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OKADAIC ACID STIMULATES NERVE GROWTH FACTOR-SENSITIVE Makio National

Received

April

Mogi

THE ACTIVITY S6 KINASE

and Gordon

624-629

OF THE OF PC12 CELLS

Guroff

Section on Growth Factors Institute of Child Health and Human National Institutes of Health Bethesda, MD 20892

Development

12, 1991

: PC12 pheochromocytoma cells contain at least two different and separable kinases that phosphorylate the S6 protein of the ribosomes. The activity of one of these S6 kinases is increased by treatment of the cells with nerve growth factor and of the other by treatment with epidermal growth factor. Okadaic acid increases the activity of the nerve growth factor-sensitive S6 kinase. The data suggest that the nerve growth factor-sensitive S6 kinase is activated by phosphorylation on serine or threonine residues and is inactivated by either phosphatase 1 or phosphatase 2A, probably the latter. 0 1991P.caderuc PES5, Inc. SUMMARY

Okadaic acid (OA) is a polyether derivative of a 38-carbon fatty acid that was originally isolated from Halichondria okadai, and has been found to be a potent tumor a black sea sponge, It has been shown that this substance is an promoter (1, 2). effective and specific inhibitor of protein phosphatases type 2A former being 100x more sensitive to (PP-2A) and type 1, the inhibition than the latter (3, 4). OA can enter intact cells and, adipocytes or hepatocytes, produces marked when added to hyperphosphorylation of a number of cellular proteins, and, subsequently, modulation of a variety of metabolic processes (3), in a manner consistent with its inhibitory action on phosphatases. The activity of the kinase(s) phosphorylating the ribosomal protein 56 is regulated by several cellular effecters and may represent a common component of a number of signalling pathways. Although the exact molecular mechanism by which S6 kinase activity Abbreviations used: OA, okadaic acid: NGF, nerve growth factor; EGF, epidermal growth factor: EGTA, ethylene glycol bis ( 0 NV-tetraacetic acid: SDS, sodium aminoethyl ether) N, N, N', dodecyl sulfate: PP-ZA, protein phosphatase DTT, type 2A; dithiothreitol; FPLC, fast protein liquid chromatography. 0006-291X/91 Copyright All rights

$1.50

0 1991 b! Academic Press. k. of reproduction in arty j?wm reserved.

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is regulated is unknown, a number of studies indicate that one or more phosphorylations may be involved. Our previous work (5, 6) that has shown nerve growth factor increases the (NGF) phosphorylation of S6 in PC12 cells by increasing the activity of a specific S6 kinase that has properties different than those of the mitogen-stimulated S6 kinases from other cells, and of the EGFstimulated S6 kinase from PC12. This work has also shown (5) that the action of NGF on S6 kinase can be reversed by treatment of the partially purified enzyme with alkaline phosphatase. Therefore, it was of interest to determine if the phosphatase inhibitor, okadaic *acid (OA), could modulate the action of NGF on this enzyme. In the present study, we have found that treatment of PC12 cells with OA increases the activity of the S6 kinase that is also activated by treatment of the cells with NGF. MATERIALS AND METHODS: Okadaic acid was purchased from Wako PC12 cells were grown and maintained as previously Chemical Co. Unless otherwise indicated, the cells were treated described (6) in the culture flasks at 37'C with NGF (50 rig/ml) for 30 minutes, or OA (500 nM) for 60 minutes. EGF (30 rig/ml) for 20 minutes, After treatment, the cells were collected and washed by centrifugation (1,100 x g, 5 minutes) and resuspended in 500 pl of lysis buffer (10 mM Tris-HCl, pH 7.2, containing 150 mM NaCl, 2 mM EGTA, 1 mM Na,V04, 0.1 mM Na,MoO,, 1mM phenylmethylsulfonylfluoride, 2 mM dithiothreitol (DTT), 1% Triton X-100, lO,ug/ml leupeptin, and 10 Ng/ml aprotinin). The resuspended cells were dispersed using a Vortex, kept on ice for 15 minutes, and the lysate centrifuged (14,000 x g, 5 minutes). The supernatant fraction was assayed for S6 kinase activity. S6 kinase activity was assayed using purified 40s ribosomal subunits from rat liver as a substrate, as described previously samples were analyzed by SDS-polyacrylamide gel The (6). electrophoresis on 12% gels according to the method of Laemmli (7). The protein bands corresponding to S6 were excised and the amount of radioactivity incorporated into the protein determined by Cerenkov counting. The Heparin-Sepharose chromatography was carried out at 4'C. cell-free extracts from control cells and from treated cells were diluted with equal volumes of buffer A (20 mM Tris-HCl, pH 6.8, 1 mM EGTA, 2 mM EDTA, 10 mM MgCl,, 2 mM DTT, 0.1% Triton X-100, 0.1 mM phenylmethylsulfonylfluoride, 10% glycerol, 1 mM benzamidineHCl, and O.lpg/ml leupeptin) and filtered through Millex-GS (0.22 pm Millipore filters). The samples were then loaded directly onto 2 ml columns of heparin-Sepharose CL-6B equilibrated with buffer A. The column was washed with buffer A and the proteins eluted with a linear gradient of NaCl from 0 to 0.75 M in buffer A at a flow rate of 0.5 ml/min by fast protein liquid chromatography (FPLC). Both Mono-S and Mono-Q column chromatography were performed by the same general procedure except that the elution was done with a linear gradient of 0 to 0.5 M NaCl. Fractions from were assayed immediately for S6 kinase each chromatography activity. 625

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RESULTS AND DISCUSSION: Treatment of PC12 cells with OA produced a substantial increase in S6 kinase activity. This increase was dose-dependent (Fig. 1, left), with half-maximal stimulation at about 0.4 uM and maximal stimulation above ~JJM. The increase was also time-dependent (Fig. 1, right), first appearing after a 30 minute lag period, reaching a maximum after approximately 1 hour, and staying at this plateau for at least another hour. In order to determine which of the S6 kinases known to be present in PC12 cells was stimulated, the characteristics of the stimulated enzyme were examined. Since previous work (5) has shown that p -glycerophosphate is a potent inhibitor of the NGFstimulated 56 kinase, the effect of/3 -glycerophosphate on the OAstimulated activity was examined. It was found that the addition of /3-glycerophosphate to cell-free preparations from OA-treated PC12 cells inhibited the activity of the OA-stimulated 56 kinase in a dose-dependent manner, with complete inhibition occurring at /-glycerophosphate (data not shown). 100 mM To confirm that OA treatment results in a stimulation of the NGF-sensitive S6 kinase, the two 56 kinases known to be present in PC12 cells (6) were separated on heparin-Sepharose. The experiment revealed that the pattern of 56 kinase activity after stimulation of the cells with OA (Fig. 2, middle, filled circles) was the same as that after stimulation with NGF (Fig. 2, top, filled circles), and quite different than that seen after stimulation with epidermal growth factor (EGF; data not shown). Further, it could be seen that addition of p -glycerophosphate (80 mM) and EGTA (20 mM) to

30

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Figure

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1. Dose-dependency and time course of activation kinase activity in PC12 cells by okadaic acid. PC12 cells, grown in 70 cm2 culture flasks, incubated at 37’C for concentrations of okadaic grown in 70 cm2 culture

okadaic acid Extracts were as described

studies identical

1

hour acid. flasks,

the

we;e

incubated

PC12 cells.

(500 n&f) at 37°C for the indicated prepared and assayed for S6 kinase

in has been results.

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Right:

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Effect of fi -glycerophosphate and EGTA on the S6 kinase activities from PC12 cells separated on heparinSepharose. Cell-free extracts (5 mg of protein) from nerve growth factor-treated (NGF; 50 rig/ml, 30 minutes) or from okadaic acid-treated (OA; 500 nM, 60 minutes) or untreated (Control) PC12 cells were prepared as described in Materials and Methods in the absence (filled circles) or the presence (open circles) of P-glycerophosphate (80 mM) and EGTA (20 mM). The extracts were filtered through Millex-GS filters (22 pm; Millipore) and applied to heparin-Sepharose CL-6B columns (0.9 x 3.0 cm) equilibrated with buffer A. The column was washed with buffer A and eluted, at a flow rate of 0.5 ml/min, with a linear gradient of from 0 to 0.75 M NaCl in the same Solid line: NaCl concentration. buffer. Peaks I and II have previously been shown (6) to contain S6 kinase activities stimulated by NGF and EGF, respectively.

Figure

2.

Figure

3. Elution profile of 56 kinase activity from PC12 cells after analysis on Mono-S/FPLC cation exchange chromatography. Cell-free extracts (2.4 mg of protein) from cells treated with nerve growth factor (NGF; 50 30 minutes), okadaic acid (OA; 500 nM, 60 w/ml, minutes), or epidennal growth factor (EGF; 30 rig/ml, 20 minutes) were applied to a Mono-S cation exchange column. The column was eluted with a linear gradient of from 0 to 0.5 M NaCl. Column fractions (0.5 ml) were assayed The data shown are from a single for 56 kinase activity. experiment: the experiment was done three times with similar results.

lysis

buffer

used

for

the

preparation

of

OA-treated

cells

inhibited the activity in peak I (Fig. 2, middle, open circles), the NGF-sensitive kinase (6), just as it did in the lysis buffer used for cells treated with NGF (Fig. 2, top, open circles). 627

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A further confirmation of the action of OA on the NGFsensitive S6 kinase was obtained when lysates from cells treated with OA were analyzed by ion-exchange chromatography and compared with similar analyses of lysates from cells treated with either NGF or EGF. Lysates from cells treated with NGF, OA, or EGF were chromatographed on the cation-exchanger Mono-S (Fig. 3) and the fractions assayed for S6 kinase activity. The data show that the S6 kinase profile from lysates from cells treated with NGF and those treated with OA are quite similar, while those from cells treated with EGF are different. Comparable extracts analyzed on the anion-exchanger Mono-Q (data not presented) also showed that the profile from lysates from cells treated with NGF and those from cells treated with OA are similar, while those from cells treated with EGF are different. In each case the peak containing the activity stimulated by treatment with OA also contained the activity stimulated by NGF. In other experiments not presented here, the activity stimulated by OA was not inhibited by treatment with H-7, an inhibitor of protein kinase C, by W-7, a calmodulin antagonist, by sodium vanadate, an inhibitor of tyrosine kinases, or by the heatstable protein inhibitor of CAMP-dependent kinases. These data indicate, at least in a preliminary way, that the OA-sensitive S6 kinase is neither protein kinase C, protein kinase A, tyrosine kinase, nor a calcium/calmodulin protein kinase. Since similar results have been obtained in studies on the NGF-sensitive 56 kinase, these data are further support for the suggestion that the OA-sensitive kinase and the NGF-sensitive 56 kinase are the same. Thus, the data presented show that OA increases an S6 kinase that is inhibitable byp -glycerophosphate, as is the NGF-sensitive kinase, one that co-migrates with the NGF-sensitive S6 kinase on heparin-Sepharose, Mono-S, and Mono-Q chromatography, and one that is not inhibited by a number of inhibitors that also do not inhibit the NGF-sensitive S6 kinase. Clearly, the simplest interpretation of the data is that QA treatment stimulates the NGF-sensitive kinase. Haystead et al. (8) have reported that the treatment of adipocytes with OA resulted in a several-fold stimulation of 56 kinase. It is possible that OA is actually stimulating an enzyme further back in the cascade leading to the activation of the S6 kinase and not the 56 kinase itself. The activity of microtubuleassociated protein kinase in both PC12 cells and fibroblastic 3y1 cells is increased by treatment with OA (9, 10) and this enzyme 628

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has been implicated as an S6 kinase kinase (11). It is not likely, however, that OA, under the present conditions, is inhibiting the dephosphorylation of S6 itself, because OA is added to the intact cells and the 56 substrate, in the form of the 40s ribosomes from rat liver, is not added until after the cells are washed extensively, lysed, and the lysate diluted into the incubation mixture. Since OA is not known to have a direct effect on any protein kinase activity, its action in this system is most certainly due to an inhibition of endogenous protein phosphatases acting on the S6 kinase. This phosphatase is presumably PP-2A since the concentration of OA in the present experiments was 0.5-1-O NM, enough for complete inhibition of PP-2A, but not enough for complete inhibition of protein phosphatase 1 (3, 4). Thus, results presented in this report provide further evidence for the role of serine/threonine phosphorylation in the activation of S6 kinases, specifically the S6 kinase in PC12 cells sensitive to the differentiating agent NGF. Since OA greatly enhances the 56 kinase activity, over and above that seen with NGF alone, the data further suggest that it is PP-2A that limits the increase in activity in the NGF-treated cell and is responsible for the eventual return of S6 kinase activity to baseline. REFERENCES 1. 2.

3. 4. 5. 6. 7. 8. 9. 10. 11.

Tachibana, K., Scheuer, P.J., Tsukitani, Y., Kikuchi, H., van Engen, D., Clardy, J., Gopichand, Y., and Schmitz, F.J. (1981) J. Am. Chem. Sot. 103, 2469-2471. Suganuma, K., Fujiki,. H., Suguri, H., Yoshisawa, S., Hirota, M., Ojika, M., Wakamatsu, K., Yamaka, K., and M. t Nakayasu, Sugimura, T. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 176817781. Haystead, T.A.J., Sim, A.T.R., Carling, D., Honner, R-C., Tsukitani, Y., Cohen, P., and Hardie, D.G. (1989) Nature 337, 78-81. Bialojan, C. and Takai, A. (1988) Biochem. J. 256, 283-290. Matsuda, Y. and Guroff, G. (1987) J. Biol. Chem. 262, 28322844. Mutoh, T., Rudkin, B.B., Koizumi, S., and Guroff, G. (1988) J. I3iol. Chem. 263, 15853-15856. Laemmli, U.K. (1970) Nature 227, 680-685. Haystead, T.A.J., Weiel, J.E., Litchfield, D.W., Tsukitani, Y ., Fischer, E.H., and Krebs, E.G. (1990) J. Biol. Chem. 265, 16571-16580. Miyasaka, T., Miyasaka, J., and Saltiel, A.R. (1990) Biochem. Biophys. Res. Commun. 168, 1237-1243. Gotoh, Y., Nishida, E., and Sakai, H. (1990) Eur. J. Biochem. 193,. 671-674. Sturgill, T.W., Ray, L.B., Erikson, E., and Maller, J.L. (1988) Nature 334, 715-718. 629

Okadaic acid stimulates the activity of the nerve growth factor-sensitive S6 kinase of PC12 cells.

PC12 pheochromocytoma cells contain at least two different and separable kinases that phosphorylate the S6 protein of the ribosomes. The activity of o...
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