Vol. 189, No. 2, 1992 December 15, 1992
BIOCHEMICAL
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Nonphorbol tumor promoters okadaic acid and calyculin-A induce membrane translocation of protein kinase C Rayudu Gopalakrishna*,
Zhen H. Chen, and Usha Gundimeda
Department of Pharmacology and Nutrition, School of Medicine, University of Southern California, Los Angeles, CA 90033 Received
October
13,
1992
Summary: The cell-permeable inhibitors of type 1 and 2A protein phosphatases, okadaic acid and calyculin-A, induced a redistribution of protein kinase C (PIE) activity and immunoreactivity (40 to 60%) from cytosol to membrane in some cell types. Calyculin-A was loo-fold more potent than okadaic acid and required only 5 to 10 nM concentrations to induce this PKC translocation. The concentration of these agents required to induce the redistribution of PKC correlated with the potency of these agents to inhibit both type 1 and 2A protein phosphatases. There was a lag period of 15 to 30 min before the onset of PKC translocation, as this process might have been induced by indirect cellular events triggered by inhibitions of protein phosphatases (I and 2A). Taken together these results suggest that although the okadaic acid class of tumor promoters and phorbol ester-related agents bind to two different cellular receptors having counteracting enzymic activities, they share a common mechanism of action, namely the induction of cytosol to membrane translocation of PKC. o 1992Academc
Tumor promoters such as phorbol esters activate protein kinase C (PKC) by apparently substituting for the endogenous regulator diacylglycerol (l-3). These terpenoid tumor promoters also induce a cytosol to membrane translocation of PKC which may represent the activation state of PKC (4-6). We have shown previously that oxidant tumor promoters such as hydrogen peroxide and periodate also can induce the activation of PKC by promoting the oxidative modification of the regulatory domain of the enzyme (7,8). Furthermore, others have shown an induction of the membrane translocation of PKC in cells treated with oxidant-generating systems (9). Thus, the oxidant tumor promoters, even though structurally unrelated to terpenoid tumor promoters, share some actions with the terpenoid tumor promoters. *To whom all correspondence
should be addressed.
Abbreviations used: PKC, protein kinase C; PP-1, type 1 protein phosphatase; PP-2A, type 2A protein phosphatase; PDBu, phorbol 12,13-dibutyrate, TPA, 12-0tetradecanoyl-phorbol 13-acetate. 0006-291X/92 Copyright All rights
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Tumor promoters such as okadaic acid, calyculin-A, and microcystin-LR have no direct effect on the isolated PKC or other protein kinases (10-12). Nonetheless, they induce hyperphosphorylation of some cellular proteins by apparently inhibiting the protein phosphatases type 1 (PP-1) and type 2A (PP-2A), which may eventually lead to tumor promotion (10-12). Furthermore, these agents may elicit many cellular actions of phorbol esters (13-16) including the stimulation of the release of arachidonic acid and its metabolites (17). Calyculin-A was shown to increase intracellular Ca2+ by activating voltage-gated Ca 2+ channels (18). Conceivably, the second messengers generated in response to these agents may amplify further the actions of these tumor promoters. The inhibition of protein phosphatases is not alone enough to induce the hyperphosphorylation of proteins, but a partial activation of protein kinases is also required. Hence, we have determined whether PKC may be activated by any indirect mechanism in intact cells treated with the okadaic acid class of agents. Since an increase in membranebound PKC reveals the activated state of PKC in intact cells, we have determined the subcellular distribution of PKC in the cells treated with these agents. Our results suggest that the okadaic acid-related tumor promoters, although they are structurally unrelated to phorbol esters and bind to different cellular receptors, share a common mechanism of action with phorbol esters by inducing a cytosol to membrane translocation of PKC. MATERIALS
AND
METHODS
Materials: Okadaic acid, I-nor-okadaone, calyculin-A, and microcystin-LR were obtained from the LC Services Corporation. The immunostaining kit with alkaline phosphatase-conjugated anti-rabbit goat y-globulins was purchased from Bio-Rad. Isolation of PKC from cvtosol and membrane fractions: Membrane bindings of PKC in homogenates and with the isolated membrane were carried out as described previously (5). The cytosolic and detergent-solubilized membrane fractions (2.5 ml) were applied to 0.5-ml DEAE-cellulose (DE-52) columns previously equilibrated with buffer A (20 mM Tris-HCl, pH 7.5/l mM EDTA/O.l mM DTT). After washing the column with 2 ml of buffer A, the bound PKC was eluted with 1.25 ml of 0.1 M NaCl in buffer A (19). To achieve a reliable estimation of PKC activity without interference by PP-1 and PP2A, the inhibitor microcystin-LR (100 nM) was included in all the PKC assays. PKC assay and PDBu binding: These determinations were carried out by using multiwell plate filtration assays as described elsewhere (20). The unique aspects of these methods were that both the incubations and filtrations were carried out in the same multiwell plate. Briefly, PKC reaction samples were incubated in 96-well plates with fitted filtration discs made of cellulose acetate membranes (HA type, Millipore). The histone Hl was precipitated and filtered with 10% TCA and the radioactivity associated with the filters was counted. For the determination of PKC-associated phorbol ester binding, the samples were incubated with [3H]-phorbol 12, 13-dibutyrate (PDBu) in the microwells, the ligand bound PKC was adsorbed onto DEAE-Sephadex beads, and the beads then were filtered. The radioactivity associated with the DEAE-Sephadex beads retained on the filter was counted. PKC activity was expressed as units, where one unit of enzyme transfers 1 nmol of phosphate to histone Hl per min at 30°C. Immunoblotting of PKC: The cytosolic and membrane fractions were electrophoresed on 12% SDS-polyacrylamide gels and the proteins were blotted on an Immobilon-P membrane (Millipore). The immunoreactive PKC was detected by using polyclonal 951
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antibodies which recognize ~1,p, and y isoenzymesof PKC. These antibodies were raisedin rabbits by injecting the C-terminal “activating” peptideof PKC (residues530558) conjugatedto keyholelimpet hemocyanin(21). RESULTS In normal rat kidney (NRK) cells treatedwith okadaic acid (500 nM), a timedependentdecreasein PKC activity in the cytosol and a concomitant increasein the membrane-associatedPKC activity was observed(Fig. 1). The cytosol to membrane redistributionof PKC becamemaximal at 40 to 60 min after okadaicacid treatment. As an appropriatecontrol, an inactive analogueof okadaic acid, 1-nor-okadaone(1 PM), having no PP-1 and PP-2A inhibitory activity and no tumor promoting activity was used. This inactive analogueof okadaicacid did not induce an appreciableredistribution
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Fig. 1, Time course of the cytosol to membrane redistribution of PKC in cells treated with okadaic acid class of agents. Confluent NRK cells (20 x 106) were treated with calyculin-A (10 nM), okadaic acid (500 nM), or 1-nor-okadaone (1 j.tM) for the indicated time periods. Both the cytosolic and detergent-solubilized membrane fractions were prepared from the control and treated cells. Then PKC phosphotransferase activity was determined in triplicates by using the multiwell filtration approach as described in the Materials and Methods. Fig. 2, Effect of various concentrations of okadaic acid and calyculin-A on the cytosol to membrane redistribution of PKC. Confluent NRK cells (approx. 20 x 106) were treated with the indicated concentrations of okadaic acid and calyculin-A for a 60-min time period. The cells treated with higher (>lO nM) concentrations of calyculin-A were detached and aggregated. These detached cells were collected by centrifugation. Both PKC activity and [3H]PDBu binding determinations were carried out in the cytosolic and detergent-solubilized membrane fractions. 952
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Fip. 3, Immunoblots of PKC from NRK cells treated with okadaic acid. The cytosolic and detergent-solubilized fractions (Fig.1) were subjected to SDSpolyacrylamide electrophoresis and then transferred to an Immobilon-P membrane. The anti-PKC antibodies that recognize all three isoenzymes of PKC (a, p, y) were used to detect immunoreactive PKC. Immunoreactive bands were visualized by alkaline phosphatase conjugated anti-rabbit goat y-globulins (Bio-rad).
of PKC. Another inhibitor of PP-1 and PP-2A, calyculin-A, also induced membrane translocation of PKC similar to okadaic acid. However, the concentration of calyculin-A required was found to be low (2 to 10 nM) for inducing the optimal translocation of PKC. Whereas under the same conditions okadaic acid was required at 50- to lOO-fold higher (250 to 500 nM) concentrations (Fig. 2). Moreover, in all these cases PKCassociated [3H]PDBu binding also showed a similar pattern of redistribution from cytosol to membrane. Any changes in subcellular redistribution of activators or inactivators of PKC could also lead to apparent changes in the subcellular distribution of PKC even in the absence of a true redistribution of the enzyme. Hence, we have measured the immunoreactivity of PKC by immunoblotting to determine whether there was any physical redistribution of the enzyme. Anti-PKC antibodies that recognize all three isoenzymes of PKC were used to detect immunoreactive PKC on the western blots. As shown in Fig. 3, PKC immunoreactivity decreased in the cytosolic fraction of okadaic acid-treated cells and correspondingly increased in the membrane fraction correlating with the observed changes in PKC activity and PDBu binding. This suggested that a physical redistribution of PKC from the cytosoi to membrane had in deed occurred. Okadaic acid and calyculin-A also induced the cytosol to membrane translocation of PKC in L2 mouse lung carcinoma, DU 145 human prostatic carcinoma, and T47D human breast carcinoma cell lines. To induce the optimal membrane translocation of PKC in all these cell lines, the concentration of okadaic acid needed was high (>lOO nM) and the concentration of calyculin-A needed was low (cl0 nM). Nevertheless, in cell lines such as Kirsten sarcoma virus-transformed NRK (KNRK) and C6 rat glioma cells, no change in subcellular distribution of PKC was observed by treating cells even with higher concentrations (0.1 to 1 pM) of okadaic acid and calyculin-A. Microcystin LR, another inhibitor of PP-1 and PP-2A, failed to show any translocation in all the cell lines probably due to its cell impermeability. Differences in PKC membrane translocations induced bv the okadaic acid class of agents and uhorbol esters; The pattern of PKC membrane translocation induced by these 953
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Fig. & Comparison of the cytosol to membrane redistribution of PKC induced by TPA and okadaic acid. Confluent L2 lung carcinoma cells (approx. 25 x 106) were treated with TPA (100 nM) or okadaic acid (500 nM) for the indicated time periods and PKC activity present in the cytosolic and detergent-solubilized membrane fractions were determined.
two different classes of tumor promoters was compared in L2 lung carcinoma cells. The phorbol ester, TPA, induced a dramatic (90%) decrease in PKC activity in the cytosol within 5 min after treatment, and nearly 75% of the decreased activity in the cytosol was recovered in the membrane fraction during this period (Fig. 4). However, with okadaic acid treatment there was a lag period of 15 to 30 min before the onset of PKC redistribution. Although maximal membrane association was observed after 40 to 60 min in okadaic acid-treated cells, the extent of PKC decreased from the cytosol or increased in the membrane was only 50 to 60% of that observed with TPA. Furthermore, in the TPA-treated cells the membrane associated PKC was dramatically downregulated after 1 h. On the contrary, in the cells treated with okadaic acid no appreciable decrease in total PKC (cytosol and membrane) was observed. Moreover, in the cells treated with okadaic acid, after a l-h period, PKC present in the membrane fraction gradually decreased while in the cytosolic fraction it increased. This was probably due to a redistribution of PKC from the membrane back to the cytosol. In cell types such as KNRK and C6 where both okadaic acid and calyculin-A failed to induce the membrane association of PKC, TPA was able to induce nearly an 80% redistribution of the PKC activity from the cytosol to membrane. Indirect actions of okadaic acid and calvculin-A; In our previous studies we have shown that TPA could induce the membrane association of PKC by binding directly to 954
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the PKC-lipid complex in experiments carried out using the crude cell homogenates as well as isolated membranes and purified PKC. Although both okadaic acid and calyculin-A have no direct effect on purified PKC (lo-12), whether these agents could induce a translocation of PKC by influencing the native form of PKC was tested by adding these agents directly to cell homogenates. Only TPA in the presence of Ca2+ produced the membrane association of PKC, while okadaic acid and calyculin-A failed to induce any membrane association of PKC in either crude cell homogenates or with the isolated membranes incubated with purified PKC (data not shown).
DISCUSSION The induction of PKC translocation by okadaic acid and calyculin-A, two structurally unrelated agents with PP-1 and PP-2A inhibitory activity, and the lack of induction of PKC translocation by the inactive analogue, 1-nor-okadaone, having no PP1 and PP-2A inhibitory activity suggest that the inhibition of PP-1 and PP-2A might have eventually lead to the induction of membrane translocation of PKC. Furthermore, the concentration of these agents required to induce PKC translocation in various cell types correlated with the potency of these agents to inhibit PP-1 and PP-2A. The okadaic acid has a differential sensitivity for PP-1 ‘and PP-2A, and the reported I&-J values are 70 nM and 1 nM, respectively (11). On the other hand, calyculin-A has more or less equal affinity for PP-1 and PP-2A, and the I& values are 2 and 1 nM, respectively (11). The requirement of higher concentrations of okadaic acid (>lOO nM) for the induction of PKC translocation in our study suggested that the inhibition of PP2A alone may not be enough, but also the inhibition PP-1 may be required. The lag period before the onset of redistribution of PKC in the cells treated with okadaic acid or calyculin-A, and the lack of ability of these agents to induce membrane association of PKC in homogenates suggested that other indirect events that were initiated by the initial inhibition of PP-1 and PP-2A by okadaic acid might have eventually triggered the PKC membrane translocation process. The okadaic acid- and calyculin A-induced membrane redistributions of PKC were low in magnitude, slow in onset, transient in duration, resulted in an unpronounced downregulation of PKC, and induced by an indirect cellular mechanism. In all these aspects it differed from the membrane translocation of PKC induced by phorbol esters. It was not clear why okadaic acid and calyculin-A failed to induce the translocation of PKC in KNRK and C6 cells in which TPA can induce this process. It is possible that okadaic acid and calyculin-A were not able to generate the intracellular regulators, which induce the PKC membrane translocation. Alternatively, the okadaic acid class of agents indirectly induced the translocation of a specific isoenzyme of PKC which may be either absent or low in KNRK and C6 cells. The membrane association of PKC induced by the okadaic acid class of agents resembled the PKC membrane translocation induced by certain hormones (6) in some aspects; it was less pronounced, transient, and there was no dramatic down regulation of PKC. Nonetheless, hormone-induced translocation of PKC is a rapid event compared to the PKC translocation induced by okadaic acid (6). The hormone-induced translocation of PKC is believed to be mediated by an enhanced breakdown of phosphatidylinositol 955
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resulting in an elevation of diacylglycerol and intracellular Ca2+ (6). However, others have reported a decrease in the phosphatidylinositol breakdown, both the basal and hormone-stimulated, in the cells treated with okadaic acid (22). Arachidonic acid and its metabolites generated by transmembrane signalling also have been shown to activate and induce the translocation of PKC (23). Okadaic acid was reported to stimulate the release of arachidonic acid and its metabolites (17). Furthermore, calyculin-A was shown to increase intracellular Ca2+ by activating voltage-gated Ca2+ channels (18). We do not know at the present whether these second messengers, Ca2+ and arachidonic acid, are involved in the induction of the PKC membrane translocation. Since the membrane association of PKC reflects the activation state of PKC (6), it is conceivable that PKC may be activated by an indirect mechanism in the cells treated with the okadaic acid class of tumor promoters. Therefore, together with a decrease in the activities of PP-1 and PP-2A, an increase in the activity of PKC can lead to hyperphosphorylation of cellular proteins. Our previous studies revealed that although oxidant tumor promoters are structurally unrelated to terpenoid tumor promoters, they induced the activation/ membrane translocation of PKC(7,8). The current observations further extend this generalization that yet another distinct class of tumor promoters, even though it acts on different cellular receptors, induced the membrane translocation of PKC. Conceivably, structurally unrelated tumor promoters of diverse origin may induce the cytosol to membrane translocation (activation) of PKC, which, in part, may mediate tumor promotion events.
Acknowledgments: We thank Dr. Wayne B. Anderson, National Cancer Institute, Bethesda, for providing the NRK and KNRK cell lines, and Mr. Drake Jantzen for his excellent technical assistance. This work was supported by USPHS Grant CA 47142 from National Cancer Institute, Grant RT 388 from the Tobacco-Related Diseases Research Program, University of California, and a Research Grant from the Wright Foundation. REFERENCES 1. Kikkawa, U., Kishimoto, A., and Nishizuka, Y. (1989) Ann. Rev. Biochem. 58, 31-44. 2. Sharkey, N., Leach, K., and Blumberg, P. M. (1984) Proc. Natl. Acad. Sci. USA. 81, 607-610. 3. Niedel, J. E., Kuhn, L. J., and Vandenbark, G. R. (1983) Proc. Natl. Acad. Sci. USA. 80,36-40. 4. Kraft, A. S., and Anderson, W. B. (1983) Nature, 301, 621-623. 5. Gopalakrishna, R., Barsky, S. H., Thomas, T. P., and Anderson, W. B. (1986) J. Biol. Chem. 261, 16,438-16,445. 6. Thomas, T. P., Gopalakrishna, R., and Anderson, W. B. (1987) Methods Enzymol. 141,399-411. 7. Gopalakrishna, R.. and Anderson, W. B. (1989) Proc. Natl. Acad. Sci. USA. 86, 6758-6762. 8. Gopalakrishna, R., and Anderson, W. B. (1991) Arch. Biochem. Biophys. 285, 382-387. 9. Larsson, R., and Cerutti, P. (1989) Cancer Res. 49, 5627-5632.
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10. Bialojan, C., and Takai, A. (1988) Biochem. J. 256,286,283-290. 11. Ishihara, H., Martin, B. L., Braughtigan, D. L., Karaki, H., Ozaki, H., Kato, Y., Fusertani, N., Watabe, S., Hashimoto, K., Uemura, D., and Hartshorne, D. J. (1989) Biochem. Biophys. Res. Commun. 159,871-877. 12. Cohen, P. (1989) Ann. Rev. Biochem. 58,453-508. 13. Suganuma, M., Fujiki, H., Fuuya-Suguri, H., Yoshizawa, S., Yusumoto, S., Kato, Y., Fusetani, N., and Sugimura, T., (1990) Cancer Res. 50, 3521-3525. 14. Sakai, A., and Fujiki, H. (1991) Jpn. J. Cancer Res. 82,518-523. 15. Yatsunami, J., Fujiki, H., Suganuma, M., Yoshizawa, S., Ericksson, J. E., Olson, M. 0. J., and Goldman, R. D. (1991) Biochem. Biophys. Res. Commun. 177, 11661170. 16. Kim, S-J., Lafyatis, R., Kim, K. Y., Angel, P., Fujiki, H., Karin, M., Spom, M. B., and Roberts, A. B. (1990) Cell Reg. 1,269-278. 17. Levine, L., Fujiki, H., Yamada, K., Ojiki, M., Gjika, H. B., and van Vunakis, H. (1988) Toxicon, 26, 1123-l 128. 18. Ishihara, H., Ozaki, H., Sato, K., Hori, M., Karaki, H., Watabe, S., Kato, Y., Fusetani, N., Hasimoto, K., Uemura, D., and Hartshorne, D. J. (1989) J. Pharmacol. Exp. Ther. 250, 388-396 19. Gopalakrishna, R., and Barsky, S. H. (1988) Proc. Natl. Acad. Sci. USA. 85, 612616. 20. Gopalakrishna, R., Chen, Z. H., Gundimeda, U., Wilson, J. C., and Anderson, W. B. (1992) Anal. Biochem. (in press) 21. Makowske, M., Ballester, R., Cayre, Y., and Rosen, 0. M. (1988) J. Biol. Chem. 263,3402-3410. 22. Garcia-Sainz, J. A., Macias-Silva, M., and Romero-Avila, M. T. (1991) Biochem. Biophys. Res. Commun. 179, 852-856. 23. McPhail, L. C., Clayton, C. C., and Snyderman, R. (1988) Science 224, 622-624.
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Nonphorbol tumor promoters okadaic acid and calyculin-A induce membrane translocation of protein kinase C Rayudu Gopalakrishna*,
Zhen H. Chen, and Usha Gundimeda
Department of Pharmacology and Nutrition, School of Medicine, University of Southern California, Los Angeles, CA 90033 Received
October
13,
1992
Summary: The cell-permeable inhibitors of type 1 and 2A protein phosphatases, okadaic acid and calyculin-A, induced a redistribution of protein kinase C (PIE) activity and immunoreactivity (40 to 60%) from cytosol to membrane in some cell types. Calyculin-A was loo-fold more potent than okadaic acid and required only 5 to 10 nM concentrations to induce this PKC translocation. The concentration of these agents required to induce the redistribution of PKC correlated with the potency of these agents to inhibit both type 1 and 2A protein phosphatases. There was a lag period of 15 to 30 min before the onset of PKC translocation, as this process might have been induced by indirect cellular events triggered by inhibitions of protein phosphatases (I and 2A). Taken together these results suggest that although the okadaic acid class of tumor promoters and phorbol ester-related agents bind to two different cellular receptors having counteracting enzymic activities, they share a common mechanism of action, namely the induction of cytosol to membrane translocation of PKC. o 1992Academc
Tumor promoters such as phorbol esters activate protein kinase C (PKC) by apparently substituting for the endogenous regulator diacylglycerol (l-3). These terpenoid tumor promoters also induce a cytosol to membrane translocation of PKC which may represent the activation state of PKC (4-6). We have shown previously that oxidant tumor promoters such as hydrogen peroxide and periodate also can induce the activation of PKC by promoting the oxidative modification of the regulatory domain of the enzyme (7,8). Furthermore, others have shown an induction of the membrane translocation of PKC in cells treated with oxidant-generating systems (9). Thus, the oxidant tumor promoters, even though structurally unrelated to terpenoid tumor promoters, share some actions with the terpenoid tumor promoters. *To whom all correspondence
should be addressed.
Abbreviations used: PKC, protein kinase C; PP-1, type 1 protein phosphatase; PP-2A, type 2A protein phosphatase; PDBu, phorbol 12,13-dibutyrate, TPA, 12-0tetradecanoyl-phorbol 13-acetate. 0006-291X/92 Copyright All rights
$4.00
0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
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Tumor promoters such as okadaic acid, calyculin-A, and microcystin-LR have no direct effect on the isolated PKC or other protein kinases (10-12). Nonetheless, they induce hyperphosphorylation of some cellular proteins by apparently inhibiting the protein phosphatases type 1 (PP-1) and type 2A (PP-2A), which may eventually lead to tumor promotion (10-12). Furthermore, these agents may elicit many cellular actions of phorbol esters (13-16) including the stimulation of the release of arachidonic acid and its metabolites (17). Calyculin-A was shown to increase intracellular Ca2+ by activating voltage-gated Ca 2+ channels (18). Conceivably, the second messengers generated in response to these agents may amplify further the actions of these tumor promoters. The inhibition of protein phosphatases is not alone enough to induce the hyperphosphorylation of proteins, but a partial activation of protein kinases is also required. Hence, we have determined whether PKC may be activated by any indirect mechanism in intact cells treated with the okadaic acid class of agents. Since an increase in membranebound PKC reveals the activated state of PKC in intact cells, we have determined the subcellular distribution of PKC in the cells treated with these agents. Our results suggest that the okadaic acid-related tumor promoters, although they are structurally unrelated to phorbol esters and bind to different cellular receptors, share a common mechanism of action with phorbol esters by inducing a cytosol to membrane translocation of PKC. MATERIALS
AND
METHODS
Materials: Okadaic acid, I-nor-okadaone, calyculin-A, and microcystin-LR were obtained from the LC Services Corporation. The immunostaining kit with alkaline phosphatase-conjugated anti-rabbit goat y-globulins was purchased from Bio-Rad. Isolation of PKC from cvtosol and membrane fractions: Membrane bindings of PKC in homogenates and with the isolated membrane were carried out as described previously (5). The cytosolic and detergent-solubilized membrane fractions (2.5 ml) were applied to 0.5-ml DEAE-cellulose (DE-52) columns previously equilibrated with buffer A (20 mM Tris-HCl, pH 7.5/l mM EDTA/O.l mM DTT). After washing the column with 2 ml of buffer A, the bound PKC was eluted with 1.25 ml of 0.1 M NaCl in buffer A (19). To achieve a reliable estimation of PKC activity without interference by PP-1 and PP2A, the inhibitor microcystin-LR (100 nM) was included in all the PKC assays. PKC assay and PDBu binding: These determinations were carried out by using multiwell plate filtration assays as described elsewhere (20). The unique aspects of these methods were that both the incubations and filtrations were carried out in the same multiwell plate. Briefly, PKC reaction samples were incubated in 96-well plates with fitted filtration discs made of cellulose acetate membranes (HA type, Millipore). The histone Hl was precipitated and filtered with 10% TCA and the radioactivity associated with the filters was counted. For the determination of PKC-associated phorbol ester binding, the samples were incubated with [3H]-phorbol 12, 13-dibutyrate (PDBu) in the microwells, the ligand bound PKC was adsorbed onto DEAE-Sephadex beads, and the beads then were filtered. The radioactivity associated with the DEAE-Sephadex beads retained on the filter was counted. PKC activity was expressed as units, where one unit of enzyme transfers 1 nmol of phosphate to histone Hl per min at 30°C. Immunoblotting of PKC: The cytosolic and membrane fractions were electrophoresed on 12% SDS-polyacrylamide gels and the proteins were blotted on an Immobilon-P membrane (Millipore). The immunoreactive PKC was detected by using polyclonal 951
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antibodies which recognize ~1,p, and y isoenzymesof PKC. These antibodies were raisedin rabbits by injecting the C-terminal “activating” peptideof PKC (residues530558) conjugatedto keyholelimpet hemocyanin(21). RESULTS In normal rat kidney (NRK) cells treatedwith okadaic acid (500 nM), a timedependentdecreasein PKC activity in the cytosol and a concomitant increasein the membrane-associatedPKC activity was observed(Fig. 1). The cytosol to membrane redistributionof PKC becamemaximal at 40 to 60 min after okadaicacid treatment. As an appropriatecontrol, an inactive analogueof okadaic acid, 1-nor-okadaone(1 PM), having no PP-1 and PP-2A inhibitory activity and no tumor promoting activity was used. This inactive analogueof okadaicacid did not induce an appreciableredistribution
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Fig. 1, Time course of the cytosol to membrane redistribution of PKC in cells treated with okadaic acid class of agents. Confluent NRK cells (20 x 106) were treated with calyculin-A (10 nM), okadaic acid (500 nM), or 1-nor-okadaone (1 j.tM) for the indicated time periods. Both the cytosolic and detergent-solubilized membrane fractions were prepared from the control and treated cells. Then PKC phosphotransferase activity was determined in triplicates by using the multiwell filtration approach as described in the Materials and Methods. Fig. 2, Effect of various concentrations of okadaic acid and calyculin-A on the cytosol to membrane redistribution of PKC. Confluent NRK cells (approx. 20 x 106) were treated with the indicated concentrations of okadaic acid and calyculin-A for a 60-min time period. The cells treated with higher (>lO nM) concentrations of calyculin-A were detached and aggregated. These detached cells were collected by centrifugation. Both PKC activity and [3H]PDBu binding determinations were carried out in the cytosolic and detergent-solubilized membrane fractions. 952
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Membrane
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Fip. 3, Immunoblots of PKC from NRK cells treated with okadaic acid. The cytosolic and detergent-solubilized fractions (Fig.1) were subjected to SDSpolyacrylamide electrophoresis and then transferred to an Immobilon-P membrane. The anti-PKC antibodies that recognize all three isoenzymes of PKC (a, p, y) were used to detect immunoreactive PKC. Immunoreactive bands were visualized by alkaline phosphatase conjugated anti-rabbit goat y-globulins (Bio-rad).
of PKC. Another inhibitor of PP-1 and PP-2A, calyculin-A, also induced membrane translocation of PKC similar to okadaic acid. However, the concentration of calyculin-A required was found to be low (2 to 10 nM) for inducing the optimal translocation of PKC. Whereas under the same conditions okadaic acid was required at 50- to lOO-fold higher (250 to 500 nM) concentrations (Fig. 2). Moreover, in all these cases PKCassociated [3H]PDBu binding also showed a similar pattern of redistribution from cytosol to membrane. Any changes in subcellular redistribution of activators or inactivators of PKC could also lead to apparent changes in the subcellular distribution of PKC even in the absence of a true redistribution of the enzyme. Hence, we have measured the immunoreactivity of PKC by immunoblotting to determine whether there was any physical redistribution of the enzyme. Anti-PKC antibodies that recognize all three isoenzymes of PKC were used to detect immunoreactive PKC on the western blots. As shown in Fig. 3, PKC immunoreactivity decreased in the cytosolic fraction of okadaic acid-treated cells and correspondingly increased in the membrane fraction correlating with the observed changes in PKC activity and PDBu binding. This suggested that a physical redistribution of PKC from the cytosoi to membrane had in deed occurred. Okadaic acid and calyculin-A also induced the cytosol to membrane translocation of PKC in L2 mouse lung carcinoma, DU 145 human prostatic carcinoma, and T47D human breast carcinoma cell lines. To induce the optimal membrane translocation of PKC in all these cell lines, the concentration of okadaic acid needed was high (>lOO nM) and the concentration of calyculin-A needed was low (cl0 nM). Nevertheless, in cell lines such as Kirsten sarcoma virus-transformed NRK (KNRK) and C6 rat glioma cells, no change in subcellular distribution of PKC was observed by treating cells even with higher concentrations (0.1 to 1 pM) of okadaic acid and calyculin-A. Microcystin LR, another inhibitor of PP-1 and PP-2A, failed to show any translocation in all the cell lines probably due to its cell impermeability. Differences in PKC membrane translocations induced bv the okadaic acid class of agents and uhorbol esters; The pattern of PKC membrane translocation induced by these 953
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Membrane
Okadaic acid
0.0 ' 0
1
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I 3
(h)
Fig. & Comparison of the cytosol to membrane redistribution of PKC induced by TPA and okadaic acid. Confluent L2 lung carcinoma cells (approx. 25 x 106) were treated with TPA (100 nM) or okadaic acid (500 nM) for the indicated time periods and PKC activity present in the cytosolic and detergent-solubilized membrane fractions were determined.
two different classes of tumor promoters was compared in L2 lung carcinoma cells. The phorbol ester, TPA, induced a dramatic (90%) decrease in PKC activity in the cytosol within 5 min after treatment, and nearly 75% of the decreased activity in the cytosol was recovered in the membrane fraction during this period (Fig. 4). However, with okadaic acid treatment there was a lag period of 15 to 30 min before the onset of PKC redistribution. Although maximal membrane association was observed after 40 to 60 min in okadaic acid-treated cells, the extent of PKC decreased from the cytosol or increased in the membrane was only 50 to 60% of that observed with TPA. Furthermore, in the TPA-treated cells the membrane associated PKC was dramatically downregulated after 1 h. On the contrary, in the cells treated with okadaic acid no appreciable decrease in total PKC (cytosol and membrane) was observed. Moreover, in the cells treated with okadaic acid, after a l-h period, PKC present in the membrane fraction gradually decreased while in the cytosolic fraction it increased. This was probably due to a redistribution of PKC from the membrane back to the cytosol. In cell types such as KNRK and C6 where both okadaic acid and calyculin-A failed to induce the membrane association of PKC, TPA was able to induce nearly an 80% redistribution of the PKC activity from the cytosol to membrane. Indirect actions of okadaic acid and calvculin-A; In our previous studies we have shown that TPA could induce the membrane association of PKC by binding directly to 954
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the PKC-lipid complex in experiments carried out using the crude cell homogenates as well as isolated membranes and purified PKC. Although both okadaic acid and calyculin-A have no direct effect on purified PKC (lo-12), whether these agents could induce a translocation of PKC by influencing the native form of PKC was tested by adding these agents directly to cell homogenates. Only TPA in the presence of Ca2+ produced the membrane association of PKC, while okadaic acid and calyculin-A failed to induce any membrane association of PKC in either crude cell homogenates or with the isolated membranes incubated with purified PKC (data not shown).
DISCUSSION The induction of PKC translocation by okadaic acid and calyculin-A, two structurally unrelated agents with PP-1 and PP-2A inhibitory activity, and the lack of induction of PKC translocation by the inactive analogue, 1-nor-okadaone, having no PP1 and PP-2A inhibitory activity suggest that the inhibition of PP-1 and PP-2A might have eventually lead to the induction of membrane translocation of PKC. Furthermore, the concentration of these agents required to induce PKC translocation in various cell types correlated with the potency of these agents to inhibit PP-1 and PP-2A. The okadaic acid has a differential sensitivity for PP-1 ‘and PP-2A, and the reported I&-J values are 70 nM and 1 nM, respectively (11). On the other hand, calyculin-A has more or less equal affinity for PP-1 and PP-2A, and the I& values are 2 and 1 nM, respectively (11). The requirement of higher concentrations of okadaic acid (>lOO nM) for the induction of PKC translocation in our study suggested that the inhibition of PP2A alone may not be enough, but also the inhibition PP-1 may be required. The lag period before the onset of redistribution of PKC in the cells treated with okadaic acid or calyculin-A, and the lack of ability of these agents to induce membrane association of PKC in homogenates suggested that other indirect events that were initiated by the initial inhibition of PP-1 and PP-2A by okadaic acid might have eventually triggered the PKC membrane translocation process. The okadaic acid- and calyculin A-induced membrane redistributions of PKC were low in magnitude, slow in onset, transient in duration, resulted in an unpronounced downregulation of PKC, and induced by an indirect cellular mechanism. In all these aspects it differed from the membrane translocation of PKC induced by phorbol esters. It was not clear why okadaic acid and calyculin-A failed to induce the translocation of PKC in KNRK and C6 cells in which TPA can induce this process. It is possible that okadaic acid and calyculin-A were not able to generate the intracellular regulators, which induce the PKC membrane translocation. Alternatively, the okadaic acid class of agents indirectly induced the translocation of a specific isoenzyme of PKC which may be either absent or low in KNRK and C6 cells. The membrane association of PKC induced by the okadaic acid class of agents resembled the PKC membrane translocation induced by certain hormones (6) in some aspects; it was less pronounced, transient, and there was no dramatic down regulation of PKC. Nonetheless, hormone-induced translocation of PKC is a rapid event compared to the PKC translocation induced by okadaic acid (6). The hormone-induced translocation of PKC is believed to be mediated by an enhanced breakdown of phosphatidylinositol 955
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resulting in an elevation of diacylglycerol and intracellular Ca2+ (6). However, others have reported a decrease in the phosphatidylinositol breakdown, both the basal and hormone-stimulated, in the cells treated with okadaic acid (22). Arachidonic acid and its metabolites generated by transmembrane signalling also have been shown to activate and induce the translocation of PKC (23). Okadaic acid was reported to stimulate the release of arachidonic acid and its metabolites (17). Furthermore, calyculin-A was shown to increase intracellular Ca2+ by activating voltage-gated Ca2+ channels (18). We do not know at the present whether these second messengers, Ca2+ and arachidonic acid, are involved in the induction of the PKC membrane translocation. Since the membrane association of PKC reflects the activation state of PKC (6), it is conceivable that PKC may be activated by an indirect mechanism in the cells treated with the okadaic acid class of tumor promoters. Therefore, together with a decrease in the activities of PP-1 and PP-2A, an increase in the activity of PKC can lead to hyperphosphorylation of cellular proteins. Our previous studies revealed that although oxidant tumor promoters are structurally unrelated to terpenoid tumor promoters, they induced the activation/ membrane translocation of PKC(7,8). The current observations further extend this generalization that yet another distinct class of tumor promoters, even though it acts on different cellular receptors, induced the membrane translocation of PKC. Conceivably, structurally unrelated tumor promoters of diverse origin may induce the cytosol to membrane translocation (activation) of PKC, which, in part, may mediate tumor promotion events.
Acknowledgments: We thank Dr. Wayne B. Anderson, National Cancer Institute, Bethesda, for providing the NRK and KNRK cell lines, and Mr. Drake Jantzen for his excellent technical assistance. This work was supported by USPHS Grant CA 47142 from National Cancer Institute, Grant RT 388 from the Tobacco-Related Diseases Research Program, University of California, and a Research Grant from the Wright Foundation. REFERENCES 1. Kikkawa, U., Kishimoto, A., and Nishizuka, Y. (1989) Ann. Rev. Biochem. 58, 31-44. 2. Sharkey, N., Leach, K., and Blumberg, P. M. (1984) Proc. Natl. Acad. Sci. USA. 81, 607-610. 3. Niedel, J. E., Kuhn, L. J., and Vandenbark, G. R. (1983) Proc. Natl. Acad. Sci. USA. 80,36-40. 4. Kraft, A. S., and Anderson, W. B. (1983) Nature, 301, 621-623. 5. Gopalakrishna, R., Barsky, S. H., Thomas, T. P., and Anderson, W. B. (1986) J. Biol. Chem. 261, 16,438-16,445. 6. Thomas, T. P., Gopalakrishna, R., and Anderson, W. B. (1987) Methods Enzymol. 141,399-411. 7. Gopalakrishna, R.. and Anderson, W. B. (1989) Proc. Natl. Acad. Sci. USA. 86, 6758-6762. 8. Gopalakrishna, R., and Anderson, W. B. (1991) Arch. Biochem. Biophys. 285, 382-387. 9. Larsson, R., and Cerutti, P. (1989) Cancer Res. 49, 5627-5632.
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