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Insulin Stimulates p70 S6 Kinase in the Nucleus of Cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
234, 681–685 (1997)
RC976699
Insulin Stimulates p70 S6 Kinase in the Nucleus of Cells Sung-Jin Kim1 and C. Ronald Kahn2 Research Division, Joslin Diabetes Center, Boston, Massachusetts, and Department of Medicine, Harvard Medical School, Boston, Massachusetts
Received April 25, 1997
Insulin action on both cytoplasmic and nuclear processes is dependent on activation of p70 S6 kinase (p70S6K). In CHO cells expressing human insulin receptors, Western blotting revealed the presence of p70S6K in the cell nucleus at a level of about 32 % of that in the cytoplasm. Following insulin treatment, there was a retardation in mobility nuclear p70S6K in SDS-PAGE indicative of a change in phosphorylation of the enzyme, but no change in the amount of enzyme. Stimulation was maximal after 10 min of insulin treatment and decreased gradually at 30 min. There was also a rapid doubling of nuclear p70S6K activity in immunocomplex assays followed by a return to baseline by 30 min. Simultaneously, insulin stimulated cytoplasmic p70S6K by almost 10-fold at 10 min, and activity remained high up to 30 min. Tetradecanoylphorbol acetate (TPA) and fetal calf serum also stimulated nuclear p70S6K as judged by gel mobility shift. TPA also promoted a decrease in cytosolic p70S6K and an increase in nuclear enzyme suggestive of translocation of the enzyme. Rapamycin, a selective inhibitor of p70S6K, and the casein kinase II inhibitor DRB blocked insulin-stimulated nuclear and cytosolic p70S6K. Thus, nuclear p70S6K is regulated by insulin, serum and TPA. The insulin effect is downstream of rapamycin and DRB-sensitive targets and occurs without translocation of the enzyme. q 1997 Academic Press
The insulin signal transduction is a complex network which results in stimulation of the inuslin receptor tyrosine kinase and activation of at least two major intracellular systems (1). One pathway involves Ras, Raf, MEK, MAP kinase and the 90 kDa S6 kinase rsk, whereas the second involves phosphatidlylinositol 31 Current address: Department of Pharmacology, School of Dentistry and Institute of Oral Biology, Kyung-Hee University, Seoul, Korea. 2 To whom correspondence should be addressed. Research Division, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. Fax: (617) 732-2593. E-mail: [email protected].
kinase (PI 3-kinase) and p70 S6 kinase (p70S6K) (1,2). While the roles of MAP kinase and rsk in both cytoplasmic and nuclear insulin signaling have been extensively studied (3,4), the role of p70S6K in insulin signaling is less clear. p70S6K is a serine/threonine kinase capable of phosphorylating ribosomal protein S6. The enzyme occurs in two isoforms derived from a common gene by differential splicing: one has an Mr Å 70,000 and is termed p70S6K, and the other has an Mr Å 85,000 and has been termed p85S6K (5,6). The molecular sequence of these isozymes is same except the p85 S6 kinase contains a 23 amino acid extension at its Nterminus which has been suggested to enhance nuclear localization (6). Activation of p70/ p85S6K requires multiple serine/threonine phosphorylations on an auto-inhibitory pseudosubstrate domain, however, the protein kinase(s) involved in this phosphorylation and activation remain unknown (7). The immunosuppressant rapamycin selectively inhibits p70S6K activation without inhibiting the MAP kinases or p90 rsk (8). Highly specific PI 3-kinase inhibitors, such as wortmannin or LY294002, on the other hand, block activation of p70S6K suggesting that p70S6K lies downstream of PI 3-kinase (9,10). p70S6K has recently been shown to complex with and be activated by the Rho family of GTPases, including Cdc42 and Rac1 (11). Previous studies have shown that in some cells following stimulation by insulin and several growth factors, p90 rsk and the MAP kinases are translocated to the nucleus where they may participate in phosphorylation of transcription factors and other proteins (12). In some cells, insulin is also capable of activating these enzymes in the nucleus without apparent translocation1. p70S6K is located in the nucleus, as well as in cytosol (13,14), and has been shown to play a role in hormonal regulation of glycogen synthase (15,16), protein synthesis (17), and gene expression (18,19). In the present study, we have explored the possibility of insulin-dependent p70S6K signaling in the nucleus of CHO cells expressing the human insulin receptor. We find that nuclear p70S6K is rapidly activated by insulin and
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that this occurs without apparent translocation of the enzyme via rapamycin and DRB-sensitive pathways. EXPERIMENTAL PROCEDURES Materials. Pork insulin was obtained from Elanco Products Co. (Indianapolis, IN). The reagents used for polyacrylamide gel electrophoresis were from Bio-Rad. Nitrocelluose filters (0.45 mm) were purchased from Schleicher & Schuell. Antibody to p70S6K was kindly provided by Dr. John Blenis, Harvard Medical School, Boston, MA. Ribosomal 40S subunits were purified from rat liver as described previously (20). All other chemicals were purchased from Sigma. Cell culture and isolation of nuclei. CHO cells expressing the human insulin receptor [CHO(Hirc)] were grown in F-12 medium with 10% fetal calf serum in humidified atmosphere of 5% CO2 at 377 C. Prior to experiments, insulin was removed from the culture medium for 12 hrs. Nuclei were isolated as previously described (21,22). Cells were serum starved for 18 hrs and stimulated with 100 nM insulin, 100 nM TPA or 10% fetal calf serum for the indicated times. Media was removed, and cultures were washed with ice-cold phosphate buffered saline. The cells were scraped into a buffer containing 20 mM Hepes, 50 mM MgCl2 , 25 mM KCl, 2 mM PMSF, 0.1 mg/ml aprotinin, 1 mM sodium vanadate, 1 mM sodium molybdate, 10 mM b-glycerophosphate, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA and 0.25 M sucrose. Cells were disrupted by passage through a 1 ml tuberculin syringe with a microfine IV needle five times. The cell homogenates were transferred to thick-walled polyallomer tubes and centrifuged at 1,000 1 g for 10 min at 47 C. The pellets were resuspended in 1 ml of isolation buffer, and 2 ml of isolation buffer supplemented with 1.6M sucrose was layered below. After centrifugation at 100,000 1 g for 35 min at 47C, the resulting pellets were subjected to a brief sonication at 47 C to disrupt the nuclei and then stored at 0707C. Measurements of lactate dehydrogenase, a cytosolic marker enzyme, and the content of DNA in the nucleus indicted that the nuclear preparation was more than 96% pure (data not shown). Western blot analysis. Electrotransfer of proteins from the gels to nitrocelluose paper (Schleicher & Schuell) was carried out for 1 hr at 100 V (constant) as described by Towbin et al (23). The filter papers were preincubated for 1 hr at 237C with PBS containing 0.1% Tween 20 and 3% bovine serum albumin, and washed with PBS containing 0.1% Tween 20 three times for 10 min each. The blots were probed with primary antibodies for 1 hr at 237C. The blots were then incubated with HRP-conjugated anti-rabbit IgG for 30 min and washed with PBS containing Tween 20 five times for 10 min each. The detection of immobilized specific antigens was carried out by enhanced chemiluminescence (ECL, New England Nuclear). Immune-complex kinase assay. Equal amounts of nuclear and postnuclear preparation were incubated with anti-p70S6K antibody for 1 hr at 47C. Pansorbin was added to the mixture and incubated for 30 min at 47C, following which the samples were subjected to centrifugation to isolate the immunocomplexes. The complexes were used in an in vitro kinase assay with ribosomal S6 as a substrate as previously described (24). The kinase assay mixtures were subjected to SDS-PAGE, and the phosphorylated ribosomal S6 was identified by autoradiography. The kinase activity, as reflected by the level of phosphorylation of S6 protein, was quantitated by scanning densitometry.
RESULTS Stimulation of Nuclear p70S6K Activity by Insulin Following stimulation with insulin, tetradecanocylphorbol acetate (TPA) or fetal calf serum, CHO(Hirc)
FIG. 1. Gel retardation analysis of p70S6K by insulin. Following treatment with 100 nM insulin, 100 nM TPA or 10% FCS for the indicated times, CHO (Hirc) cells were subjected to subcellular fractionation as described under Experimental Procedures. Equal amounts (10 mg) of nuclear and postnuclear fractions were applied to 7.5% SDS-polyacrylamide gels, followed by electrophoresis and transfer to nitrocellulose membranes. The membranes were probed with antibody against p70S6K and detected using a ECL system according to a protocol provided by the manufacturer (ICN). Fig. 1A is representative of two separate experiments. Relative migration distances and percent maximal shifts of p70S6K in nuclear and postnuclear fraction were expressed as a function of insulin treatment time, and are presented in Figs. 1B and 1C as the mean of two separate experiments.
cells were subjected to subcellular fractionation, and p70S6K phosphorylation was studied by gel mobility shifts on SDS-polyacrylamide gel. Activation of p70S6K requires phosphorylation of the enzyme resulting in a slower migration on SDS-polyacrylamide gel (25). Under basal conditions, both the nuclear and the postnuclear (cytosolic) prepartion contained a significant amount of p70S6K (Fig. 1A). With equal amounts of protein loaded, the relative concentration of p70S6K in the nucleus was 32 % of that in the cytosol. Treatment of
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cells with 100 nM insulin resulted in a mobility shift of the p70S6K present in both the nuclear and cytosolic (postnuclear) fractions (Fig. 1A). There was no apparent translocation of the enzyme as judged by a lack of change in the protein levels of the p70S6K in these fractions. Quantitation of the gel shift by scanning densitometry revealed that phosphorylation of cytosolic p70S6K rose dramatically by 10 min and increased slightly further at 30 min. In contrast, nuclear p70S6K was maximally shifted within 10 min of insulin treatment, after which there was a slow decrease by 30 min to 60% of the maximal mobility shift (Figs. 1B and 1C). Maximal phosphorylation of cytosolic p70S6K appeared to be higher than that of nuclear p70S6K as evidenced by the greater degree of retardation on SDS-PAGE (Fig. 1A; also see Fig. 3). TPA and fetal calf serum also stimulated a mobility shift in nuclear p70S6K of about equal magnitude to that produced by insulin. In addition, with TPA the level of cytosolic p70S6K protein appeared to be reduced, and this was associated with an increased signal in the nuclear fraction. This suggests that TPA, but not insulin or serum, causes a translocation of the enzyme from cytosol to nucleus. In all cases, p85S6K was also observed as a very minor band in the nucleus which showed similar mobility shifts to the more prominent p70S6K band. To confirm the activation of nuclear p70S6K by insulin, direct immunocomplex kinase assays were em-
FIG. 3. Comparison of the phosphorylation status of insulinstimulated p70S6K from nuclear and postnuclear fractions. Following preincubation with or without rapamycin (20 ng/ml) for 5 min, CHO (Hirc) cells were stimulated with insulin (100 nM) for 10 min after which nuclear and postnuclear fractions were isolated. Equal amounts (10 mg) of each fraction were subjected to electrophoresis, and followed by Western blot analysis using antibody against p70S6K. p70S6K was detected with ECL.
ployed. Insulin stimulated cytosolic p70S6K activities by 10-fold in 10 min, and this was followed by a slight decrease to 8-fold stimulation at 30 min (Fig. 2, top). In contrast, nuclear p70S6K activity was stimulated by only 1.8-fold over basal by insulin, however, activation was very rapid, reaching its peak by 5 min (Fig. 2, bottom). Activation of nuclear p70S6K was also transient decreasing back to basal levels within 30 min. Thus, both the magnitude and the kinetics of activation of nuclear p70S6K by insulin were different from that of cytosolic p70S6K. Inhibition of the Nuclear p70S6K Activity by Rapamycin and DRB
FIG. 2. Stimulation of p70S6K activity by insulin. Following insulin treatment (100 nM) for the indicated times, nuclei and postnuclear supernatant were isolated. The fractions were subjected to immunoprecipitation with anti- p70S6K antibody. p70S6K assays were carried out with the immune-complex assay using ribosomal S6 as a substrate. The reaction mixture was subjected to SDS-PAGE followed by autoradiography. The kinase activities have been quantitated by scanning densitometry of the S6 protein.
Rapamycin is a specific inhibitor of p70S6K activation which acts on an upstream protein (target of rapamycin) involved in activation of cytosolic S6 kinase (26,27). To explore the mechanism of activation of nuclear p70S6K, cells were pre-incubated with rapamycin prior to insulin stimulation, and then subjected to subcellular fractionation and SDS-PAGE. Rapamycin pretreatment almost completely blocked the insulin stimulated mobility shift of p70S6K in both nuclear and postnuclear fractions, indicating that the target of rapamycin was involved in both processes (Fig. 3). Since casein kinase II phosphorylates p70S6K in vitro (28), we also explored the possibility that this enzyme was involved in the activation of p70S6K by insulin using the casein kinase II inhibitor 5,6-dichlorobenzimidazole riboside (DRB) (29) (Fig. 4). DRB pretreatment partially blocked the insulin stimulated mobility shift of p70S6K in both nuclear and cytosolic fractions, suggesting that a casein kinase II-dependent pathway may participate in regulation of p70S6K signaling. However, under the conditions used, this inhibitory effect was not as great as that of rapamycin.
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FIG. 4. DRB and rapamycin inhibit insulin-stimulated p70S6K in CHO(Hirc) cells. Following preincubation with or without rapamycin (20 ng/ml) for 5 min or 5,6-dichlorobenziimidazole riboside (DRB, 1 mM) for 10 min, CHO(Hirc) cells were stimulated with 100 nM insulin for indicated times, following which nuclear and postnuclear fractions were isolated. Equal amounts (10 mg) of each fraction were subjected to electrophoresis, followed by Western blot analysis using antibody against p70S6K and detected with ECL.
DISCUSSION Insulin actions are mediated by multiple protein kinases including raf kinase, MEK, MAP kinases, p90 rsk, p70S6K and PI 3-kinase. It has been suggested that p70S6K plays an essential role in the response of many cells to a variety of growth factors and insulin. In the case of insulin, p70S6K has been shown to be involved in regulation of protein synthesis (2,6), glycogen synthesis (15,16), mitogenesis (6) and GLUT1 and hexokinase II gene expression (18,19). p70S6K has also been shown to play a role in regulation of the cAMP-responsive activator CREM (14). In this communication, we have shown that p70S6K is present in the nucleus, that the enzyme in the nucleus is rapidly activated by insulin, and that the nuclear p70S6K appears to have some differences in regulation, such as time course, from the cytosolic p70S6K. To begin, a significant amount of p70S6K is present in the nucleus of unstimulated CHO (Hirc) cells. This is similar to our findings that in CHO and other cell types, even in the basal state, several serine kinases, including MEK, casein kinase II, and the MAP kinases ERK I and ERK II are present in the nucleus at significant concentrations (30). The method employed in the isolation of nuclei in the present and previous studies is widely used by many investigators, and the purity of the nuclear preparation is greater than 96% as evidenced by cytosolic marker enzyme assays (data not shown). Thus, contamination of cytosolic p70S6K in the nuclear preparation could not account for the presence of the enzyme. Interestingly, in CHO cells we find that it is the p70S6K, rather than the p85S6K, which is prominent in the nucleus despite the absence of a known nuclear localization signal in the p70 form of the protein (28). Activation of nuclear p70S6K by insulin is rapid, but the extent of stimulation is lower than cytosolic p70S6K.
Activation of the nuclear enzyme is also more transient, suggesting differences in the mechanism of activation of nuclear and cytosolic p70S6K. Nuclear p70S6K is also activated by insulin in differentiated 3T3-F442A cells with kinetics similar to that of CHO cells (data not shown). Stimulation of cells by phorbol esters and fetal calf serum causes retardation of nuclear p70S6K on SDS-PAGE indicative of increased phosphorylation and stimulation of p70S6K by these factors as well. TPA also caused an apparant translocation of the enzyme with a decrease in level of protein in the cytoplasm and an increase in p70S6K in the nucleus. The differences in molecular retardation of cytosolic and nuclear p70S6K by TPA and fetal calf serum support the notion that the changes observed in the nuclear preparation are not simply due to contamination by cytosol. Both nuclear and cytosolic p70S6K were inhibited by rapamycin, suggesting the rapamycin-sensitive target lies upstream of divergence to nuclear and cytosolic p70S6K signaling pathway. The finding that DRB inhibits nuclear and cytosolic p70S6K supports the hypothesis that casein kinase II may also lie upstream of p70S6K activation and may play an important role in the regulation of p70S6K in CHO (Hirc) cells. However, it remains to be determined whether the rapamycin-sensitive target lies upstream or downstream of DRB-regulated target in the insulin-regulated p70S6K signaling pathway. Considering the recent observation that casein kinase II phosphorylates p70S6K in vitro (28), and our previous observation that insulin stimulates nuclear casein kinase II activity, it is possible that casein kinase II may directly phospholylate and activate cellular p70S6K in vivo. Other approaches will be necessary to understand the role of casein kinase II in the regulation of p70S6K. In summary, insulin rapidly phosphorylates and activates nuclear p70S6K. The activation of the nuclear p70S6K in response to insulin occurs faster than the activation of cytosolic p70S6K, although the extent of phosphorylation of nuclear p70S6K is lower than that of cytosolic p70S6K. Both nuclear and cytosolic p70S6K are inhibited to the almost same extent by rapamycin and DRB. The rapid activation of nuclear p70S6K in response to insulin may serve as a molecular switch to turn on nuclear insulin-sensitive targets, such as transcription factors, DNA binding proteins and enzymes involved in gene expression.
ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health, DK 33201 and by the NIH-sponsored Diabetes and Endocrinology Research Center, P30 DK36836. SJK acknowledges support from Kyung-Hee University. The authors thank Dr. John Blenis for providing the anti-p70S6K antibody.
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REFERENCES 1. Cheatham, B., and Kahn, C. R. (1995) Endocr. Rev. 16, 117– 142. 2. Weng, Q. P., Andrabi, K., Kozlowski, M. T., Grove, J. R., and Avruch, J. (1995) Mol. Cell Biol. 15, 2333–2340. 3. Sturgill, T. W., Ray, L. B., Erikson, E., and Maller, J. L. (1988) Nature 334, 715–718. 4. Chung, J., Chen, R. H., and Blenis, J. (1991) Mol. Cell Biol. 11, 1868–1874. 5. Reinhard, C., Thomas, G., and Kozma, S. C. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4052–4056. 6. Thomas, G. (1993) Biochem. Soc. Trans. 21, 901–904. 7. Ferrari, S., Bannwarth, W., Morley, S. J., Totty, N. F., and Thomas, G. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7282–7286. 8. Kozma, S. C., McGlynn, E., Siegmann, M., Reinhard, C., Ferrari, S., and Thomas, G. (1993) J. Biol. Chem. 268, 7134–7138. 9. Okada, T., Sakuma, L., Fukui, Y., Hazeki, O., and Ui, M. (1994) J. Biochem. 269(5), 3563–3567. 10. Vlahos, C. J., Matter, W. F., Hui, K. Y., and Brown, R. F. (1994) J. Biochem. 269(7), 5241–5248. 11. Chou, M. M., and Blenis, J. (1996) Cell 85, 573–583. 12. Chen, R., Sarnecki, C., and Blenis, J. (1992) Mol. Cell Biol. 12, 915–927. 13. Reinhard, C., Fernandez, A., Lamb, N. J., and Thomas, G. (1994) EMBO J. 13, 1557–1565. 14. de Groot, R. P., Ballou, L. M., and Sassone-Corsi, P. (1994) Cell 78, 81–91.
15. Azpiazu, I., Saltiel, A. R., DePaoli Roach, A. A., and Lawrence, J. C., Jr. (1996) J. Biol. Chem. 271, 5033–5039. 16. Shepherd, P. R., Nave, B. T., and Siddle, K. (1995) Biochem. J. 305, 25–28. 17. Sadoshima, J., and Izumo, S. (1995) Circ. Res. 77, 1040–1052. 18. Osawa, H., Sutherland, C., Robey, R. B., Printz, R. L., and Granner, D. K. (1996) J. Biol. Chem. 271, 16690–16694. 19. Taha, C., Mitsumoto, Y., Liu, Z., Skolnik, E. Y., and Klip, A. (1995) J. Biol. Chem. 270, 24678–24681. 20. Terao, K., and Ogata, K. (1970) Biochem. Biophys. Res. Commun. 38, 80–85. 21. Lee, K. A., Bindereif, A., and Green, M. R. (1988) Gene. Anal. Tech. 5, 22–31. 22. Blobel, G., and Potter, V. R. (1966) Science 154, 1662–1665. 23. Towbin, H., Staehlin, J., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4350–4354. 24. Chen, R. H., and Blenis, J. (1990) Mol. Cell Biol. 10, 3204–3215. 25. Monfar, M., Lemon, K. P., Grammer, T. C., Cheatham, L., Chung, J., Vlahos, C. J., and Blenis, J. (1995) Mol. Cell Biol. 15, 326–337. 26. Kuo, C. J., Chung, J., Florentino, D. F., Flanagan, W. M., Blenis, J., and Crabtree, G. R. (1992) Nature 358, 70–73. 27. Chung, J., Kuo, C. J., Crabtree, G. R., and Blenis, J. (1992) Cell 69, 1227–1236. 28. Coffer, P. J., and Woodgett, J. R. (1994) Biochem. Biophys. Res. Commun. 198, 780–786. 29. Pinna, L. A. (1990) Biochim. Biophys. Acta 1054, 267–284. 30. Kim, S. J., and Kahn, C. R. (1997) Biochem. J. 323, 621–627.
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234, 681–685 (1997)
RC976699
Insulin Stimulates p70 S6 Kinase in the Nucleus of Cells Sung-Jin Kim1 and C. Ronald Kahn2 Research Division, Joslin Diabetes Center, Boston, Massachusetts, and Department of Medicine, Harvard Medical School, Boston, Massachusetts
Received April 25, 1997
Insulin action on both cytoplasmic and nuclear processes is dependent on activation of p70 S6 kinase (p70S6K). In CHO cells expressing human insulin receptors, Western blotting revealed the presence of p70S6K in the cell nucleus at a level of about 32 % of that in the cytoplasm. Following insulin treatment, there was a retardation in mobility nuclear p70S6K in SDS-PAGE indicative of a change in phosphorylation of the enzyme, but no change in the amount of enzyme. Stimulation was maximal after 10 min of insulin treatment and decreased gradually at 30 min. There was also a rapid doubling of nuclear p70S6K activity in immunocomplex assays followed by a return to baseline by 30 min. Simultaneously, insulin stimulated cytoplasmic p70S6K by almost 10-fold at 10 min, and activity remained high up to 30 min. Tetradecanoylphorbol acetate (TPA) and fetal calf serum also stimulated nuclear p70S6K as judged by gel mobility shift. TPA also promoted a decrease in cytosolic p70S6K and an increase in nuclear enzyme suggestive of translocation of the enzyme. Rapamycin, a selective inhibitor of p70S6K, and the casein kinase II inhibitor DRB blocked insulin-stimulated nuclear and cytosolic p70S6K. Thus, nuclear p70S6K is regulated by insulin, serum and TPA. The insulin effect is downstream of rapamycin and DRB-sensitive targets and occurs without translocation of the enzyme. q 1997 Academic Press
The insulin signal transduction is a complex network which results in stimulation of the inuslin receptor tyrosine kinase and activation of at least two major intracellular systems (1). One pathway involves Ras, Raf, MEK, MAP kinase and the 90 kDa S6 kinase rsk, whereas the second involves phosphatidlylinositol 31 Current address: Department of Pharmacology, School of Dentistry and Institute of Oral Biology, Kyung-Hee University, Seoul, Korea. 2 To whom correspondence should be addressed. Research Division, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. Fax: (617) 732-2593. E-mail: [email protected].
kinase (PI 3-kinase) and p70 S6 kinase (p70S6K) (1,2). While the roles of MAP kinase and rsk in both cytoplasmic and nuclear insulin signaling have been extensively studied (3,4), the role of p70S6K in insulin signaling is less clear. p70S6K is a serine/threonine kinase capable of phosphorylating ribosomal protein S6. The enzyme occurs in two isoforms derived from a common gene by differential splicing: one has an Mr Å 70,000 and is termed p70S6K, and the other has an Mr Å 85,000 and has been termed p85S6K (5,6). The molecular sequence of these isozymes is same except the p85 S6 kinase contains a 23 amino acid extension at its Nterminus which has been suggested to enhance nuclear localization (6). Activation of p70/ p85S6K requires multiple serine/threonine phosphorylations on an auto-inhibitory pseudosubstrate domain, however, the protein kinase(s) involved in this phosphorylation and activation remain unknown (7). The immunosuppressant rapamycin selectively inhibits p70S6K activation without inhibiting the MAP kinases or p90 rsk (8). Highly specific PI 3-kinase inhibitors, such as wortmannin or LY294002, on the other hand, block activation of p70S6K suggesting that p70S6K lies downstream of PI 3-kinase (9,10). p70S6K has recently been shown to complex with and be activated by the Rho family of GTPases, including Cdc42 and Rac1 (11). Previous studies have shown that in some cells following stimulation by insulin and several growth factors, p90 rsk and the MAP kinases are translocated to the nucleus where they may participate in phosphorylation of transcription factors and other proteins (12). In some cells, insulin is also capable of activating these enzymes in the nucleus without apparent translocation1. p70S6K is located in the nucleus, as well as in cytosol (13,14), and has been shown to play a role in hormonal regulation of glycogen synthase (15,16), protein synthesis (17), and gene expression (18,19). In the present study, we have explored the possibility of insulin-dependent p70S6K signaling in the nucleus of CHO cells expressing the human insulin receptor. We find that nuclear p70S6K is rapidly activated by insulin and
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that this occurs without apparent translocation of the enzyme via rapamycin and DRB-sensitive pathways. EXPERIMENTAL PROCEDURES Materials. Pork insulin was obtained from Elanco Products Co. (Indianapolis, IN). The reagents used for polyacrylamide gel electrophoresis were from Bio-Rad. Nitrocelluose filters (0.45 mm) were purchased from Schleicher & Schuell. Antibody to p70S6K was kindly provided by Dr. John Blenis, Harvard Medical School, Boston, MA. Ribosomal 40S subunits were purified from rat liver as described previously (20). All other chemicals were purchased from Sigma. Cell culture and isolation of nuclei. CHO cells expressing the human insulin receptor [CHO(Hirc)] were grown in F-12 medium with 10% fetal calf serum in humidified atmosphere of 5% CO2 at 377 C. Prior to experiments, insulin was removed from the culture medium for 12 hrs. Nuclei were isolated as previously described (21,22). Cells were serum starved for 18 hrs and stimulated with 100 nM insulin, 100 nM TPA or 10% fetal calf serum for the indicated times. Media was removed, and cultures were washed with ice-cold phosphate buffered saline. The cells were scraped into a buffer containing 20 mM Hepes, 50 mM MgCl2 , 25 mM KCl, 2 mM PMSF, 0.1 mg/ml aprotinin, 1 mM sodium vanadate, 1 mM sodium molybdate, 10 mM b-glycerophosphate, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA and 0.25 M sucrose. Cells were disrupted by passage through a 1 ml tuberculin syringe with a microfine IV needle five times. The cell homogenates were transferred to thick-walled polyallomer tubes and centrifuged at 1,000 1 g for 10 min at 47 C. The pellets were resuspended in 1 ml of isolation buffer, and 2 ml of isolation buffer supplemented with 1.6M sucrose was layered below. After centrifugation at 100,000 1 g for 35 min at 47C, the resulting pellets were subjected to a brief sonication at 47 C to disrupt the nuclei and then stored at 0707C. Measurements of lactate dehydrogenase, a cytosolic marker enzyme, and the content of DNA in the nucleus indicted that the nuclear preparation was more than 96% pure (data not shown). Western blot analysis. Electrotransfer of proteins from the gels to nitrocelluose paper (Schleicher & Schuell) was carried out for 1 hr at 100 V (constant) as described by Towbin et al (23). The filter papers were preincubated for 1 hr at 237C with PBS containing 0.1% Tween 20 and 3% bovine serum albumin, and washed with PBS containing 0.1% Tween 20 three times for 10 min each. The blots were probed with primary antibodies for 1 hr at 237C. The blots were then incubated with HRP-conjugated anti-rabbit IgG for 30 min and washed with PBS containing Tween 20 five times for 10 min each. The detection of immobilized specific antigens was carried out by enhanced chemiluminescence (ECL, New England Nuclear). Immune-complex kinase assay. Equal amounts of nuclear and postnuclear preparation were incubated with anti-p70S6K antibody for 1 hr at 47C. Pansorbin was added to the mixture and incubated for 30 min at 47C, following which the samples were subjected to centrifugation to isolate the immunocomplexes. The complexes were used in an in vitro kinase assay with ribosomal S6 as a substrate as previously described (24). The kinase assay mixtures were subjected to SDS-PAGE, and the phosphorylated ribosomal S6 was identified by autoradiography. The kinase activity, as reflected by the level of phosphorylation of S6 protein, was quantitated by scanning densitometry.
RESULTS Stimulation of Nuclear p70S6K Activity by Insulin Following stimulation with insulin, tetradecanocylphorbol acetate (TPA) or fetal calf serum, CHO(Hirc)
FIG. 1. Gel retardation analysis of p70S6K by insulin. Following treatment with 100 nM insulin, 100 nM TPA or 10% FCS for the indicated times, CHO (Hirc) cells were subjected to subcellular fractionation as described under Experimental Procedures. Equal amounts (10 mg) of nuclear and postnuclear fractions were applied to 7.5% SDS-polyacrylamide gels, followed by electrophoresis and transfer to nitrocellulose membranes. The membranes were probed with antibody against p70S6K and detected using a ECL system according to a protocol provided by the manufacturer (ICN). Fig. 1A is representative of two separate experiments. Relative migration distances and percent maximal shifts of p70S6K in nuclear and postnuclear fraction were expressed as a function of insulin treatment time, and are presented in Figs. 1B and 1C as the mean of two separate experiments.
cells were subjected to subcellular fractionation, and p70S6K phosphorylation was studied by gel mobility shifts on SDS-polyacrylamide gel. Activation of p70S6K requires phosphorylation of the enzyme resulting in a slower migration on SDS-polyacrylamide gel (25). Under basal conditions, both the nuclear and the postnuclear (cytosolic) prepartion contained a significant amount of p70S6K (Fig. 1A). With equal amounts of protein loaded, the relative concentration of p70S6K in the nucleus was 32 % of that in the cytosol. Treatment of
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cells with 100 nM insulin resulted in a mobility shift of the p70S6K present in both the nuclear and cytosolic (postnuclear) fractions (Fig. 1A). There was no apparent translocation of the enzyme as judged by a lack of change in the protein levels of the p70S6K in these fractions. Quantitation of the gel shift by scanning densitometry revealed that phosphorylation of cytosolic p70S6K rose dramatically by 10 min and increased slightly further at 30 min. In contrast, nuclear p70S6K was maximally shifted within 10 min of insulin treatment, after which there was a slow decrease by 30 min to 60% of the maximal mobility shift (Figs. 1B and 1C). Maximal phosphorylation of cytosolic p70S6K appeared to be higher than that of nuclear p70S6K as evidenced by the greater degree of retardation on SDS-PAGE (Fig. 1A; also see Fig. 3). TPA and fetal calf serum also stimulated a mobility shift in nuclear p70S6K of about equal magnitude to that produced by insulin. In addition, with TPA the level of cytosolic p70S6K protein appeared to be reduced, and this was associated with an increased signal in the nuclear fraction. This suggests that TPA, but not insulin or serum, causes a translocation of the enzyme from cytosol to nucleus. In all cases, p85S6K was also observed as a very minor band in the nucleus which showed similar mobility shifts to the more prominent p70S6K band. To confirm the activation of nuclear p70S6K by insulin, direct immunocomplex kinase assays were em-
FIG. 3. Comparison of the phosphorylation status of insulinstimulated p70S6K from nuclear and postnuclear fractions. Following preincubation with or without rapamycin (20 ng/ml) for 5 min, CHO (Hirc) cells were stimulated with insulin (100 nM) for 10 min after which nuclear and postnuclear fractions were isolated. Equal amounts (10 mg) of each fraction were subjected to electrophoresis, and followed by Western blot analysis using antibody against p70S6K. p70S6K was detected with ECL.
ployed. Insulin stimulated cytosolic p70S6K activities by 10-fold in 10 min, and this was followed by a slight decrease to 8-fold stimulation at 30 min (Fig. 2, top). In contrast, nuclear p70S6K activity was stimulated by only 1.8-fold over basal by insulin, however, activation was very rapid, reaching its peak by 5 min (Fig. 2, bottom). Activation of nuclear p70S6K was also transient decreasing back to basal levels within 30 min. Thus, both the magnitude and the kinetics of activation of nuclear p70S6K by insulin were different from that of cytosolic p70S6K. Inhibition of the Nuclear p70S6K Activity by Rapamycin and DRB
FIG. 2. Stimulation of p70S6K activity by insulin. Following insulin treatment (100 nM) for the indicated times, nuclei and postnuclear supernatant were isolated. The fractions were subjected to immunoprecipitation with anti- p70S6K antibody. p70S6K assays were carried out with the immune-complex assay using ribosomal S6 as a substrate. The reaction mixture was subjected to SDS-PAGE followed by autoradiography. The kinase activities have been quantitated by scanning densitometry of the S6 protein.
Rapamycin is a specific inhibitor of p70S6K activation which acts on an upstream protein (target of rapamycin) involved in activation of cytosolic S6 kinase (26,27). To explore the mechanism of activation of nuclear p70S6K, cells were pre-incubated with rapamycin prior to insulin stimulation, and then subjected to subcellular fractionation and SDS-PAGE. Rapamycin pretreatment almost completely blocked the insulin stimulated mobility shift of p70S6K in both nuclear and postnuclear fractions, indicating that the target of rapamycin was involved in both processes (Fig. 3). Since casein kinase II phosphorylates p70S6K in vitro (28), we also explored the possibility that this enzyme was involved in the activation of p70S6K by insulin using the casein kinase II inhibitor 5,6-dichlorobenzimidazole riboside (DRB) (29) (Fig. 4). DRB pretreatment partially blocked the insulin stimulated mobility shift of p70S6K in both nuclear and cytosolic fractions, suggesting that a casein kinase II-dependent pathway may participate in regulation of p70S6K signaling. However, under the conditions used, this inhibitory effect was not as great as that of rapamycin.
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FIG. 4. DRB and rapamycin inhibit insulin-stimulated p70S6K in CHO(Hirc) cells. Following preincubation with or without rapamycin (20 ng/ml) for 5 min or 5,6-dichlorobenziimidazole riboside (DRB, 1 mM) for 10 min, CHO(Hirc) cells were stimulated with 100 nM insulin for indicated times, following which nuclear and postnuclear fractions were isolated. Equal amounts (10 mg) of each fraction were subjected to electrophoresis, followed by Western blot analysis using antibody against p70S6K and detected with ECL.
DISCUSSION Insulin actions are mediated by multiple protein kinases including raf kinase, MEK, MAP kinases, p90 rsk, p70S6K and PI 3-kinase. It has been suggested that p70S6K plays an essential role in the response of many cells to a variety of growth factors and insulin. In the case of insulin, p70S6K has been shown to be involved in regulation of protein synthesis (2,6), glycogen synthesis (15,16), mitogenesis (6) and GLUT1 and hexokinase II gene expression (18,19). p70S6K has also been shown to play a role in regulation of the cAMP-responsive activator CREM (14). In this communication, we have shown that p70S6K is present in the nucleus, that the enzyme in the nucleus is rapidly activated by insulin, and that the nuclear p70S6K appears to have some differences in regulation, such as time course, from the cytosolic p70S6K. To begin, a significant amount of p70S6K is present in the nucleus of unstimulated CHO (Hirc) cells. This is similar to our findings that in CHO and other cell types, even in the basal state, several serine kinases, including MEK, casein kinase II, and the MAP kinases ERK I and ERK II are present in the nucleus at significant concentrations (30). The method employed in the isolation of nuclei in the present and previous studies is widely used by many investigators, and the purity of the nuclear preparation is greater than 96% as evidenced by cytosolic marker enzyme assays (data not shown). Thus, contamination of cytosolic p70S6K in the nuclear preparation could not account for the presence of the enzyme. Interestingly, in CHO cells we find that it is the p70S6K, rather than the p85S6K, which is prominent in the nucleus despite the absence of a known nuclear localization signal in the p70 form of the protein (28). Activation of nuclear p70S6K by insulin is rapid, but the extent of stimulation is lower than cytosolic p70S6K.
Activation of the nuclear enzyme is also more transient, suggesting differences in the mechanism of activation of nuclear and cytosolic p70S6K. Nuclear p70S6K is also activated by insulin in differentiated 3T3-F442A cells with kinetics similar to that of CHO cells (data not shown). Stimulation of cells by phorbol esters and fetal calf serum causes retardation of nuclear p70S6K on SDS-PAGE indicative of increased phosphorylation and stimulation of p70S6K by these factors as well. TPA also caused an apparant translocation of the enzyme with a decrease in level of protein in the cytoplasm and an increase in p70S6K in the nucleus. The differences in molecular retardation of cytosolic and nuclear p70S6K by TPA and fetal calf serum support the notion that the changes observed in the nuclear preparation are not simply due to contamination by cytosol. Both nuclear and cytosolic p70S6K were inhibited by rapamycin, suggesting the rapamycin-sensitive target lies upstream of divergence to nuclear and cytosolic p70S6K signaling pathway. The finding that DRB inhibits nuclear and cytosolic p70S6K supports the hypothesis that casein kinase II may also lie upstream of p70S6K activation and may play an important role in the regulation of p70S6K in CHO (Hirc) cells. However, it remains to be determined whether the rapamycin-sensitive target lies upstream or downstream of DRB-regulated target in the insulin-regulated p70S6K signaling pathway. Considering the recent observation that casein kinase II phosphorylates p70S6K in vitro (28), and our previous observation that insulin stimulates nuclear casein kinase II activity, it is possible that casein kinase II may directly phospholylate and activate cellular p70S6K in vivo. Other approaches will be necessary to understand the role of casein kinase II in the regulation of p70S6K. In summary, insulin rapidly phosphorylates and activates nuclear p70S6K. The activation of the nuclear p70S6K in response to insulin occurs faster than the activation of cytosolic p70S6K, although the extent of phosphorylation of nuclear p70S6K is lower than that of cytosolic p70S6K. Both nuclear and cytosolic p70S6K are inhibited to the almost same extent by rapamycin and DRB. The rapid activation of nuclear p70S6K in response to insulin may serve as a molecular switch to turn on nuclear insulin-sensitive targets, such as transcription factors, DNA binding proteins and enzymes involved in gene expression.
ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health, DK 33201 and by the NIH-sponsored Diabetes and Endocrinology Research Center, P30 DK36836. SJK acknowledges support from Kyung-Hee University. The authors thank Dr. John Blenis for providing the anti-p70S6K antibody.
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