments, effects that were morphologically distinct from those induced by phalloidin, another hepatotoxic compound. As we previously reported, phalloidin is not an inhibitor of protein phosphatase 1 or 2A (6). Eriksson et al. (23,24) also reported that inhibition of protein phosphatases is associated with hepatocyte deformation due to reorganization of microfilaments, not to actin polymerization. As for the structure —function relationship of microcystin molecules, 3-amino-9-methoxy-2,6,8,-trimethy 1- 10-phenyldeca4,6-dienoic acid and two carboxylic acids are important for inhibition of and binding to protein phosphatases 1 and 2A (25,26). These results were recently confirmed in a report by Namikoshi et al. (27) that a (C 3 HTO) monoester of the acarboxyl on the glutamic acid of MC-LR did not show any toxicity. Hyperphosphorylation of cytokeratins is an additional suitable model for the structure-function relationship of the microcystins. The function of phosphorylated cytokeratins in hepatocytes is not well clarified. Since phosphorylation of vimentin is induced in the M phase by activation of pM"* 2 kinase in baby hamster kidney cells (28), this new inhibitor of protein phosphatases 1 and 2A, MC-LR, will provide further information in relation to cell cycle regulation in hepatocytes. Acknowledgements This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture and a grant from the Ministry of Health and Welfare for a Comprehensive 10-Year Strategy for Cancer Control, Japan, and grants from the Foundation for Promotion of Cancer Research, the Princess Takamatsu Cancer Research Fund, the Smoking Research Fund and the Uehara Memorial Life Science Foundation.
References l.Botes.D.P., Tuinman.A.A., Wessels.P.L., Viljoen.C.C, Kruger.H., Williams.D.H., Santikarn.S., Smith.R.J. and Hammond.S.J. (1984) The structure of cyanoginosin-LA, a cyclic heptapeptide toxin from the cyanobacterium Microcystis aeruginosa. J. Chem. Soc. Perkin Trans., 1, 2311-2318. 2. Carmichael.W.W. (1988) Toxins of freshwater algae. In Anthony,T.T. (cd.), Handbook of Natural Toxins; Marine Toxins and Venoms. Vol. 3, Marcel Dekker, Inc., New York, pp. 121-147. 3.Watanabe,M.F., Oishi.S., Harada,K.-L, Matsuura.K., Kawai.H. and Suzuki,M. (1988) Toxins contained in Microcystis species of cyanobacteria (blue-green algae). Toxicon, 26, 1017-1025. 4. Namikoshi,M., Rinehart.K.L., Sakai.R., Sivonen,K. and Carmichael.W.W. (1990) Structures of three new cyclic heptapeptide hepatotoxins produced by the cyanobacterium (blue-green alga) Nostoc sp. strain 152. J. Org. Chem., 55, 6135-6139. 5. Matsushima.R.N., Ohta.T., Nishiwaki.S., Suganuma,M., Kohyama.K., Ishikawa.T., Carmichael.W.W. and Fujiki.H. (1992) Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR. J. Cancer Res. Clin. Oncol., 118, 420-424. 6. Yoshizawa.S., Matsushima.R., Watanabe.M.F., Harada,K.-I., Ichihara.A., Carmichael.W.W. and Fujiki.H. (1990) Inhibition of protein phosphatases by microcystin and nodularin associated with hepatotoxicity. J. Cancer Res. CUn. OncoL, 116, 609-614. 7. YatsunamiJ., Fujiki,H., Suganuma.M., Yoshizawa.S., ErikssonJ.E., Olson,M.OJ. and Goldman,R.D. (1991) Vimentin is hyperphosphorylated in primary human fibroblasts treated with okadaic acid. Biodtem. Biophys. Res. Commun., 177, 1165-1170. 8. Baribault.H., Blouin.R., Bourgon.L. and Marceau.N. (1989) Epidermal growth factor-induced selective phosphorylation of cultured rat hepatocyte 55-KD cytokeratin before filament reorganization and DNA synthesis. J. Cell BioL, 109, 1665-1676. 9. KrishnamurthyJ., Carmichael.W.W. and Sarver.E.W. (1986) Toxic peptides from freshwater cyanobacteria (blue—green algae). I. Isolation, purification and characterization of peptides from Microcystis aeruginosa and Anabena flos-aquae. Toxicon, 24, 865—873. 10. Galey.F.D., Beasly,V.R., Carmichacl,W.W., Kleppe.G., Hooser.S.B. and
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Discussion MC-LR, which was recently shown to be a potent liver tumor promoter, provides a new mechanism of tumor promotion in rat liver (5,6). MC-LR is a potent inhibitor of protein phosphatases 1 and 2A and induces increased phosphorylation of various proteins in primary cultured rat hepatocytes (6). We named the increased phosphorylation the apparent 'activation' of protein kinases (16), because protein kinases are not directly activated by MC-LR, which belongs to the okadaic acid class of tumor promoters. This paper reported that cytokeratins 8 and 18 were hyperphosphorylated in primary cultured rat hepatocytes treated with MC-LR dose dependently. Preliminary results with a similar system were recently discussed by Falconer and Yeung (17). We further extended the study of cytokeratins 8 and 18 to demonstrate that they are also substrates for in vitro phosphorylation by protein kinases in a cytosolic fraction of rat liver. Cytokeratin is a type of intermediate filament, others include vimentin, desmin and lamins, to name a few (18). The types of intermediate filaments that are mainly phosphorylated by the okadaic acid class tumor promoters are dependent on cell types. Hyperphosphorylated vimentin was found in primary human fibroblasts treated with okadaic acid and DTX-1 (7) and various cytokeratins were found in human keratinocytes treated with okadaic acid and DTX-1 (Yatsunami et al., manuscript in preparation). In addition to these intermediate filaments, a heat shock protein, (HSP) 27, was also hyperphosphorylated in human keratinocytes treated with okadaic acid (Yatsunami etal., manuscript in preparation). Although MC-LR, like other okadaic acid class compounds, has a simple mechanism of action due to inhibition of protein phosphatases 1 and 2A, target proteins are numerous, as shown in Figure 1. The increase of phosphorylation is influenced by various factors; the amount of protein, the turn-over rate of a phosphate on a protein, and so on.
PhospborylatkM of cytokeratins
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Haschek,W.M. (1987) Blue-green algae (Microcystis aeruginosa) hepatotoxicosis in daily cows. Am. J. Vet. Res.. 48, 1415-1420. 11. Suganuma,M., Fujilri,H., Suguri,H., Yoshizawa.S., Hirota,M., Nakayasu.M., Ojika.M., Wakamatsu.K., Yamada.K. and Sugimura.T. (1988) Okadaic acid: An additional rion-phorbol-12-tetradecanoate-13-acetate-type tumor promoter. Proc. Nail. Acad Set. USA, 85, 1768-1771. 12. Seglen.P.O. (1976) Preparation of isolated rat liver cells. Methods Cell Bid., 13, 2 9 - 8 3 . 13. Laemmli.U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. 14. Achtstaetter.T., Matzfeld.M., Quinlan.R.A., Parmelee,D.C. and Franke.W.W. (1986) Separation of cytokeratin peptides by gel electrophoretic and chromatographic techniques and their identification by immunoblotting. Methods EnzymoL, 134, 355-371. 15. O'Farrell.P.Z., Goodman.H.M. and O'Farrell.P.H. (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell, 12, 1133-1142. 16. Sassa.T., Richter.W.W., Uda.N., Suganuma.M., Suguri.H., Yoshizawa,S., Hirota,M. and Fujiki.H. (1989) Apparent 'activation' of protein kinases by okadaic acid class tumor promoters. Biochem. Biophys. Res. Commuru, 159, 939-944. 17. Falconer.I.R. and Yeung.D.S.K. (1992) Cytoskeletal changes in hepatocytes induced by Microcystis toxins and their relation to hyperphosphorylation of cell proteins. Chem.-Biol. Interactions, 81, 181-196. 18. Steinert.P.M. and Roop,D.R. (1988) Molecular and cellular biology of intermediate filaments. Annu. Rev. Biochem., 57, 593—625. 19. Kawahara.H., Cadrin.M. and French.S.W. (1990) Ethanol-induced phosphorylation of cytokeratin in cultured hepatocytes. Lift Set, 47, 859—863. 20. Fujiii,H., Suganuma.M., Nishiwaki.S., Yoshizawa.S., Yatsunami.J., Matsushima.R., Furuya.H., Okabe.S., Matsunaga.S. and Sugimura.T. (1992) Specific mechanistic aspects of animal tumor promoters: The okadaic acid pathway. In D'Amato.R., Slaga.T.J., Farland.W. and Henry.C. (eds), Relevance of Animal Studies to Evaluation of the Human Cancer Risk. John Wiley & Sons Inc., New York, pp. 337-350. 21.Katsuma,Y., Marceau.N., Ohta.M. and French.S.W. (1988) Cytokeratin intermediate filaments of rat hepatocytes: different cytoskeletal domains and their three-dimensional structure. Hepatology, 8, 559—568. 22.Hooser,S.B., Beasley.V.R., Wahe.L.L., Kuhlenschmidt.M.S., Carmichael,W.W. and Haschek.W.M. (1991) Actin filament alterations in rat hepatocytes induced in vivo and in vitro by microcystin-LR, a hepatotoxin from the blue—green alga, Microcystis aeruginosa. Vet. Pathoi, 28, 259-266. 23. ErikssonJ.E., Paatero.G.I.L., MeriluotoJ.A.O., Codd.G.A., Kass.G.E.N., Nkotera.P. and Orrenius.S. (1989) Rapid microfUament reorganization induced in isolated rat hepatocytes by microcystin-LR, a cyclic peptide toxin. Exp. Cell Res., 185, 86-100. 24. ErikssonJ.E., Toivola.D., MeriluotoJ.A.O., Karaki.H., Han.Y.G. and Hartshome,D. (1990) Hepatocyte deformation induced by cyanobacteria] toxins reflects inhibition of protein phosphatases. Biochem. Biophys. Res. Commun., 173, 1347-1353. 25. Matsushima.R.N., Nishiwaki.S., Ohta,T., Yoshizawa.S., Suganuma.M., Harada,K.-I., Watanabe.M.F. and Fujiki.H. (1991) Structure-function relationships of microcystins, liver tumor promoters, in interaction with protein phosphatase. Jpn. J. Cancer Res., 82, 993-996. 26. Taylor.C, Quinn.RJ., McCulloch.R., Matsushima.R.N. and Fujiki.H. (1992) An alternative computer model of the 3-dimensional structures of microcystinLR and nodularin rationalising their interactions with protein phosphatases 1 and 2A. BioMed Chem. Lett.. 2, 299-302. 27. Namikoshi.M., Rinehart.K.L. and Sakai.R. (1992) Identification of 12 hepatotoxins from a homer lake bloom of the cyanobacteria Microcystis aeruginosa, Microcystis viridis, and Microcystis wesenbergii: nine new microcystins. J. Org. Chem., 57, 866-872. 28. Chou,Y.H., BischoffJ.R., Beach.D. and Goldman.R.D. (1990) Intermediate filament reorganization during mitosis is mediated by rAW3*2 phosphorylation of vimentin. Cell, 62, 1063-1071. 29. Hsu,S.-M., Raine.L. andFanger.H. (1981>Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem., 29, 577-580. Received on March 11, 1992; revised on August 25, 1992; accepted on September 1, 1992
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Hyperphosphorylation of cytokeratins 8 and 18 by microcystin-LR, a new liver tumor promoter, in primary cultured rat hepatocytes
Tetsuya Ohta, Rie Nishiwaki, Jun Yatsunami, Atsumasa Komori, Masami Suganuma and Hirota Fujiki1 Cancer Prevention EHvision, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104, Japan
Microcystin-LR (MC-LR), an inhibitor of protein phosphatases 1 and 2A, is a potent tumor promoter in rat liver initiated with diethylnltrosamine. To understand its biochemical process in hepatocytes, primary cultured rat hepatocytes were treated with MC-LR. MC-LR (1 /xM) induced phosphorylation of various proteins. Two 55 and 49 kDa proteins were phospborylated at a 3-fold higher rate than other proteins, and these proteins were identified to be cytokeratins 8 and 18 respectively, by immunoprecipitation and Western blot analysis using monoclonal anti-cytokeratin 8 and 18 antibodies. The basic cytokeratins 8 and 18 showed pi 6.4 and 5.4 respectively, in two-dimensional gel electrophoresis. MC-LR dose dependently increased phosphorylation of cytokeratins 8 and 18 in a cell-free system by incubation with a cytosolic fraction of rat liver containing both protein kinases and protein phosphatases 1 and 2A, and with [7-32P]ATP. Cytokeratins 8 and 18 were target proteins for phosphorylation induced by inhibition of protein phosphatases 1 and 2A, in vitro and in rat hepatocytes. Thus, the treatment of rat hepatocytes with MC-LR induced hyperphosphorylation of cytokeratins 8 and 18 associated with morphological changes, indicating that intermediate filament networks were rearranged in the cytoplasm. The hyperphosphorylation of cytokeratins is a significant biochemical process associated with liver tumor promotion. Introduction Microcystin-LR (MC-LR*), which contains leucine and arginine in two variable L-amino acids of cyclic heptapeptide is one of the microcystins isolated from blue-green algae, such as Microcystis, Anabena, Oscillatoria and Nostoc (1 —4). MC-LR had a potent tumor-promoting activity in rat liver initiated with diethylnitrosamine by repeated i.p. injections of a few ng per injection (5). Since MC-LR is a potent inhibitor of protein phosphatases 1 and 2A, the tumor-promoting activity in the liver is induced by the same mechanism of action as that of okadaic acid in mouse skin (5). We previously reported that the microcystins increased phosphorylation of various proteins in primary cultured rat hepatocytes, due to inhibition of protein phosphatases 1 and 2A (6). This paper reports the results of further experiments for characterizing the nature of proteins phosphorylated by MC-LR in rat hepatocytes. We recently reported that hyperphosphorylation of vimentin in primary human fibroblasts was induced by okadaic acid and dinophysistoxin-1 (35-methylokadaic acid, DTX-1) (7). In •Abbreviations: MC-LR, microcystin-LR; DTX-1, dinophysistoxin-1; PMSF, phenylmethanesulfonyl fluoride; HSP 27, heat shock protein 27. © Oxford University Press
Materials and methods Materials MC-LR was purified from lyophilized cells of laboratory cultured Microcystis aeruginosa pcc-7820 and natural bloom material dominated by M.aeruginosa, collected from a farm pond near Monroe, WI in 1985 (9,10). DTX-1 was purified from the black sponge, Halichondria okadai (11). Cytokeratins 8 and 18 were purchased from Boehringer Mannheim Yamanouchi Co., Japan. Monoclonal anticytokeratin antibodies were purchased: K8.13 from ICN Bkxnedicals Inc., Lisle, IL, LP3K from Cymbus Bioscience Ltd, Southampton, UK and PKK3 from Labsystems, Helsinki, Finland. Preparation of primary cultured rat hepatocytes Hepatocytes were isolated from male Fischer 344 rat liver digested with 0.05% collagenase, as described previously (12). Cells (2 x 106) were placed in a culture dish containing 2 nil of William's E medium enriched with 10% fetal calf serum, 1 nM dexamethasone, 600 ng/ml kanamycin and 250 ng/ml Fungizone, and were incubated for 30 h at 37°C in a 5% COj incubator. Hyperphosphorylation of proteins in primary cultured rat hepatocytes One day after preparation of primary cultured rat hepatocytes, the medium was replaced by phosphate-deficient Dulbecco's modified Eagle's medium and incubated for 14 h. Then [32P]orthophosphate was added to the medium at a concentration of 3.7 MBq/ml and incubated for 3 h. Cells were treated with either MC-LR or DTX-1 for 2 h, and further sonicated in lysis buffer [1 % deoxycholic acid, 1% NP-40, 7 mM NaCl, 1 mM M g d j , 7 mM Tris-HCl pH 7.4, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 jig/ml antipain]. After centrifugation, supematants were subjected to 10% SDS-PAGE (13). Incorporation of ^ P into the proteins was determined by autoradiography. Immunoprecipitation Aliquots (500/d) of cell h/sates were reacted with anti-cytokeratin armbody K8.13. The immunocomplex was absorbed to protein A sepharose 4B (Pharmacia) and washed with lysis buffer. The immunoprecipitates were separated on 10% SDS-PAGE, and further subjected to autoradiography. Western blot analysis After the treatment of rat hepatocytes with MC-LR as described above, the cell lysates were subjected to SDS-PAGE, and transferred to a nitrocellulose membrane. The membranes were reacted with a monoclonal anli-cytokeratin antibody (K8.13, LP3K or PKK3) and were stained by the immunoperoxidase method using the avidin—biotin complex (Vectastain ABC kit, Vector Laboratories, Burlingame).
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'To whom correspondence should be addressed
primary human keratinocytes, hyperphosphorylation of various cytokeratins was induced by the okadaic acid class tumor promoters, which are all potent inhibitors of protein phosphatases 1 and 2A (J.Yatsunami etal., manuscript in preparation). Vimentin and cytokeratins, which are types of intermediate filaments, are target proteins for phosphorylation induced by inhibition of protein phosphatases 1 and 2A. We assumed that the hvperphosphorylated 55 and 49 kDa proteins in rat hepatocytes treated with MC-LR were cytokeratins 8 and 18, by estimation of their mol wts (8). Immunoprecipitation with the monoclonal anti-cytokeratin 8 and 18 antibodies and two-dimensional gel electrophoresis revealed that cytokeratins 8 and 18 were hyperphosphorylated in primary cultured rat hepatocytes treated with either MC-LR or DTX-1 as a positive control. In addition, MC-LR dose dependently increased phosphorylation of cytokeratins 8 and 18 in vitro by incubation with a cytosolic fraction of rat liver. The role of hyperphosphorylation of cytokeratins in tumor promotion of rat liver is discussed.
T.OfaU et al.
1
kDa
2
3
S7-
68-
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- 5 5 kDa - 4 9 kDa
31 -
55 kDa 49 kDa
Fig. 1. Hyperphosphorylation of 55 and 49 kDa proteins induced by MCLR and DTX-1. Control (lane 1), treated with 10 nM, 100 nM, 1 /»M and 10 yM MC-LR (lanes 2, 3, 4, 5) and 1 t>M DTX-1 (lane 6). The radioactivity of several proteins was measured by a BAS-2000 imageanalyzer. At a concentration of 10 pM MC-LR, phosphorylation of the 55 and 49 kDa proteins was —3-fold higher than that of the control. Two-dimensional gel electrophoresis After labeling with [32P]orthophosphate, rat hepatocytes were treated with MCLR for 1 h in a culture system. The cytoskeletal fraction was isolated according to the method described by Achtstactter el al. (14), and subjected to twodimensional gel electrophorcsis (15). Incorporation of 32 P into cytoskeletal proteins was determined by autoradiography. Phosphorylation of cytokeratins 8 and 18 in a cell-free system A cytosolic fraction of rat liver (50 /ig) containing both protein phosphatases 1 and 2A and protein kinases, [y-^PJATP (2.8 x 104 Bq) and either cytokerau'n 8 or 18 (15 /jg) as a substrate was incubated with various concentrations of MCLR for 10 min at 30°C (16). The reaction was stopped by addition of SDS sample buffer and heat treatment, and the reaction mixture was subjected to 10% SDS-PAGE. The radioactivity of cytokeratins 8 and 18 was measured by BAS-2000 image-analyzer (Fuji Film Co., Japan). The phosphorylation of cytokeratins 8 or 18 in the absence of MC-LR was expressed as 1.0.
Results Hyperphosphorylation of proteins in primary cultured rat hepatocytes Treatment of rat hepatocytes with MC-LR at various concentrations from 10 nM to 10 /tM induced phosphorylation of — 10 proteins with various mol wts (Figure 1). In particular, phosphorylation of two 55 and 49 kDa proteins was ~ 3-fold higher than that of other proteins, at a concentration of 10 /tM MC-LR (Figure 1, lane 5), demonstrating that these two proteins are hyperphosphorylated. The phosphorylation pattern by MCLR was almost the same as that by DTX-1 (Figure 1, lane 6), indicating that the phosphorylation of various proteins was induced by inhibition of protein phosphatases 1 and 2A in rat hepatocytes, because MC-LR and DTX-1 are both potent inhibitors of protein phosphatases 1 and 2A. The hyperphosphorylated proteins were found in both die soluble and insoluble fractions of lysis buffer (data not shown), indicating that these proteins become soluble in lysis buffer after phosphorylation, like phosphorylated cytokeratins in human keratinocytes treated with okadaic acid and DTX-1 (J.Yatsunami et al, manuscript in preparation). Before characterization of these two proteins, we further studied several effects on the cells, associated with hyperphosphorylation of proteins. Treatment with 1 /tM MC-LR or DTX-1 induced morphological changes in primary cultured rat hepatocytes from the normal round form to a bleb form, indicating that cytoskeletal 2444
Fig. 2. Autoradiogram of the lmmunoprecipitates of hyperphosphorylated 55 and 49 kDa proteins. Control (lane 1), treated with 10 /iM MC-LR (lane 2) and 1 ?M DTX-1 (lane 3).
networks were rearranged from the cell periphery to the cytoplasm, due to phosphorylation of cellular proteins (data not shown). Hyperphosphorylation of cytokeratins 8 and 18 in primary cultured rat hepatocytes Lysates of the cells treated with 10 /tM MC-LR or 1 /tM DTX-1 were immunoprecipitated with monoclonal anti-cytokeratin antibody K8.13 and resulted in precipitating hyperphosphorylated 55 and 49 kDa proteins (Figure 2). Since the anti-cytokeratin antibody used for the experiment cross-reacted with cytokeratins 1, 5, 6, 7, 8, 10, 11 and 18, the results indicated that hyperphosphorylated proteins are cytokeratins, probably cytokeratins 8 and 18, as estimated from their mol wts and from their prevalance in the hepatocytes. To identify the two cytokeratins, Western blot analysis of the cell lysates was performed with anti-cytokeratin 8 and 18 antibodies. As Figure 3, lanes 1 and 2, shows, the results with the monoclonal anti-cytokeratin antibody K8.13 confirmed the previous results that the hyperphosphorylated 55 and 49 kDa proteins were cytokeratins. Moreover, the monoclonal anticytokeratin 8 antibody, LP3K, specifically reacted with the 55 kDa protein in the cell lysates from the control cells and the cells treated with 1 /iM MG-LR as well (Figure 3, lanes 3 and 4). Similarly, the monoclonal anti-cytokeratin 18 antibody, PKK3, specifically cross-reacted with the 49 kDa protein in the cell lysates (Figure 3, lanes 5 and 6). Distinct from the immunoprecipitation of the cytokeratins, a cytoskeletal fraction was obtained from primary cultured rat hepatocytes treated with 1 /tM MC-LR or with a vehicle as a control. Since cytoskeletal fractions of hepatocytes contain mainly the two cytokeratins 8 and 18, two-dimensional gel electrophoresis clearly gave protein spots of 55 and 49 kDa associated with various pis (Figure 4). For example, a basic spot of 55 kDa protein showed pi 6.4 and that of 49 kDa protein
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Pbosphorylatfon of cytokeratins
• 55 kDa • 49 kDa
55 kDa —
«-49kDa
1-b
1-a •IEF S D S
18
2-a
2-b
18 18
Fig. 4. Two-dimensional gel electrophorcsis of 32P-labeled cytoskeletal fraction of the primary cultured rat hepatocytes. Coomassie brilliant blue staining (1-a, 2-a), autoradiogram (1-b, 2-b). MC-LR (1 iiM) treated (1-a, 1-b), control (2-a, 2-b). The most basic non-phosphorylated 55 kDa cytokeratin showed a pi of 6.4 and that of the 49 kDa cytokeratin showed a pi of 5.4. The 55 and 49 kDa cytokeratin spots were determined by Western blot analysis (data not shown).
showed pi 5.4. All spots of the 55 and 49 kDa proteins reacted with anti-cytokeratin antibody K8.13 in Western blot analysis (data not shown), supporting the evidence that the 55 kDa protein is cytokeratin 8 and the 49 kDa protein is cytokeratin 18. Their autoradiograms showed these two cytokeratins were phosphoproteins. Interestingly, the cytoskeletal fraction of the cells treated with 1 /iM MC-LR contained cytokeratins 8 and 18 that were more strongly phosphorylated than that of the control cells (Figure 4, 1-b and 2-b). From these results, we concluded that the hyperphosphorylated 55 and 49 kDa proteins are cytokeratins 8 and 18 respectively. Phosphorylation of cytokeratins 8 and 18 in a cell-free system To test the possibility that cytokeratins 8 and 18 are substrates for phosphorylation mediated through inhibition of protein phosphatases 1 and 2 A by MC-LR or DTX-1, cytokeratins 8 and 18 were phosphorylated in a cell-free system. Figure 5 shows that cytokeratins 8 and 18 were phosphorylated by protein kinases in a cytosolic fraction of rat liver beyond a basal phosphorylation level, and their phosphorylation level was increased by MC-LR, due to inhibition of protein phosphatases 1 and 2A, also present in the cytosolic fraction. Interestingly, the phosphorylation of cytokeratin 8 was increased by MC-LR dose dependency, whereas that of cytokeratin 18 was saturated at concentrations from 10 nM to 1 /tM. At a concentration of 1 fiM MC-LR,
10°
10'
10'
10 3
Concentration ( nM ) Fig. S. Phosphorylation of cytokeratins 8 (O) and 18 ( • ) in a cell-free system. Concentrations of MC-LR were 0, 1, 10 and 100 nM and 1 jiM. The phosphorylation of cytokeratins 8 or 18 in the absence of MC-LR was expressed as 1.0.
cytokeratins 8 and 18 were phosphorylated at rates - 5 and 2.5 times greater than the basal phosphorylation level, indicating that cytokeratin 8 has many more serine/threonine phosphorylation sites than cytokeratin 18. 2445
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Fig. 3. Western blot analysis of the 55 and 49 kDa cytokeratins with monoclonal anti-cytokeratin antibodies. Immunoblotting was performed with monoclonal anti-cytokeratin antibodies K8.13 (lanes 1 and 2), LP3K (lanes 3 and 4) and PKK3 (lanes 5 and 6) according to the avidin-biotin complex method (29). Cytosolic fractions of cells were treated with a vehicle Canes 1, 3 and 5) and with 1 /M MC-LR (lanes 2, 4 and 6).
T.Ohta et al.
It has not been well investigated which protein kinases are involved in increased phosphorylation of cytokeratins in rat hepatocytes. The phosphorylation of cytokeratins 8 and 18 are induced in primary cultured rat hepatocytes treated with ethanol, vasopressin and 12-Otetradecanoylphorbol-13-acetate, indicating that protein kinase C is one of the responsible protein kinases (19). Epidermal growth factor treatment also results in hyperphosphorylation of the two cytokeratins in cultured rat hepatocytes (8). Baribault et al. (8) found that phosphorylation of cytokeratin 8 was much higher than that of cytokeratin 18 in non-stimulated hepatocytes. These results are in agreement with our in vitro phosphorylation by a cytosolic fraction. Intermediate filaments are significant target proteins for the okadaic acid pathway. We think that the turn-over rate of phosphorylation/dephosphorylation in intermediate filaments is more rapid than that of other proteins, such as HSP 27. Since hyperphosphorylation was found in fibroblasts and keratinocytes as well as in hepatocytes in this experiment, hyperphosphorylation of intermediate filaments induced by inhibition of protein phosphatases 1 and 2A is a general biochemical reaction that occurs in various cells (20). Hyperphosphorylation of cytokeratins is significant for tumor promotion in many respects. It was reported that intermediate filaments in the cell cortex formed a distinct sheet of matted filaments which enveloped entire hepatocytes (21). Therefore, it is easily explained why hyperphosphorylation of cytokeratins is associated with morphological changes in hepatocytes. The microcystins are potent hepatotoxic compounds, but their effects on cultured hepatocytes have not been fully investigated. Hooser et al. (22) reported that MC-LR induced plasma 2446
membrane blebbing and marked reorganization of actin microfilaments, effects that were morphologically distinct from those induced by phalloidin, another hepatotoxic compound. As we previously reported, phalloidin is not an inhibitor of protein phosphatase 1 or 2A (6). Eriksson et al. (23,24) also reported that inhibition of protein phosphatases is associated with hepatocyte deformation due to reorganization of microfilaments, not to actin polymerization. As for the structure —function relationship of microcystin molecules, 3-amino-9-methoxy-2,6,8,-trimethy 1- 10-phenyldeca4,6-dienoic acid and two carboxylic acids are important for inhibition of and binding to protein phosphatases 1 and 2A (25,26). These results were recently confirmed in a report by Namikoshi et al. (27) that a (C 3 HTO) monoester of the acarboxyl on the glutamic acid of MC-LR did not show any toxicity. Hyperphosphorylation of cytokeratins is an additional suitable model for the structure-function relationship of the microcystins. The function of phosphorylated cytokeratins in hepatocytes is not well clarified. Since phosphorylation of vimentin is induced in the M phase by activation of pM"* 2 kinase in baby hamster kidney cells (28), this new inhibitor of protein phosphatases 1 and 2A, MC-LR, will provide further information in relation to cell cycle regulation in hepatocytes. Acknowledgements This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture and a grant from the Ministry of Health and Welfare for a Comprehensive 10-Year Strategy for Cancer Control, Japan, and grants from the Foundation for Promotion of Cancer Research, the Princess Takamatsu Cancer Research Fund, the Smoking Research Fund and the Uehara Memorial Life Science Foundation.
References l.Botes.D.P., Tuinman.A.A., Wessels.P.L., Viljoen.C.C, Kruger.H., Williams.D.H., Santikarn.S., Smith.R.J. and Hammond.S.J. (1984) The structure of cyanoginosin-LA, a cyclic heptapeptide toxin from the cyanobacterium Microcystis aeruginosa. J. Chem. Soc. Perkin Trans., 1, 2311-2318. 2. Carmichael.W.W. (1988) Toxins of freshwater algae. In Anthony,T.T. (cd.), Handbook of Natural Toxins; Marine Toxins and Venoms. Vol. 3, Marcel Dekker, Inc., New York, pp. 121-147. 3.Watanabe,M.F., Oishi.S., Harada,K.-L, Matsuura.K., Kawai.H. and Suzuki,M. (1988) Toxins contained in Microcystis species of cyanobacteria (blue-green algae). Toxicon, 26, 1017-1025. 4. Namikoshi,M., Rinehart.K.L., Sakai.R., Sivonen,K. and Carmichael.W.W. (1990) Structures of three new cyclic heptapeptide hepatotoxins produced by the cyanobacterium (blue-green alga) Nostoc sp. strain 152. J. Org. Chem., 55, 6135-6139. 5. Matsushima.R.N., Ohta.T., Nishiwaki.S., Suganuma,M., Kohyama.K., Ishikawa.T., Carmichael.W.W. and Fujiki.H. (1992) Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR. J. Cancer Res. Clin. Oncol., 118, 420-424. 6. Yoshizawa.S., Matsushima.R., Watanabe.M.F., Harada,K.-I., Ichihara.A., Carmichael.W.W. and Fujiki.H. (1990) Inhibition of protein phosphatases by microcystin and nodularin associated with hepatotoxicity. J. Cancer Res. CUn. OncoL, 116, 609-614. 7. YatsunamiJ., Fujiki,H., Suganuma.M., Yoshizawa.S., ErikssonJ.E., Olson,M.OJ. and Goldman,R.D. (1991) Vimentin is hyperphosphorylated in primary human fibroblasts treated with okadaic acid. Biodtem. Biophys. Res. Commun., 177, 1165-1170. 8. Baribault.H., Blouin.R., Bourgon.L. and Marceau.N. (1989) Epidermal growth factor-induced selective phosphorylation of cultured rat hepatocyte 55-KD cytokeratin before filament reorganization and DNA synthesis. J. Cell BioL, 109, 1665-1676. 9. KrishnamurthyJ., Carmichael.W.W. and Sarver.E.W. (1986) Toxic peptides from freshwater cyanobacteria (blue—green algae). I. Isolation, purification and characterization of peptides from Microcystis aeruginosa and Anabena flos-aquae. Toxicon, 24, 865—873. 10. Galey.F.D., Beasly,V.R., Carmichacl,W.W., Kleppe.G., Hooser.S.B. and
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Discussion MC-LR, which was recently shown to be a potent liver tumor promoter, provides a new mechanism of tumor promotion in rat liver (5,6). MC-LR is a potent inhibitor of protein phosphatases 1 and 2A and induces increased phosphorylation of various proteins in primary cultured rat hepatocytes (6). We named the increased phosphorylation the apparent 'activation' of protein kinases (16), because protein kinases are not directly activated by MC-LR, which belongs to the okadaic acid class of tumor promoters. This paper reported that cytokeratins 8 and 18 were hyperphosphorylated in primary cultured rat hepatocytes treated with MC-LR dose dependently. Preliminary results with a similar system were recently discussed by Falconer and Yeung (17). We further extended the study of cytokeratins 8 and 18 to demonstrate that they are also substrates for in vitro phosphorylation by protein kinases in a cytosolic fraction of rat liver. Cytokeratin is a type of intermediate filament, others include vimentin, desmin and lamins, to name a few (18). The types of intermediate filaments that are mainly phosphorylated by the okadaic acid class tumor promoters are dependent on cell types. Hyperphosphorylated vimentin was found in primary human fibroblasts treated with okadaic acid and DTX-1 (7) and various cytokeratins were found in human keratinocytes treated with okadaic acid and DTX-1 (Yatsunami et al., manuscript in preparation). In addition to these intermediate filaments, a heat shock protein, (HSP) 27, was also hyperphosphorylated in human keratinocytes treated with okadaic acid (Yatsunami etal., manuscript in preparation). Although MC-LR, like other okadaic acid class compounds, has a simple mechanism of action due to inhibition of protein phosphatases 1 and 2A, target proteins are numerous, as shown in Figure 1. The increase of phosphorylation is influenced by various factors; the amount of protein, the turn-over rate of a phosphate on a protein, and so on.
PhospborylatkM of cytokeratins
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