©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inactivation of Glycogen Synthase Kinase-3 by Epidermal Growth Factor Is Mediated by Mitogen-activated Protein Kinase/p90 Ribosomal Protein S6 Kinase Signaling Pathway in NIH/3T3 Cells (*)

(Received for publication, October 18, 1994; and in revised form, November 18, 1994)

Hagit Eldar-Finkelman (1)(§) Rony Seger (4) Jackie R. Vandenheede (5)(¶) Edwin G. Krebs (1) (2) (3)(**)

From the  (1)Departments of Pharmacology and (2)Biochemistry and (3)Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, the (4)Department of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot 76100, Israel, and (5)Afdeling Biochemie Facultei der Geneeskunde, Katholieke Universiteit Leuven, B 300 Leuven, Belgium

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The role of the p90 ribosomal protein S6 kinase/mitogen-activated protein kinase (RSK/MAPK) signaling pathway in regulating glycogen synthase kinase-3 (GSK-3) activity was investigated. In vitro studies showed that GSK-3 was inactivated by 50% upon incubation with RSK purified from epidermal growth factor (EGF)-stimulated NIH/3T3 cells. Subsequently, the effect of EGF on GSK-3 activity was measured in NIH/3T3 cells that stably overexpressed mutated forms of MAPK kinase (MAPKK). The activation of RSK by EGF was markedly decreased in cell lines expressing the dominant negative MAPKK mutants S222A and K97A and was increased in cells expressing the S222E mutant as compared with control cell lines. EGF induced a rapid decrease in GSK-3beta activity (50%) in control and S222E cells; however, only 25 and 10% inhibition in GSK-3beta activity was observed in cell lines expressing the dominant negative mutants K97A and S222A, respectively, suggesting that inhibition of GSK-3 was partially blocked in these cells. Taken together, these results suggest that the action of EGF on GSK-3 inactivation is mediated by the RSK/MAPK signaling pathway in NIH/3T3 cells and provide evidence for a mechanism regulating GSK-3 activity in intact cells.


INTRODUCTION

Glycogen synthase kinase-3 (GSK-3) (^1)was originally discovered as a protein kinase that phosphorylates and inactivates glycogen synthase (2) and was found to be identical to factor A that activates the MgATP-dependent form of protein phosphatase-1(3, 4) . GSK-3 was further identified as a multisubstrate protein kinase acting on many substrates such as ATP citrate lyase(5) , the regulatory subunit of cyclic AMP-dependent protein kinase (RII)(6) , transcription factors such as c-jun, c-myc, c-myb, and CREB(7, 8, 9, 10) , the eukaryotic initiation factor (eIF-2B)(11) , and the microtubule-associated protein, tau(12) . Molecular cloning from a rat brain library revealed two GSK-3 isoforms termed alpha and beta, which are 98% identical in their kinase domains(13) . Mammalian GSK-3beta showed close homology to the Drosophilazesta-white 3/shaggy gene product involved in fruit fly development(14, 15) . In the budding yeast Saccharomyces cerevisiae the MCK1 and MDS1 gene products, playing an important role in the chromosome segregation process, also showed high homology with shaggy and GSK-3(16) . Taken together, these data suggest that GSK-3 may play an important role in cell proliferation, differentiation, and development.

Little is known about the regulation of GSK-3 by growth factors and hormones. Initially it was shown that insulin rapidly decreased the activity of a protein kinase in rat adipocytes, which was subsequently identified as GSK-3(17, 18) . It is currently accepted that GSK-3 activity is regulated by phosphorylation. Protein kinase C was reported to phosphorylate GSK-3beta but not GSK-3alpha in vitro, decreasing its activity by 50%(19) . The ribosomal protein S6 kinase, RSK, a major substrate of MAPK, was also shown to directly phosphorylate and inactivate both GSK-3 isoforms in vitro(20, 21) . More recently, GSK-3 was shown to be activated by tyrosine phosphorylation(22, 23) ; the phosphotyrosine site was identified as tyrosine 216(22) , a position analogous with the TEY motif found in MAPKs. Like MAPK, GSK-3beta appears to be a dual specific enzyme, but unlike MAPK, it is differentially regulated by tyrosine and serine/threonine phosphorylation(24) . Still, a key question is how the enzyme is regulated in vivo.

In these studies we have examined the regulation of GSK-3 activity by EGF in NIH/3T3 cell lines that overexpress mutated forms of MAPK kinase (MAPKK)(1) . These MAPKK mutants included mutations at serine 222, one of the sites essential for enzyme activity(25, 26) , to glutamic acid or alanine (S222E and S222A, respectively), and a mutation at lysine 97 (K97A). We report that EGF induced a rapid decrease in GSK-3beta activity in control and S222E mutant cells. However, in cell lines expressing the dominant negative mutants S222A and K97A, which showed inhibition of RSK activation by EGF, the decrease in GSK-3beta activity was partially blocked. Our studies suggest that EGF-induced GSK-3 inactivation is regulated by the RSK/MAPK signaling pathway.


MATERIALS AND METHODS

Proteins and Peptides

Synthetic peptides were prepared by the peptide synthesis facility of the Howard Hughes Medical Institute, Seattle, WA. These included: RRLSSLRA (S6 peptide); YRRAAVPPSPSLSRHSSPHQSEDEEE (GS1 peptide), which is patterned after the GSK-3 phosphorylation sites in glycogen synthase except that an alanine is substituted for a serine in position 5 in order to avoid having a consensus sequence for cAMP-dependent protein kinase in the peptide; and TTYADFIASGRTGRRNAIHD (protein kinase inhibitor peptide). The GS1 peptide was first incubated with casein kinase-2 and MgATP, which resulted in the incorporation of 1 mol of P(i)/mol of GS1. Heparin (10 µg/ml) was added to the peptide to block any residual casein kinase-2 activity present in the ``P-GS1'' preparation. Casein kinase-2 was purified to homogeneity from bovine testis as described(27) . The protein phosphatase-1 inhibitor, I2, and GSK-3 purified from rabbit skeletal muscle were kindly provided by Curt Diltz and Dr. Edmond H. Fisher (Department of Biochemistry, University of Washington).

Antibodies

R-8829E, a rabbit polyclonal antibody, was raised against the synthetic peptide ESSILAQRRVRKLPSTTL corresponding to the C terminus of RSK; it is possible that this antibody will cross-react with RSK, due to a similarity in the sequences of these isoforms. Rabbit polyclonal antibodies against GSK-3beta were previously described(28) ; these antibodies were raised against the synthetic peptide TNNAASASASNST.

Cell Culture and Protein Kinase Assays

MAPKK-transfected NIH/3T3 cells (1) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. For experiments, subconfluent cells were serum-starved for 18 h with Dulbecco's modified Eagle's medium supplemented with 0.1% fetal calf serum. Following EGF treatment for the indicated times, cells were washed twice with ice-cold phosphate-buffered saline and scraped into Buffer H (50 mM beta-glycerophosphate, pH 7.3, 1.5 mM EGTA, 1 mM EDTA, 2 mMDL-dithiothreitol, 0.2 mM sodium orthovanadate, 1 mM benzamidine, 10 µg/ml aprotinin, 20 µg/ml leupeptin, 1 mM NaF, 0.5 µg/ml microcystine (Life Technologies, Inc.), and 2 µg/ml pepstatin A (Sigma)). Cell lysates were sonicated and centrifuged at 100,000 times g for 20 min at 4 °C, and subsequent steps were also performed at this temperature. The resulting supernatants were passed through DE52 (Whatman) minicolumns that were equilibrated with Buffer H and washed once with Buffer H containing 0.02 M NaCl. ``Flow-through'' and wash were collected and diluted to equal protein concentrations in Buffer H (50-100 µg) and incubated with antibodies against RSK or GSK-3beta prebound to Sepharose-protein A (Sigma) for 2 h with frequent agitation. The immunoprecipitates were washed twice with 50 mM Tris, pH 7.3, 0.5 M LiCl, and 1 mMDL-dithiothreitol and twice with 50 mM Tris, pH 7.3. The kinase reaction was started by addition of substrate (P-GS1 or S6 peptide) and [-P]ATP. The reaction mixture for GSK-3 kinase assays included: 50 mM Tris, pH 7.2, 250 µM [-P]ATP (0.25 mCi/ml), 10 mM MgCl(2), 0.5 mg/ml bovine serum albumin, 2 µg/ml heparin, and 60 µM P-GS1 peptide in a final volume of 30 µl. One unit of kinase activity is referred to as the amount of enzyme causing the incorporation of 1 pmol of P(i)/min into P-GS1. The reaction mixture for RSK kinase has been described elsewhere (1) . Following 15 min of incubation at 30 °C, the reaction mixture was spotted on phosphocellulose paper (Whatman p81) and processed as described previously(1) . In a different set of experiments inhibitor 2 (10 µg) was incubated with GSK-3beta immunoprecipitates in the presence of 10 mM MgCl(2) and 100 µM [-P]ATP for 15 min at 30 °C, heated with Laemmli sample buffer, and subjected to 15% SDS-polyacrylamide gel electrophoresis. Gels were dried and autoradiographed.

Immunoblots

Equal amounts of lysate protein were immunoprecipitated with specific antibodies against GSK-3beta (described in the previous section). Immunoprecipitates were washed as described, boiled with Laemmli sample buffer, and subjected to 12% SDS-polyacrylamide gel electrophoresis. Gels were blotted onto polyvinylidene difluoride membranes (Millipore) and incubated with GSK-3beta antibodies for 15 h at 4 °C. Detection was carried out using horseradish peroxidase-labeled protein A (Amersham Corp.).


RESULTS

Inactivation of GSK-3 by RSK in Vitro

The time course of the phosphorylation of P-GS1 peptide catalyzed by purified GSK-3 was determined in the presence or absence of activated RSK added in the form of an immunoprecipitate derived from lysates of EGF-treated NIH/3T3 cells (Fig. 1). As can be seen, the rate of phosphorylation of the peptide substrate was inhibited (approximately 50%) from the onset in the presence of RSK (opencircles). No inhibition occurred when GSK-3 was incubated under similar conditions with a preimmune serum immunoprecipitate (closedcircles). Incubation of GSK-3 with RSK partially purified from EGF-stimulated NIH/3T3 cells gave similar observations (data not shown). The results obtained confirm the previous reports showing that RSK purified from skeletal muscle is capable of phosphorylating and inactivating both isoforms of GSK-3(20, 21) .


Figure 1: Effect of RSK on GSK-3-catalyzed phosphorylation of P-GS1. The P-GS1 peptide (final concentration, 60 µM) was incubated with purified GSK-3 (5 µl of 8 units/ml) together with Mg-[-P]ATP and RSK (opencircles) that had been immunoprecipitated from EGF-stimulated NIH/3T3 cell lysates or from lysates treated with preimmune serum (closedcircles). Phosphorylation of the peptide was determined by counting at the indicated times. Results are presented as the amount of phosphate incorporated into the P-GS1 peptide present in the reaction mixture.



Inactivation of GSK-3 by EGF in MAPKK-transfected Cell Lines

NIH/3T3 cell lines expressing wild-type or each of several mutant forms were recently obtained in this laboratory (1) and studied to determine the activation responses of components of the MAPK cascade to stimulation by EGF. S222A and K97A mutations were shown to inhibit the activation of MAPKK, MAPK, and RSK while the S222E mutants showed enhanced activations by the growth factor(1) . Activation of RSK by EGF was now examined in more detail in these cells (Fig. 2). It is apparent that there is a rapid (8-fold) activation of RSK in control cells peaking within 4-6 min and then falling off. In the S222E cell line the peak of activity is approximately double that of the controls. The biphasic profile of RSK activation was comparable in all cell lines and was similar to that seen for the activation of MAPKK and MAPK as previously reported for these cells(1) .


Figure 2: Activation of RSK in EGF-stimulated MAPKK-transfected cell lines. MAPKK-transfected cells were treated with EGF (30 ng/ml) for the indicated times. RSK activity was assayed by the immunocomplex protein kinase assay using S6 peptide as the substrate. Results are the means of duplicates from one representative experiment. Opencircles, control cells; closedcircles, S222E cells; opensquares, K97A cells; closedsquares, S222A cells.



The fact that RSK was differentially activated in the MAPKK-transfected cell lines (Fig. 2) prompted us to examine the effect of EGF on GSK-3 activity in these cells. The presence of GSK-3 in these cells was detected by Western blot analyses performed on cell lysates that were immunoprecipitated with specific antibodies against GSK-3beta(28) . A single band of 45 kDa, equally distributed in all cell lines, was detected (Fig. 3) and indicated that these antibodies specifically immunoprecipitated GSK-3beta; such precipitates have been shown to serve as a reliable tool for measuring GSK-3 activity in crude cell preparations(23, 28) , and this technique was then applied here. Transfected cell lines were treated with EGF (30 ng/ml) for different periods of times. GSK-3beta was immunoprecipitated from cell lysates, and their GSK-3 kinase activity was measured using P-GS1 peptide as the substrate. As shown in Fig. 4A, EGF caused a rapid decrease in GSK-3beta activity (50% decrease after 4-5 min) in both control and S222E cell lines. However, a decrease of only 10 and 25% in GSK-3beta activity was observed in S222A and K97A cells, respectively (Fig. 4A), indicating that inactivation of GSK-3beta was impaired in these cells. In addition, the recovery of GSK-3beta activity was quicker and more complete in S222A and K97A cells than in control or S222E cell lines. GSK-3beta activity was also assayed using inhibitor-2 (I2) as the substrate (Fig. 4B). Consistent with the previous results, phosphorylation of I2 by GSK-3beta was markedly decreased in control and S222E cell lines treated with EGF (Fig. 4B, lanes1-4). Little or no effect on I2 phosphorylation was detected in S222A and K97A cells (Fig. 4, lanes 5-8). (In this particular experiment, the basal activity of GSK-3beta in S222A cells was somewhat low relative to the other cell lines (Fig. 4B, lanes7 and 8). Generally, however, the basal levels of GSK-3beta activity were comparable in all cell lines.) Although higher levels of RSK activity were detected in S222E cells (Fig. 2), EGF-induced GSK-3 inactivation was found to be comparable with that of the control cells. We cannot provide a complete explanation for this observation, but it is possible that in a complex cascade such as that operating here, a 1:1 relationship between the activity of upstream and downstream kinases may not always exist.


Figure 3: Expression of GSK-3beta in MAPKK-transfected cell lines. Cell lysates, normalized for the amount of protein, were incubated with specific antibodies against GSK-3beta as described under ``Materials and Methods.'' Washed immunoprecipitates were subjected to 10% SDS-gel electrophoresis, blotted onto polyvinylidene difluoride membranes incubated with anti-GSK-3beta antibodies, and detected with horseradish peroxidase-labeled protein A. Lane1, control cells; lane2, S222E cells; lane3, K97A cells; lane4, S222A cells.




Figure 4: Inactivation of GSK-3beta in EGF-stimulated MAPKK-transfected cell lines. NIH/3T3 cells were treated with EGF for the indicated times. GSK-3beta was immunoprecipitated from cell lysates as described under ``Materials and Methods.'' A, GSK-3 activity was determined in the immunoprecipitates using P-GS1 peptide as the substrate and is given as the percentage of enzyme activity measured in non-stimulated cells. Results are the mean of five independent experiments; S.D. was less than 10%. Opencircles, control cells; closedcircles, S222E cells; opensquares, K97A cells; closedsquares S222A cells. B, cells were treated with EGF for 6 min, and GSK-3beta was immunoprecipitated from cell lysates as described under ``Materials and Methods.'' I2 (10 µg) was added to the immunoprecipitates, and its phosphorylation was analyzed as described under ``Materials and Methods.'' Lanes 1 and 2, control cells; lanes3 and 4, S222E cells; lanes5 and 6, K97A cells; lanes7 and 8, S222A cells.




DISCUSSION

The fact that GSK-3 is constitutively active in resting cells (22, 23) is consistent with the concept that this enzyme might be negatively regulated by growth factors. Little is known, however, about the mechanism that might be involved in such regulation. Nor was it known whether growth factors or hormones other than insulin could cause GSK-3 inactivation. Here we show that EGF causes a rapid decrease in GSK-3beta activity in NIH/3T3 cells and that EGF-induced GSK-3beta inactivation is partially prevented in cells expressing the dominant negative mutants (S222A and K97A) of MAPKK. Since the activation of RSK, MAPK, and MAPKK by EGF was markedly suppressed in these cells (1) (Fig. 2), these results suggest that EGF action on GSK-3 inactivation is mediated by the MAPK signaling pathway. These data alone cannot be used, however, to determine which kinase (i.e. MAPKK, MAPK, RSK, or an as yet unknown downstream kinase) is directly involved in GSK-3 inactivation, but in vitro studies suggest that RSK is the component inactivating GSK-3 (20, 21) (Fig. 1). These data do not exclude the possibility that other mechanisms might also be involved in the regulation of GSK-3. For example, as noted earlier, tyrosine phosphorylation has also been shown to regulate the enzyme(22, 24) , suggesting that activation of tyrosine phosphatase could play a role in GSK-3 inactivation. Indeed, a decrease in phosphotyrosine content was reported to cause inactivation of the enzyme in A431 cells(23) .

The question as to whether the Ras/MAPK signaling pathway mediates the action of insulin and related factors such as EGF (also see (32) ) on glycogen metabolism is still not clear. Studies in rat adipocytes suggested that the MAPK signaling pathway is not sufficient for mediating the action of insulin on glycogen synthase and glucose transport(29, 30) . In contrast, expression of activated mutants of Ras in 3T3-L1 adipocytes was shown to mimic the effect of insulin on membrane trafficking of glucose transporters(31) . If it is assumed that GSK-3 does play an important role in regulating glycogen synthase activity, our studies could imply that the RSK/MAPK signaling pathway mediates the action of hormone or growth factor regulation of the enzyme. However, it is quite possible that inactivation of GSK-3 through the MAPK cascade is not sufficient by itself to induce these changes. Additional studies are needed to clarify these points.

Another laboratory has reported similar results to those presented here; this came to our attention during the review process. These studies indicate that insulin-like growth factor and EGF induce inactivation of GSK-3 in intact cells and show that the MAPK cascade appears to play a role in regulating GSK-3 activity(33, 34, 35) . This conclusion is drawn on the basis of utilizing wortmannin to block MAPK activation and its effect on GSK-3.


FOOTNOTES

*
This work was supported by Grant DK 42528 from the National Institutes of Health and by a grant from the Muscular Dystrophy Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by an American Heart Association training fellowship.

Research Director of the Belgian ``National Fonds voor Wetenschappelijk Onderzoek.''

**
To whom correspondence should be addressed: Dept. of Pharmacology, SL-15, University of Washington, Seattle, WA 98195. Tel.: 206-543-8500; Fax: 206-543-0858.

(^1)
The abbreviations used are: GSK-3, glycogen synthase kinase-3; EGF, epidermal growth factor; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; RSK, p90 ribosomal protein S6 kinase; I2, inhibitor-2.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.