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Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

	Abstract

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The ERK MAP kinase and PI3-kinase/Akt pathways are major intracellular signaling modules, which are known to regulate diverse cellular processes including cell proliferation, survival and malignant transformation. However, it has not been fully understood how these two pathways interact with each other. Here, we demonstrate that inhibition of the ERK pathway by the MEK inhibitor U0126 or PD98059 significantly potentiates EGF- and FGF-induced Akt phosphorylation at both Thr308 and Ser473. We also show that hyperactivation of the ERK pathway greatly attenuates EGF- and FGF-induced Akt phosphorylation. Furthermore, the enhanced Akt phosphorylation induced by U0126 is inhibited by the PI3-kinase inhibitor LY294002, and is accompanied by the up-regulation of Ras activity. These results suggest that the ERK pathway inhibition enhances Akt phosphorylation through the Ras/PI3-kinase pathway. Thus, our results demonstrate that the ERK pathway negatively modulates the PI3-kinase/Akt pathway in response to growth factor stimulation.

	Introduction

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Extracellular signal-regulated kinase (ERK), also known as p42/p44 mitogen-activated protein (MAP) kinase, plays a pivotal role in regulation of diverse cellular processes, including cell proliferation, differentiation, migration and survival (Nishida & Gotoh 1993; Lewis et al. 1998; Chang & Karin 2001; Pearson et al. 2001). The ERK MAP kinase pathway is activated in response to a wide range of extracellular signals such as growth factors. When growth factors associate and activate receptor tyrosine kinases (RTKs), adaptor proteins such as Shc and Grb2 bind to RTKs. Then, the GDP/GTP exchange factor (GEF) for Ras, Son of Sevenless (Sos), which binds to Grb2, activates Ras at the plasma membrane. Ras activation triggers the sequential activation of Raf, the MAP kinase kinase MEK, and ERK. Activated ERK phosphorylates a number of substrates to regulate diverse cellular responses.

The phosphatidylinositol 3-kinase (PI3-kinase) pathway is also a major intracellular signaling module that regulates multiple cellular processes, including cell proliferation, survival, and cellular responses to insulin and nutrients (Fruman et al. 1998; Katso et al. 2001; Cantley 2002). Growth factors activate PI3 kinase via their specific RTKs, and activated PI3 kinase phosphorylates phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), converting PI(4,5)P2 to PI(3,4,5)P3, a major lipid second messenger (Czech 2003). PI(3,4,5)P3 binds to Akt, also known as protein kinase B (PKB), an important downstream effecter of the PI3-kinase pathway, and provokes a conformational change in Akt, which allows phosphorylation of Akt at Thr308 by 3-phosphoinositide-dependent protein kinase-1 (PDK1) (Vanhaesebroeck & Alessi 2000; Biondi 2004). In addition, Ser473 phosphorylation by PDK2 is also required for full activation of Akt, although the identity of PDK2 still remains controversial (Dong & Liu 2005). Akt phosphorylates numerous substrates that are involved in various cellular functions (Liang & Slingerland 2003; Fresno Vara et al. 2004).

As both the MEK/ERK and PI3-kinase/Akt pathways are responsible for the regulation of numerous biological processes, it is of great interest to know whether these two pathways interact with each other. In this context, there are several reports demonstrating that PI3 kinase plays an important role in activation of the MEK/ERK pathway (Sajan et al. 1999; Yart et al. 2001; Hayashi et al. 2006). It has also been reported that the GTP-bound active form of Ras activates PI3 kinase (Rodriguez-Viciana et al. 1994; Jimenez et al. 2002). Nevertheless, much remains to be studied about the signaling crosstalk between the MEK/ERK and PI3-kinase/Akt pathways.

In this study, we show that inhibition of the MEK/ERK pathway potentiates growth factor-induced phosphorylation of Akt, and that ERK activation results in attenuation of Akt phosphorylation induced by growth factor stimulation. We also show that PI3-kinase activity is required for the enhanced Akt phosphorylation induced by the inhibition of the MEK/ERK pathway. Moreover, this enhanced Akt phosphorylation is accompanied by enhanced activation of Ras. These results suggest that the MEK/ERK pathway can modulate Akt phosphorylation via the Ras/PI3-kinase pathway.

	Results

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Inhibition of the MEK/ERK cascade enhances growth factor-induced Akt phosphorylation

To investigate whether the activity of the ERK pathway affects the PI3-kinase/Akt pathway, we monitored Akt phosphorylation after FGF treatment of NIH3T3 cells in the presence of the MEK inhibitor, U0126 or PD98059. Compared with the control, the pretreatment of cells with U0126 markedly enhanced FGF-induced phosphorylation of Akt at both Thr308 and Ser473 (Fig. 1A). PD98059, which inhibits ERK phosphorylation to a lesser extent than U0126 under the conditions used, also enhanced Akt phosphorylation but to a lesser extent (Fig. 1A). These results indicate that the inhibition of ERK activation potentiates FGF-induced Akt phosphorylation in NIH3T3 cells. We next examined the effect of the inhibition of the MEK/ERK pathway on growth factor-induced Akt phosphorylation in more detail. NIH3T3 cells were pretreated with U0126 and then stimulated with FGF or EGF. Phosphorylation of Akt at both Thr308 and Ser473 were monitored up to 120 min after growth factor stimulation. As a result, FGF-induced phosphorylation of Akt at Thr308 and Ser473 were both enhanced markedly by U0126 (Fig. 1B). EGF-induced phosphorylation of Akt at both Thr308 and Ser473 were also markedly enhanced by U0126 (Fig. 1C). These results suggest that ERK activity has an inhibitory effect on growth factor-induced phosphorylation of Akt at both Thr308 and Ser473.

View larger version (40K): [in this window] [in a new window]   Figure 1  Inhibition of the MEK/ERK cascade potentiates growth factor-induced phosphorylation of Akt. (A) NIH3T3 cells pretreated with 20 µM U0126 or 100 µM PD98059 (at –30 min) were stimulated with 50 ng/mL FGF. Phosphorylation levels of ERK1/2 and those of Akt at Thr308 and Ser473 were analyzed by immunoblotting with the indicated antibodies. (B, C) NIH3T3 cells pretreated with 20 µM U0126 were stimulated with (B) 50 ng/mL FGF or (C) 30 nM EGF. Phosphorylation levels of ERK1/2 and Akt and protein levels of PTEN were analyzed. Three independent experiments gave similar results.

We then examined whether activation of the MEK/ERK pathway attenuates Akt phosphorylation induced by growth factor stimulation. We made use of the B-Raf:ER (estrogen receptor) NIH3T3 cells, in which B-Raf conjugated with ER is stably transfected (Pritchard et al. 1995). B-Raf is known to function as a specific activator of MEK. In B-Raf:ER cells, the addition of 4-hydroxytamoxifen (4-HT), an ER ligand, leads to immediate activation of ERK (Pritchard et al. 1995, Fig. 2A). As a control, we treated B-Raf:ER cells with FGF in the absence of 4-HT. Phosphorylation of Akt at Thr308 and Ser473 took place within 10 min (Fig. 2B, EtOH). Pretreatment of cells with 4-HT significantly attenuated the FGF-induced phosphorylation of Akt at both Thr308 and Ser473 (Fig. 2B, 4-HT). EGF-induced Akt phosphorylation at either Thr308 or Ser473 was also attenuated by pretreatment with 4-HT (Fig. 2C). These results indicate that growth factor-induced phosphorylation of Akt at Thr308 and Ser473 are down-regulated under conditions where the MEK/ERK pathway is activated.

View larger version (60K): [in this window] [in a new window]   Figure 2  Activation of the MEK/ERK cascade down-regulates growth factor-induced phosphorylation of Akt. (A) B-Raf:ER cells were treated with 1 µM 4-HT or solvent (EtOH) alone. ERK phosphorylation was analyzed by immunoblotting. (B, C) B-Raf:ER cells were pretreated with 1 µM 4-HT (at –20 min) and stimulated with (B) 50 ng/mL FGF or (C) 30 nM EGF. Phosphorylation levels of ERK1/2 and those of Akt at Thr308 and Ser473 were analyzed by immunoblotting with the indicated antibodies. Three independent experiments gave similar results.

It is intriguing to investigate the mechanisms underlying the MEK/ERK pathway-dependent modulation of Akt phosphorylation. We first examined whether de novo transcription is required for the up-regulation of Akt phosphorylation by the MEK inhibitor U0126. NIH3T3 cells were pretreated with U0126 and then stimulated with FGF in the presence or absence of actinomycin D, a general transcription inhibitor. As a result, U0126 pretreatment enhanced FGF-induced phosphorylation of Akt even in the presence of actinomycin D (Fig. 3). Actinomycin D alone did not affect the phosphorylation of Akt induced by FGF. These results indicate that de novo transcription is not required for the MEK/ERK pathway-dependent down-regulation of Akt phosphorylation.

View larger version (42K): [in this window] [in a new window]   Figure 3  Inhibition of de novo transcription does not affect U0126-induced up-regulation of Akt phosphorylation by FGF. NIH3T3 cells pretreated with 5 µg/mL actinomycin D, 20 µM U0126, or both reagents (at –30 min) were stimulated with 50 ng/mL FGF. Phosphorylation levels of ERK1/2 and those of Akt at Thr308 and Ser473 were analyzed by immunoblotting with the indicated antibodies. Two independent experiments gave similar results.

We next examined the possible involvement of PTEN in the MEK/ERK pathway-dependent down-regulation of growth factor-induced phosphorylation of Akt. PTEN is known to function as a negative regulator of the PI3-kinase/Akt pathway by dephosphorylating the lipid second messenger PI(3,4,5)P3 (Maehama & Dixon 1999). We monitored endogenous PTEN protein levels after growth factor stimulation of NIH3T3 cells in the presence or absence of U0126. The results show that PTEN protein levels are not affected by U0126 treatment (Fig. 1B,C), and thus indicate that the enhanced Akt phosphorylation by the MEK inhibition is not due to down-regulation of PTEN protein levels.

Inhibition of the MEK/ERK cascade results in the activation of Ras

It has been reported that the PI3-kinase/Akt pathway is activated by Ras in a GTP-dependent manner. Activated Ras binds to and activates the p110 catalytic subunit of PI3 kinase. So we hypothesized that the MEK/ERK pathway-dependent modulation of Akt phosphorylation is mediated by Ras. To investigate whether Ras activity is altered by the inhibition of the MEK/ERK cascade, NIH3T3 cells were treated with FGF in the presence or absence of U126 and then the amount of the GTP form of Ras was determined. The results show that U0126 pretreatment led to the strong enhancement of FGF-induced activation of Ras as well as Akt phosphorylation (Fig. 4). Similar results were obtained with EGF treatment (data not shown). These results demonstrate that the inhibition of the MEK/ERK cascade results in enhanced activation of Ras, and suggest that the activated Ras may mediate the MEK/ERK pathway inhibition-dependent enhancement of Akt phosphorylation through the activation of PI3 kinase.

View larger version (67K): [in this window] [in a new window]   Figure 4  Inhibition of the MEK/ERK cascade enhances FGF-induced Ras activation in parallel with Akt phosphorylation. NIH3T3 cells pretreated with 20 µM U0126 were stimulated with 50 ng/mL FGF. The GTP-bound active form of Ras was precipitated by using GST-RBD and the precipitate was immunoblotted with anti-Ras antibody. Phosphorylation levels of ERK1/2 and those of Akt at Thr308 and Ser473 were also analyzed by immunoblotting with the indicated antibodies. Similar results were obtained from two independent experiments.

There are several reports demonstrating that ERK phosphorylates Sos, induces its dissociation from Grb2, and thereby promotes inactivation of Ras (Rozakis-Adcock et al. 1995; Corbalan-Garcia et al. 1996; Porfiri & McCormick 1996; Foschi et al. 1997; Nakayama et al. 2008). To examine whether Sos mediates the MEK/ERK pathway-dependent down-regulation of Akt, we used a phosphorylation-sites mutant of Sos, Sos-4A, in which the four ERK phosphorylation sites, Ser1118, Ser1153, Ser1164 and Ser1179, were substituted with alanines. Sos-4A has been reported to be resistant to ERK-dependent dissociation of Sos from Grb2 (Corbalan-Garcia et al. 1996). NIH3T3 cells were co-transfected with HA-tagged Akt and either myc-tagged Sos-WT (wild-type) or myc-tagged Sos-4A. The cells were then treated with FGF, and phosphorylation of Akt was monitored. As a result, FGF-induced phosphorylation of Akt was enhanced by co-expression of Sos (Fig. 5). Importantly, Sos-4A had a stronger effect on the enhancement of Akt phosphorylation than Sos-WT (Fig. 5). This result strongly suggests that the MEK/ERK pathway-dependent down-regulation of Akt phosphorylation is mediated, at least in part, through ERK-mediated phosphorylation of Sos that leads to inactivation of Ras.

View larger version (29K): [in this window] [in a new window]   Figure 5 ERK-dependent phosphorylation of Sos leads to down-regulation of Akt phosphorylation NIH3T3 cells were co-transfected with HA-tagged Akt and either myc-tagged Sos-WT or 4A, and then treated with 25 ng/mL FGF HA-tagged Akt was immunoprecipitated by using anti-HA antibody and the precipitate was immunoblotted with the indicated antibodies The whole cell lysate was immunoblotted with anti-myc antibody.

Thus, it is likely that inhibition of the MEK/ERK cascade enhances Akt phosphorylation via the Sos/Ras/PI3-kinase pathway. It is, however, also possible that the inhibition of ERK activation potentiates Akt phosphorylation through other mechanism(s) not involving PI3 kinase. To verify that PI3-kinase activity is required for the enhanced Akt phosphorylation by the MEK inhibition, we utilized a specific PI3-kinase inhibitor LY294002. Pretreatment of cells with LY294002 almost completely suppressed FGF-induced phosphorylation of Akt either in the presence or absence of the MEK inhibitor U0126 (Fig. 6). These results indicate that PI3 kinase mediates the up-regulation of Akt phosphorylation induced by the inhibition of the MEK/ERK cascade.

View larger version (46K): [in this window] [in a new window]   Figure 6  LY294002 inhibits U0126-induced up-regulation of Akt phosphorylation by FGF. NIH3T3 cells pretreated with 20 µM LY294002, 20 µM U0126, or both reagents (at –30 min) were stimulated with 50 ng/mL FGF. Phosphorylation levels of ERK1/2 and those of Akt at Thr308 and Ser473 were analyzed by immunoblotting with the indicated antibodies. Similar results were obtained from two independent experiments.

	Discussion

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Both the ERK MAP kinase and PI3-kinase/Akt pathways play an important role in the regulation of cell proliferation and cell survival. It has been reported that ERK inhibits apoptosis by phosphorylating Bim and Mcl-1 (Reginato et al. 2003; Collins et al. 2005), and that ERK promotes cell cycle progression by inducing eventual phosphorylation of Rb. The PI3-kinse/Akt pathway also regulates cell survival by inhibiting apoptosis (Yao & Cooper 1995). Several factors such as GSK-3, GLUT4, p21/Waf1, and mTOR, which are involved in cell growth regulation, are reported to be the downstream targets of the PI3-kinase/Akt pathway (Osaki et al. 2004). Also in cell-cycle progression, it has been reported that both pathways are required for induction of cyclin D1 and initiation of DNA replication (Assoian & Schwartz 2001; Kim et al. 2004). Our results clearly demonstrate that growth factor-induced activation of the ERK MAP kinase pathway leads to immediate modulation of the PI3-kinase/Akt pathway that is also stimulated by growth factors. Although the physiological significance of this modulation remains to be elucidated, it is possible that ERK prevents excessive activation of the PI3-kinase/Akt pathway that may cause impairment of cellular functions. Another possibility is that the feedback regulation from ERK to Akt helps to set the direction of growth factor signaling to the proper and/or limited output response depending on the situation. There is a report demonstrating that the ERK MAP kinase and PI3-kinase/Akt pathways have distinct roles at distinct times in G2/M cell cycle progression (Roberts et al. 2002). The balance of activities of these two pathways may be important for the proper regulation of cell proliferation, and the ERK MAP kinase pathway may have a role in regulating the activity of the PI3-kinase/Akt pathway suitable for the appropriate cellular function under certain circumstances.

	Experimental procedures

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Cell culture and growth factor stimulation

NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum (CS) and maintained in 5% CO₂ at 37 °C. The cells were seeded in 35-mm culture dishes at a density of 1.5 x 10⁵ cells per dish. One day later, the medium was exchanged to DMEM without serum and the cells were incubated for 21 h before stimulation. B-Raf:ER cells were cultured in low-glucose DMEM (Gibco) containing 10% fetal bovine serum (FBS) and maintained in 5% CO₂ at 37 °C. The cells were seeded as with NIH3T3 cells. One day later, the medium was exchanged to DMEM containing 1% FBS and the cells were incubated for 42 h before stimulation. On growth factor stimulation, recombinant human EGF (BD Biosciences) or recombinant human basic FGF (R&D systems) was added to the medium and the cells were incubated for the indicated durations. Pretreatment with U0126 (Promega), PD98059 (Cell Signaling), LY294002 (Cell Signaling), or actinomycin D (Sigma) is performed 30 min before growth factor stimulation. Pretreatment with 4-HT (Sigma) is performed 20 min before growth factor stimulation.

Plasmids

The open reading frame of rat Akt was cloned into pSR-HA vector. Myc-tagged Sos-WT and Sos-4A were described previously (Nakayama et al. 2008).

Immunoblotting analysis

The cells were lysed in Laemmli sample buffer at the indicated time points and boiled for 10 min. After the cell lysates were subjected to SDS-PAGE, proteins were transferred to PVDF membrane (Immobilon-P; Millipore). Membranes were then incubated with the indicated antibodies: anti-Akt, anti-phospho Akt Thr308, anti-phospho Akt Ser473, anti-PTEN, anti-phospho-ERK1/2 antibodies (Cell Signaling); anti-ERK1 (K-23), anti-HA (Y-11), anti-myc (A-14) antibodies (Santacruz); anti-Ras antibody (Upstate). Immunoreactive bands were detected by the ECL Western blotting detection system (Amersham Corp.).

Detection of the GTP-bound active form of Ras

GTP-bound active form of Ras was quantified by detecting the amount of Ras capable of binding to Ras-binding domain (RBD) of Raf-1, basically as described previously (de Rooij & Bos 1997). Briefly, the cells were lysed in incubation buffer (25 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 5 mM NaF, 1 mM sodium pyrophosphate, 1% Nonidet P-40, 10% glycerol, 20 µg/mL aprotinin, 4 µg/mL leupeptin, 1 mM sodium orthovanadate), and the GTP-bound form of Ras was precipitated with GST-RBD coupled to glutathione-Sepharose beads. The precipitate was subjected to immunoblotting with anti-Ras antibody.

Transfection

NIH3T3 cells were seeded in 60-mm culture dishes at a density of 3.0 x 10⁵ cells per dish. One day later, cells were transfected with 250 ng of HA-tagged Akt and 1 µg of myc-tagged Sos-WT or 4A. The total amount of DNA per dish was adjusted to 1.25 µg by adding pcDNA3 empty vector. One day after transfection, culture medium was replaced to DMEM containing 0.1% CS, and then the cells were incubated for 24 h before FGF stimulation.

Immunoprecipitation

The cells were lysed in incubation buffer and cell lysates were centrifuged at 12 000 g for 20 min. The supernatant was then mixed and incubated with anti-HA antibody (16B12; Covance) and protein G-Sepharose beads (GE Healthcare) for 2 h at 4 °C. The beads were then washed twice with the incubation buffer. After resolution by SDS-PAGE, the precipitates were analyzed by immunoblotting.

	Acknowledgements

 
This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to E.N.).

	Footnotes

 
Communicated by: Yoshimi Takai

^aPresent address: Department of Medical Life Systems, Faculty of Life and Medical Sciences, Doshisha University, 1-3 Tatara Miyakodani, Kyotanabe City, Kyoto 606-0394, Japan

^* Correspondence: ytsuchiy{at}mail.doshisha.ac.jp or L50174{at}sakura.kudpc.kyoto-u.ac.jp

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Received: 13 May 2008
Accepted: 15 June 2008

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