help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Calleja, V.
Right arrow Articles by Van Obberghen, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Calleja, V.
Right arrow Articles by Van Obberghen, E.
Endocrinology Vol. 138, No. 3 1111-1120
Copyright © 1997 by The Endocrine Society

Articles

The Effect of Cyclic Adenosine Monophosphate on the Mitogen-Activated Protein Kinase Pathway Depends on Both the Cell Type and the Type of Tyrosine Kinase-Receptor1

Véronique Calleja, Pilar Ruiz Enríquez2, Chantal Filloux, Pascal Peraldi, Véronique Baron and Emmanuel Van Obberghen

INSERM U145, Faculté de Médecine, Avenue de Valombrose, 06107, Nice cedex 02, France

Address all correspondence and requests for reprints to: E. Van Obberghen, M.D., Ph.D., INSERM U145, Faculté de Médecine, Avenue de Valombrose, 06107 Nice Cedex 02, France. E-mail: vanobberg{at}unice.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mitogen-activated protein kinase (MAP kinase) is a key participant in growth factor-stimulated intracellular events such as proliferation and differentiation. We and others have previously described a cross-talk between the MAP kinase pathway and the cAMP pathway. Indeed, in several cell lines and, in particular in fibroblasts, an increase in the level of cAMP produced an inhibition of MAP kinase together with decreased cell proliferation. In contrast, in PC12 cells, cAMP induced an increase in the NGF-induced activation of MAP kinase concomitantly with augmented NGF-induced differentiation. Therefore, it has been proposed that the cellular context is important for the nature of the cAMP effects on growth factor-stimulated MAP kinase activity. Here we show that the type of tyrosine kinase receptor stimulated also participates in the nature of the cAMP effect. Thus, in NIH3T3 fibroblasts expressing NGF receptors (NIH3T3/trk cells) we found that cAMP potentiates NGF-stimulated ERK1 and MEK1 activities, whereas in NIH3T3 fibroblasts expressing insulin receptors (NIH3T3/IR cells) we saw no effect of cAMP on the activation of insulin-stimulated ERK1 and MEK1. In PC12 cells and in Rat1 fibroblasts expressing insulin receptors (PC12/IR and Rat1/IR cells) we observed, respectively, a potentiation and an inhibition of insulin-stimulated ERK1 activity. In addition, cAMP does not seem to modify the basal nor growth factor-stimulated Shc or IRS-1 tyrosine phosphorylation in the different cell lines studied. Finally, we observed that cAMP inhibited serum- and insulin-induced, but not NGF-induced, cell proliferation in NIH3T3 cells. However, cAMP potentiated insulin-stimulated cell differentiation in PC12/IR cells. These results led us to conclude that the cAMP effect on cell proliferation in NIH3T3 fibroblasts and PC12/IR cells appears to be correlated, in part, with the effect of cAMP on the MAP kinase pathway, but by itself this pathway cannot fully account for these observations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LIGAND BINDING to tyrosine kinase receptors leads to receptor autophosphorylation on several tyrosine residues, and to phosphorylation of cellular substrates. For most receptors, autophosphorylation creates docking sites for proteins containing SH2 (Src Homology 2) domains, such as Shc (Src homology collagen), PI 3-K (phosphatidylinositol 3-kinase), or PLC{gamma} (phospholipase C{gamma}) leading to the formation of multicomponent signaling complexes (1). The insulin receptor and the IGF-1 receptor autophosphorylate but use mainly IRS-like (insulin receptor substrate-like) proteins as docking molecules. These phosphorylated IRS-like proteins, IRS-1, IRS-2 and Gab-1, bind to several SH2 domain-containing proteins (2, 3). Phosphorylated Shc and IRS-1 are both able to interact with Grb2 (growth factor receptor-bound protein 2), a docking molecule composed of one SH2 and two SH3 domains (4, 5). Grb2 is constitutively associated with the guanine nucleotide exchange factor Sos (Son of sevenless), which promotes the exchange of Ras-GDP (inactive) to Ras-GTP (active) (6). Therefore, Grb2 associated with Shc or IRS-1 allows the recruitment of the complex to the cell membrane, where Ras is localized. Activated Ras is then able to associate and activate, by a poorly understood mechanism, the serine/threonine kinase Raf 1 (7, 8, 9). Raf 1 is a member of the family of mitogen-activated protein kinase-kinase (MAPKK) kinases that activate MAPK kinases or MEKs; MEK1 and MEK2, (MAP kinase/ERK Kinases), by a double serine phosphorylation (10, 11, 12). MEKs are dual specific kinases (13, 14, 15, 16), which in turn activate MAP kinases by phosphorylation on both threonine and tyrosine (17, 18). The MAP kinases, p44MAPK and p42MAPK, also named extracellular-signal regulated kinases (ERK1 and 2 respectively), are serine/threonine protein kinases, which have been implicated in many cellular responses to growth factors. Numerous signaling molecules are activators of the MAP kinase cascade. These include, in addition to the tyrosine kinase receptors (19, 20, 21), phorbol esters such as PMA (22), which stimulate protein kinase C (PKC) and ligands for the serpentine receptors, such as thrombin which acts through G proteins (23).

Previous studies have demonstrated a cross-talk between the MAP kinase pathway and the cAMP signaling pathway. It has been shown that cAMP attenuated tyrosine kinase receptor-stimulated MAP kinase in cells such as smooth muscle cells treated with PDGF BB (24), fibroblasts stimulated by EGF, LPA (25, 26, 27), PDGF, or insulin (28) and adipocytes stimulated by insulin (29). This inhibitory action occurs concomitantly with a decrease in the biological effects induced by these receptors. This inhibition appears to be mediated by protein kinase A (PKA). Indeed, PKA was shown to phosphorylate Raf 1, inducing a decrease in the association between Ras and Raf 1, which leads to a reduced Raf 1 activity (25, 27).

In contrast to all other systems, we have previously shown that in PC12 cells an elevation in intracellular cAMP increased in a more than additive manner the effects of NGF, IGF-I and PMA on ERK1 and MEK1 activities. Further, cAMP increased by 3-fold NGF-stimulated neurite formation in this cell line (30). Surprisingly, the activity of B-Raf, the predominant isoform of Raf in PC12 cells, was inhibited. Like in other cell types, the phosphorylation of B-Raf by PKA was responsible for reducing its ability to associate with Ras, resulting in a decrease in its activity (31). Hence, the mechanism(s) by which cAMP potentiated growth factor-stimulated MAP kinase in PC12 cells remains unknown. The differences between the cAMP effect on ERK1 activity in NGF-stimulated PC12 cells compared to its effect in other cell types (i.e. potentiation vs. inhibition) could be due to the PC12 cell context per se. Alternatively, it could result, at least in part, from the nature of the tyrosine kinase receptors.

To approach this issue, we studied the effects of overexpression of NGF receptors in a cellular context different from that of PC12 cells, i.e. NIH3T3 cells (NIH3T3/trk). Conversely, we used PC12 cells expressing insulin receptors (PC12/IR cells) (32). Finally, we looked at NIH3T3 cells and Rat1 cells overexpressing the insulin receptor (NIH3T3/IR, Rat1/IR respectively) to compare the cAMP effect on the insulin-stimulated MAP kinase pathway in different cells. In this series of cell lines we looked at the cAMP effect on: 1) ERK1 and MEK1 activities; 2) Shc and IRS-1 tyrosine phosphorylation; and 3) cell growth and differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies
Peptides corresponding to amino acids 356–367 of rat ERK1 (TARFQPGAPEAP), and to the 17 N-terminal amino acids of human MEK1 (PKKKPTPIQLNPAPDGS) were synthesized by Neosystem (Strasbourg, France). The peptides were coupled to keyhole limpet hemocyanin and used to generate rabbit polyclonal antisera (30).

The anti-IRS-1 antibody is a rabbit polyclonal antiserum directed against the 14 C-terminal amino acids of IRS-1 (CASINFQKQPEDRQ) (33). The mouse monoclonal antiphosphotyrosine antibody (clone 4G10), and the rabbit anti-Shc polyclonal antibody raised against amino acids 366–473 of a recombinant fusion protein Shc-SH2 coupled to GST, were purchased from UBI (Lake Placid, NY).

Materials
Sodium orthovanadate, BSA, leupeptin, phenylmethylsulfonyl fluoride (PMSF), myelin basic protein (MBP) from bovine brain, protein A-sepharose, Triton X-100, 8-(4-chlorophenylthio)-cAMP (CPT), forskolin were purchased from Sigma (St. Louis, Ml). NGF was from Promega (Madison, Wl), insulin (Actrapid) from Novo-Nordisk (Copenhagen, Denmark) and PDGF-BB was from Pepro Tech Inc. (Rocky Hill, NJ). Dimethylsulfoxide (DMSO) was used as a carrier for forskolin at a final concentration of 0.04% (vol/vol). [{gamma}-32P] ATP was purchased from ICN (France).

Cell culture
NIH3T3 cells transfected with a plasmid pCO12-trk encoding the full-length human NGF receptor (TrkA) (NIH3T3/trk) expressing 105 receptors/cell were a generous gift from Dr. Alan Saltiel (Department of Signal Transduction, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, MI) (34). They were cultured in DMEM supplemented with glutamine, 10% (vol/vol) FCS, and 200 µg/ml geneticin.

Rat pheochromocytoma PC12 cells expressing the human insulin receptor (PC12/IR) (5.105 receptors/cell) were a generous gift from Prof. Joseph Schlessinger (Department of Pharmacology, University Medical Center, New York, NY) (32). The cells were cultured in DMEM supplemented with glutamine, 10% (vol/vol) horse serum (Hyclone), 10% (vol/vol) FCS, and 200 µg/ml geneticin.

NIH3T3 mouse fibroblasts overexpressing the human insulin receptor (106 receptors/cell) (NIH3T3/IR), were a generous gift of Prof. Jonathan Whittaker (Division of Endocrinology, Department of Medicine, Health Science Center, Stony Brook, New York, NY) (35). The cells were cultured in DMEM supplemented with glutamine (2 mM) and 10% (vol/vol) FCS.

Rat1 fibroblasts expressing the human insulin receptor (106 receptors/cell) (Rat1/IR), were cultured in DMEM/HAM F12 (1:1, vol/vol) supplemented with glutamine (2 mM), and 10% (vol/vol) FCS, were kindly provided by Dr. Axel Ullrich (Max Planck Institute, Munich, Germany) (36).

The fibroblast cell lines and the PC12/IR cells were cultured for 2 days and then starved for 48 h (fibroblasts) or overnight (PC12/IR cells) in media containing 0.2% (wt/vol) BSA (fibroblasts) or 0.2% BSA (wt/vol) plus 0.5% serum (vol/vol) (PC12/IR cells), before stimulation.

Immunoprecipitation of ERK1 or MEK1 activated in intact cells
All cell lines were plated into 12-well dishes for ERK1 or MEK1 assays. Following starvation, the cells were incubated for various times with the indicated effectors. The cells were washed twice with stop buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM Na4P2O7, 2 mM Na3VO4, 100 mM NaF) and solubilized for 15 min at 4 C in the same buffer containing 1% (vol/vol) Triton X-100, 100 U/ml aprotinin, 20 µM leupeptin and 0.2 mg/ml PMSF (solubilization buffer). The cell lysates were clarified by centrifugation at 18,000 x g for 15 min at 4 C and then incubated for 2 h at 4 C with antibodies to ERK1 or MEK1 preabsorbed on protein A-Sepharose beads. Pellets were then washed three times with the solubilization buffer.

ERK1 activity
Pellets containing immunoprecipitated ERK1 were washed twice with 50 mM HEPES, 150 mM NaCl, 10% (vol/vol) glycerol, 0.1% (vol/vol) Triton X-100, 0.2 mM Na3VO4. They were resuspended in 50 µl of the same buffer containing 100 units/ml aprotinin, 20 µM leupeptin, and 0.2 mg/ml PMSF. Phosphorylation of MBP was initiated by addition of 150 µg/ml MBP, 10 mM Mg acetate, 1 mM dithiothreitol (DTT), and [{gamma}-32P]ATP (5 µM, 33 Ci/mmol). The phosphorylation reaction was allowed to proceed for 30 min at room temperature and stopped by spotting 50 µl onto Whatman P-81 filter papers which were then dropped into 1% (vol/vol) orthophosphoric acid. The papers were washed several times, rinsed once in ethanol, air dried, and the radioactivity was determined by Cerenkov-counting. Background values obtained from a mixture lacking cell lysate were subtracted from all values.

MEK1 activity
Immunoprecipitated MEK1 activity was measured by an in vitro reconstitution assay using recombinant rat ERK1 (11, 37) and MBP. Briefly, pellets containing immunoprecipitated MEK1 were washed twice in 50 mM HEPES, pH 7.4, and resuspended with 50 ml of 50 mM HEPES, 100 U/ml aprotinin, 20 mM leupeptin, 0.2 mg/ml PMSF pH 7.4, containing 1 µg of recombinant rat ERK1. The phosphorylation cascade was initiated by addition of [{gamma}-32P]ATP (50 µM, 50 Ci/mmol), 150 µg/ml MBP, 15 mM MgCl2, 2 mM EGTA. The phosphorylation reaction was allowed to proceed for 20 min at room temperature and was stopped as described above for the ERK1 assay. The background consisting of all reagents except for cell lysate was subtracted from all values.

Tyrosine phosphorylation of Shc and IRS-1
The cells were plated into 6-well dishes for phosphorylation assay of Shc and IRS-1. After stimulation by the indicated agonists the cells were solubilized as described above. Lysates were incubated for 4 h with pellets of protein A-sepharose coupled to anti-IRS-1 antibody or anti-Shc antibody. The pellets were washed three times with 50 mM HEPES, 150 mM NaCl, 0.1% (vol/vol) Triton X-100 and resuspended in Laemmli sample buffer (3% (wt/vol) SDS, 70 mM Tris, 11% (vol/vol) glycerol) containing bromophenol blue (0.05%) and ß-mercaptoethanol 0.05% (vol/vol). The proteins were separated on SDS/PAGE using a 7.5% resolving gel. The proteins were then transferred to polyvinyliden difluoride (PVDF) membranes. The membranes were incubated in blocking buffer (10 mM Tris, 140 mM NaCl/5% (wt/vol) BSA) for 2 h at 22 C, then overnight at 4 C with the mouse antiphosphotyrosine antibodies. The membranes were washed several times for 1 h with 10 mM Tris, 140 mM NaCl/0.5% (vol/vol) NP40 and were incubated with rabbit antimouse antibody for 1 h. After further washing for 45 min, the membranes were incubated for 1 h at 22 C with [125I] conjugated protein A (5 x 105 cpm/ml in blocking buffer) and then washed again. The tyrosine-phosphorylated proteins were visualized by autoradiography.

In some experiments, the tyrosine-phosphorylated Shc proteins from treated or untreated cells were immunoprecipitated by the antiphosphotyrosine antibody and revealed by Western blotting with the anti-Shc antibody.

Cell proliferation in NIH3T3 fibroblasts and differentiation in PC12/IR cells
NIH3T3/trk and NIH3T3/IR cells were plated into 6-well dishes and PC12/IR cells in collagen coated 6-well dishes at a density of 40,000 cells per well. After 2 days, the cells were starved for 48 h and then stimulated by NGF (100 ng/ml), insulin (10-7 M), serum (10%), forskolin (10-5 M) or CPT-cAMP (1 mM) alone or in combination as indicated. The NIH3T3 cells were counted 4 days after stimulation and the PC12/IR cells were photographed after 48-h stimulation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To approach the relative contributions of the cellular environment and of the receptor type in the cAMP-effect on the MAP kinase pathway, we used different cell lines overexpressing the NGF receptor (NIH3T3/trk) or the insulin receptor (NIH3T3/IR, Rat1/IR, and PC12/IR). We studied ERK1 and MEK1 activation, and Shc and IRS-1 tyrosine phosphorylation. The agonists used to increase the level of intracellular cAMP were CPT-cAMP, which is a permeable and nonmetabolizable analog of cAMP, and forskolin, which directly activates adenylate cyclase.

Effect of cAMP on insulin- or NGF-stimulated ERK1 and MEK1 activities
NIH3T3/trk cells were stimulated by NGF for different periods of time in the presence or absence of forskolin. As shown in Fig. 1AGo, NGF stimulation of ERK1 was rapid, with a maximal effect at 5 min (2.2-fold), and was sustained for at least 1 h. Forskolin alone had no effect on ERK1 activity. When NGF and forskolin were added together, ERK1 activity was enhanced at 5 and 10 min compared with the effect of NGF alone (3.2-fold). Thus, increasing the intracellular cAMP level results in potentiation of NGF-stimulated ERK1 activity in NIH3T3 fibroblasts expressing NGF receptors.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 1. Effect of cAMP on ERK1 and MEK1 in NIH3T3/trk cells. A, ERK1 activity; B, MEK1 activity. Quiescent cells were stimulated with NGF (100 ng/ml), forskolin (10-5 M), or both for the indicated periods. ERK1 and MEK1 activities were then analyzed as described in the Materials and Methods section by measuring incorporation of [{gamma}-32P] ATP into MBP. The results were expressed as a percentage of maximal NGF stimulation. Data are means ± SD of ten experiments (P <= 0.0005 at 5 min) (A) and 3 experiments (B), performed in triplicate.

 
We next looked at MEK1. After stimulation of the cells by the ligands for increasing periods of time, MEK1 was immunoprecipitated and recombinant ERK1 was added as a substrate. Activation of recombinant ERK1 was then detected by incorporation of radiolabeled Pi into MBP. As shown in Fig. 1BGo, a transient stimulation of MEK1 by NGF was observed with a maximum at 5 min and a decrease at 15 min. However, in contrast to MAP kinase we observed a slight increase from 15 min up to 1 h probably due to a difference in regulation of both enzymes. The forskolin effect on MEK1 activity was similar to that observed on ERK1, i.e. forskolin alone had no effect, but forskolin plus NGF enhanced the NGF action. We conclude that in NIH3T3 cells expressing the NGF receptor, forskolin potentiates the NGF effect on both MEK1 and ERK1.

In NIH3T3/IR cells, insulin-stimulation of ERK1 was transient with a peak at 5 min (2.4-fold) (Fig. 2AGo). At later times a sustained activity of approximately 25% of maximum was observed until at least 1 h. Interestingly, forskolin did not alter the effect of insulin on ERK1 activity (2.5-fold). Similarly to the findings with ERK1, we observed that MEK1 (Fig. 2BGo) was transiently stimulated by insulin with a peak at 5 min; the activity decreased at 15 min and was followed by a sustained activation of approximately 30% of maximum until at least 1 h. Forskolin alone did not stimulate MEK1, and addition of forskolin to insulin did not modify the insulin-stimulated MEK1 activity. Taken together our results indicate that in NIH3T3 cells the cAMP effect on ERK1 and MEK1 vary depending on the type of stimulated tyrosine kinase receptor, i.e. the NGF receptor vs. the insulin receptor.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 2. Effect of cAMP on ERK1 and MEK1 in NIH3T3/IR cells. ERK1 activity (A) and MEK1 activity (B) were measured as described in the legend to Fig. 1Go after stimulation by forskolin (10-5 M) or by insulin (10-7 M) with or without forskolin. The results were expressed as a percentage of maximal insulin stimulation. Data are means ± SD of six experiments, performed in triplicate.

 
In PC12/IR cells (Fig. 3AGo), insulin stimulation of ERK1 was transient with a maximum at 5 min (8-fold). When CPT-cAMP and insulin were added together, ERK1 activation was increased at each time point (12-fold at 5 min). CPT-cAMP alone induces a slight ERK1 activation (2-fold). The stimulation of MEK1 correlated with that of ERK1 because it was transient with a maximum between 2 and 5 min, and potentiated by CPT-cAMP (Fig. 3BGo).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 3. Effect of cAMP on ERK1 and MEK1 in PC12/IR cells. A, ERK1 activity; B, MEK1 activity. Quiescent cells were stimulated by CPT-cAMP (10-3 M), insulin (10-7 M) with or without CPT-AMP for the indicated periods. ERK1 and MEK1 activities were then measured as described in the legend to Fig. 1Go. The results were expressed as a percentage of maximal insulin stimulation. Data are means ± SD of five experiments, performed in triplicate.

 
We next studied the effect of a cAMP increase in Rat1 fibroblasts overexpressing the insulin receptor (Rat1/IR). As shown in Fig. 4AGo, insulin induced a sustained ERK1 activation with a maximum at 5 min (5-fold) and lasting 1 h. Forskolin induced a complete inhibition of ERK1. Concerning MEK1 (Fig. 4BGo), the inhibition induced by forskolin had a similar time-course as that obtained with ERK1.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 4. Effect of cAMP on ERK1 and MEK1 in Rat1/IR cells. ERK1 (A) and MEK1 (B) activities were measured as described in the legend to Fig. 1Go after stimulation by forskolin (10-5 M) or by insulin (10-7 M) with or without forskolin. The results were expressed as a percentage of maximal insulin stimulation. Data are means ± SD of three experiments, performed in triplicate.

 
In conclusion, the cellular context appears to have an important impact on the nature of the cAMP effect. Indeed, the cAMP action on the MAP kinase cascade varied depending on the cells in which the insulin receptor is activated. Further, the nature of the receptor itself (NGF vs. insulin) seems also to be important, because in a same cell context (NIH3T3 fibroblasts), cAMP affects differently the MAP kinase cascade.

Effect of cAMP on PDGF-stimulated ERK1 activity in NIH3T3/trk and NIH3T3/IR cells
To verify that the difference in the cAMP effect on MAP kinase between NIH3T3/trk and NIH3T3/IR cells did not stem from a clonal variation, we studied the cAMP effect on an endogenous receptor tyrosine kinase expressed in both cell lines, the PDGF receptor. NIH3T3/trk cells and NIH3T3/IR cells were stimulated for 5 min by PDGF in absence or presence of forskolin, and then ERK1 activity was measured. In both cell lines, forskolin had no effect on the maximal ERK1 activity stimulated by PDGF (Fig. 5Go).



View larger version (59K):
[in this window]
[in a new window]
  		Figure 5. Effect of cAMP on PDGF-stimulated ERK1 in NIH3T3/trk and NIH3T3/IR cells. The ERK1 activity was measured as described in the legend to Fig. 1Go after 5 min of stimulation by PDGF (10 ng/ml) with or without forskolin (10-5 M). The results were expressed as fold-stimulation over basal. Data are means ± SD of three experiments for NIH3T3/trk cells and four experiments for NIH3T3/IR cells, performed in triplicate.

 
Effect of cAMP on tyrosine phosphorylation of Shc
Once phosphorylated by a tyrosine kinase receptor, both Shc and IRS-1 are able to bind the complex Grb2-Sos, leading to Ras activation and consequently stimulation of the MAP kinase cascade. Therefore, we searched for a possible modulation in tyrosine phosphorylation of these proteins by cAMP agonists. Tyrosine phosphorylation of Shc was measured after treatment of cells by effectors for the indicated time periods. In some experiments the tyrosine-phosphorylated Shc was immunoprecipitated from the cell lysates using an anti-phosphotyrosine antibody (Fig. 6Go, A, B, and D). Immunoprecipitated proteins were then separated by polyacrylamide gel electrophoresis, and after transfer, immunoblotted with anti-Shc antibodies. In panel C, Shc was immunoprecipitated using an anti-Shc antibody, and its phosphorylation was visualized by blotting with an antiphosphotyrosine antibody.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 6. Effect of cAMP on Shc tyrosine phosphorylation in different cell-lines. Quiescent NIH3T3/trk, PC12/IR, NIH3T3/IR and Rat1/IR cells were stimulated with NGF (100 ng/ml) (A), insulin (10-7 M) (B–D), forskolin (10-5 M) (A, C and D) or CPT-cAMP (10-3 M) (B), alone or in combination as indicated. The solubilized proteins were then immunopurified with an antiphosphotyrosine antibody preabsorbed on protein G-sepharose (A, B, and D) or an anti-Shc antibody preabsorbed on protein A-sepharose (C) and separated on a 7.5% polyacrylamide gel electrophoresis. The gels were then transferred to a PVDF membrane, and the Shc proteins were detected by immunoblotting with an anti-Shc antibody (A, B, and D) or an antiphosphotyrosine antibody (C) followed by exposure to [125I] protein A. The blots were submitted to autoradiography.

 
In all the cell lines studied, two proteins were found which corresponded to the p52Shc and p46Shc isoforms, except in Rat1/IR cells (D) where only p52Shc was observed. p66Shc was not detectable. Tyrosine phosphorylation of p52Shc and p46Shc was increased by NGF (A) or insulin (B–D) treatment. Forskolin alone had no effect on Shc tyrosine phosphorylation. Further, addition of forskolin or CPT-cAMP in combination with NGF or insulin did not modify the growth factor-induced tyrosine phosphorylation of Shc. This suggests that the inhibition or potentiation of ERK1 and MEK1 activities due to cAMP did not occur at the Shc level.

Effect of cAMP on tyrosine phosphorylation of IRS-1
IRS-1 tyrosine phosphorylation was studied only in cells overexpressing insulin receptors: PC12/IR, NIH3T3/IR and Rat1/IR cells (Fig. 7Go, A–C, respectively). After stimulation of the cells, IRS-1 was immunoprecipitated and tyrosine phosphorylation was revealed by immunoblotting with an antiphosphotyrosine antibody. In each cell line studied, tyrosine phosphorylation of IRS-1 was maximal at 5 min. It was persistant for at least 1 h in NIH3T3/IR and Rat1/IR cells, but decreased at 30 min in PC12 cells. CPT-cAMP (A) or forskolin (B, C) did not modify IRS-1 tyrosine phosphorylation, and did not alter the insulin effect except in Rat1/IR cells (C). In these cells (C), addition of forskolin to insulin induced a slight, but significant, decrease in IRS-1 tyrosine phosphorylation at 10 min (20%, P <= 0.025, n = 4).



View larger version (29K):
[in this window]
[in a new window]
  		Figure 7. Effect of cAMP on IRS-1 tyrosine phosphorylation in PC12/IR, NIH3T3/IR, and Rat1/IR cells. Quiescent cells were stimulated with insulin (10-7 M), forskolin (10-5 M) (B and C), or CPT-cAMP (10-3 M) (A) added separately or together. The solubilized proteins were then immunopurified with an anti-IRS-1 antibody preabsorbed on protein A-sepharose and separated on a 7.5% polyacrylamide gel electrophoresis. The gels were then transferred to a PVDF membrane, and phosphorylated IRS-1 was detected by immunoblotting with an antiphosphotyrosine antibody followed by exposure to [125I] protein A. The blots were submitted to autoradiography.

 
Effect of cAMP on NIH3T3 cells proliferation and on PC12/IR cells differentiation
To determine whether the effect of cAMP on MEK1 and ERK1 could be of physiological relevance, we studied the effect of cAMP-elevating agents on cell growth in NIH3T3 cells and on cell differentiation in PC12/IR cells. To search for a cAMP effect on cell growth, cells were plated into six-well dishes, starved for 48 h, and then stimulated by the indicated effectors. The cells were counted 4 days after stimulation as described in the Methods section. As shown in Table 1Go, in NIH3T3/trk cells, forskolin did not stimulate cell proliferation. Serum-stimulation, taken as a control, induced a 6-fold increase in proliferation (this value correspond to our 100% proliferation). When forskolin was added to serum we observed a 30% inhibition. NGF-stimulation induced an increase in proliferation (2- to 3-fold) after 4 days that was not modified by addition of forskolin. In NIH3T3/IR cells, after 4 days serum led to an approximately 10-fold stimulation (our 100% proliferation) and forskolin decreased the serum-stimulated proliferation by approximately 40%. Insulin stimulated cellular proliferation (3-fold), and we found an inhibition of approximately 35% by concomitant addition of forskolin to insulin.


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of cAMP on cellular proliferation in NIH3T3/trk and NIH3T3/IR fibroblasts

 
Stimulation of PC12/IR cells by insulin induces cell differentiation (32) (fig. 8CGo). Therefore, we looked at the effect of CPT-cAMP on insulin-induced differentiation of PC12/IR cells. CPT-cAMP alone was able to produce cell differentiation. However, the neurite size was less important than with insulin stimulation (Fig. 8BGo). When CPT-cAMP was added to insulin we observed an increase in the number of differentiated cells and in the length of the neurites compared with insulin alone (Fig. 8DGo).



View larger version (188K):
[in this window]
[in a new window]
  		Figure 8. Effect of cAMP on PC12/IR cells differentiation. The PC12/IR cells were plated on collagen-coated six-well dishes at a density of 40,000 cells per well. Two days later, the cells were starved overnight in DMEM 0.2% (wt/vol) BSA, 0.5% (vol/vol) serum (A), and stimulated with insulin (C) (10-7 M) and CPT-cAMP (10-3 M) (B) alone or in combination (D) as indicated. The pictures were taken 48 h after stimulation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report we compared, in different cell systems, the cAMP effect on ERK1 and MEK1 stimulated by NGF and insulin. We also studied early steps of insulin and NGF signaling, such as IRS-1 and Shc tyrosine phosphorylation. Finally, the effect of cAMP on proliferation of NIH3T3 cells and differentiation of PC12/IR cells was investigated. The results are summarized in Table 2Go.


View this table:
[in this window]
[in a new window]
  		Table 2. Effect of cAMP on different growth factor-stimulated events in the different cell lines studied

 
We show that in NIH3T3/trk fibroblasts, forskolin potentiated the NGF-stimulated ERK1 and MEK1 activities. In contrast, in NIH3T3/IR cells cAMP had no effect on the insulin-stimulated ERK1 and MEK1. This difference between the two cell lines was also observed using CPT-cAMP or other agents which raise cAMP (cholera toxin, IBMX) (data not shown). Moreover, it does not stem from clonal variation between NIH3T3/trk and NIH3T3/IR cells because both cell lines gave similar results, i.e. no effect of cAMP on the PDGF-induced ERK1 stimulation. Further, despite a difference in receptor number (105 NGF receptors/cell in NIH3T3/trk cells vs. 106 receptors/cell in NIH3T3/IR cells), stimulation by NGF is sufficient to maximally activate the MAP kinase (2.2-fold) in a manner similar to the insulin receptor (2.4-fold). Dose-response experiments showed that the peptide concentrations used in our work (100 ng/ml NGF and 10-7 M insulin) induced a maximal effect. Moreover, we found that at each growth factor concentration cAMP was without effect on insulin action, whereas it potentiated NGF action. In conclusion, the differences in the cAMP effect on MAP kinase is likely to be a consequence of qualitative differences between NGF and insulin signaling. Hence, our results indicate that in a given cell system regulation of the MAP kinase pathway by cAMP depends, at least in part, on which tyrosine kinase receptor is activated.

In Rat1/IR cells, cAMP produced a total inhibition of insulin-stimulated ERK1 and MEK1. These data are in accordance with previous published observations in Rat1 fibroblasts exposed to EGF, LPA (25, 26, 27), or PDGF (28). In PC12/IR cells, we found an increase in insulin-stimulated ERK1 and MEK1 after treatment by CPT-cAMP. This is similar to our previous work in native PC12 cells stimulated by NGF and other effectors (30). Recently, we also reported a comparable potentiating effect of cAMP on glucose-activated ERK1 in pancreatic ß-cells (38). Likewise, another group showed an additive effect of cAMP on IGF-1-stimulated MAP kinase activity in primary cultures of pars tuberalis cells (39). Our observations in NIH3T3/IR cells are at variance with those obtained by Burgering et al., who found an inhibition of ERK2 by a cAMP-elevating agent after stimulation by EGF and insulin in NIH3T3 expressing insulin receptors (A14 cells) (28). The reason for this discrepancy is not clear but could be explained by differences between the cell lines. This reinforces the notion attributing an important role of the cell context in the cAMP actions on the MAP kinase cascade.

To look for a putative target regulated by cAMP, we studied tyrosine phosphorylation of Shc and IRS-1. Generally speaking, in our cell lines treatment by a cAMP agonist had no effect on the basal or growth factor-stimulated tyrosine phosphorylation of Shc and IRS1. In Rat1 cells, phosphorylation of IRS-1 was decreased at 10 min; however, this decrease did not appear to be correlated with the total inhibition of ERK1 observed in Fig. 4AGo. Taking our observations together, we conclude therefore that in our cell lines the target for cAMP is likely to be located downstream of Shc and IRS-1 in the MAP kinase pathway, or on a parallel pathway that could impinge on MEK1 and ERK1. The MAP kinase cascade has been shown to be inhibited by increased cAMP levels in several cell systems such as PDGF-stimulated smooth muscle cells (24), and insulin-stimulated adipocytes (29). However, the mechanism by which cAMP inhibits the MAP kinase pathway is only partially known (25, 26, 27, 28, 40). It has been suggested that the cAMP-dependent protein kinase (PKA) could decrease the interaction between Ras and Raf, which is the first functional module of the MAP kinase cascade. The small G protein Rap has been proposed as a potential molecule responsible for such an inhibition because it has been described as acting upstream of Raf and MEK, and downstream of Ras in Rat1 cells (41). In addition, it is phosphorylated by PKA in vitro and in vivo (42, 43). Rap would compete with Ras for the interaction with Raf 1, but in contrast to Ras it would not be able to activate Raf 1 (24). Moreover, PKA phosphorylates Raf 1 itself, thereby inhibiting its interaction with Ras and hence its kinase activity (44). We and others also found that cAMP inhibited B-Raf, the major Raf isoform in PC12 cells, and this despite its potentiating effect on NGF-stimulated ERK1 activity in these cells (31, 45).

The mechanism(s) underlying the potentiating effect of cAMP on MAP kinase is (are) still unknown. One simple explanation could be that, in some cell lines, growth factors could activate, in addition to Raf, an unidentified MEK kinase which would be positively modulated by PKA. This MEK kinase could be present in PC12 cells, NIH3T3/trk and NIH3T3/IR cells, but absent in Rat1 fibroblasts or other systems where cAMP does not stimulate the ERK1. Several MEK activators different from Raf or MEKK (46) have been cloned recently (47, 48, 49), but the effect of PKA or cAMP on these MEK activators has not been evaluated so far. It is also possible that the MAP kinase activity could be regulated by cAMP through a signaling pathway triggered by tyrosine kinase receptors such as PI 3-K or PLC {gamma}. This mechanism could explain the difference between NIH3T3/trk and NIH3T3/IR if we consider that each tyrosine kinase receptor could interact and/or activate the different mediators implicated in signaling with a specific strength and time course. Accordingly, it has been described that trkA and EGF receptors have a distinct affinity for PLC {gamma} that could account for a differential effect of growth factors on MAP kinase activation and on biological effects such as transformation of fibroblasts (50). Therefore, the differential activation by insulin receptors and trkA of a given set of molecules implicated in intracellular signaling and submitted or not to regulation by cAMP could explain the different effects of cAMP on the MAP kinase cascade.

We next investigated whether the cAMP modulation of the MAP kinase pathway could be correlated with changes in biological responses such as cellular proliferation in NIH3T3 fibroblasts or differentiation in PC12/IR cells. Results from fibroblasts showed a slight induction of cell proliferation by insulin or NGF (approximately 30–40% of serum stimulation) that was not modified by addition of forskolin in NIH3T3/trk cells but diminished by 35% in NIH3T3/IR cells. In PC12/IR cells, stimulation with insulin induced a slow growth of dendritic processes after 24 h that was accelerated by addition of CPT-cAMP, as was recently described by our group for NGF (30). Thus, the cAMP effect on proliferation does not correlate with its effect on MAP kinase. Thus, we conclude that the MAP kinase pathway by itself cannot fully account for the biological effect induced by cAMP. Several reports are compatible with this view. In thyrocytes, cAMP activated proliferation independently of MAP kinase (51) (for review see Ref.52). Similarly, McKenzie et al. showed that preincubation of CCL39 cells with cAMP before serum stimulation inhibited cellular proliferation without inhibition of serum- and {alpha} thrombin-stimulated MAP kinase activity (53). A possible candidate responsible for the cAMP effect on cellular proliferation and acting independently of the MAP kinase pathway could be p70S6k, which has been shown to be crucial for the cell cycle progression through the G1 phase (54). In contrast to p90S6k, p70S6k is considered to be the major physiological S6 kinase in mammalian cells and is not activated by MAP kinase. Phosphorylation of the ribosomal protein S6 by p70S6k increases protein synthesis, which is essential for induction of cellular proliferation by growth factors (for review see Ref.55). Our hypothesis is supported by the observation that treatment of lymphoid cells by agents increasing intracellular cAMP levels inhibited IL2-induced activation of p70S6k, and concomitantly cellular proliferation without affecting the MAP kinase cascade (56) (for a review see Ref.57). We could imagine that cAMP affects cell proliferation and differentiation by a concomitant action on the MAP kinase cascade and on the p70s6k pathway.

Another possibility to explain the cAMP effect on cell growth could be that cAMP acts directly on the cell cycle, through G1 cyclins and their associated cyclin-dependent kinases (cdks), which are responsible for progression in the G1 phase. It has been reported that cAMP induced G1 phase arrest in CSF-1-stimulated macrophages through p27Kip1, which is an inhibitor of the cyclin-dependent kinase 4. cAMP has been found to increase the level of p27Kip1 leading to saturation of the complex cyclin D-cdk4 by p27Kip1, and thus preventing phosphorylation of the complex by the cdk-activating kinase (CAK) (58).

In summary, the present study further illustrates that specificity in signaling by tyrosine kinase receptors and a given final biological response are generated by a complex interplay between the intracellular content and agents to which the cells are exposed.


   Acknowledgments
 
We acknowledge Robert Ballotti for scientific discussion, Virginie Leblanc for technical assistance, and Georges Visciano for illustrations.


   Footnotes
 
1 This research was supported by l’Institut National de la Santé et de la Recherche Médicale (INSERM), Association pour la Recherche sur le Cancer (ARC) Grant 6432, Université de Nice-Sophia Antipolis, and Groupe Lipha (Lyon, France) Contract 93123 and La Ligue Nationale Contre le Cancer (Axe oncogenèse). Back

2 Supported by a postdoctoral fellowship of Spanish Ministry of Education and by La Ligue Contre le Cancer (Département du Var). Back

Received August 19, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ullrich A, Schlessinger J 1990 Signal tranduction by receptor tyrosine kinase activity. Cell 61:203–212[CrossRef][Medline]
  2. Waters SB, Pessin JE 1996 Insulin receptor substrate 1 and 2 (IRS1 and IRS2): what a tangled web we weave. Trends Cell Biol 6:1–4[CrossRef][Medline]
  3. Holgado-Madruga M, Emlet DR, Moscatello DK, Godwin AK, Wong AJ 1996 A Grb2-associated docking protein in EGF-and insulin-receptor signalling. Nature (London) 379:560–564[CrossRef][Medline]
  4. Rozakis-Adcock M, Fernley R, Wade J, Pawson T, Bowtell D 1993 The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1. Nature (London) 363:83–85[CrossRef][Medline]
  5. Lowenstein EJ, Daly RJ, Batzer AG, Li W, Margolis B, Lammers R, Ullrich A, Skolnik EY, Bar-Sagi D, Schlessinger J 1992 The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell 70:431–442[CrossRef][Medline]
  6. Li N, Batzer A, Daly R, Yajnik V, Skolnik E, Chardin P, Bar-Sagi D, Margolis B, Schlessinger J 1993 Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signaling. Nature (London) 363:85–88[CrossRef][Medline]
  7. Koide H, Satoh T, Nakafuku M, Kaziro Y 1993 GTP-dependent association of Raf-1 with Ha-Ras: identification of Raf as a target downstream of Ras in mammalian cells. Proc Natl Acad Sci USA 90:8683–8686[Abstract/Free Full Text]
  8. Warne PH, Viciana PR, Downward J 1993 Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro. Nature (London) 364:352–355[CrossRef][Medline]
  9. Moodie SA, Willumsen BM, Weber MJ, Wolfman A 1993 Complexes of ras-GTP with Raf-1 and mitogen-activated protein kinase kinase. Science 260:1658–1661[Abstract/Free Full Text]
  10. Huang W, Alessandrini A, Crews CM, Erikson RL 1993 Raf-1 forms a stable complex with Mek-1 and activates Mek-1 by serine phosphorylation. Proc Natl Acad Sci USA 90:10947–10951[Abstract/Free Full Text]
  11. Kyriakis JM, App H, Zhang X-F, Banerjee P, Brautigan DL, Rapp UR, Avruch J 1992 Raf-1 activates MAP kinase-kinase. Nature (London) 358:417–421[CrossRef][Medline]
  12. Alessi DR, Saito Y, Campbell DG, Cohen P, Sithanandam G, Rapp U, Ashworth A, Marshall CJ, Cowley S 1994 Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1. EMBO J 13:1610–1619[Medline]
  13. Seger R, Seger D, Lozeman FJ, Ahn NG, Graves LM, Campbell JS, Ericsson L, Harrylock M, Jensen AM, Krebs EG 1992 Human T-cell mitogen-activated protein kinase kinases are related to yeast signal transduction kinases. J Biol Chem 267:25628–25631[Abstract/Free Full Text]
  14. Seger R, Ahn NG, Posada J, Munar ES, Jensen AM, Cooper JA, Cobb MH, Krebs EG 1992 Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells. J Biol Chem 267:14373–14381[Abstract/Free Full Text]
  15. Nakielny S, Cohen P, Wu J, Sturgill T 1992 MAP kinase activator from insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase. EMBO J 11:2123–2129[Medline]
  16. Crews CM, Erikson RL 1992 Purification of a murine protein-tyrosine/threonine kinase that phosphorylates and activates the Erk-1 gene product: relationship to the fission yeast byr1 gene product. Proc Natl Acad Sci USA 89:8205–8209[Abstract/Free Full Text]
  17. Ray LB, Sturgill TW 1988 Insulin-stimulated microtubule-associated protein kinase is phosphorylated on tyrosine and threonine in vivo. Proc Natl Acad Sci USA 85:3753–3757[Abstract/Free Full Text]
  18. Payne DM, Rossomando AJ, Martino P, Erickson AK, Her J-H, Shabanowitz J, Hunt DF, Weber MJ, Sturgill TW 1991 Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J 10:885–892[Medline]
  19. Ahn NG, Seger R, Bratlien RL, Diltz CD, Tonks NK, Krebs EG 1991 Multiple components in an epidermal growth factor-stimulated protein kinase cascade. J Biol Chem 266:4220–4227[Abstract/Free Full Text]
  20. Gomez N, Cohen P 1991 Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases. Nature (London) 353:170–173[CrossRef][Medline]
  21. Boulton TG, Nye SH, Robbins DJ, Ip NY, Radziejewska E, Morgenbesser SD, DePinho RA, Panayotatos N, Cobb MH, Yancopoulos GD 1991 ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 65:663–675[CrossRef][Medline]
  22. Hoshi M, Nishida E, Sakai H 1988 Activation of a Ca2+-inhibitable protein kinase that phosphorylates microtubule-associated protein 2 in vitro by growth factors, phorbol esters, and serum in quiescent cultured human fibroblasts. J Biol Chem 262:5396–5401
  23. Kohno M, Pouysségur J 1986 Alpha-thrombin-induced tyrosine phosphorylation of 43,000- and 41,000-Mr proteins is independent of cytoplasmic alkalinization in quiescent fibroblasts. Biochem J 238:451–457[Medline]
  24. Graves LM, Bornfeldt KE, Raines EW, Potts BC, Macdonald SG, Ross R, Krebs EG 1993 Protein kinase A antagonizes platelet-derived growth factor-induced signaling by mitogen-activated protein kinase in human arterial smooth muscle cells. Proc Natl Acad Sci USA 90:10300–10304[Abstract/Free Full Text]
  25. Cook SJ, McCormick F 1993 Inhibition by cAMP of Ras-dependent activation of Raf. Science 262:1069–1072[Abstract/Free Full Text]
  26. Hordijk PL, Verlaan I, Jalink K, Van Corven EJ, Moolenaar WH 1994 cAMP Abrogates the p21ras-Mitogen-activated protein kinase pathway in fibroblasts. J Biol Chem 269:3534–3538[Abstract/Free Full Text]
  27. Wu J, Dent P, Jelinek T, Wolfman A, Weber MJ, Sturgill TW 1993 Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate. Science 262:1065–1068[Abstract/Free Full Text]
  28. Burgering BMT, Pronk GJ, Van Weeren PC, Chardin P, Bos JL 1993 cAMP antagonizes p21ras-directed activation of extracellular signal-regulated kinase 2 and phosphorylation of mSos nucleotide exchange factor. EMBO J 12:4211–4220[Medline]
  29. Sevetson BR, Kong X, Jr JCL 1993 Increasing cAMP attenuates activation of mitogen-activated protein kinase. Proc Natl Acad Sci USA 90:10305–10309[Abstract/Free Full Text]
  30. Frödin M, Peraldi P, Van Obberghen E 1994 Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells. J Biol Chem 269:6207–6214[Abstract/Free Full Text]
  31. Peraldi P, Frödin M, Barnier JV, Calleja V, Scimeca J-C, Filloux C, Calothy G, Van Obberghen E 1994 Regulation of the MAP kinase cascade in PC12 cells: B-Raf activates MEK (MAP kinase or ERK kinase) and is inhibited by cAMP. FEBS Lett 357:290–296
  32. Dikic I, Schlessinger J, Lax I 1994 PC12 cells overexpressing the insulin receptor undergo insulin-dependent neuronal differentiation. Cur Biol 4:702–708[CrossRef][Medline]
  33. Ricort J-M, Tanti J-F, Van Obberghen E, Le Marchand-Brustel Y 1996 Different effects of insulin and platelet-derived-growth-factor on phosphatidylinositol 3-kinase at the subcellular level in 3T3–L1 adipocytes. A possible explanation for their specific effects on glucose transport. Eur J Biochem 239:17–22[Medline]
  34. Ohmishi M, Pang L, Decker SJ, Saltiel AR 1992 Nerve growth factor stimulates the activities of the raf-1 and the mitogen-activated protein kinases via the trk protooncogene. J Biol Chem 267:14604–14610[Abstract/Free Full Text]
  35. Hofmann C, White MF, Whittaker J 1989 Human insulin receptors expressed in insulin-insensitive mouse fibroblasts couple with extant cellular effector systems to confer insulin sensitivity and responsiveness. Endocrinology 124:257–263[Abstract]
  36. Ullrich A, Bell JR, Chen EY, Herrera R, Petruzzelli LM, Dull TJ, Gray A, Coussens L, Liao YC, Tsubokawa M, Mason A, Seeburg PH, Grunfeld C, Rosen OM, Ramachandran J 1985 Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. Nature (London) 313:756–761[CrossRef][Medline]
  37. Kyriakis JM, Force TL, Rapp UR, Bonventre JV, Avruch J 1993 Mitogen regulation of c-Raf-1 protein kinase activity toward mitogen-activated protein kinase-kinase. J Biol Chem 268:16009–16019[Abstract/Free Full Text]
  38. Frödin M, Sekine N, Roche E, Filloux C, Prentki M, Wollheim CB, Van Obberghen E 1994 Glucose, other secretagogues, and nerve growth factor stimulate mitogen-activated protein kinase in the insulin-secreting ß-cell line, INS-1. J Biol Chem 270:7882–7889[Abstract/Free Full Text]
  39. Hazlerigg DG, Thompson M, Hastings MH, Morgan PJ 1996 Regulation of mitogen-activated protein kinase in the pars tuberalis of the ovine pituitary: interactions between melatonin, insulin-like growth factor-1, and forskolin. Endocrinology 137:210–218[Abstract]
  40. Nagasaka Y, Kaku K, Nakamura R, Kaneko T 1994 cAMP inhibits the insulin-stimulated mitogen-activated protein kinase pathway in rat hepatoma H4EII cells. Biochem Biophys Res Commun 202:1104–1112[CrossRef][Medline]
  41. Cook SJ, Rubinfeld B, Albert I, McCormick F 1993 RapV12 antagonizes Ras-dependent activation of ERK1 and ERK2 by LPA and EGF in Rat-1 fibroblasts. EMBO J 12:3475–3485[Medline]
  42. Polakis P, Rubinfeld B, McCormick F 1992 Phosphorylation of rap1GAP in vivo and by cAMP-dependent kinase and the cell cycle p34cdc2 kinase in vitro. J Biol Chem 267:10780–10785[Abstract/Free Full Text]
  43. Kawata M, Kikuchi A, Hoshijima M, Yamamoto K, Hashimoto E, Yamamura H, Takai Y 1989 Phosphorylation of smg p21, a ras p21-like GTP-binding protein, by cyclic AMP-dependent protein kinase in a cell-free system and in response to prostaglandin E1 in intact human platelets. J Biol Chem 264:15688–15695[Abstract/Free Full Text]
  44. Häfner S, Adler HS, Mischak H, Janosch P, Heidecker G, Wolfman A, Pippig S, Lohse M, Ueffing M, Kolch W 1994 Mechanism of inhibition of Raf-1 by protein kinase A. Mol Cell Biol 14:6696–6703[Abstract/Free Full Text]
  45. Vaillancourt RR, Gardner AM, Johnson GL 1994 B-Raf-dependent regulation of the MEK-1/mitogen-activated protein kinase pathway in PC12 cells and regulation by cAMP. Mol Cell Biol 14:6522–6530[Abstract/Free Full Text]
  46. Lange-Carter CA, Pleiman CM, Gardner AM, Blumer KJ, Johnson GL 1993 A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf. Science 260:315–319[Abstract/Free Full Text]
  47. Yamaguchi K, Shirakabe K, Shibuya H, Irie K, Oishi I, Ueno N, Taniguchi T, Nishida E, Matsumoto K 1995 Identification of a member of the MAPKKK family as a potential mediator of TGF-ß signal transduction. Science 270:2008–2011[Abstract/Free Full Text]
  48. Zheng C-F, Ohmichi M, Saltiel AR, Guan K-L 1994 Growth factor induced MEK activation is primarily mediated by an activator different from c-raf. Biochemistry 33:5595–5599[CrossRef][Medline]
  49. Blank JL, Gerwins P, Elliott EM, Sather S, Johnson GL 1996 Molecular cloning of mitogen-activated protein/ERK kinase kinases (MEKK) 2 and 3. J Biol Chem 271:5361–5368[Abstract/Free Full Text]
  50. Obermeier A, Tinhofer I, Grunicke HH, Ullrich A 1996 Transforming potentials of epidermal growth factor and nerve growth factor receptors inversely correlate with their phospholipase C{gamma} affinity and signal activation. EMBO J 15:73–82[Medline]
  51. Lamy F, Wilkin F, Baptist M, Posada J, Roger PP, Dumont JE 1993 Phosphorylation of mitogen-activated protein kinases is involved in the epidermal growth factor and phorbol ester, but not in the thyrotropin/cAMP, thyroid mitogenic pathway. J Biol Chem 268:8398–8401[Abstract/Free Full Text]
  52. Dumont JE, Jauniaux J-C, Roger PP 1989 The cyclic AMP-mediated stimulation of cell proliferation. Trends Biochem Sci 14:67–71[CrossRef][Medline]
  53. McKenzie FR, Pouysségur J 1996 cAMP-mediated growth inhibition in fibroblasts is not mediated via mitogen-activated protein (MAP) kinase (ERK) inhibition. J Biol Chem 271:13476–13483[Abstract/Free Full Text]
  54. Lane HA, Fernandez A, Lamb NJC, Thomas G 1993 p70s6k function is essential for G1 progression. Nature (London) 363:170–172[CrossRef][Medline]
  55. Proud CG 1996 p70 S6 kinase: an enigma with variation. Trends Biochem Sci 21:181–185[CrossRef][Medline]
  56. Monfar M, Lemon KP, Grammer TC, Cheatham L, Chung J, Vlahos CJ, Blenis J 1995 Activation of pp70/85 S6 kinases in interleukin-2-responsive lymphoid cells is mediated by phosphatidylinositol 3-kinase and inhibited by cyclic AMP. Mol Cell Biol 15:326–337[Abstract]
  57. Chou MM, Blenis J 1995 The p70kDa S6 kinase: regulation of a kinase with multiple roles in mitogenic signalling. Curr Opin Cell Biol 7:806–814[CrossRef][Medline]
  58. Kato J-Y, Matsuoka M, Polyac K, Massagué J, Sherr CJ 1994 Cyclic AMP-induced G1 phase arrest mediated by an inhibitor (p27Kip1) of cyclin-dependent kinase 4 activation. Cell 79:487–496[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
G. Boguslawski, P. W. McGlynn, K. A. Harvey, and A. T. Kovala
SU1498, an Inhibitor of Vascular Endothelial Growth Factor Receptor 2, Causes Accumulation of Phosphorylated ERK Kinases and Inhibits Their Activity in Vivo and in Vitro
J. Biol. Chem., February 13, 2004; 279(7): 5716 - 5724.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
K. Balmanno, T. Millar, M. McMahon, and S. J. Cook
{Delta}Raf-1:ER* Bypasses the Cyclic AMP Block of Extracellular Signal-Regulated Kinase 1 and 2 Activation but Not CDK2 Activation or Cell Cycle Reentry
Mol. Cell. Biol., December 15, 2003; 23(24): 9303 - 9317.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. W. Pearson and M. H. Cobb
Cell Condition-dependent Regulation of ERK5 by cAMP
J. Biol. Chem., December 6, 2002; 277(50): 48094 - 48098.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
L. J. McCawley, S. Li, M. Benavidez, J. Halbleib, E. V. Wattenberg, and L. G. Hudson
Elevation of Intracellular cAMP Inhibits Growth Factor-Mediated Matrix Metalloproteinase-9 Induction and Keratinocyte Migration
Mol. Pharmacol., July 1, 2000; 58(1): 145 - 151.
[Abstract] [Full Text]

Home page
 Mol. Endocrinol.Home page
C. M. Boney, P. A. Gruppuso, R. A. Faris, and A. R. Frackelton Jr.
The Critical Role of Shc in Insulin-Like Growth Factor-I-Mediated Mitogenesis and Differentiation in 3T3-L1 Preadipocytes
Mol. Endocrinol., June 1, 2000; 14(6): 805 - 813.
[Abstract] [Full Text]

Home page
 J. Pharmacol. Exp. Ther.Home page
M. Kimura and M. Ogihara
Stimulation by Transforming Growth Factor-alpha of DNA Synthesis and Proliferation of Adult Rat Hepatocytes in Primary Cultures: Modulation by alpha - and beta -Adrenoceptor Agonists
J. Pharmacol. Exp. Ther., October 1, 1999; 291(1): 171 - 180.
[Abstract] [Full Text]

Home page
 Endocr. Rev.Home page
J. J. Evans
Modulation of Gonadotropin Levels by Peptides Acting at the Anterior Pituitary Gland
Endocr. Rev., February 1, 1999; 20(1): 46 - 67.
[Abstract] [Full Text]

Home page
 EndocrinologyHome page
A. Kimura, M. Ohmichi, T. Takeda, H. Kurachi, H. Ikegami, K. Koike, K. Masuhara, J. Hayakawa, T. Kanzaki, M. Kobayashi, et al.
Mitogen-Activated Protein Kinase Cascade Is Involved in Endothelin-1-Induced Rat Puerperal Uterine Contraction
Endocrinology, February 1, 1999; 140(2): 722 - 731.
[Abstract] [Full Text]

Home page
 Cancer Res.Home page
T. Putz, Z. Culig, I. E. Eder, C. Nessler-Menardi, G. Bartsch, H. Grunicke, F. Uberall, and H. Klocker
Epidermal Growth Factor (EGF) Receptor Blockade Inhibits the Action of EGF, Insulin-like Growth Factor I, and a Protein Kinase A Activator on the Mitogen-activated Protein Kinase Pathway in Prostate Cancer Cell Lines
Cancer Res., January 1, 1999; 59(1): 227 - 233.
[Abstract] [Full Text] [PDF]

Home page