A Dominant Role for the Raf-MEK Pathway in Forskolin, 12-O-Tetradecanoyl-phorbol Acetate, and Platelet-Derived Growth Factor-Induced CREB (cAMP-Responsive Element-Binding Protein) Activation, Uncoupled from Serine 133 Phosphorylation in NIH 3T3 Cells

Ole Morten Seternes, Bjarne Johansen and Ugo Moens1


1 Department of Gene Biology Institute of Medical Biology University of Tromsø N-9037 Tromsø, Norway


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study we describe that platelet-derived growth factor (PDGF), 12-0-tetradecanoyl-phorbol-acetate (TPA), and forskolin induced CREB (cAMP-responsive element-binding protein) Ser-133 phosphorylation with comparable magnitude and kinetics in NIH 3T3 cells. While forskolin was the most potent activator of CREB, TPA or PDGF modestly increased CREB activity. The role of protein kinase C, protein kinase A, and the Raf-MEK kinase pathway in the activation and Ser-133 phosphorylation of CREB by these three stimuli was investigated. We found that inhibition of the Raf-MEK kinase pathway efficiently blocks transcriptional activation of CREB by all three stimuli. This dominant involvement of Raf-MEK in CREB transcriptional activation seems to be uncoupled from CREB Ser-133 phosphorylation. We further demonstrate that although inhibition of Raf-MEK represses forskolin-induced CREB activation, forskolin by itself failed to activate ERK1/2 and Elk-1 mediated transcription. These results suggest that a basal level of Raf-MEK activity is necessary for both PDGF- and forskolin-induced CREB activation, independent of CREB Ser-133 phosphorylation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Regulation of gene expression in response to extracellular stimuli occurs often through signal transduction pathways that involve a cascade of biochemical events eventually leading to phosphorylation of specific transcription factors (1). One of the best characterized signaling pathways is the cAMP/protein kinase A (PKA) pathway. Genes whose activity can be induced by increased intracellular cAMP levels often contain so-called cAMP response elements (CRE) in their promoter region to which the cAMP-responsive element binding protein (CREB), a member of the large CREB/CREM/ATF family of transcription factors, can bind (2, 3, 4). PKA has been shown to phosphorylate CREB at the serine residue 133 (Ser-133) in vivo, indicating that CREB mediates cAMP-induced gene expression (5). This residue seems to be crucial for transcriptional activation of CREB since replacing Ser-133 by alanine rendered CREB unresponsive to both cAMP and PKA, and this mutated form of CREB failed to activate transcription of CRE-responsive promoters (5). Several recent studies have shown that the cAMP/PKA pathway is not the only signaling pathway able to induce Ser-133 phosphorylation and transcriptional activation of CREB. CREB is phosphorylated and activated upon nerve growth factor, epidermal growth factor, fibroblast growth factor, hepatocyte growth factor/scatter factor/mast/stem cell growth factor, endothelin-1, UV irradiation, cross-linking surface Ig, and arginine vasopressin treatment (6, 7, 8, 9, 10, 11). Once phosphorylated at Ser-133, CREB can make contact with a large transcriptional coactivator termed CREB-binding protein (CBP). The importance of this interaction for phospho-CREB-activated transcription has been shown by several laboratories (12, 13, 14, 15, 16).

Although Ser-133 phosphorylation of CREB is necessary for CREB to interact with CBP and activate transcription, Ser-133 phosphorylation alone is not sufficient for transcriptional activation in vivo. For example, Ca2+/Cam kinase II can phosphorylate CREB at Ser-133 (8, 17, 18), but this kinase is unable to activate transcription even though CREB phosphorylated by Cam kinase II can physically interact with CBP (19). The authors showed that Cam kinase II also phosphorylated CREB at Ser-142 and that this phosphorylation inhibited the transcriptional activity of CREB. Treatment of Jurkat T-cells with 12-O-tetradecanoyl-phorbol-acetate (TPA) resulted in Ser-133-phosphorylated CREB protein that was transcriptionally inactive as measured in a CREBGAL4 system. Simultaneous costimulation, however, with suboptimal doses of the cAMP agonist forskolin resulted in a transcriptionally active CREB (20). Finally, CREB became phosphorylated, but not activated, upon stimulation of CD28 on T lymphocytes. Cotreatment with TPA or anti-CD3 allowed CREB activation (21).

We investigated whether platelet-derived growth factor (PDGF), TPA, and forskolin could induce Ser-133 phosphorylation and transcriptional activation of CREB in NIH 3T3 cells. Our results show that all agents can induce CREB phosphorylation with comparable kinetics and magnitude, but the transcriptional activity of CREB induced by TPA and PDGF was less efficient than that measured after forskolin stimulation. The role of PKC, PKA, and the Raf/mitogen-activated protein (MAP) kinase pathways in the activation of CREB by each of these stimuli was investigated. Our results indicate that the Raf/MAP kinase pathway is the main mediator of PDGF-induced CREB activation. Forskolin-induced CREB activation is mediated by the PKA but also by the Raf/MAP kinase pathway. PDGF- or forskolin-induced Ser-133 phosphorylation of CREB, however, was not affected by the MEK-specific inhibitor PD98059. Inhibition of PKC, PKA, or the Raf-MAP kinase-signaling pathway all abrogated the transcription activity of CREB induced by TPA. Together, these results fail to establish a direct correlation between CREB Ser-133 phosphorylation and transcriptional activation and demonstrate that different pathways can activate CREB in vivo.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Growth Factors, TPA, and Forskolin Induce Phosphorylation of CREB at Ser-133 in NIH 3T3 Cells with Comparable Levels and Kinetics as the cAMP-Elevating Agent Forskolin
CREB was originally characterized as a cAMP-responsive transcription factor whose transcriptional activation is mediated via PKA phosphorylation at Ser-133 (2). Several laboratories have reported that growth factor stimulation and cellular stress can induce Ser-133 phosphorylation of CREB as well as activate CREB (6, 9, 10, 20, 22). We wanted to compare the ability of different growth stimuli and the adenylate cyclase activator forskolin to induce CREB Ser-133 phosphorylation in murine fibroblasts NIH 3T3 cells. Forskolin has been shown to stimulate both Ser-133 phosphorylation and CREB activation in NIH 3T3 cells (23, 24, 25). Whole-cell extracts were prepared from NIH 3T3 cells serum-starved for 20–24 h and from serum-starved cells stimulated with either 10 µM forskolin, 10 ng/ml PDGF, 10% newborn calf serum (NCS) or 50 ng/ml TPA for 15, 30, 60, or 180 min, respectively. Western blot analysis with an antibody that recognizes the Ser-133 phosphorylated epitope of CREB were performed. Forskolin rapidly induced Ser-133 phosphorylation of CREB with the peak between 15 and 30 min, followed by a gradual decline in phosphorylation after 60 and 180 min (Fig. 1Go). Stimulation with NCS, PDGF, and TPA induced CREB phosphorylation with comparable magnitude and time kinetics as forskolin. Due to the structural similarities between CREB and ATF-1, this antibody also reacts with the Ser-63-phosphorylated form of ATF-1, the phosphorylation kinetics of which parallels that observed for phospho-CREB.



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Figure 1. Forskolin, NCS, PDGF, and TPA Stimulate CREB Ser-133 Phosphorylation

NIH 3T3 cells (35-mm wells) were serum starved for 20–24 h and treated with either 10 µM forskolin, 10% NCS, 10 ng/ml PDGF, or 50 ng/ml TPA for the times indicated before harvesting directly in 50 µl SDS-sample buffer. Extract (20 µl) was separated by 10% SDS-PAGE, electroblotted onto polyvinylidene fluoride membranes, and probed with an antibody specific for Ser-133-phosphorylated CREB (P-CREB) and the Ser-63 phosphorylated form of ATF-1. Total CREB expression was analyzed by reprobing the stripped blots with an antibody recognizing CREB regardless of its phosphorylation status (lower panel). The positions of P-CREB and phospho-ATF-1 are indicated.

 
PDGF and TPA, but Not Serum, Moderately Induce the Transcriptional Activity of CREB
Ser-133 phosphorylation has been regarded as the rate-limiting step in transcriptional activation by CREB. Since PDGF, NCS, or TPA induced Ser-133 phosphorylation with levels comparable to forskolin-treated cells, we wanted to investigate whether these mitogens also could induce CREB-mediated transcription. Therefore, NIH 3T3 cells were cotransfected with a reporter plasmid containing the luciferase gene under control of the minimal promoter of the adenovirus E1b gene and five binding motifs for the yeast transcription factor GAL4 and with a GAL4-CREB expression plasmid. This chimeric protein consists of the DNA-binding domain of GAL4 fused to full-length CREB. Treating such cotransfected cells with forskolin resulted in a more than 20-fold increase in luciferase activity compared with unstimulated cells (Fig. 2AGo). Neither increasing the concentration of forskolin (up to 100 µM) nor cotreatment with the phospodiesterase inhibitor isobutymethylxanthine (0.5 mM) elevated this induction level (results not shown). TPA and PDGF were less potent inducers of luciferase activity with only a 3- and 5-fold enhancement of luciferase expression, respectively. NCS failed to induce the transcriptional activity of GAL4-CREB but was able to induce phosphorylation of CREB (Fig. 2AGo). Costimulation with either TPA or PDGF had no negative influence on forskolin-induced CREB activation (our unpublished data), thus indicating that no concomitant inhibitory signal could be the reason for the relative low induction of CREB activity observed after PDGF and TPA stimulation. Cotransfection with a plasmid expressing only the GAL4-DNA-binding domain did not induce any transactivation with either stimuli (data not shown). Similar experiments were performed with a mutant GAL4-CREB hybrid in which serine-133 was replaced by an alanine. This single-point mutation abolishes the PKA-induced transcriptional activity of CREB (5). None of the stimuli tested here could induce transcription activity of the CREB Ala-133 mutant (Fig. 2BGo). Our results confirm that Ser-133 is crucial for transcriptional activation of CREB induced by forskolin, PDGF, or TPA in NIH 3T3 cells, but fail to illustrate a direct correlation between the degree of CREB Ser-133 phosphorylation and transcriptional activation.



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Figure 2. Activation of CREBGAL4-Mediated Transcription

A, Growth stimuli are inefficient activators of CREB-mediated transcription. NIH 3T3 cells (35-mm wells) were transfected with 1 µg p5GE1bLuc plus 0.5 µg pCMVCREBGAL4 together with 0.2 µg pCH110 to correct for transfection efficiency. Cells were serum starved for 20 h posttransfection before stimulation with either 10 µM forskolin, 10% NCS, 10 ng/ml PDGF, or 50 ng/ml TPA for 3 h. The mean luciferase/ß-gal ratio was calculated (± SD, n = 6) The activity from unstimulated cells transfected with CREBGAL4 was arbitrary set as 1. B, Transfections and stimulation were done as in panel A except that ALA 133 CREBGAL4 was used as transactivator.

 
PKA Is Involved in Forskolin- and TPA-, but Not PGDF-Induced, Activation of CREB
Since PKA seems to be the major kinase responsible for Ser-133 phosphorylation and activation of CREB induced by a wide variety of stimuli, we tested whether forskolin, TPA, or PGDF-induced CREB activation in NIH 3T3 cells was PKA dependent. Previous studies have shown that both Ca2+- and PKC-mediated CREB activation in several cell lines is dependent on PKA (15). This experiment was also justified by the recent report of a cAMP-independent route for activation of PKA. The authors showed that the C{alpha} catalytic subunit of PKA resides in an inactive complex with the transcription factor NF-{kappa}B (26). This catalytic subunit could be released by agents that induce phosphorylation/degradation of I{kappa}B, e.g. by TPA which is a potential inducer of I{kappa}B phosphorylation/degradation. NIH 3T3 cells were transfected with a plasmid that expresses a heat-stable inhibitor of PKA (PKI). PKI has been previously described as a specific and potent inhibitor of PKA activation and PKA-induced gene expression (27). To exclude squelching by the distinct promoters of the different plasmids, transfections were also done with a plasmid that expressed a nonfunctional PKI protein (PKImut). Overexpression of PKI strongly repressed forskolin (80% inhibition)- and TPA (50% reduction)-induced CREBGAL4 gene expression (Fig. 3AGo). In contrast, PKI expression had no effect on PDGF-induced CREBGAL4 activity. Expression of PKI did not reduce the levels of CREBGAL4 fusion protein as determined by Western blotting (results not shown). Thus TPA- and forskolin-, but not PDGF-mediated, CREB activation seems to be dependent on PKA.



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Figure 3. PKA Inhibitor Protein PKI Represses TPA- and Forskolin-Induced CREB Activation

A, NIH 3T3 cells were transfected with 1 µg p5GE1bLuc plus 0.5 µg pCREBGAL4 and either 1 µg pPKImut or 1 µg pPKI. After 20 h maintenance in medium containing 0.5% serum, the transfected cells were stimulated with 50 ng/ml TPA, 10 ng/ml PDGF, or 10 µM forskolin (F) for 3 h. The mean luciferase activities (± SD, n = 3–6) are presented. B, NIH 3T3 cells transfected with 1 µg p5GE1bLuc plus 0.5 µg GAL4ElkC and either 1 µg pPKImut or 1 µg pPKI. After transfection the cells were kept for 20 h in 0.5% NCS and then treated with 50 ng/ml TPA for 3 h. Each bar represents the mean luciferase/ß-gal ratio (± SD, n = 3). Activity from untreated cells transfected with GAL4 ElkC was arbitrarily set as 1.

 
Previous studies have indicated that TPA activates the Raf/MAP kinase signaling pathway, a signaling pathway that has been implicated in CREB activation by nerve growth factor (NGF) (22, 28, 29). It is therefore theoretically possible that PKI exerts its negative effect on TPA-induced CREB activation through disturbance of TPA-induced activation of the MAP kinase pathway. To investigate whether the inhibitory effect of PKI on TPA-induced CREB activation targets TPA ability to activate ERK1/2, we cotransfected cells with a GAL4-Elk-1 expression plasmid together with the PKI expression vectors. The transcription factor Elk-1 is a direct substrate for ERK1/2, and ERK phosphorylation of the C-terminal part of Elk-1 leads to transcriptional activation (30). Stimulation with TPA resulted in a 30-fold increase in Elk-1-mediated expression, while no influence on either basal or TPA-induced GAL4-Elk-1 activity was observed in the presence of PKI (Fig. 3BGo). These observations strongly suggest that PKI does not interfere with TPA-induced activation of the MAP kinase pathway.

PDGF- or Forskolin-Mediated CREB Transactivation and Ser-133 Phosphorylation Are PKC Independent
Ser-133 of CREB also forms the target for a putative PKC phosphorylation motif. Indeed, PKC phosphorylates CREB at Ser-133 in vitro with similar efficiency as PKA (20, 31). Phorbol esters such as TPA are believed to activate certain forms of PKC by mimicking the second messenger diacylglycerol (32). PDGF can activate classic PKCs (cPKC) through activation of phospholipase C{gamma}, which leads to generation of diacylglycerol and subsequently cPKC activation (33). Cross-talk between the cAMP and PKC pathways is well illustrated (34). This suggested that PKC could be a kinase that was indirectly responsible for TPA-, forskolin-, and PDGF-mediated transcriptional activation of CREB. To test this, NIH 3T3 cells were pretreated for 1 h with 0.5 µM of the PKC-specific inhibitor GF109203X, able to block both classical PKC subtypes and the novel PKC{delta} and -{epsilon}, but not -{zeta} (35, 36) before stimulation with TPA, forskolin, or PDGF. Pretreatment with GF109203X completely abolished TPA-induced CREBGAL4 activity but had no influence on PDGF-induced CREB activity (Fig. 4Go and results not shown). GF109203X alone had no effect on basal luciferase activity (Fig. 4Go). We also examined whether the PKC inhibitor GF109203X abrogated phosphorylation of CREB (Fig. 5Go). GF109203X clearly reduced TPA-induced CREB Ser-133 phosphorylation, e.g. compare lanes 2 and 6, but interfered imperceptibly with PDGF-induced phosphorylation, e.g. lanes 3 and 7, or forskolin-induced phosphorylation (data not shown). The compound had no effect on either forskolin-induced CREB activation or Ser-133 phosphorylation (data not shown). This indicates that PDGF- and forskolin-induced CREB activation/Ser-133 phosphorylation are independent of the phorbol ester-induced PKCs. The lack of involvement of cPKCs in PDGF-mediated CREB phosphorylation was also shown by depletion of PKC by exhausting NIH 3T3 cells for 20 h with 400 ng/ml TPA before stimulation. This pretreatment with TPA had little effect on subsequent PDGF-stimulated Ser-133 phosphorylation, whereas it completely blocked TPA-induced CREB phosphorylation (results not shown).



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Figure 4. PKC Mediates TPA-Induced CREB Activation

NIH 3T3 cells were transfected as described in the legend of Fig. 2BGo. Where indicated, cells were pretreated with 500 nM PKC inhibitor GF109203X for 30 min before stimulation with TPA or PDGF. Cells were harvested 3 h later, and luciferase and ß-galactosidase activities were determined as described in Materials and Methods. Each bar represents the mean luciferase/ß-galactosidase ratio (± SD, n = 3–6). The activity in unstimulated CREBGAL4-transfected cells was set as 1.

 


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Figure 5. Both PKC Inhibitor GF109203X and MEK Inhibitor PD98059 Act Differently on TPA- and PDGF-Induced CREB Ser-133 Phosphorylation

Serum-starved (20 h) NIH 3T3 cells were pretreated with either PD98059 (50 µM) or GF109203X (0.5 µM) for 60 min and then stimulated with 50 ng/ml TPA or 10 ng/ml PDGF for 30 min. The cells were directly lysed in SDS-sample buffer, and the lysates were boiled for 3 to 5 min. Lysate (20 µl) was separated on 10% SDS-PAGE. The gel was blotted and Ser-133-phosphorylated CREB and Ser-69-phosphorylated ATF-1 were visualized as described in the Materials and Methods. Total CREB level in each sample was determined by stripping and reprobing the membrane with an antibody that recognizes CREB regardless of its phosphorylation status (lower panel). A representative experiment is shown.

 
The Raf-MEK Pathway Is Involved in Forskolin-, TPA-, and PGDF-Induced CREB Activation
Growth factors, including PDGF, and phorbol esters are both potent activators of the MAP kinases, ERK1 and ERK2, in NIH 3T3 cells (our unpublished results and Ref. 28). Growth factors such as nerve growth factor and epidermal growth factor have been shown to induce CREB phosphorylation in a Ras/Raf-dependent manner, and MAPKAP-kinase 1b (also known as RSK2) and MAPKAP-K-2 have been identified as these CREB kinases (22). MAPKAP-K2 is activated by the p38 MAP kinase, while MAPKAP-K1b is directly activated by the MAP kinases ERK1/2 (37), and the only known activators of ERK1/2 are the dual specificity kinases MEK1 and MEK2. Activation of ERK1/2 by phorbol esters in NIH 3T3 cells is believed to occur through Raf, which in turn activates MEK1/MEK2 (29). To test whether PKC-induced CREB activation occurs through the Raf-MEK-MAP kinase pathway, we first cotransfected with an expression plasmid for a dominant negative mutant of c-Raf (Raf 1–130) expressing only the N-terminal regulatory domain of c-Raf-1 (38). Expression of Raf 1–130 inhibited TPA-induced CREB activation by more than 70%, while it repressed the forskolin-induced CREBGAL4 activity by almost 70% (Fig. 6Go). Coexpression of a dominant negative mutant of c-Raf did not inhibit basal CREBGAL4 transcriptional activity or ß-galactosidase expression from the cotransfected pCH110 plasmid (results not shown), which argues against a general effect of the inhibitors on either transcription or translation of the reporter gene.



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Figure 6. Raf Is Involved in Forskolin- and TPA-Induced CREB Activation

NIH 3T3 cells were transfected as described in Fig. 2BGo, except that the cells were either pretreated with 50 µM PD98059 for 90 min or cotransfected with 1 µg of dominant negative Raf expression plasmid (pRaf 1–130) before stimulation with either 10 µM forskolin or 50 ng/ml TPA for 3 h. Luciferase and ß-gal activities were measured as in Fig. 2BGo. Each bar represents the mean luciferase/ß-gal ratio (± SD, n = 6). Luciferase activity corrected for ß-gal activity in unstimulated cells was set as 1.

 
To further explore the involvement of the Ras/Raf/MAP kinase pathway in CREB activation by PGDF, TPA, and forskolin, we made use of the recently developed flavone compound PD98059, an efficient and specific inhibitor of MEK1/2 (39, 40). TPA-induced, as well as forskolin-mediated, CREBGAL4 activation was reduced by 60% in the presence of PD98059 (see Fig. 7Go). The real inhibitory effect of PD98059 on TPA- or forskolin-induced CREB activation is probably more since PD98059 alone slightly stimulated CREB-mediated luciferase activity. Pretreatment with this MEK inhibitor strongly repressed PDGF-induced CREBGAL4 activation (Fig. 7Go). PDGF has also been reported to activate ERK1/2 independent of MEK via phosphatidylinositol-3-kinases (PI3K) (41). Pretreatment of cells with wortmannin, a specific inhibitor of PI3K did not influence PDGF-induced CREB activation (data not shown), indicating that CREB activation by PDGF involved MEK rather than PI3K.



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Figure 7. Forskolin-, TPA-, and PDGF-Stimulated CREBGAL4 Activity Is MEK Dependent

NIH 3T3 cells were transfected with pG5E1bLuc and pCREBGAL4 as in Fig. 2BGo, except that some of the cells were pretreated with 50 µM PD98059 for 60 min, before stimulation with either 10 µM forskolin, 50 ng/ml TPA, or 10 ng/ml PDGF for 3 h. Relative luciferase activity was calculated as explained in the legend of Fig. 2BGo. Each bar represents the mean (± SD, n = 3).

 
The results above show for the first time that the Raf-MAP kinase pathway is involved in forskolin-mediated CREB activation. Studies have demonstrated that forskolin and PKA can stimulate ERK1/2 activation via B-Raf in PC12 cells (42). To determine whether forskolin stimulation resulted in ERK1/2 activation, we transfected NIH 3T3 cells with an Elk-1 GAL4 fusion protein where the C-terminal part of Elk-1 (containing the ERK1/2 phosphorylation sites) was fused to the DNA-binding domain of GAL 4. Forskolin was unable to stimulate transcriptional activity from GAL4-Elk-1 (Fig. 8Go), indicating that ERK1/2 was not activated by forskolin. This is agreement with our earlier finding that forskolin was unable to stimulate ERK1/2 activation in NIH 3T3 cells (43). The only known downstream targets for MEK are the MAP kinases ERK1/2. MEK activates ERK1/2 by dual phosphorylation of ERK1/2 on both tyrosine and threonine. The MAP kinases can efficiently be deactivated by a class of dual specificity phosphatases that are able to specifically dephosphorylate the tyrosine and threonine residues phosphorylated by MEK (reviewed in Ref. 44). CL 100 and Pyst-1 are two members of this family of phosphatases. While CL 100 is able to dephosphorylate/deactivate several MAP kinase types (p38, SAPK, and ERKs), Pyst-1 seems to be more specific toward ERK1/2. To determine whether blocking downstream effectors of MEK could also repress forskolin-mediated CREBGAL-4 activity, we cotransfected expression vectors for CL100 and Pyst-1 together with CREBGAL4 and p5GE1bLuc. Coexpression of either phosphatases efficiently repressed both basal and forskolin-induced CREBGAL4-mediated luciferase activity (Fig. 9Go). Taken together, the Raf/MAP kinase pathway is involved in forskolin-mediated CREB activation, but forskolin by itself is not able to activate this pathway. The inhibitory effect of the MEK inhibitor PD98059 on forskolin-induced CREB activation could therefore be the result of a negative effect on PKA activation by forskolin. We tested whether PD98059 interfered with forskolin-induced PKA activation. The results are shown in Fig. 10AGo. Exposure to 10 µM forskolin resulted in a 3-fold increase in PKA activity compared with unstimulated cells, but no effect on either basal (not shown) or forskolin-induced PKA activity was measured. These results support the findings that the Raf/MAP kinase is involved in forskolin-induced CREB activation.



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Figure 8. Forskolin Fails to Stimulate Transcription from GAL4ElkC

NIH 3T3 cells were transfected with 1.0 µg p5GE1bLuc and 0.5 GALElkC together with 0.2 µg pCH110. After serum starvation for 20 h, the cells were treated with 10 µM forskolin (F) or left untreated (U) and 3 h later the cells were harvested. The results represent the mean (± SD) of one experiment with three independent parallels. Relative luciferase activities were calculated as described in Fig. 2BGo.

 


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Figure 9. MAP-Kinase Phosphatases Inhibit Forskolin-Induced CREB Activation

Cells were transfected and treated as in Fig. 2AGo except that either 1 µg pSG5 (Vector), 1 µg CL100, or 1 µg Pyst 1 expression plasmid was added.

 


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Figure 10. MEK Inhibitor Does Not Interfere with Forskolin-Induced PKA Activation or CREB Ser-133 Phosphorylation

A, PKA activity measured in whole-cell extracts from untreated, forskolin-treated, or forskolin plus PD98059-treated cells. After maintenance in medium containing 0.5% serum, the cells were incubated with 50 µM PD98059 for 90 min before stimulation with 10 µM forskolin for 30 min. Preparation of cell extracts and PKA assays were done as described in Materials and Methods. The results are the mean of the PKA activity in three to six separate extracts measured in duplicate. B, PD98059 does not alter forskolin-induced CREB Ser-133 phosphorylation. Western blot analyses with a CREB phospho Ser-133-specific antibody of cells pretreated for 90 min with 50 µM PD98059 and stimulated for 30 min with 10 µM forskolin were performed as described in the legend to Fig. 1Go. The blot was stripped and reprobed with an antibody recognizing CREB regardless of its phosphorylation status (lower panel). The positions of phospho-CREB (P-CREB), phospho-ATF-1(P-ATF-1), and CREB are indicated.

 
Next we examined whether PD98059 affected Ser-133 phosphorylation of CREB. TPA-induced Ser-133 phosphorylation of CREB was reduced in PD98059-treated cells (Fig. 5Go, lanes 2 and 4). However, PD98059 treatment did not repress forskolin-induced Ser-133 phosphorylation (Fig. 10BGo). In contrast to the efficient repression of PDGF-mediated CREB activity by the MEK inhibitor, PDGF-induced CREB Ser-133 phosphorylation was poorly affected by pretreatment with the compound (Fig. 5Go, lanes 3 and 5).

The Q2 Domain of CREB Is Activated by Forskolin, TPA, and PDGF
CREB is regarded as a bipartite transcription factor where both a kinase- inducible domain (KID, amino acids 101–160) and a glutamine-rich domain termed Q2 (amino acids 160–284) are necessary for full CREB activation in response to external stimuli (45). Earlier investigators have shown that the Q2 domain fused to a heterolog DNA-binding domain (GAL4 from yeast) functions as a constitutive transcriptional activator. As we found that the Raf-MEK pathway was necessary for full CREB activation in response to forskolin, TPA, and PDGF independently of the KID domain, we asked whether the Q2 domain could be a possible target. To test this, the cells were transfected with a GAL4 Q2 construct in which amino acids 160–284 of CREB were fused to the DNA-binding domain of GAL4 and stimulated with forskolin, TPA, and PDGF. The GAL4Q2 protein was activated by all three stimuli (Fig. 11AGo). The magnitude of activation by the different stimuli was similar to what was observed with the full-length CREB (Fig. 2AGo). Interestingly, GAL4Q2 was not activated by overexpression of PKA at doses that transactivated the full-length CREB 20- to 30-fold (Fig. 11AGo and results not shown). PKA activity actually decreased GAL4Q2 activity. We found also that the MEK-specific inhibitor PD98059 was able to repress basal GAL4Q2 activity (Fig. 11BGo).



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Figure 11. The Glutamine-Rich Q2 Domain of CREB Can Mediate Forskolin-, PDGF-, or TPA-Induced Transcription

A, TPA, PDGF, and forskolin, but not coexpression of PKA, efficiently activates Q2. NIH 3T3 cells were transfected as in Fig. 2Go except that a GAL4Q2 expression vector was used instead of CREBGAL4. After serum starvation for 20–24 h, the cells were treated for 3 h with 10 µM forskolin (F), 10 ng/ml PDGF or 50 ng/ml TPA (T), or left untreated (U). Some cells were also cotransfected with 1.0 µg expression plasmid for the catalytic subunit of PKA{alpha} (PKA). B, PD98059 represses basal pGAL4 Q2 activity. NIH 3T3 cells were transfected as above. After serum starvation for 20–24 h, the cells were treated for 4 h with either PD98059 or vehicle (U). The results from one representative experiment are presented as ratio luciferase/ß-galactosidase activity with unstimulated GAL4 Q2 activity as 1 (n = 3).

 
In conclusion, the Ras/Raf/MAP kinase pathway is involved in CREB activation by forskolin, PGDF, and TPA, but seems to be dispensable for forskolin- or PGDF-induced Ser-133 phosphorylation because the MEK inhibitor PD98059 did not affect CREB phosphorylation in PGDF- or forskolin-treated NIH 3T3 cells.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CREB was originally isolated as a cAMP-responsive transcription factor whose transcriptional potential was activated through PKA-mediated phosphorylation of Ser-133 in the kinase-inducible domain of CREB (reviewed in Refs. 45, 46). Recent studies have shown that mitogenic stimuli are also able to induce CREB Ser-133 phosphorylation. The identities of these mitogen-regulated CREB kinases are still controversial, but both the MAP kinases, ERK1 and ERK2, and p38-activated kinases have been proposed (9, 22). In the present study we observed that in NIH 3T3 cells, PDGF, serum, and TPA induced CREB Ser-133 phosphorylation with comparable kinetics and quantitative levels as the adenylate cyclase activator forskolin. Thus our results and those of others indicate that CREB can function as an integrator of multiple signaling pathways activated by both mitogenic stimuli and cAMP agonists. However, serum could not increase the activity of CREB while PDGF and TPA could only moderately increase CREB activity as measured in the CREBGAL4 system. Other investigators have failed to detect CREB-mediated transcription by phorbol ester or growth factors despite the fact that these stimuli could induce Ser-133 phosphorylation of CREB (6, 20, 47). These reports, combined with our observation that serum failed to induce CREB-mediated transcription, suggest that Ser-133 phosphorylation per se may not be sufficient for CREB activation and that additional events, involving separate signaling pathways, may be necessary for activation of CREB-mediated transcription.

Since the PKA-, PKC-, and Raf-MEK-dependent signaling pathways have been implicated in the regulation of CREB transcriptional activation (5, 7, 48) we wanted to investigate the role of each of these signaling pathways in CREB-mediated transcription induced by either forskolin, TPA, or PDGF. The fact that recent reports have demonstrated that cAMP- and mitogen-stimulated signaling pathways mediate extensive cross-talks also at levels upstream of CREB further justified such an investigation (26, 42).

PKA has a central role in CREB-mediated transcription, not only in response to cAMP, but also for PKC and calcium-induced CREB activation (20, 49). Our results from overexpression of PKI indicated a positive role for PKA in TPA- induced CREB activation in NIH 3T3 cells. This role for PKA in mitogen-stimulated gene expression is in agreement with the previous observation by Day and co-workers (27), who found that PKI repressed TPA-induced gene expression from a PRL promoter. A positive role for PKA in MAP kinase-mediated signaling has been demonstrated involving a novel Ras-independent pathway via the Ras-related small G protein Rap-1. Rap-1 is a selective activator of B-Raf and an inhibitor of c-Raf-1 (42). We found, however, that expression of neither B-Raf nor c-Raf-1 was induced by forskolin, and the levels of B-Raf and c-Raf-1 proteins in our NIH 3T3 cells were comparable (results not shown). Moreover, if forskolin could activate the MAP kinase pathway in NIH 3T3 cells, one should expect that inhibition of PKA also would repress TPA-induced Elk-1 activation since Elk-1 is a well known target for the MAP kinase pathway (30). However, we failed to detect any effect of PKI overexpression on TPA-induced Elk-1 activation. An alternative explanation comes from the study by Zhong et al. (26), which showed that a fraction of the C{alpha} subunit of PKA resides in complex with NF-{kappa}B. This activity can be released from NF-{kappa}B independent of cAMP. Phorbol esters as TPA are well known inducers of NF-{kappa}B in different cell types (50, 51). Therefore, TPA-induced CREB activation in NIH 3T3 cells may be assigned to the kinase activity of the PKA C{alpha} subunits liberated from NF-{kappa}B. If this were true, TPA and PDGF must activate NF-{kappa}B differently in NIH 3T3 cells, since PDGF-induced CREB activation was independent of any PKA activity as shown by the use of PKI. While we found that TPA alone could moderately increase the transcriptional activity in NIH 3T3 cells, Brindle and co-workers (20) showed that suboptimal concentrations were necessary to activate CREBGAL4 in phorbol ester-stimulated Jurkat cells. A threshold level of activated PKA seems to be required to activate CREB by phorbol ester. We observed a basal level of PKA level in serum-starved NIH 3T3 cells (Fig. 10AGo). This residual PKA activity may be sufficient to obtain CREB activation by phorbol ester alone, while in Jurkat cells suboptimal concentrations of forskolin are required to obtain a critical PKA activity. Expression of PKA inhibitor protein will block this basal PKA activity and, therefore, phorbol ester-induced CREB activation.

The fact that PDGF activates CREB independently of PKA strongly suggests that alternative signaling pathways are involved in CREB-mediated transcription. Since PDGF can activate classical PKCs (cPKC) through activation of phospholipase C{gamma} and PKC has been shown to induce CREB-mediated transcription, there is a possibility that a PKC-dependent signaling pathway may be involved. Our results with the inhibitor GF109203X or PKC depletion by long-term exposure to TPA indicate that neither the classical PKCs nor the novel PKC{delta} and -{epsilon} isoforms are necessary for PDGF-induced CREB activation. It does not, however, exclude that other PDGF-activated PKC isoforms such as {zeta} or {lambda}, which are not inhibited by GF109203X or TPA down-regulation, could be responsible (52).

By the use of a dominant negative mutant of Raf or the MEK inhibitor PD98059 we found that PDGF-, TPA-, and forskolin-induced CREB activation were dependent on the Raf-MEK signaling pathway. PDGF and TPA are believed to transmit signals from the membrane to the nucleus through MEK and the downstream MAP kinases ERK1 and -2. Therefore, it was not surprising to find that inhibition of MEK blocked transcriptional activity of CREB induced by PDGF. However, the fact that PD98059 repressed both PDGF- and forskolin-induced CREB activation, but not CREB Ser-133 phosphorylation, was more intriguing. Recent studies have shown that cAMP and PKA can activate MAP kinase and Elk-1 in PC 12 cells independently of Ras via the Ras-related G protein RAP-1 (42). The observation that forskolin failed to activate Elk-1- mediated transcription, combined with our previous results which showed that forskolin was unable to activate the MAP kinases ERK1/2 in NIH 3T3 cells, means that a basal level of MEK activity rather than activation of the MEK pathway is necessary for maximal CREB activation in response to forskolin (43). Although activation of MEK without subsequent activation of ERK has been reported, these studies suggested that the ultimate function of MEK is dependent on the manner in which MEK is activated (53). One of the upstream activators of MEK is Raf-1 (54). We found that overexpression of a dominant negative mutant of Raf-1 repressed forskolin-induced CREB activation, which indicates involvement of an upstream signal to MEK induced by forskolin. It is also likely that the Ras-binding domain of Raf-1 can interact with other members of the Ras family, such as Rap-1. Therefore, the dominant negative Raf-1 mutant used in this study may interfere with Rap-1 effectors. Recent studies have pointed out that Ras has multiple effectors in addition to Raf-1, and forskolin activation of MEK may involve upstream activators other than Raf-1 (reviewed in Ref. 55). Furthermore, we demonstrated, by using the two dual specificity phosphatases, CL100 and Pyst1, a role for a downstream effector of MEK in forskolin-induced CREB activation. The fact that the phosphatase Pyst 1 deactivates ERK1/ERK2 100-fold better than other members of MAP kinases, combined with the known specificity of MEK, indicates that this effector operates at the level of the ERK1/2 or further downstream of MAPK, represented by the MAPKAP kinases (56, 57). The use of a dominant-negative mutant of MAPKAP-K1a and GF109203X (a potent inhibitor of MAPKAP-K1b), however, indicates that these kinases downstream of ERK1/2 are not involved (Fig. 3Go and data not shown; Ref. 58).

In conclusion, we found that PDGF and forskolin induce at least two signals that target CREB. One of these signals triggers CREB Ser-133 phosphorylation, while the other signal governs the transcriptional activity state of CREB. This latter signal is MEK dependent and may involve a novel signaling pathway from forskolin through MEK down to CREB. Experiments from other cell lines or in vitro transcriptional systems further support the model that more than one signal is needed for full transcriptional activation of CREB (20, 59, 60). CREB is a bipartite transcription factor consisting of a KID and a noninducible constitutive glutamine-rich Q2 domain (45). These two domains synergize to induce transcription by CREB. Further examination of the mechanism required for CREB-mediated transcription of target genes after its phosphorylation at Ser-133 led to the proposal of a two-signal model for target gene activation: a phospho-Ser-133-dependent interaction of CREB with RNA polymerase II via the coactivator CBP, and a glutamine-rich domain interaction with TFIID via hTAFII130 (60). Recruitment of CBP-RNA pol II complex per se was not sufficient for transcriptional activation, but activator-mediated recruitment of TFIID was also required for induction of signal-dependent genes (60). Our results indicate that a signal through the MAP kinase pathway may effect the activator-mediated recruitment of TFIID to the complex via Q2 domain of CREB and TAFII130. The inhibition of transcriptional activity from a GAL4 Q2 chimeric protein by the MEK inhibitor supports this assumption (Fig. 11BGo). A previous study has demonstrated that the Q2 domain of CREB functions as a constitutive activator when fused to GAL4 in F9 cells (45). However, it appears that the Q2 domain when fused to GAL4 is inducible to both growth stimuli and cAMP in NIH 3T3 cells. Whether these signals influence the Q2 domain directly or the proteins that interact with the Q2 domain at the promoter is still open for investigation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Forskolin, TPA, and PDGF were purchased from Sigma Chemical Co. (St. Louis, MO). NCS was from BioWhittaker, Inc. (Verviers, Belgium). Cell culture medium was obtained from Gibco BRL, Gaithersburg, MD). Phospho-CREB(133) and CREB antibodies, biotinylated protein molecular weight standard, and CDP-Star were all obtained from New England Biolabs, Inc. Beverly, MA). Alkaline phosphatase-conjugated swine antirabbit antibody was purchased from DAKO Corp. (Copenhagen, Denmark). Dual assay kit was acquired from Perkin Elmer-Tropix. PD98059 was from New England Biolabs, Inc. GF109203X was purchased from Calbiochem (La Jolla, CA). Heat-stable PKA inhibitor peptide PKI TTYADFIASGRTGRRNAIHD was from Sigma Chemical Co. CREBtide KRREILSRRPSYRK was synthesized by Ø. Rekdal (University of Tromsø).

Cell Culture
NIH 3T3 cells (ATCC CRL 1658, American Type Culture Collection, Manassas, VA) were maintained in DMEM supplemented with 10% (vol/vol) NCS, HEPES, NaHCO3, penicillin (100 U/ml), and 100 µg/ml streptomycin (Life Technologies, Gaithersburg, MD).

Plasmids
The GAL4 fusion protein expression vectors, pCMVCREBGAL4 and pCMVALA133 CREBGAL4, have been described previously; pCMVPKI and pCMVPKImut are improved versions of the expression plasmids of the heat-stable inhibitor of PKA and were all generously provided by R. A. Maurer (17, 27). GAL Q2 expressing the Q2 domain of CREB fused to GAL4 has been described previously and was provided by M. Montminy (45). The catalytic subunit of PKA {alpha} was expressed from a pSR vector provided by K. Tasken (43). The plasmid G5E1bLuc, which contains five binding sites for the yeast transcription factor GAL4 upstream of an adenovirus E1b TATA-box and a luciferase gene, was provided by R. J. Davis (61). Plasmid pCH110 was purchased from Pharmacia Biotech (Uppsala, Sweden) and contains the bacterial lacZ gene driven by a SV40 early promoter. The two dual-specificity phosphatases, Pyst1 and CL100, were expressed as myc-tagged proteins from the eukaryote expression vector pSG5 and were generously provided by S. M. Keyse (56). The dominant-negative mutant of c-Raf was expressed from the plasmid pRaf 1–130 provided by C. J. Der (38).

Transient Transfection and Luciferase Assay
For reporter gene assays, NIH 3T3 cells (35-mm wells) were transfected with 0.5 µg of a GAL4 fusion protein expression plasmid together with 1.0 µg of a GAL4 luciferase reporter plasmid pG5E1bLuc using the Ca-phosphate coprecipitation method (62). In some experiments, cotransfections were performed with various amounts of expression plasmids as indicated in the figure legends. The total amount of plasmid DNA in each transfection reaction was maintained constant at 4 µg by adjusting with the prokaryotic plasmid pGEM3Zf. The cells were serum starved 20–24 h posttransfection and then stimulated for 3 h with either 10 µM forskolin, 10% NCS, 10 ng/ml PDGF, or 50 ng/ml TPA before harvesting in 100 µl potassium phosphate, pH 7.8, 0.2% Triton X-100, and 0.5 mM dithiothreitol. Cotransfection with 0.2 µg of a ß-galactosidase reporter (pCH110, Pharmacia Biotech) was performed to correct for variations in transfection efficiency and sample handling. Luciferase and ß-galactosidase activities were determined in 10 µl lysate using the Dual-Assay Kit (Perkin Elmer-Tropix) and a Luminoscan RT (Labsystems OY, Helsinki, Finland) luminometer according to the manufacturer’s instructions.

Protein Kinase Assay
Whole-cell extracts were made as described previously (63). NIH 3T3 cells were serum starved for 20–24 h before they were stimulated for 30 min (as described in figure legends), and the 100-mm culture dish was placed on ice. The medium was removed and the monolayer washed two times with PBS before the cells were scraped off in 500 µl lysis buffer (10 mM Tris-HCl, pH 7.05, 50 mM NaCl, 50 mM NaF, 1% Triton X-100, 30 mM Na4P2O7, 5 µM ZnCl2, 100 µM Na3VO4, 1 mM dithiothreitol, 2.8 µg aprotinin per ml, 2.5 µg each of leupeptin and pepstatin per ml, 0.5 mM benzamidine, and 0.5 mM phenymethylsulfonyl fluoride (all from Sigma Chemical Co.). After vigorous vortexing for 45 sec at 4 C, the lysates were cleared by centrifugation at 10,000 x g for 10 min at 4 C and frozen in aliquots at 70 C. PKA activity was determined as described previously (43). Briefly, 10 µl extract were incubated in a total volume of 40 µl containing 50 mM Tris, pH 7.5, 10 mM MgCl, 100 µM({gamma}-32 P), 0.25 mg/ml BSA, and 50 µM of CREB-tide as a substrate. Reaction mixtures were incubated for 10 min at 30 C and spotted onto nitrocellulose paper. The filters were washed twice in 1% phosphoric acid and twice in water, and radioactivity was determined by scintillation counting. Total PKA activity was determined in the presence of 10 µM cAMP. PKA activity was defined as that sensitive to the inhibitor peptide PKA inhibitor peptide (1 µM). Each value represents the mean of at least three parallels.

Immunoblotting
Cells were grown in 35-mm wells in medium containing 0.5% NCS for 20–24 h and treated with 10 µM forskolin, 10 ng/ml PDGF, 10% NCS, or 50 ng/ml TPA for the times indicated. The cell extracts were prepared as described previously (43). Twenty microliters of lysate were separated by 10% SDS-PAGE, and proteins were detected by immunoblotting using phospho-CREB or CREB control antibody as described previously (43). The blots were developed using a rabbit alkaline phosphatase-conjugated antibody and the chemiluminiscence substrate CDP-Star (Perkin Elmer/Tropix). Molecular weights were estimated using a biotinylated broad range molecular weight protein standard and alkaline phosphatase-conjugated antibiotin antibody (New England Biolabs, Inc.).


    ACKNOWLEDGMENTS
 
The authors wish to thank R. J. Davis, C. J. Der, T. Johansen, S. M. Keyse, R. Maurer, M. Montminy, and K. Tasken for providing the following plasmids: p5GE1bLuc, pRaf (1–130), pGAL4-ElkC, pSGCL100, pSGPyst1, pCMVCREBGAL4, pCMVALA 133 CREBGAL4, pCMVPKI, pCMVPKImut, pGAL4-Q2, and pSRPKA-{alpha} respectively; Ø. Rekdal for synthesizing the CREB-tide; and R. Robberrecht for critically reading the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Ugo Moens, Institute of Medical Biology, Department of Gene Biology, University of Tromsø, Tromsø, Norway N-9037.

This work was supported by fundings from the Norwegian Cancer Society (D.N.K.) and the Erna and Olav Aakre Foundation.

Received for publication May 21, 1998. Revision received January 15, 1999. Accepted for publication March 16, 1999.


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