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Endocrinology, doi:10.1210/en.2006-0325
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Endocrinology Vol. 147, No. 12 6036-6045
Copyright © 2006 by The Endocrine Society

Regulation of the RAP1/RAF-1/Extracellularly Regulated Kinase-1/2 Cascade and Prolactin Release by the Phosphoinositide 3-Kinase/AKT Pathway in Pituitary Cells

David Romano, Morgane Pertuit, Ramahefarizo Rasolonjanahary, Jean-Vianney Barnier, Karine Magalon, Alain Enjalbert and Corinne Gerard

Laboratoire Interactions Cellulaires Neuroendocriniennes (D.R., M.P., R.R., K.M., A.E., C.G.), Unité Mixte de Recherche 6544, Institut Fédératif de Recherche Jean-Roche, Faculté de Médecine Nord, 13916 Marseille cedex 20, France; and Laboratoire de Neurobiologie Cellulaire et Moléculaire (J.-V.B.), 91198 Gif-sur-Yvette, France

Address all correspondence and requests for reprints to: David Romano, Laboratoire Interactions Cellulaires Neuroendocriniennes, Unité Mixte de Recherche 6544, Institut Fédératif de Recherche Jean-Roche, Faculté de Médecine Nord, 13916 Marseille cedex 20, France. E-mail: romano.d{at}nord.univ-mrs.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In pituitary cells, prolactin (PRL) synthesis and release are controlled by multiple transduction pathways. In the GH4C1 somatolactotroph cell line, we previously reported that MAPK ERK-1/2 are a point of convergence between the pathways involved in the PRL gene regulation. In the present study, we focused on the involvement of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the MAPK ERK-1/2 regulation and PRL secretion in pituitary cells. Either specific pharmacological PI3K and Akt inhibitors (LY294002, Akt I, and phosphoinositide analog-6) or Akt dominant-negative mutant (K179M) enhanced ERK-1/2 phosphorylation in unstimulated GH4C1 cells. Under the same conditions, PI3K and Akt inhibition also both increased Raf-1 kinase activity and the levels of GTP-bound (active form) monomeric G protein Rap1, which suggests that a down-regulation of the ERK-1/2 cascade is induced by the PI3K/Akt signaling pathway in unstimulated cells. On the contrary, ERK-1/2 phosphorylation, Raf-1 activity, and Rap1 activation were almost completely blocked in IGF-I-stimulated cells previously subjected to PI3K or Akt inhibition. Although the PRL promoter was not affected by either PI3K/Akt inhibition or activation, PRL release increased in response to the pharmacological PI3K/Akt inhibitors in unstimulated GH4C1 and rat pituitary primary cells. The IGF-I-stimulated PRL secretion was diminished, on the contrary, by the pharmacological PI3K/Akt inhibitors. Taken together, these findings indicate that the PI3K/Akt pathway exerts dual regulatory effects on both the Rap1/Raf-1/ERK-1/2 cascade and PRL release in pituitary cells, i.e. negative effects in unstimulated cells and positive ones in IGF-I-stimulated cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A WIDE RANGE of extracellular stimuli can activate the MAPK rapidly growing fibrosarcoma (Raf)/MAPK kinase (MEK)/ERK pathway, which transduces gene transcription, differentiative, and proliferative signals (1, 2). The signaling pathways running from activated membrane receptors to ERK-1/2 have been thoroughly studied, and the small GTPase Ras superfamily is known to play a central role in this network (3, 4). Once they have been activated, small G proteins recruit the serine/threonine kinases Raf and facilitate their activation (5), and then Raf phosphorylates and stimulates the downstream kinase MEK, which in turn exhibits a serine/threonine and tyrosine kinase activity, resulting in the phosphorylation and activation of ERK-1/2 (6).

The phosphoinositide 3-kinase (PI3K)/Akt pathway is also activated by many types of extracellular stimuli and regulates fundamental cellular functions, such as cell proliferation, growth, and survival (7, 8). PI3Ks are a family of proteins that phosphorylate phosphoinositides (9). The resulting lipid products (phosphoinositide 3-phosphate) act as second messengers and mediate most of the known cellular functions of PI3Ks. These lipids regulate the location and/or activity of a number of target proteins downstream of the PI3Ks. One such protein is the serine/threonine kinase Akt (also named protein kinase B). For Akt to be completely activated, its activation domain (Thr308) and hydrophobic motif (Ser473) both have to be phosphorylated (10, 11). Activated Akt has many cellular effects, which are mediated by the phosphorylation of downstream targets involved in apoptotic mechanisms, cell cycle progression, and the control of gene expression (8).

Several reports have described the effects of the PI3K pathway on ERK-1/2 activation, which seem to depend on the cell type and the stimulus (12). Some studies indicate that ERK-1/2 activation is PI3K/Akt dependent (13, 14, 15, 16, 17), whereas the activated PI3K/Akt pathway has also been found to be involved in the inhibition of ERKs (18, 19, 20). In addition, the PI3K/Akt pathway regulates the ERK cascade by triggering Raf-1 (21, 22, 23, 24), B-Raf (25), or MEK (26) to regulate cell survival (13, 14), proliferation, and differentiation (27, 28).

Pituitary cell lines, which have conserved their differentiation potential, are useful and reliable tools for studying the molecular mechanisms underlying the complex regulation of pituitary functions. We recently reported that, in the GH4C1 rat pituitary cell line, ERK-1/2 activation serves as a point of convergence for PRL gene regulation by neuropeptides [vasoactive intestinal polypeptide (VIP), pituitary adenylyl cyclase activating polypeptide, TRH] and growth factors [epidermal growth factor (EGF)]. In addition, we provide evidence that both monomeric G proteins Ras and Rap1 (Ras proximate) play a key differential role in this process (29). PRL gene regulation by IGF-I via Ets transcription factor also requires ERK-1/2 activation in GH4C1 cells (30). Besides ERK-1/2 activation, IGF-I has been found to stimulate the PI3K/Akt pathway in rat pituitary primary cells (31). Moreover, IGF-I has been found to stimulate prolactin (PRL) release via an ERK-dependent mechanism and to inhibit GH release via a PI3K- and ERK-dependent mechanism in teleost pituitary primary cells (32).

Although the cross talk between protein kinase C (PKC) or protein kinase A (PKA) pathways and the ERK-1/2 cascade has been extensively studied in pituitary cells (29, 33, 34) and seems to play a decisive role in the physiological function of pituitary cells, the cross talk between PI3K/Akt and ERK-1/2 pathways has never been explored. In this study, we focused on the interactions between these two pathways in somatolactotroph GH4C1 cells as well as on their putative physiological consequences. The results obtained here show that pharmacological inhibition of the PI3K/Akt pathway increased ERK activation in unstimulated GH4C1 cells. Similar effects were also observed concerning Raf-1 kinase activity, but not B-Raf activity, and the GTP-bound levels of Rap1, but not Ras. PRL release was also enhanced when the PI3K/Akt pathway was inhibited in GH4C1 and pituitary primary cells, whereas the IGF-I-induced activation of Rap1, Raf-1, ERK, and PRL release were blocked by inhibiting PI3K/Akt. This study therefore shows for the first time that the PI3K/Akt pathway has opposite effects in pituitary cells on ERK activation and a physiological response (PRL secretion), depending on the level of stimulation to which the PI3K/Akt pathway is exposed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult female Wistar rats (200–250 g) were obtained from Charles River Breeding Laboratories (L’Arbresle, France) and housed in a controlled environment with food and water available ad libitum. Animals manipulations were performed according to the recommendations of the French Ethical Committee and under the supervision of authorized investigators.

Materials
IGF-I was purchased from AbCys (Paris, France). Bacitracin, Nonidet-P40 (NP40), LY294002, Akt inhibitor (Akt I), and phosphoinositide analog-6 (PIA6) were from Calbiochem (VWR International, Strasbourg, France). TransFast transfection reagent, EGF, and MEK inhibitor U0126 were from Promega (Charbonnier, France). Protein A agarose beads were from Invitrogen (Cergy-Pontoise, France). Glutathione agarose beads, insulin, and all other reagents were purchased from Sigma-Aldrich (Meylan, France). LY294002, Akt I, and PIA6 were solubilized in dimethylsulfoxide at concentrations of x10,000, x1,000, and x1,000, respectively. The vehicle had no effect on either kinase activation or the level of PRL expression or release at these final concentrations used.

Cell culture
The somatolactotroph GH4C1 cell line (a generous gift from Dr. A. Sobel, France) grown in Ham’s F10 medium supplemented with 15% horse serum (Eurobio, Les Ulis, France), 2.5% fetal calf serum (Invitrogen), penicillin (50 U/ml), and streptomycin (50 µg/ml) was maintained at 37 C in water-saturated atmosphere containing 7% CO2. Cells were weekly subcultured and 4–5 d after the last passage, cells were serum starved in Ham’s F10 medium for 15 h before each experiment.

Anterior pituitaries were dissected rapidly after rat decapitation and dispersed as previously described (35). Cells were plated in 24-well plates for PRL release experiments (0.2 x 106/well) and maintained in culture in DMEM supplemented with 10% fetal calf serum, glutamine (2 mM), and antibiotics (penicillin and streptomycin, 0.05 mg/ml) for 5 d. Cells were serum starved in DMEM for 15 h before each experiment.

Endogenous ERK and Akt activation assay
GH4C1 cells grown in vitro for 5 d in 6-well tissue culture plates were incubated as described in the figure legends. Cells were solubilized at 4 C for 20 min in a lysis buffer [25 mM Tris (pH 7.4), 150 mM NaCl, 1% NP40, 0.25% deoxycholate (DOC), 1 mM EGTA, 1 mM 4-(2-aminoethyl)benzene sulfonyl fluoride (AEBSF), 1 mM sodium orthovanadate (Na3VO4), 1 mM sodium fluoride (NaF), 10 µg/ml leupeptin, and aprotinin]. Lysates were clarified by centrifugation at 10,000 x g for 20 min at 4 C. Forty micrograms of denatured proteins [determined using the Bio-Rad DC protein assay (Bio-Rad, Ivry-sur-Seine, France)] were resuspended in Laemmli’s sample buffer, separated on 10% SDS-PAGE, and transferred onto polyvinyl difluoride (PVDF) membrane (PerkinElmer, Courtaboeuf, France). Immunodetection of phosphorylated forms of ERK-1/2 and Akt was performed using a rabbit polyclonal double-phospho-specific MAPK and phospho-Ser473-Akt antiserum (Cell Signaling, Ozyme, France), respectively, and antirabbit IgG coupled to alkaline phosphatase as the secondary antibody. Blots were developed with the enhanced chemiluminescence Western-Star detection system (Tropix, Applera, France) and quantified with a GeneGnome (Ozyme, Yvelines, France). In all the described experiments, the total ERK-1/2 and/or Akt protein content was systematically monitored by reprobing the membrane using a rabbit polyclonal ERK-1 antiserum (Santa Cruz Biotechnology, Tebu, France) and/or a rabbit polyclonal Akt antiserum (Cell Signaling), respectively.

ERK activation assay using a transient expression system
GH4C1 cells grown in vitro for 2 d in 6-well tissue culture plates were cotransfected with 2.5 µg pCDNAI/hemagglutinin (HA)-tagged ERK1 (36) combined with 2 µg pCMV6/HA-AktK179M (a dominant negative mutant) or 2 µg of empty vector (pCMV6) using the TransFast transfection reagent according to the manufacturer’s instructions (Promega). Forty-eight hours after the transfection, serum-starved cells were treated and solubilized as described above. Denatured proteins (40 µg) were separated on 10% SDS-PAGE and immunodetection of phosphorylated HA-ERK-1 was performed as described above. In all the described experiments, the expression levels of HA-ERK-1 and HA-AktK179M were assayed using an anti-HA antibody (12CA5, Roche, Meylan, France).

In vitro Raf kinase assay and determination of Raf-1 phosphorylation
Raf-1 and B-Raf activities were measured by performing in vitro kinase assays (37, 38). Treated cells were solubilized in the following lysis buffers: 20 mM Tris (pH 8), 136 mM NaCl, 10% glycerol, 1% NP40, 0.1% sodium dodecyl sulfate (SDS), 0.5% DOC, 2 mM EDTA, 1 mM AEBSF, 2 mM Na3VO4, 10 µg/ml leupeptin and aprotinin in the case of Raf-1 and 50 mM Tris (pH 8), 100 mM NaCl, 1% Triton X-100, 5 mM EDTA, 10 mM sodium pyrophosphate, 1 mM AEBSF, 1 mM Na3VO4, 1 mM benzamidine, 1 mM dithiotreitol (DTT), 10 µg/ml leupeptin and aprotinin in that of B-Raf. Six hundred or 300 µg of total proteins from cleared lysates supernatants were incubated for 1.5 h at 4 C with specific rabbit polyclonal Raf-1 or B-Raf antibody (Santa Cruz Biotechnology), respectively, and precipitated using protein A agarose beads. Beads were washed four times in the respective lysis buffers (without SDS and DOC in the case of Raf-1) and then once in kinase assay buffer [30 mM HEPES (pH 7.5), 7 mM MnCl2, 5 mM MgCl2, 1 mM DTT] in the case of Raf-1 and buffer [40 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM DTT, 10 mM paranitrophenylphosphate] in that of B-Raf. Kinase assays were performed for 20 min at 25 C using a MEK-kinase-deficient (39) recombinant protein as the substrate and 15 and 25 µM {gamma}32P-ATP (PerkinElmer) with Raf-1 and B-Raf, respectively. Reactions were stopped by adding Laemmli’s sample buffer. Denatured samples were separated on 10% SDS-PAGE, and 32P-MEK was determined and quantified after performing autoradiographic imaging using a Molecular Imager (Bio-Rad). To determine Raf-1 phosphorylation, an immunoprecipitation assay was performed as described above. Beads were resuspended in Laemmli’s sample buffer, denatured samples were separated on 10% SDS-PAGE, and immunodetection of the phosphorylated form of Raf-1 was carried out using a rabbit polyclonal phospho-Ser338-Raf-1 antibody (Upstate, Euromedex, Souffelweyersheim, France).

Ras and Rap1 activation assay
Ras and Rap1 GTP loading was measured by performing pull-down experiments as described previously (29), using the fusion proteins glutathione-S-transferase (GST)-Raf1-Ras-binding domain (RBD) and GST-Ral-guanine dissociation stimulator (GDS)-RBD (40, 41). Treated cells were solubilized as described above in the respective lysis buffers: [50 mM Tris (pH 8), 150 mM NaCl, 10% glycerol, 1% NP40, 0.1% SDS, 0.5% DOC, 1 mM Na3VO4, 1 mM AEBSF, 1 mM benzamidine, 2 mM MgCl2, 10 µg/ml leupeptin and aprotinin] in the case of Ras-GTP determination and (50 mM Tris (pH 8), 200 mM NaCl, 10% glycerol, 1% NP40, 2 mM Na3VO4, 1 mM AEBSF, 1 mM benzamidine, 2.5 mM MgCl2, 10 µg/ml leupeptin and aprotinin) in that of Rap1-GTP determination. One or 2 mg of total proteins, respectively, from cleared lysate supernatants were incubated for 1 h at 4 C with glutathione-agarose beads freshly coupled to GST-Raf-1RBD to isolate Ras-GTP or to GST-RalGDS-RBD to isolate Rap1-GTP. Beads were washed four times in the respective lysis buffers (without SDS and DOC in the case of Ras), denatured, and separated on 12% SDS-PAGE. Immunodetection of Ras and Rap1 was performed using mouse monoclonal anti-Ras and anti-Rap1 antibodies (Transduction Laboratory, Interchim, France). In all the described experiments, the total of Ras or Rap1 content and the phosphorylation status of ERK-1/2 were systematically controlled.

Rat (r) PRL promoter assay and determination of hormone release
PRL promoter activity was assayed as described previously (29). An rPRL reporter construct pGL3 (–270rPRL) containing the sequence corresponding to the –270 to +60 region of the rat PRL gene was obtained by PCR and inserted upstream of the firefly luciferase coding sequence in the pGL3 vector (Promega). GH4C1 cells, grown in vitro in 24-well tissue culture plates for 2 d, were cotransfected with 100 ng of the pGL3 (–270rPRL) vector and 4 ng of the renilla luciferase reporter vector phRL-TK (Promega) as the internal standard, using Transfast reagent, according to the manufacturer’s instructions. Forty-eight hours after the transfection, serum-starved cells were treated for 6 h in Ham’s F10 medium containing 10–5 M bacitracin and the substances to be tested as described in the legends, after which cells were washed, lysed, and analyzed to determine the luciferase activities according to the manufacturer’s instructions (Promega).

GH4C1 cells and primary cells were plated in 24-well tissue dishes for 5 d before being treated for the secretion experiments. Serum-starved cells were treated for 6 h in Ham’s F10 or DMEM medium containing 10–5 M bacitracin and the substances to be tested, as described in the figure legends. At the end of the incubation, the culture medium was recovered and centrifuged at 400 x g. The supernatant was stored at –80 C before the PRL RIA (42). PRL RIA reagents were a generous gift from Dr. A. Parlow (National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, CA).

Data and statistical analysis
LY294002, Akt I, PIA6, and the expression of Akt mutant affect the baseline level of the following: 1) endogenous ERK-1/2 and HA-ERK-1 phosphorylation, 2) the Rap1-GTP levels, 3) the Raf-1 kinase activity, and 4) the level of PRL release in serum-starved control cells. The data obtained on the specific effects of the pharmacological inhibitors or Akt mutant on cells treated by IGF-I are expressed as percentages of the maximum effect of IGF-I remaining in the presence of LY294002, Akt I, PIA6, or Akt mutant, in line with previous studies (29). Experimental values were first expressed as percentages of control conditions (untreated cells in the absence of pharmacological treatment or mutant), and the following calculations were performed: (Tx-Ux) x 100/MT0-MU0, where Tx and Ux are the respective values obtained with treated and untreated cells in the presence of x quantity of inhibitory factor and MT0 and MU0 are maximum values obtained with treated and untreated cells, respectively, in the absence of inhibitory factor. Experiments were performed at least in triplicate. Data shown in the figures and tables either were obtained in representative experiments or were means of quadriplicate determinations, as stated in the legends. Statistical analysis was performed using the t test, with P < 0.05 denoting significant differences.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGF-I activates both ERK-1/2 and PI3K/Akt pathways in GH4C1 cells
IGF-I is known to bind to tyrosine kinase receptor and activate intracellular pathways, such as the MAPK ERK-1/2 and the PI3K cascades. We first analyzed the activation of these two pathways by IGF-I in the GH4C1 somatolactotroph cell line. A dose-dependent increase in phospho-ERK-1/2 (PERK-1/2) was found to be induced by IGF-I, reaching a maximum at 10 nM (Fig. 1AGo, upper panel). Akt phosphorylation at Ser473 (pSer473) was also dose-dependently increased by IGF-I (Fig. 1BGo, upper panel). This phosphorylation was sustained because the pSer473 level was still 45% of maximum after 60 min (data not shown). Moreover, pSer473 was completely abolished by 5 µM LY294002 (LY), a specific PI3K inhibitor, in both unstimulated and IGF-I-stimulated cells (Fig. 1BGo, upper panel). Finally, we observed that Akt was not phosphorylated by VIP, TRH or EGF (Fig. 1CGo, upper panel), which are known to induce ERK-1/2 phosphorylation and regulate pituitary functions. The fact that no change occurred in the total levels of the ERK-1/2 and Akt proteins with any of the treatments applied suggests that the increase in the phosphorylation rate was not due to a change in the total level of these kinases (Fig. 1Go, A–C, lower panels).


Figure 1
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  		FIG. 1. Activation of ERK and PI3K/Akt cascades by IGF-I in GH4C1 cells. A, Serum-starved GH4C1 cells were incubated for 5 min in the presence and absence of increasing concentrations of IGF-I as indicated. Forty micrograms of cell lysates were resolved on a denaturing 10% polyacrylamide gel before being transferred onto PVDF membrane. PERK-1/2 was detected using a phospho-specific polyclonal antibody (upper panel) and total ERK-1/2 was recognized by a specific polyclonal antibody (lower panel). B, Serum-starved GH4C1 cells were preincubated for 30 min with and without 5 µM LY294002 (LY) and incubated for 5 min in the presence and absence of IGF-I at the doses indicated with and without LY. Forty micrograms of cell lysates were resolved on a denaturing 10% polyacrylamide gel and transferred onto PVDF membrane. Phospho-Ser473Akt was detected using a phospho-specific polyclonal antibody (upper panel), and total Akt was recognized by a specific polyclonal antibody (lower panel). C, Serum-starved GH4C1 cells were incubated for 5 min in the presence and absence (control, C) of IGF-I (10 nM), VIP (0.1 µM), TRH (1 µM), or EGF (1 nM). Phospho-Ser473Akt (upper panel) and total Akt (lower panel) were detected as described in B. One representative experiment is shown here of at least three independent determinations performed under each condition.

 
Cross talk between PI3K/Akt and ERK-1/2 pathways
To determine the effects of the PI3K/Akt pathway on ERK-1/2 activation, GH4C1 cells were treated with increasing doses of LY294002. As shown in Fig. 2AGo, in unstimulated cells, ERK-1 (Fig. 2AGo, empty squares) and ERK-2 (Fig. 2AGo, full squares) phosphorylation was dose-dependently enhanced by the PI3K inhibitor, reaching a maximum 5-fold activation at the highest dose (5 µM). In view of the low LY294002 doses used in these experiments, it seems most unlikely that the pharmacological inhibitor may have had any nonspecific effects. The possible involvement of Akt was tested using two other pharmacological agents, the Akt inhibitor (Akt I, 10 µM) (43) and the PIA6 (10 µM) (44). As with LY294002, both inhibitors again enhanced the PERK-1 (Fig. 2BGo, white bars) and PERK-2 (Fig. 2BGo, black bars) levels in unstimulated cells. These results indicate that the PI3K/Akt pathway exerts an inhibitory role on the ERK-1/2 activation levels in unstimulated GH4C1 cells.


Figure 2
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  		FIG. 2. Increase in ERK-1/2 phosphorylation in response to pharmacological inhibition of PI3K and Akt in GH4C1 cells. Serum-starved GH4C1 cells were preincubated for 30 min with and without increasing concentrations of LY294002 (A), with LY294002 (LY, 5 µM), Akt inhibitor (Akt I, 10 µM), or PIA6 (10 µM) (B), and with and without LY294002 (LY, 5 µM) (C) and incubated for 5 min in the presence and absence of LY, Akt I, or PIA6 (A and B) or IGF-I (10 nM), VIP (0.1 µM), TRH (1 µM), and EGF (1 nM) (C) with and without LY294002 (LY, 5 µM). Cell lysates and Western blottings (phospho-ERK-1 and 2, empty and full squares and bars, respectively) were performed as described in Materials and Methods. Data were expressed as percentages of the control values (untreated cells) (A–C) and maximum activation (D) induced by each agonist as described in Materials and Methods. A and B, Values (means ± SEM) of independent determinations (n = 3) are given for each condition. **, P < 0.01 and ***, P < 0.001, compared with the control conditions in the absence of any treatment. C and D, A representative experiment is given.

 
The effects of the pharmacological inhibitors were then tested in IGF-I-stimulated cells. ERK-1/2 phosphorylation was only slightly increased by IGF-I after treatment with LY294002, compared with the effect of LY294002 alone, whereas VIP-, TRH-, and EGF-induced ERK-1/2 phosphorylation was enhanced after LY294002 treatment (Fig. 2CGo). Considering the effect of the pharmacological inhibitor in unstimulated cells, the specific effect of LY294002 treatment in agonist-stimulated cells (as described in Materials and Methods) is shown in Fig. 2DGo. IGF-I-induced ERK phosphorylation is strongly depressed by LY294002, whereas VIP-, TRH-, and EGF-induced ERK phosphorylation were not affected. Moreover, both Akt inhibitors (Akt I and PIA6) dramatically reduced IGF-I-induced ERK phosphorylation (Table 1Go). These results suggest that ERK-1/2 stimulation by IGF-I requires the PI3K/Akt pathway in the GH4C1 cell line, whereas this stimulation by VIP, TRH, and EGF is not dependent on the PI3K/Akt pathway. In addition, this cross talk seems to be ERK specific because other MAPK pathways, such as p38 MAPK and c-Jun N-terminal kinase, were not affected by either PI3K/Akt inhibition or IGF-I stimulation (data not shown).


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  		TABLE 1. Effects of PI3K/Akt inhibition on IGF-I-induced stimulation of ERK-1/2, Raf-1, and Rap1

 
Lastly, to directly determine whether Akt affects unstimulated and IGF-I-stimulated PERK-1/2 levels, we investigated ERK regulation in GH4C1 cells transiently cotransfected with a dominant-negative Akt mutant, Akt-K179M, and a tagged form of ERK-1, HA-ERK-1. We previously established that the chimeric HA-ERK-1 is functionally expressed in GH4C1 cells (29). The use of transfected ERK-1 improved the overall reproducibility of these assays. HA-ERK-1 is activated by IGF-I because a similar increase occurred in the phosphorylated form of HA-ERK-1 and in the endogenous kinase ERK-1 after IGF-I treatment (Fig. 3Go, upper panel). In cells transfected with the catalytically inactive form Akt-K179M, a 2-fold increase in phosphorylated HA-ERK-1 was observed (Fig. 3Go, upper panel) in unstimulated cells, whereas HA-ERK-1 phosphorylation was weakly increased in the presence of Akt-K179M in cells treated with IGF-I, compared with the effect of the mutant alone (Fig. 3Go, upper panel). After quantification of the specific effect of Akt-K179M in IGF-I stimulated cells, IGF-I-induced phosphorylation of HA-ERK-1 was inhibited by 64 ± 15% (n = 3). Taken together, these results show the occurrence of cross talk between the PI3K/Akt signaling pathway and the ERK-1/2 cascade in GH4C1 cells, resulting in opposite effects: the PI3K/Akt pathway regulates ERK-1/2 phosphorylation negatively in unstimulated cells, whereas it is necessary for IGF-I-induced ERK-1/2 phosphorylation to occur.


Figure 3
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  		FIG. 3. Involvement of Akt in HA-ERK-1 regulation. GH4C1 cells were cotransfected with 2.5 µg cDNA of HA-ERK-1 and 2 µg cDNA of the dominant-negative Akt-K179M or the empty vector. Two days after transfection, serum-starved cells were incubated for 5 min in the presence and absence of IGF-I (10 nM). Cell lysates and Western blots were performed as described in Materials and Methods. Phosphorylated forms of HA-ERK-1 and endogenous ERK-1/2 were detected with the phospho-specific ERK-1/2 antibody (upper panel). Expression levels of Akt mutant and HA-tagged ERK-1 were detected in the presence of anti-HA monoclonal antibody 12CA5 (middle and lower panels, respectively).

 
The PI3K/Akt pathway regulates Raf-1 but not B-Raf
Previous studies have shown that the cross talk between the PI3K/Akt and ERK-1/2 pathways occurs at the level of Raf kinases (21, 24). To determine whether Raf kinases are involved in the regulation of ERK-1/2 phosphorylation by the PI3K/Akt pathway in somatolactotroph cells, in vitro kinase assays were performed on the two immunoprecipitated isoforms expressed in GH4C1 cells, Raf-1 and B-Raf. In unstimulated GH4C1 cells, PI3K and Akt inhibition by LY294002 (5 µM), Akt I (10 µM), and PIA6 (10 µM), respectively, resulted in an increase in Raf-1 kinase activity (Fig. 4Go, A and C, white bars). Under the same conditions, B-Raf kinase activity was not affected by any of these treatments (Fig. 4Go, B and C, gray bars). Interestingly, IGF-I (10 nM) enhanced Raf-1 kinase activity but not B-Raf kinase activity (Fig. 4CGo). Moreover, as indicated in Table 1Go, in the presence of the pharmacological PI3K/Akt inhibitors, IGF-I-stimulated Raf-1 kinase activity decreased dramatically. These findings were confirmed by analyzing the phosphorylation state of Raf-1. When fully activated, Raf-1 is phosphorylated at Ser338 residue (45). As indicated in Table 2Go, in unstimulated cells, Ser338 phosphorylation was enhanced in immunoprecipitated Raf-1 after treatment with LY294002 (5 µM), Akt I (10 µM), and PIA6 (10 µM), respectively, whereas IGF-I-induced Ser338 phosphorylation was blocked by LY294002, Akt I, and PIA6. These findings support the idea that ERK-1/2 cascade regulation by the PI3K/Akt pathway involves Raf-1 but not B-Raf.


Figure 4
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  		FIG. 4. Involvement of PI3K and Akt in endogenous Raf-1, but not B-Raf, kinase activity. Serum-starved GH4C1 cells were preincubated for 30 min with and without LY294002 (LY, 5 µM), Akt I (10 µM), or PIA6 (10 µM) before being incubated for 5 min in the presence and absence of IGF-I (10 nM) with and without LY, Akt I, or PIA6. Six hundred or 300 µg of cell lysates were immunoprecipitated with 2 µg of Raf-1 antibody (A and C, white bars) or 1 µg of B-Raf antibody (B and C, gray bars), respectively. Kinase assays were performed as described in Materials and Methods using a recombinant MEK-kinase-deficient agent as the substrate. C, Data obtained in three independent determinations under each condition, expressed as percentages of the control values (untreated cells), are means ± SEM (**, P < 0.01; ***, P < 0.001).

 

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  		TABLE 2. Effects of the PI3K/Akt pathway on Raf-1 phosphorylation at serine 338

 
The PI3K/Akt pathway regulates Rap1 GTPase but not Ras
In previous studies, we established that the differential recruitment of the GTPases Ras and Rap1 contributes decisively to ERK-1/2 activation in the GH4C1 somatolactotroph cell line (29). Because monomeric G proteins have been found to participate differentially in ERK-1/2 regulation by the PI3K/Akt pathway in PC12 cells (46), we investigated the effects of pharmacological PI3K and Akt inhibitors on the levels of the active Ras and Rap1 forms in somatolactotroph cells. To examine the GTP-bound status of Ras and Rap1, GST fusion proteins containing the RBD of Raf-1 and the Rap1-binding domain of RalGDS were used, respectively, as described previously (29, 40, 47). Cells were treated or not with IGF-I in the presence or absence of the PI3K/Akt pharmacological inhibitors, and pull-down experiments were carried out on cell lysates.

In unstimulated cells, the levels of the active Rap1 form (Rap1-GTP) increased in response to LY294002 (5 µM) and Akt I. (10 µM) treatments (Fig. 5Go, A, upper panel, and C, white bars), whereas the Ras-GTP level was not affected by either of these treatments (Fig. 5Go, B, upper panel, and C, gray bars). IGF-I also enhanced (2.5-fold) the Rap1-GTP level (Fig. 5Go, A, upper panel, and C, white bar), whereas its activatory effect on Ras was less pronounced (Fig. 5Go, B, upper panel, and C, gray bar). Finally, IGF-I-induced Rap1 activation was inhibited by both PI3K/Akt pharmacological inhibitors, whereas Ras activation was not significantly affected (Table 1Go). In all the experiments performed, similar total Ras and Rap1 protein levels were obtained in cell lysates with all the treatments tested (Fig. 5Go, A and B, lower panels). These results strongly suggest that Rap1 is involved in the regulation of ERK-1/2 by the PI3K/Akt pathway.


Figure 5
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  		FIG. 5. Involvement of PI3K and Akt in endogenous Rap1, but not Ras, activation. Serum-starved GH4C1 cells were preincubated for 30 min with and without LY294002 (LY, 5 µM) and Akt I (10 µM) and incubated for 5 min in the presence and absence of IGF-I (10 nM) with and without LY or Akt I. Two or 1 mg of cell lysates was incubated in the presence of GST-RalGDS-RBD or GST-Raf-1-RBD fusion protein coupled to glutathione-agarose beads to isolate Rap1-GTP (A and C, white bars) or Ras-GTP (B and C, gray bars), respectively. Isolated proteins were resolved on a denaturing polyacrylamide gel and transferred onto PVDF membrane. Rap1-GTP and Ras-GTP were detected with their respective monoclonal antibodies (A and B, upper panels). To check that total Rap1 and Ras were not affected by the various treatments, 40 µg of cell lysates were resolved on a denaturing polyacrylamide gel, transferred onto PVDF membrane, and the total proteins detected using the same monoclonal antibodies (A and B, lower panels). C, Data obtained by performing three independent determinations under each condition, expressed as percentages of the control values (untreated cells), are means ± SEM (*, P < 0.05; ***, P < 0.001).

 
Regulation of PRL transcription and secretion by the PI3K/Akt pathway
We previously established that the ERK-1/2 cascade is involved in PRL promoter regulation in somatolactotroph cell lines (29). Moreover, IGF-I is known to stimulate PRL release in pituitary primary cell cultures (32, 48). We therefore investigated whether PI3K/Akt signaling modulates PRL synthesis and release. To examine the effects of the PI3K/Akt pathway on PRL synthesis, GH4C1 cells were transiently transfected with the PRL proximal promoter-luciferase reporter construct. Luciferase activity was not found to be significantly affected by either IGF-I stimulation or PI3K/Akt inhibition, whereas it was reduced by the MEK inhibitor U0126 (Fig. 6AGo) as previously demonstrated (29). In unstimulated GH4C1 cells, PRL secretion was significantly enhanced by LY294002 (P < 0.001 vs. control cells), Akt I (P < 0.05), and PIA6 (P < 0.001) (Fig. 6BGo, gray bars). Similar results (P < 0.05) were observed in primary cells (Fig. 6BGo, striped bars). Moreover, the LY294002-induced PRL secretion was abolished (P < 0.01 vs. LY294002-treated cells) by the MEK inhibitor U0126 (Fig. 6BGo), suggesting that the PI3K inhibition induced an increase in PRL secretion by an ERK-dependent mechanism in unstimulated pituitary cells. IGF-I-stimulated PRL secretion (Fig. 6BGo) is strongly reduced by the pharmacological PI3K/Akt inhibitors (P < 0.001 vs. IGF-I-treated cells), the MEK inhibitor U0126 (P < 0.001), and both inhibitors (LY294002+U0126) added simultaneously (P < 0.001) in GH4C1 cells (Fig. 6CGo, gray bars). We also observed qualitatively similar results (P < 0.05) in IGF-I-stimulated primary cells (Fig. 6Go, B and C, striped bars). These results show that the regulation exerted by the PI3K/Akt pathway on the PRL secretion seems to involve the ERK-1/2 cascade.


Figure 6
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  		FIG. 6. Involvement of PI3K and Akt in PRL secretion but not PRL promoter activity. A, GH4C1 cells seeded into 24-well cell culture plates were cotransfected with 100 ng/well of pGL3(–270 rPRL) and 4 ng/well of phRL-TK. After 2 d in vitro, serum-starved cells were preincubated for 30 min with and without LY294002 (LY, 5 µM), Akt I (10 µM), or PIA6 (10 µM) and incubated for 6 h in the presence and absence of IGF-I (10 nM) with and without LY, Akt I, PIA6, or U0126 (10 µM). Luciferase activity was measured using a dual luciferase reporter assay system. B, To determine the PRL levels released, serum-starved GH4C1 cells (gray bars) and rat pituitary primary cells (striped bars) were preincubated for 30 min with and without LY294002 (LY, 5 µM), Akt I (10 µM), or PIA6 (10 µM) and incubated for 6 h in the presence and absence of IGF-I (10 nM) with and without U0126 (10 µM), LY, Akt I, or PIA6, and a PRL RIA was then performed. PRL levels in control media were 20.5 ± 0.5 and 221 ± 6 ng/well in GH4C1 and primary cells, respectively. Each assay was performed in quadriplicate. A representative experiment of at least three independent experiments for each condition is given. Data, expressed as percentages of the control values (A and B) or maximum effect induced by IGF-I treatment (C), are means ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study show the existence of a functional IGF-I sensitive PI3K/Akt pathway in the GH4C1 pituitary cell line, as previously observed in rat pituitary primary cells (31). In addition, it was established here for the first time that the mechanism whereby the PI3K/Akt pathway regulates the MAPK ERK-1/2 likely involves Rap1 GTPase and Raf-1 kinase. Cross talk between the PI3K/Akt and ERK-1/2 pathways has been studied in several cell types and seems to depend on the signal type and intensity (27). In some cells, the PI3K/Akt pathway is involved in maintaining the basal PERK-1/2 levels (13, 14). Here we observed, on the contrary, that in the GH4C1 cell line the inhibition of PI3K/Akt signaling by either specific pharmacological inhibitors (LY294002, Akt I, PIA6) or a dominant-negative mutant (Akt-K179M) enhances ERK-1/2 phosphorylation/activation in unstimulated cells, which suggests that the weakly activated PI3K/Akt pathway seems to constitutively inhibit the ERK-1/2 cascade in the GH4C1 cell line. By contrast, the finding that IGF-I-induced ERK-1/2 phosphorylation is blocked by PI3K/Akt inhibition suggests that strongly activated PI3K/Akt signaling plays a crucial role in the stimulation of the ERK-1/2 cascade by IGF-I in somatolactotroph cells. To our knowledge, this is the first time these opposite effects of the PI3K/Akt pathway, depending on its activation level, on the ERK-1/2 cascade have been reported. The fact that other MAPK pathways, such as p38 MAPK and JNK/stress-activated protein kinase, are not sensitive to PI3K/Akt inhibition or activation suggests that PI3K/Akt interacts specifically with the ERK-1/2 cascade. It was established in addition that Akt is not activated by EGF, VIP, and TRH (Fig. 1CGo), and ERK-1/2 activation by these agonists is not affected by PI3K/Akt inhibition (Fig. 2Go, C and D). The involvement of PI3K/Akt in IGF-I-induced ERK regulation therefore seems to be stimulus specific. Moreover, ERK regulation by IGF-I does not involve the PKC pathway because IGF-I-induced ERK phosphorylation is not affected by the PKC inhibitor bisindolylmaleimide I (data not shown), whereas we have previously shown that TRH-induced ERK phosphorylation is mediated by a Gq/PKC pathway and is blocked by the PKC inhibitor in GH4C1 cells (49).

Interactions between the PI3K/Akt and ERK-1/2 pathways have been found to occur, in particular at the level of Akt and Raf kinases (25, 50). Akt phosphorylates B-Raf in its amino-terminal regulatory domain and thus inhibits its activity (25). In addition, while this work was in progress, another group reported that mutations in the Akt phosphorylation motif of B-Raf increase its in vitro enzymatic activity (51). In the GH4C1 cell line, we observed that B-Raf activity is not affected by the PI3K/Akt pathway, whereas Raf-1 activity is regulated by the PI3K/Akt pathway in both the presence and absence of IGF-I. As previously found to occur in the human breast cancer cell line MCF-7, Raf-1 phosphorylation depends on the level of activation of Akt (24). In GH4C1 cells, weakly activated Akt suffices to abolish Raf-1 activity, probably via the inhibitory phosphorylation site Ser259, whereas Akt highly activated by IGF-I triggers Raf-1 activity by indirectly promoting phosphorylation of the activation site Ser338 (Table 2Go). The effects observed on ERK-1/2 phosphorylation may therefore be the downstream outcomes of a dual regulation of Raf-1 by the PI3K/Akt pathway.

In a previous study (29), we reported that Ras and Rap1 are differentially involved in the ERK-dependent regulation of the PRL gene in GH4C1 cells. In addition, Fernandez et al. (52) recently suggested that IGF-I-induced cAMP response element-dependent transcription may be mediated by the ERK pathway and may involve Rap1 in GH4C1 cells, as observed on overexpressing Rap1GAP1, which acts by maintaining Rap1 in its inactive GDP-bound form. In line with these results, it was established in the present study that IGF-I can directly and strongly activate Rap1 by enhancing the level of its GTP-bound form, whereas Ras is only weakly activated. In line with our own findings on ERK-1/2 and Raf-1 activation, inhibition of the PI3K/Akt pathway almost completely blocked IGF-I-induced Rap1 activation. Moreover, in unstimulated cells, as observed in the case of Raf-1 kinase activity and ERK-1/2 phosphorylation, PI3K/Akt inhibition increased the Rap1-GTP levels but had no effect on the Ras-GTP levels. Although the molecular mechanism underlying Rap1 regulation by the PI3K/Akt pathway has not yet been elucidated, the present findings show for the first time in a neuroendocrine model that the ERK-1/2 cascade may be regulated by PI3K/Akt signaling at a point upstream of Raf-1 kinase, involving the monomeric G protein Rap1. Interestingly, Liu et al. (53) recently established the requirement of the PI3K in Insulin-induced Ras/ERK activation by a PKC-independent pathway in several endocrine models. Our results show that both PI3K and its major effector Akt are involved in IGF-I-induced ERK activation and affect Rap1 activation, accounting for a specific signal induced by IGF-I distinct from the insulin pathway. It has been reported that Rap1 may be directly phosphorylated at Ser180 by cAMP-dependent protein kinase A (54). Rap1 has also been described as a suppressor of Ras-dependent Raf-1 activity because it sequesters Raf-1, and phosphorylation seems to affect its binding activity to Raf-1 (55). Indirectly, the phosphorylation of C3G (a Rap1-specific nucleotide exchange factor) and/or CrkII (the docking protein associated with C3G) may affect their mutual affinity and/or their affinity for Rap1 and thus influence the Rap1 GDP/GTP exchange cycle (56).

Interactions between ERK-1/2 and PI3K/Akt pathways have been thought to constitute a specific physiological response (27, 28). Both pathways are known to be involved in the regulation of pituitary functions such as promoter regulation and hormone secretion (29, 30, 32, 33, 34). Previous studies on various models, such as primary rat and fish anterior pituitary and human prolactinoma cell cultures, have suggested that IGF-I may be a specific secretagogue responsible for PRL release. It has by now clearly established that IGF-I stimulates PRL release without affecting its synthesis in pituitary cells (48, 57, 58, 59), and this effect seems to be an ERK-dependent mechanism (32). As previously reported in studies on human pituitary tumors (59) and rat pituitary cell lines (48), we did not detect any effect of either IGF-I or PI3K/Akt inhibition on the transcriptional activity of the PRL gene promoter coupled to a transiently transfected reporter gene (Fig. 6AGo) or the endogenous PRL mRNA levels (data not shown) in GH4C1 cells. However, in unstimulated GH4C1 and primary rat pituitary cells, PRL release was enhanced by PI3K/Akt inhibition. Moreover, the PI3K pathway regulated PRL secretion by an ERK-dependent mechanism because the LY294002-induced PRL secretion was reverted by the specific MEK inhibitor U0126 in both GH4C1 and primary cells.

It can therefore be concluded that, in the absence of any stimulus, the inhibitory effects of the PI3K/Akt pathway on the ERK-1/2 cascade affect PRL secretion. As observed in the case of the Rap1/Raf-1/ERK-1/2 cascade, IGF-I-stimulated PRL secretion was blocked by the pharmacological PI3K/Akt inhibitors. Consequently, the IGF-I-induced ERK activation and full PRL release require an intact and active PI3K/Akt pathway in pituitary cells. Moreover, this process seems to be highly IGF-I specific because other stimulating factors of PRL release such as VIP, pituitary adenylyl cyclase activating polypeptide, TRH, and EGF, which strongly activate the ERK-1/2 cascade in the GH4C1 cells (49), do not stimulate the PI3K/Akt pathway in these cells (Fig. 1CGo). Although these agonists also regulate PRL gene transcription via an ERK-dependent mechanism in GH4C1 cells (29), IGF-I has no effect on the PRL promoter activity, which suggests that the PI3K/Akt pathway may cross talk with restricted cytoplasmic ERK-1/2 pools via specific scaffolding proteins.

In other cell types, the negative regulation of the ERK-1/2 cascade exerted by the PI3K/Akt pathway has been described as a prodifferentiative effect at the expense of cellular proliferation (27, 28). However, the present data indicate that, in pituitary cells, a weak PI3K/Akt signal inhibits a differentiated pituitary function, i.e. PRL secretion. Because the PI3K/Akt pathway has been found to mediate prosurvival signals, this negative regulation of both ERK-1/2 cascade and PRL secretion may reflect the recruitment of the cellular components required to promote survival in the absence of any extracellular stimulus. The differentiative signals mediated by the MAPK ERK-1/2 cascade (29, 34) may therefore be switched off under these conditions and reactivated in response to a physiological stimulus such as IGF-I.


   Acknowledgments
 
We thank Dr. M. E. Greenberg and Dr. D. R. Kaplan (Children’s Hospital, Boston, MA, and Hospital for Sick Children, Toronto, Canada) for their gift of plasmid pCMV6-HA-AktK179M. We also thank Dr. J. L. Bos and M. Van Triest (University Medical Center Utrecht, Utrecht, The Netherlands) for their gift of plasmids containing GST-Raf1-RBD and GST-RalGDS-RBD and for providing the protocols to prepare the fusion proteins. pcDNA.I/HA-ERK1 was a generous gift from G. Pagès (Centre Antoine Lacassagne, Nice, France). We also thank Dr. I. Pellegrini-Bouiller for preparing the rPRL reporter construct pGL3(–270rPRL).


   Footnotes
 
This work was supported by the Centre National pour la Recherche Scientifique and grants from the Association pour le Développement des Recherches Biologiques et Médicales and the Fondation pour la Recherche Médicale.

Disclosure summary: D.R., M.P., R.R., J.-V.B., K.M., A.E. and C.G. have nothing to declare.

First Published Online August 24, 2006

Abbreviations: AEBSF, 4-(2-Aminoethyl)benzene sulfonyl fluoride; Akt I, Akt inhibitor; DOC, deoxycholate; DTT, dithiotreitol; EGF, epidermal growth factor; GDS, guanine dissociation stimulator; GST, glutathione-S-transferase; HA, hemagglutinin; MEK, MAPK kinase; NP40, Nonidet-P40; PERK, phospho-ERK; PIA6, phosphoinositide analog-6; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PRL, prolactin; r, rat; Raf, rapidly growing fibrosarcoma; Rap1, Ras proximate; PVDF, polyvinyl difluoride; RBD, Ras-binding domain; SDS, sodium dodecyl sulfate; VIP, vasoactive intestinal polypeptide.

Received March 13, 2006.

Accepted for publication August 14, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. O’Neill E, Kolch W 2004 Conferring specificity on the ubiquitous Raf/MEK signalling pathway. Br J Cancer 90:283–288[CrossRef][Medline]
  2. Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, Cobb MH 2001 Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22:153–183[Abstract/Free Full Text]
  3. Downward J 2003 Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer 3:11–22[CrossRef][Medline]
  4. Zwartkruis FJ, Bos JL 1999 Ras and Rap1: two highly related small GTPases with distinct function. Exp Cell Res 253:157–165[CrossRef][Medline]
  5. Kerkhoff E, Rapp UR 2001 The Ras-Raf relationship: an unfinished puzzle. Adv Enzyme Regul 41:261–267[CrossRef][Medline]
  6. Wellbrock C, Karasarides M, Marais R 2004 The RAF proteins take centre stage. Nat Rev Mol Cell Biol 5:875–885[CrossRef][Medline]
  7. Vivanco I, Sawyers CL 2002 The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat Rev Cancer 2:489–501[CrossRef][Medline]
  8. Yang ZZ, Tschopp O, Baudry A, Dummler B, Hynx D, Hemmings BA 2004 Physiological functions of protein kinase B/Akt. Biochem Soc Trans 32:350–354[CrossRef][Medline]
  9. Vanhaesebroeck B, Ali K, Bilancio A, Geering B, Foukas LC 2005 Signalling by PI3K isoforms: insights from gene-targeted mice. Trends Biochem Sci 30:194–204[CrossRef][Medline]
  10. Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, Hemmings BA 1996 Mechanism of activation of protein kinase B by insulin and IGF-I. EMBO J 15:6541–6551[Medline]
  11. Stokoe D, Stephens LR, Copeland T, Gaffney PR, Reese CB, Painter GF, Holmes AB, McCormick F, Hawkins PT 1997 Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. Science 277:567–570[Abstract/Free Full Text]
  12. Duckworth BC, Cantley LC 1997 Conditional inhibition of the mitogen-activated protein kinase cascade by wortmannin. Dependence on signal strength. J Biol Chem 272:27665–27670[Abstract/Free Full Text]
  13. Versteeg HH, Evertzen MW, van Deventer SJ, Peppelenbosch MP 2000 The role of phosphatidylinositide-3-kinase in basal mitogen-activated protein kinase activity and cell survival. FEBS Lett 465:69–73[CrossRef][Medline]
  14. Wennstrom S, Downward J 1999 Role of phosphoinositide 3-kinase in activation of ras and mitogen-activated protein kinase by epidermal growth factor. Mol Cell Biol 19:4279–4288[Abstract/Free Full Text]
  15. Kim SE, Cho JY, Kim KS, Lee SJ, Lee KH, Choi KY 2004 Drosophila PI3 kinase and Akt involved in insulin-stimulated proliferation and ERK pathway activation in Schneider cells. Cell Signal 16:1309–1317[CrossRef][Medline]
  16. Schmidt EK, Fichelson S, Feller SM 2004 PI3 kinase is important for Ras, MEK and Erk activation of Epo-stimulated human erythroid progenitors. BMC Biol 2:7[CrossRef][Medline]
  17. Yart A, Laffargue M, Mayeux P, Chretien S, Peres C, Tonks N, Roche S, Payrastre B, Chap H, Raynal P 2001 A critical role for phosphoinositide 3-kinase upstream of Gab1 and SHP2 in the activation of ras and mitogen-activated protein kinases by epidermal growth factor. J Biol Chem 276:8856–8864[Abstract/Free Full Text]
  18. Choi WS, Sung CK 2004 Inhibition of phosphatidylinositol-3-kinase enhances insulin stimulation of insulin receptor substrate 1 tyrosine phosphorylation and extracellular signal-regulated kinases in mouse R-fibroblasts. J Recept Signal Transduct Res 24:67–83[CrossRef][Medline]
  19. Laprise P, Langlois MJ, Boucher MJ, Jobin C, Rivard N 2004 Down-regulation of MEK/ERK signaling by E-cadherin-dependent PI3K/Akt pathway in differentiating intestinal epithelial cells. J Cell Physiol 199:32–39[CrossRef][Medline]
  20. van der Heide LP, Hoekman MF, Biessels GJ, Gispen WH 2003 Insulin inhibits extracellular regulated kinase 1/2 phosphorylation in a phosphatidylinositol 3-kinase (PI3) kinase-dependent manner in Neuro2a cells. J Neurochem 86:86–91[CrossRef][Medline]
  21. Chaudhary A, King WG, Mattaliano MD, Frost JA, Diaz B, Morrison DK, Cobb MH, Marshall MS, Brugge JS 2000 Phosphatidylinositol 3-kinase regulates Raf1 through Pak phosphorylation of serine 338. Curr Biol 10:551–554[CrossRef][Medline]
  22. Sun H, King AJ, Diaz HB, Marshall MS 2000 Regulation of the protein kinase Raf-1 by oncogenic Ras through phosphatidylinositol 3-kinase, Cdc42/Rac and Pak. Curr Biol 10:281–284[CrossRef][Medline]
  23. Zimmermann S, Moelling K 1999 Phosphorylation and regulation of Raf by Akt (protein kinase B). Science 286:1741–1744[Abstract/Free Full Text]
  24. Moelling K, Schad K, Bosse M, Zimmermann S, Schweneker M 2002 Regulation of Raf-Akt cross-talk. J Biol Chem 277:31099–31106[Abstract/Free Full Text]
  25. Guan KL, Figueroa C, Brtva TR, Zhu T, Taylor J, Barber TD, Vojtek AB 2000 Negative regulation of the serine/threonine kinase B-Raf by Akt. J Biol Chem 275:27354–27359[Abstract/Free Full Text]
  26. Bondeva T, Pirola L, Bulgarelli-Leva G, Rubio I, Wetzker R, Wymann MP 1998 Bifurcation of lipid and protein kinase signals of PI3K{gamma} to the protein kinases PKB and MAPK. Science 282:293–296[Abstract/Free Full Text]
  27. Reusch HP, Zimmermann S, Schaefer M, Paul M, Moelling K 2001 Regulation of Raf by Akt controls growth and differentiation in vascular smooth muscle cells. J Biol Chem 276:33630–33637[Abstract/Free Full Text]
  28. Rommel C, Clarke BA, Zimmermann S, Nunez L, Rossman R, Reid K, Moelling K, Yancopoulos GD, Glass DJ 1999 Differentiation stage-specific inhibition of the Raf-MEK-ERK pathway by Akt. Science 286:1738–1741[Abstract/Free Full Text]
  29. Romano D, Magalon K, Ciampini A, Talet C, Enjalbert A, Gerard C 2003 Differential involvement of the Ras and Rap1 small GTPases in vasoactive intestinal and pituitary adenylyl cyclase activating polypeptides control of the prolactin gene. J Biol Chem 278:51386–51394[Abstract/Free Full Text]
  30. Castillo AI, Tolon RM, Aranda A 1998 Insulin-like growth factor-1 stimulates rat prolactin gene expression by a Ras, ETS and phosphatidylinositol 3-kinase dependent mechanism. Oncogene 16:1981–1991[CrossRef][Medline]
  31. Fernandez M, Sanchez-Franco F, Palacios N, Sanchez I, Fernandez C, Cacicedo L 2004 IGF-I inhibits apoptosis through the activation of the phosphatidylinositol 3-kinase/Akt pathway in pituitary cells. J Mol Endocrinol 33:155–163[Abstract]
  32. Fruchtman S, Gift B, Howes B, Borski R 2001 Insulin-like growth factor-I augments prolactin and inhibits growth hormone release through distinct as well as overlapping cellular signaling pathways. Comp Biochem Physiol B Biochem Mol Biol 129:237–242[CrossRef][Medline]
  33. Ohmichi M, Sawada T, Kanda Y, Koike K, Hirota K, Miyake A, Saltiel AR 1994 Thyrotropin-releasing hormone stimulates MAP kinase activity in GH3 cells by divergent pathways. Evidence of a role for early tyrosine phosphorylation. J Biol Chem 269:3783–3788[Abstract/Free Full Text]
  34. Kievit P, Lauten JD, Maurer RA 2001 Analysis of the role of the mitogen-activated protein kinase in mediating cyclic-adenosine 3',5'-monophosphate effects on prolactin promoter activity. Mol Endocrinol 15:614–624[Abstract/Free Full Text]
  35. Rasolonjanahary R, Gerard C, Dufour MN, Homburger V, Enjalbert A, Guillon G 2002 Evidence for a direct negative coupling between dopamine-D2 receptors and PLC by heterotrimeric Gi1/2 proteins in rat anterior pituitary cell membranes. Endocrinology 143:747–754[Abstract/Free Full Text]
  36. Meloche S, Pages G, Pouyssegur J 1992 Functional expression and growth factor activation of an epitope-tagged p44 mitogen-activated protein kinase, p44mapk. Mol Biol Cell 3:63–71[Abstract]
  37. Morrison DK 1995 Activation of Raf-1 by Ras in intact cells. Methods Enzymol 255:301–310[Medline]
  38. Papin C, Eychene A, Brunet A, Pages G, Pouyssegur J, Calothy G, Barnier JV 1995 B-Raf protein isoforms interact with and phosphorylate Mek-1 on serine residues 218 and 222. Oncogene 10:1647–1651[Medline]
  39. Mansour SJ, Matten WT, Hermann AS, Candia JM, Rong S, Fukasawa K, Vande Woude GF, Ahn NG 1994 Transformation of mammalian cells by constitutively active MAP kinase kinase. Science 265:966–970[Abstract/Free Full Text]
  40. de Rooij J, Bos JL 1997 Minimal Ras-binding domain of Raf1 can be used as an activation-specific probe for Ras. Oncogene 14:623–625[CrossRef][Medline]
  41. Franke B, Akkerman JW, Bos JL 1997 Rapid Ca2+-mediated activation of Rap1 in human platelets. EMBO J 16:252–259[CrossRef][Medline]
  42. Niswender GD, Chen CL, Midgley Jr AR, Meites J, Ellis S 1969 Radioimmunoassay for rat prolactin. Proc Soc Exp Biol Med 130:793–797[Medline]
  43. Hu Y, Qiao L, Wang S, Rong SB, Meuillet EJ, Berggren M, Gallegos A, Powis G, Kozikowski AP 2000 3-(Hydroxymethyl)-bearing phosphatidylinositol ether lipid analogues and carbonate surrogates block PI3-K, Akt, and cancer cell growth. J Med Chem 43:3045–3051[CrossRef][Medline]
  44. Castillo SS, Brognard J, Petukhov PA, Zhang C, Tsurutani J, Granville CA, Li M, Jung M, West KA, Gills JG, Kozikowski AP, Dennis PA 2004 Preferential inhibition of Akt and killing of Akt-dependent cancer cells by rationally designed phosphatidylinositol ether lipid analogues. Cancer Res 64:2782–2792[Abstract/Free Full Text]
  45. Diaz B, Barnard D, Filson A, MacDonald S, King A, Marshall M 1997 Phosphorylation of Raf-1 serine 338-serine 339 is an essential regulatory event for Ras-dependent activation and biological signaling. Mol Cell Biol 17:4509–4516[Abstract]
  46. York RD, Molliver DC, Grewal SS, Stenberg PE, McCleskey EW, Stork PJ 2000 Role of phosphoinositide 3-kinase and endocytosis in nerve growth factor-induced extracellular signal-regulated kinase activation via Ras and Rap1. Mol Cell Biol 20:8069–8083[Abstract/Free Full Text]
  47. Herrmann C, Horn G, Spaargaren M, Wittinghofer A 1996 Differential interaction of the ras family GTP-binding proteins H-Ras, Rap1A, and R-Ras with the putative effector molecules Raf kinase and Ral-guanine nucleotide exchange factor. J Biol Chem 271:6794–6800[Abstract/Free Full Text]
  48. Lara JI, Lorenzo MJ, Cacicedo L, Tolon RM, Balsa JA, Lopez-Fernandez J, Sanchez-Franco F 1994 Induction of vasoactive intestinal peptide gene expression and prolactin secretion by insulin-like growth factor I in rat pituitary cells: evidence for an autoparacrine regulatory system. Endocrinology 135:2526–2532[Abstract]
  49. Le Pechon-Vallee C, Magalon K, Rasolonjanahary R, Enjalbert A, Gerard C 2000 Vasoactive intestinal polypeptide and pituitary adenylate cyclase-activating polypeptides stimulate mitogen-activated protein kinase in the pituitary cell line GH4C1 by a 3',5'-cyclic adenosine monophosphate pathway. Neuroendocrinology 72:46–56[CrossRef][Medline]
  50. Jun T, Gjoerup O, Roberts TM 1999 Tangled webs: evidence of cross-talk between c-Raf-1 and Akt. Sci STKE 1999:PE1
  51. Ikenoue T, Kanai F, Hikiba Y, Tanaka Y, Imamura J, Ohta M, Jazag A, Guleng B, Asaoka Y, Tateishi K, Kawakami T, Matsumura M, Kawabe T, Omata M 2005 Functional consequences of mutations in a putative Akt phosphorylation motif of B-raf in human cancers. Mol Carcinog 43:59–63[Medline]
  52. Fernandez M, Sanchez-Franco F, Palacios N, Sanchez I, Cacicedo L 2005 IGF-I and vasoactive intestinal peptide (VIP) regulate cAMP-response element-binding protein (CREB)-dependent transcription via the mitogen-activated protein kinase (MAPK) pathway in pituitary cells: requirement of Rap1. J Mol Endocrinol 34:699–712[Abstract/Free Full Text]
  53. Liu L, Xie Y, Lou L 2006 PI3K is required for insulin-stimulated but not EGF-stimulated ERK1/2 activation. Eur J Cell Biol 85:367–374[CrossRef][Medline]
  54. Lou L, Urbani J, Ribeiro-Neto F, Altschuler DL 2002 cAMP inhibition of Akt is mediated by activated and phosphorylated Rap1b. J Biol Chem 277:32799–32806[Abstract/Free Full Text]
  55. Hu CD, Kariya K, Okada T, Qi X, Song C, Kataoka T 1999 Effect of phosphorylation on activities of Rap1A to interact with Raf-1 and to suppress Ras-dependent Raf-1 activation. J Biol Chem 274:48–51[Abstract/Free Full Text]
  56. Quilliam LA, Rebhun JF, Castro AF 2002 A growing family of guanine nucleotide exchange factors is responsible for activation of Ras-family GTPases. Prog Nucleic Acids Res Mol Biol 71:391–444[Medline]
  57. Fruchtman S, Jackson L, Borski R 2000 Insulin-like growth factor I disparately regulates prolactin and growth hormone synthesis and secretion: studies using the teleost pituitary model. Endocrinology 141:2886–2894[Abstract/Free Full Text]
  58. Kajimura S, Uchida K, Yada T, Hirano T, Aida K, Gordon Grau E 2002 Effects of insulin-like growth factors (IGF-I and -II) on growth hormone and prolactin release and gene expression in euryhaline tilapia, Oreochromis mossambicus. Gen Comp Endocrinol 127:223–231[CrossRef][Medline]
  59. Atkin SL, Landolt AM, Foy P, Jeffreys RV, Hipkin L, White MC 1994 Effects of insulin-like growth factor-I on growth hormone and prolactin secretion and cell proliferation of human somatotrophinomas and prolactinomas in vitro. Clin Endocrinol (Oxf) 41:503–509[Medline]



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