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J. Biol. Chem., Vol. 277, Issue 34, 31099-31106, August 23, 2002

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Regulation of Raf-Akt Cross-talk

Karin Moelling, Karen Schad^§, Magnus Bosse, Sven Zimmermann^¶, and Marc Schweneker

From the Institute of Medical Virology, University of Zurich, 8028 Zurich, Switzerland

Received for publication, December 15, 2001, and in revised form, May 9, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have recently shown that the Ras-Raf-MEK-ERK and phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathways can cross-talk in the human breast cancer cell line MCF-7. High Raf activity induces growth arrest and differentiation in these cells, whereas high PI3K/Akt activity correlates with cell survival and proliferation. Here we show that the Raf-Akt cross-talk is regulated in a concentration- and ligand-dependent manner. High doses of insulin-like growth factor I (IGF-I) activate Akt quickly and strongly enough to suppress Raf kinase activity via phosphorylation of Ser-259, whereas low doses of IGF-I do not trigger this cross-talk but are still mitogenic. Phorbol 12-myristate 13-acetate, a differentiation-inducing stimulus, potently activates the Ras-Raf-MEK-ERK pathway but only weakly activates PI3K/Akt and does not trigger the cross-talk. Thus, the herein analyzed parameters such as ligand type, concentration, and time course may contribute to the cellular response of either proliferation or differentiation. This is highly relevant to understanding cellular transformation and may be of use in areas like tissue engineering.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Signal transduction via activated Ras mediates several apparently conflicting cellular responses such as proliferation, apoptosis, growth arrest, differentiation, and senescence, depending on the duration and strength of the external stimulus and on the cell type. For a recent review see Kolch (1). A downstream effector of Ras is Raf-1. Raf-1 is a serine/threonine kinase (2) that can be activated by a variety of extracellular stimuli, among them insulin-like growth factor I (IGF-I)¹ that activates the type 1 IGF surface receptor, a receptor tyrosine kinase mainly implicated in the induction of proliferation (3).

Raf-1 activation itself is complex and involves membrane recruitment, phosphorylation on serine/threonine residues as well as on tyrosine residues, and binding of 14-3-3 protein family members. Activated Raf-1 phosphorylates and activates the MEK-ERK kinase pathway (4, 5). Downstream effectors of ERKs are nuclear transcription factors such as Myc and Elk (6-11), which trigger biological responses via direct impact on gene expression.

Another important pathway that is triggered by IGF-I or insulin via phosphorylation of insulin receptor substrate, IRS-1, is the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. PI3K is activated by binding of its p85 regulatory subunit to tyrosine-phosphorylated IRS-1. Activation of PI3K increases the amounts of membrane-localized phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate. One of the crucial downstream targets of PI3K is the serine/threonine kinase Akt (12). Akt is recruited to the membrane by direct binding of its pleckstrin homology domain to the PI3K-produced phospholipids (13). Upstream kinases such as 3-phosphoinositide-dependent protein kinase 1 (PDK1) activate Akt by phosphorylation on Thr-308 (14) and Ser-473 (15).

Active Akt causes a variety of biological effects, including suppression of apoptosis by phosphorylation and inactivation of several targets along pro-apoptotic pathways such as the Bcl-2 family member BAD (16, 17) or caspase-9 (18). Moreover, it regulates glucose uptake by a largely uncharacterized mechanism and controls the activity of glycogen synthase kinase 3 (GSK3) (19). For a comprehensive review see Scheid and Woodgett (20).

Whereas only one direct downstream target is known for Raf-1 many proven or putative targets exist for Akt. The targets share a characteristic phosphorylation sequence within a highly conserved motif (RXRXX(S/T)), which we have previously characterized within the Raf-1 sequence. It is located in the regulatory domain of Raf-1 with Ser-259 being the target within the Akt phosphorylation motif (RQRSTS²⁵⁹). Based on these findings we have recently established that the Ras-Raf-MEK-ERK and the PI3K-Akt pathways cross-talk on the level of Raf-1 and Akt (21-23).

We have shown that Akt directly phosphorylated Raf-1 on Ser-259 and resulted in a decrease in Raf-1 activity (21). The inhibition of Raf-1 is due to the phosphorylation-dependent binding of the 14-3-3 protein, a negative regulator of Raf-1. In MCF-7 cells, the inactivation of the cross-talk between the two pathways switched the biological response from proliferation to cell cycle arrest. Stimulation with 100 ng/ml IGF-I and the concomitant pharmacological inhibition of PI3K (and indirectly of Akt) with LY294002 led to an increase of Raf-1-kinase and ERK activity because the inhibitory effect of the cross-talk was suppressed.

The cross-talk between the Ras-Raf-MEK-ERK and the PI3K-Akt pathways was also demonstrated in other cellular systems. In the work of Rommel et al. (22), the relative contributions of the Ras-Raf-MEK-ERK and the PI3K-Akt pathway activities were assessed during myotube differentiation of C2C12 cells. Enforced activation of Akt or inhibition of the MEK-ERK pathway promoted differentiation and therefore myotube formation, whereas enforced Raf-1 activity blocked differentiation in the precursor myoblast stage. Intriguingly, the cross-talk was only observed in post-differentiation myotubes and not in myoblasts, where Akt activity or its inhibition had no influence on the Ras-Raf-MEK-ERK pathway. This suggests a regulation of the cross-talk, in this particular case with a cell stage specificity.

Recently, a cross-talk between Akt and Raf has been also shown in neonatal vascular smooth muscle (VSM) cells from rat (23). Early passage cells expressed various levels of -actin and muscle myosin depending on their developmental or differentiation stage. We showed that platelet-derived growth factor, a potent mitogen, induced a sustained activation of PI3K-Akt and allowed only a transient activation of Ras-Raf-MEK-ERK, because activated Akt was able to phosphorylate and terminate Raf-1 kinase activity. This cross-talk correlated with proliferation of VSM cells. Thrombin in turn induced differentiation markers such as myosin indicating differentiation of VSM cells. Thrombin induced a strong and biphasic phosphorylation of ERK-1/2, whereas Akt was only partially and transiently phosphorylated.

Cross-talk or description of the balance between PKB/Akt and Ras-Raf-MEK-ERK signaling has recently been shown by other groups to depend on the kind of agonist and the cellular background. Besides the work done in MCF-7, C2C12, and VSM cells (21-23), Guan et al. (24) showed a cross-talk between Akt and B-Raf in HEK293 cells. However, both pathways have also been shown to act in parallel. Hence, in intestinal epithelial cells PI3K and Raf synergized for cellular proliferation and transformation (25) whereas certain tumor cells or primary tumors featured constitutively overexpressed Akt (26, 27). In rat PC12 cells, B-Raf and c-Raf synergized for sustained ERK activity, which correlated with differentiation as has been described earlier (28, 29). A similar synergy led to differentiation of megakaryocytes (UT7 cells) by thrombopoietin (30). Ras-Raf signaling dominated for cell proliferation in epidermal growth factor-stimulated COS cells (31) similar to the situation observed in myotubes (22) or 3T3L1 preadipocytes.² Raf and Akt may also synergize for survival (29, 32). Depending on the cellular background and type of stimulus, Raf alone can be antiapoptotic even independently of its kinase activity by interaction with apoptosis signal-regulating kinase 1 (ASK-1) (33), or it can be proapoptotic (34).

The role of the MAP kinase in MCF-7 cells stimulated by insulin or TPA/PMA has been analyzed by others (35, 36). Alblas et al. (35) characterized the kinetics of induction of immediate early genes and showed that insulin induced JNK whereas TPA did not. The inhibitory effect of TPA on cell cycle entry could be reverted by the MAP kinase inhibitor PD98059, indicating that ERK effectors function as inhibitors of proliferation in MCF-7 cells.

Here we analyzed the kinetics of the two pathways, Ras-Raf-MEK-ERK and PI3K-Akt, upon stimulation by IGF-I and PMA using different ligand concentrations. We focused on analyzing the cross-talk between the two pathways.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- The MCF-7 cell line (human mammary gland carcinoma; purchased from ATCC, Manassas, VA) was maintained in Dulbecco's modified Eagle's medium/F-12 medium (Invitrogen) supplemented with 5% charcoal-treated fetal calf serum (Seratec Co., Goettingen, Germany), 100 units/ml penicillin, and 100 mg/ml streptomycin (both Invitrogen).

Cell Lysis-- MCF-7 cells were seeded onto 100-mm Falcon tissue culture dishes and grown to a confluency of about 75%. 24 h before stimulation the cells were starved in phenol red- and serum-free Dulbecco's modified Eagle's medium/F-12. The medium was changed a second time 12 h before stimulation. Cells were stimulated for the indicated times with either 100 ng/ml IGF-I (Calbiochem, San Diego, CA), 10 ng/ml IGF-I, or 100 ng/ml PMA (Calbiochem) with or without prior incubation with 20 µM LY294002 (Calbiochem). Cells were lysed for 10 min in radioimmunoprecipitation assay buffer (20 mM Tris-HCl, pH 7.5, 135 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 10% glycerol, 1 mM dithiothreitol, 25 mM -glycerol phosphate, 25 mM NaF, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 10 µM pepstatin, Trasylol (500,000 Kallikrein® inactivator units) 1:1000, 5 µg/ml leupeptin). Lysates were cleared by centrifugation, and protein concentrations of the supernatants were determined using the Protein Assay Kit II (Bio-Rad).

Western Blotting-- Equal amounts of protein were separated in 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Amersham Biosciences). For protein detection, primary antibodies used were specific for Raf phosphorylated on Ser-259, for activated Akt or for activated ERK (Cell Signaling Technologies, Beverly, MA). Primary antibodies were detected by enhanced chemiluminescence (Amersham Biosciences). Membranes were stripped, blocked, and reprobed with antibodies to Raf (R19120; BD Biosciences, Franklin Lakes, NJ) or to ERK (C14; Santa Cruz Biotechnology, Santa Cruz, CA).

Raf Kinase Assay-- Stimulation was performed as described, and cleared lysates were subjected to immunoprecipitation with antibodies to Raf (21-23). Beads were resuspended in 30 µl of kinase buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM dithiothreitol, 20 mM -glycerol phosphate, 5 mM MnCl₂) supplemented with 10 µM ATP (Roche Diagnostics, Basel, Switzerland), 5 µCi of [-³²P]ATP (Amersham Biosciences), and 27.8 µg/ml unactivated MEK1 (Upstate Biotechnology, Charlottesville, VA). The Raf kinase prefers Mn²⁺ over Mg²⁺, in contrast to the ERK kinase and protein kinase C (2, see Upstate Biotechnology Catalogue 2001), thus increasing the Raf kinase specificity. The kinase reaction was performed for 30 min at 30 °C, the reaction was stopped with 10 µl of 4× SDS sample buffer (250 mM Tris-HCl, pH 6.8, 40% glycerol, 8% SDS, 0.4% bromphenol blue, 200 µl/ml -mercaptoethanol). The proteins were separated on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane, and MEK phosphorylation was assayed using a PhosphorImager (Amersham Biosciences).

ERK in Vitro Kinase Assay-- Cells were lysed as described previously (21-23). Subsequently, ERK-2 was precipitated from cellular lysates using an ERK-2 specific antibody (C-14, Santa Cruz Biotechnology). Kinase reactions were performed for 15 min at 30 °C in 30 µl of kinase buffer, adjusted to 20 µM ATP, 1 µCi of [-³²P]ATP, and 10 µg of myelin basic protein (Sigma). The kinase reactions were separated on 12.5% SDS-gels. Finally, the dried gels were exposed and quantified using a PhosphorImager.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Akt-Raf Cross-talk Takes Place in Proliferating Cells-- Growth properties of proliferating cells have been determined for MCF-7 cells treated with low and high concentrations of IGF-I in their growth medium. After about 24 h of treatment cells incubated with both low IGF-I (10 ng/ml) and high IGF-I (100 ng/ml) started to proliferate. At later time points, the proliferation rate was higher at higher IGF-I concentrations (not shown). As previously shown, PMA (100 ng/ml) showed no growth-promoting effects (Fig. 1) presumably due to expression of the cell-cycle inhibitor p21^cip1 via the Ras-Raf-MEK-ERK pathway (21).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Growth properties of MCF-7 cells. MCF-7 cells were seeded in 6-well plates and incubated for 4 h. Subsequently, the cells were starved for 24 h and then (0 h) incubated in medium supplemented with 5% fetal calf serum (FCS), 10 or 100 ng/ml IGF-I, and 100 ng/ml PMA or left untreated. Medium change was performed daily. At the times indicated the cells were trypsinized and the cell number was determined. Each point was measured as duplicate. Untreated control, black line, con; 5% fetal calf serum, red line; 10 ng/ml IGF-I, green line, low; 100 ng/ml IGF, dark blue line, high; 100 ng/ml PMA, blue line.

It has been shown by others that a proliferative stimulus keeps the activity of the Ras-Raf-MEK-ERK pathway under control to prevent growth arrest by ERK-dependent up-regulation of cell cycle inhibitors (37). To investigate how this control is achieved we analyzed the activity of the two signaling pathways Ras-Raf-MEK-ERK and PI3K-Akt after stimulation with low and high doses of IGF-I. Fig. 2A shows the phosphorylation state of the Akt target site Ser-259 of Raf-1 in cells stimulated with high doses of IGF-I (100 ng/ml), which led to a rapid increase of Ser-259 phosphorylation (lane 3), visualized by a phospho-specific antibody. Inhibition of Akt by LY294002, an inhibitor of the Akt upstream kinase PI3K (38), suppresses this increase in phosphorylation (lane 4). Over time, the phosphorylation of Ser-259 diminishes (lane 5).

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Raf, ERK, and Akt activity at high IGF-I concentrations. A, effects of Akt inhibition on phosphorylation of Raf on Ser-259. MCF-7 cells were deprived of serum for 24 h, incubated for 20 min with 20 µM LY294002 where indicated, and then stimulated with 100 ng/ml IGF-I for 3 or 10 min or left untreated. Cell lysates were subjected to Western blot analysis with an antibody specific for Raf phosphorylated on Ser-259 (upper panel), stripped, and reprobed with anti-Raf antibody (lower panel). The nature of the strong band is unknown. B, inhibitory effect of Akt on Raf kinase activity. MCF-7 cells were starved by serum withdrawal for 24 h, incubated for 20 min with the PI3K inhibitor LY294002 (20 µM), and stimulated with 100 ng/ml IGF-I for the indicated time periods. Endogenous Raf protein was immunoprecipitated, and the in vitro kinase activity of Raf toward a glutathione S-transferase fusion protein of MEK (GST-MEK) was assayed. Immunocomplexes were also subjected to immunoblot analysis with an antibody specific to Raf. C, activation of Akt and ERK. MCF-7 cells were treated as described above. After lysis and immunoblot analysis, the samples were assayed for Akt and ERK activation. Activity of Akt was visualized with a phosphospecific antibody for Ser-473 (upper lane), and the activity of ERK was visualized with an antibody specific for phosphorylated Tyr-202/Tyr-204 (middle). The total amount of protein in each lane was detected by reprobing the membrane with an antibody against ERK-2 (bottom).

Because increased phosphorylation of Ser-259 of Raf-1 is usually associated with a decrease in the Raf-1 kinase activity, we next measured its activity in an in vitro kinase assay (Fig. 2B). After starvation, MCF-7 cells were stimulated for 3, 5, or 10 min with 100 ng/ml IGF-I with or without concomitant inhibition of PI3K/Akt by LY294002. Endogenous Raf was immunoprecipitated, and its activity toward a glutathione S-transferase fusion protein of MEK (GST-MEK) was assayed. The radioactivity incorporated into glutathione S-transferase-MEK was quantified by PhosphorImager analysis. High doses of IGF-I cause a transient, ~2-fold activation of Raf-1 kinase activity peaking around 3 to 5 min (lanes 3 and 5). Concomitant inhibition of PI3K-Akt increases Raf-1 kinase activity by about 50% at every time point measured.

This increase in Raf-1 kinase activity is transmitted to its downstream targets ERK-1 and -2 as demonstrated by using ERK activation-specific antibodies (Fig. 2C). The ERK activity mirrors the activity of Raf-1, although with slightly delayed kinetics due to the downstream position of ERK. Thus, the peak of ERK activation lies around 10 min (lane 7). Both the Raf-1 and ERK kinase activities are higher in the presence of the PI3K inhibitor (Fig. 2, B and C). At later time points the ERK activity decreased (lanes 9-12). Under the same conditions strong activation of Akt is visible and persistent. Even after 90 min of stimulation, no decrease in Akt activity is detectable (data not shown). At all time points analyzed, Akt activity can be potently suppressed by the PI3K inhibitor LY294002. Thus, the Raf-Akt cross-talk apparently takes place after immediate and strong activation of Akt, which phosphorylates Raf and thereby negatively controls the activity of the Ras-Raf-MEK-ERK pathway to allow proliferation.

Akt-Raf Cross-talk Is Not Triggered at Lower IGF-I Concentration-- Interestingly also stimulation with low doses of IGF-I (10 ng/ml) allowed MCF-7 cells to proliferate (see Fig. 1). However, low IGF-I induced a rather low activation of Raf-1 kinase activity (1.5- to 2-fold), which was independent of LY294002 when assayed in an in vitro kinase assay (Fig. 3B). Low IGF-I concentration did not induce detectable above basal phosphorylation of Ser-259, and not unexpectedly LY294002 did not affect this level either (Fig. 3A).

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Raf, Akt, and ERK activity at low IGF-I concentrations. A, effect of Akt inhibition on phosphorylation of Raf on Ser-259. MCF-7 cells were treated as described above except that 10 ng/ml IGF-I was used. The antibodies used were the same as described in Fig. 2A. B, inhibition of Akt has no effect on Raf activity. The assay was performed as described in Fig. 2B, but with 10 ng/ml IGF-I instead of 100 ng/ml. C, activation of Akt and ERK. Stimulation of the cells was performed as shown in Fig. 2C, but with 10 ng/ml IGF-I instead of 100 ng/ml. Antibodies for the immunoblot analysis were the same as described in Fig. 2C.

By using the same phosphorylation-specific antibodies as above, ERK and Akt activation were also measured at low doses of IGF-I (Fig. 3C). In contrast to the findings with high doses of IGF-I, we observed a much lower phosphorylation of ERK-1/2 with a peak at 5-10 min, which was inhibited by LY294002 during early time points (3-30 min). This paradox has been detected before (31, 39), and several explanations have been postulated. In agreement with others, we conclude, that PI3K must play several distinct roles in the regulation of the ERK pathway, both positive or negative. We show here that this can depend on the strength of the incoming signal (see also Ref. 31).

In keeping with previous findings, we show that low doses of mitogen do not trigger the Raf-Akt cross-talk via phosphorylation of Ser-259 and that in a weakly stimulated Ras-Raf-MEK-ERK pathway the PI3K-Akt pathway has no inhibitory effect on the Raf-1 kinase, i.e. there is no cross-talk.

Phorbol Esters Do Not Induce Akt-dependent Raf Inhibition-- After having established that the cross-talk depended on the ligand concentrations, we addressed the question of ligand specificity and the biological outcome. In contrast to exposure to proliferation-inducing IGF-I, MCF-7 cells, which were treated with phorbol esters such as PMA, underwent growth arrest (Fig. 1) driven at least in part by an ERK-dependent induction of the cell-cycle inhibitor p21^cip1 (21, 37).

PMA (100 ng/ml) induced some phosphorylation of Ser-259 on Raf-1 (Fig. 4A) over various exposure periods. Importantly, the absence or presence of LY294002 did not significantly alter the degree of phosphorylation suggesting that PI3K-Akt was not involved. Likewise, LY294002 did not significantly influence the Raf-1 kinase nor ERK activity (Fig. 4, B and C). As has been shown in various cell systems before, PMA strongly stimulated the Raf-1 kinase activity (4- to 5-fold) (Fig. 4B) and persisted at a high level for up to 90 min (data not shown). Inhibition of PI3K-Akt by LY294002 had no influence on the PMA-mediated Raf-1 kinase activation. This finding differed from the results obtained by stimulation with high doses of IGF-I (Fig. 2, B and C).

View larger version (42K): [in this window] [in a new window]   Fig. 4.   Raf, Akt, and ERK activity under growth-arresting conditions. A, assays on phosphorylation of Raf-1 at Ser-259 were performed as described for Fig. 2A except that 100 ng/ml PMA was used (lane 7 contains slightly less input). B, effect of inhibition of Akt on Raf kinase activity. Assays were performed as described in Fig. 2B using 100 ng/ml PMA. C, activation of Akt and ERK. Stimulation of the cells was performed as shown in Fig. 2C, but 100 ng/ml PMA was used. Antibodies for the immunoblot analysis were the same as described in Fig. 2C.

The ERK activation reached a very high level immediately after PMA stimulation and was sustained over all time points measured (Fig. 4C). These results confirm former findings stating that PMA is a strong inducer of ERK activity (39). Surprisingly, PMA treatment also led to some activation of Akt, however, delayed and even reduced when compared with low doses of IGF-I. Our results further showed that PMA-induced activation of Akt occurred via PI3K, because it was completely suppressed by LY294002. Yet the inhibitor had no significant influence on Raf-1 or the ERK activity, demonstrating that a cross-talk between Akt and Raf-1 did not take place during PMA-induced growth arrest.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ras-Raf-MEK-ERK and PI3K-Akt pathways have been previously analyzed after stimulation with two different ligands, IGF-I and PMA, on one single cell type (MCF-7) (21). Moreover, two different cellular stages of muscle cell differentiation (C2C12 cells) were analyzed in another study (22), and a third investigation probed again two different ligands, platelet-derived growth factor and thrombin, on neonatal vascular smooth muscle (VSM) cells (23). From these results, we concluded that a cross-talk between Akt and Raf depends on the type of ligand and the cellular background or stage of differentiation. In MCF-7 cells high Raf activity induced cellular growth arrest by activating a cell-cycle inhibitor p21^cip1. Similarly, high Raf activity correlated with differentiation of VSM cells, where a biphasic ERK activity was induced. In contrast, high Raf kinase activity was expressed in undifferentiated proliferating myoblasts.

Our results show that the Raf-Akt cross-talk is regulated in a concentration- and ligand-dependent manner in MCF-7 cells. A possible explanation for the mechanism of this regulation is provided by the kinetics of activation of the two pathways.

High doses of mitogenic stimuli activate Akt in a fast and sufficiently strong way to down-regulate the Raf kinase activity (Figs. 2 and 5A). Here, spatial factors like co-localization of the two kinases may play an important role because both are recruited to the plasma membrane already early during activation (13, 40, 41). Because Raf kinase activation and recruitment to the plasma membrane occur at slightly later time points than that of Akt, Akt can counteract the Raf-1 kinase activation by directly phosphorylating its Ser-259, which inhibits the Raf-1 kinase and thereby prevents induction of cell-cycle inhibitors (21). Thus, under these conditions the Raf-Akt cross-talk prevents ERK-dependent growth arrest and shifts the biological response toward proliferation.

Low doses of mitogen cause a lower activity of Raf-1 and the ERK kinase activities, and the cross-talk does not take place. In this case, the Akt activation is delayed but strong enough to cause an albeit slower proliferation (Fig. 1). Because no cross-talk occurs, we postulate that the observed Akt activation is not potent enough to inhibit Raf-1 (Figs. 3 and 5B). Under these circumstances the Raf activation may not have been sufficient to induce cell-cycle inhibitors in an ERK-dependent manner.

PMA, a purely growth-arresting stimulus of MCF-7 cells, causes a dramatic difference in the kinetics of Raf-MEK-ERK and PI3K-Akt activities (Figs. 4 and 5C). Because Akt is only poorly activated it is not able to down-regulate the Raf-1 kinase activity and does not counteract the induction of cell-cycle inhibitors such as p21^cip1, which was induced by PMA within 30 min and is expressed for at least 3 days as was shown for MCF-7 cells previously (21). This appears to be a prerequisite for differentiation of MCF-7 cells.

Interestingly, the growth-arrested MCF-7 cells do not undergo apoptosis in the presence of differentiation-inducing ligands such as PMA. This may be due to the weak activation of PI3K/Akt and its weak anti-apoptotic potential. A similar situation is also observed in T-lymphocytes where protein kinase C activation also mediates survival through activation of PI3K in the absence of IL-2 for example (42, 43). These instances, however, seem to be dependent on the cell type, because Akt activation by phorbol esters is not observed in other cell lines such as NIH3T3, COS7, or HEK293 cells (15).²

The influence of PI3K on ERK activity is complex. Our findings indicate a dependence on concentration, ligand type, and time course. At high insulin concentrations the ERK activity is increased by inhibition of PI3K-Akt due to the Raf-Akt cross-talk. Yet PI3K can also have the opposite effect, namely inhibition of ERK activity by LY294002 (37, 38). This is demonstrated here as well, however, only at later time points when the ERK activity is becoming weaker (Fig. 5A, 60 and 90 min).

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Schematic summary of the kinetics of the Akt and ERK activities. A, high IGF-I concentrations cause strong and persistent Akt activity in MCF-7 cells, whereas activation of ERK peaks after about 10 min and then decreases. Inhibition of PI3K and Akt by LY294002 leads to a stronger ERK activity at early time points, but at later time points activation of ERK is inhibited. B, at low IGF-I concentrations Akt and ERK activity are low, and LY294002 reduces the ERK kinase activation at early time points. C, in growth-arrested MCF-7 cells treated with PMA, Akt activity is weak, whereas activation of ERK is strong and persistent. The LY294002 inhibitor does not influence the ERK activity. Akt activity was quantified by densitometric scanning. Activation of ERK was analyzed by in vitro kinase assays and quantified by PhosphorImager analysis. The activities are shown by fold activation. ERK activity, dashed line; ERK activity plus LY294002, bold line; Akt activity, dotted line.

The weak ERK activity under low IGF-I conditions also exhibits such a paradox, a reduction of ERK activity by LY294002 is shown in Figs. 3C and 5B with no cross-talk and no influence on the Raf-1 kinase by LY294002 (Fig. 3, A and B). This would imply a previously postulated "permissive" influence of PI3K on ERK, which has also been observed by others (31, 39, 44). PI3K may lead to ERK activation through Rac or PAK and by low or intermediate growth factor concentrations that activate ERK by Ras-Raf independent pathways (31) (Fig. 6). This would imply a positive influence of PI3K on downstream effectors of the Raf-1 kinase, a phenomenon that has also been detected by others (45).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   A model for the Akt-Raf interaction in MCF-7 cells. Phosphorylation of Raf by Akt leads to cross-talk and inhibition of the Ras-Raf-MEK-ERK cascade and induction of proliferation in the presence of high IGF-I concentration (thick arrows). LY294002 relieves this block and allows Raf to induce growth arrest (21). At low IGF-I concentration (thin arrows) no Akt-Raf cross-talk takes place. PMA directly affects Raf-MEK-ERK.

MCF-7 cells have also been studied with regard to signal transduction to the nucleus. The convergence of various signaling pathways on diverse transcriptional activators may contribute to the broad spectrum of cellular responses depending on the cell type.

Not only is the balance between Akt- and Raf-dependent signal transduction pathways relevant for the cellular response, additional signaling arms may have to be considered as well. One of them is the IB-NF-B-mediated pathway, which leads to repression of gene expression following stimulation with phorbol esters (46). Furthermore, matrix attachment may lead to signaling effects via an integrin-induced pathway, which can contribute to ERK phosphorylation (45).

In summary, we have shown that ligand-type, ligand concentration, intensity of signaling, and time courses contribute to Akt-Raf cross-talks possibly converging at the level of spatial proximity of the two kinases. Such antagonistic pathways may have evolved to ensure survival of cells and to protect them from a hyperactive, apoptosis-inducing Raf-1. Even retroviruses have evolved their own mechanism to down-tune Raf-1 kinase activity to ensure cell survival by fusion to gag, which results in a low specific activity of the kinase (47).

	ACKNOWLEDGEMENTS

The excellent technical assistance of A. Schauerte is gratefully acknowledged. We thank Dr. J. Heinrich for stimulating discussions and Dr. B. A. Hemmings for the generous supply of various plasmids.

	FOOTNOTES

To whom correspondence should be addressed: Institute of Medical Virology, Gloriastrasse 30, 8028 Zurich, Switzerland. Tel.: 41-1-634-2652; Fax: 41-1-634-4967; E-mail: moelling@immv.unizh.ch.

^§ Present address: Basilea Pharmaceutica Ltd., 4002 Basel, Switzerland.

^¶ Present address: UBS AG, 8001 Zurich, Switzerland.

Published, JBC Papers in Press, June 4, 2002, DOI 10.1074/jbc.M111974200

^* This work was supported by the Swiss National Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

² K. Moelling, K. Schad, M. Bosse, S. Zimmermann, and M. Schweneker, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: IGF-I, insulin-like growth factor I; ERK, extracellular signal-regulated kinase; IRS-1, insulin-receptor substrate 1; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MEK, mitogen-activated protein kinase/ERK kinase; PI3K, phosphatidylinositol 3-kinase; PKB/Akt, protein kinase B; PMA, phorbol 12-myristate 13-acetate; TPA, 12-O-tetradecanoylphorbol-13-acetate; VSM, vascular smooth muscle.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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