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Keywords:

  • c-fos;
  • extracellular signal-regulated kinases;
  • mitogen-activated protein kinase;
  • nerve growth factor;
  • PC12 cells

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The duration of intracellular signaling is thought to be a critical component in effecting specific biological responses. This paradigm is demonstrated by growth factor activation of the extracellular signal-regulated kinase (ERK) signaling cascade in the rat pheochromocytoma cell line (PC12 cells). In this model, sustained ERK activation induced by nerve growth factor (NGF) results in differentiation, whereas transient ERK activation induced by epidermal growth factor (EGF) results in proliferation in these cells. Recently, the immediate early gene product c-fos has been proposed to be a sensor for ERK signaling duration in fibroblasts. In this study, we ask whether this is true for NGF and EGF stimulation of PC12 cells. We show that NGF, but not EGF, can regulate both c-fos stability and activation in an ERK-dependent manner in PC12 cells. This is achieved through ERK-dependent phosphorylation of c-fos. Interestingly, distinct sites regulate enhanced stability and transactivation of c-fos. Phosphorylation of Thr325 and Thr331 are required for maximal NGF-dependent transactivation of c-fos. In addition, a consensus ERK binding site (DEF domain) is also required for c-fos transactivation. However, stability is controlled by ERK-dependent phosphorylation of Ser374, while phosphorylation of Ser362 can induce conformational changes in protein structure. We also provide evidence that sustained ERK activation is required for proper post-translational regulation of c-fos following NGF treatment of PC12 cells. Because these ERK-dependent phosphorylations are required for proper c-fos function, and occur sequentially, we propose that c-fos is a sensor for ERK signaling duration in the neuronal-like cell line PC12.

Abbreviations used:
DMSO

dimethylsulfoxide

EGF

epidermal growth factor

ERK

extracellular signal-regulated kinase

NGF

nerve growth factor

PBS

phosphate-buffered saline

PBST

phosphate-buffered saline + 0.1% Tween-20

PDGF

platelet-derived growth factor

SDS–PAGE

sodium dodecyl sulfate – polyacrylamide gel electrophoresis

TAD

transactivation domain

WT

wild type

Nerve growth factor (NGF) triggers neuronal differentiation in the PC12 cell model through the sustained activation of the mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK). Neuronal differentiation by NGF is characterized by the induction of immediate early genes that encode transcription factors that promote the transcriptional activation of a set of NGF-responsive genes. Because of the requirement of sustained ERK activation for differentiation, NGF action on PC12 cells has served as a model for the role of duration of intracellular signals in dictating physiological responses. However, the mechanism by which transcription factors sense sustained ERK activation in PC12 cells is not known.

Other cell types have provided insight into how incremental changes in the duration of ERK activation can have profound effects on cellular responses (Weber et al. 1997; Bottazzi et al. 1999; Roovers et al. 1999; Adachi et al. 2002; Murphy et al. 2002; Koike et al. 2003; Werlen et al. 2003). For example, one the one hand, in Swiss 3T3 fibroblast cells, the sustained activation of ERKs is required for growth factor-induced proliferation by platelet-derived growth factor (PDGF). On the other hand, transient activation of ERKs by epidermal growth factor (EGF) is not mitogenic in these cells. One ERK target, the transcription factor c-fos, has been proposed to mediate this action (Murphy et al. 2002). c-fos is a proto-oncogene that, unlike its oncogenic counterpart, v-fos, requires additional signals to achieve maximal proliferative potential (Chen et al. 1993; Okazaki and Sagata 1995; Chen et al. 1996; Monje et al. 2003). ERK-dependent signals stimulate c-fos at multiple levels (Monje et al. 2005), but perhaps the best studied of these actions is the stimulation of c-fos transcription (Monje et al. 2003; Tanos et al. 2005). This is achieved by phosphorylation of the transcription factor Elk-1 which functions with other serum response factors to turn on the c-fos promoter (Gille et al. 1992; Hipskind et al. 1994).

Sustained ERK activation is not always associated with proliferation (Okazaki and Sagata 1995; York et al. 2000; Boss et al. 2001; Garcia et al. 2001). In the rat pheochromocytoma cell line (PC12 cells; Tischler and Greene 1975; Greene and Tischler 1976; Traverse et al. 1992; Marshall 1995), sustained activation of ERKs by NGF is required for the induction of neuronal differentiation and growth arrest (Cowley et al. 1994; Marshall 1995; Kao et al. 2001). In a recent report, c-fos was also shown to be required for NGF-dependent differentiation and neurite outgrowth of PC12 cells (Gil et al. 2004). It is possible that, despite differences in the cellular response to sustained activation of ERKs, PC12 cells and Swiss 3T3 cells share targets of sustained ERK activation.

Direct phosphorylation of c-fos protein by ERKs can also enhance c-fos function at AP-1 promoters (Sutherland et al. 1992; Monje et al. 2003). This occurs via two interdependent mechanisms. First, ERK-dependent phosphorylations on Ser362 and Ser374 within the C-terminus (called ‘priming’ phosphorylations) can stabilize c-fos, possibly by interfering with degradation signals within the c-fos protein (Okazaki and Sagata 1995; Ferrara et al. 2003). The Ser374 site has been shown to be phosphorylated by ERKs in vivo and in vitro (Chen et al. 1996). However, ERK does not phosphorylate c-fos at Ser362 in vitro (Monje et al. 2003), and additional kinases have been proposed for the phosphorylation of the Ser362 site (Tratner et al. 1992; Chen et al. 1993). RSK2, an ERK-dependent kinase, has been implicated in growth factor-induced phosphorylation of this site. Most previous studies examining these sites within c-fos utilized c-fos double mutants that were mutated at both sites (Chen et al. 1993; Okazaki and Sagata 1995; Chen et al. 1996; Murphy et al. 2002, 2004). By examining individual mutants of these sites, we show that phosphorylations at these sites have distinct contributions to c-fos stability and priming of c-fos for further phosphorylations.

Second, these phosphorylations prime additional ERK phosphorylations within the transactivation domain (TAD) of c-fos that Gutkind and colleagues have shown potentiate AP-1-dependent transcription (Monje et al. 2003). These phosphorylations on Thr325 and Thr331 (and possibly Thr232) are thought to contribute to the retarded electrophoretic mobility shift associated with elevated c-fos phosphorylation and function (Monje et al. 2003). TAD phosphorylation by ERKs may be enhanced by directing the binding of ERKs to an ERK targeting domain, also known as a DEF domain, which has been identified near these sites (Murphy et al. 2002).

In this study, we test whether c-fos is a sensor of sustained ERK activation in PC12 cells. We show that phosphorylations of Thr325 and Thr331 are required for maximal NGF-dependent transactivation of c-fos in PC12 cells. Like in fibroblasts, c-fos requires an intact DEF domain for transactivation. c-fos stabilization and conformational changes in protein structure are also regulated by ERK-dependent sites, namely Ser374 and Ser362, respectively. We provide evidence that sustained ERK activation is required for post-translational regulation of c-fos during differentiation of PC12 cells. Thus, our data suggest that both neuronal and non-neuronal cell types utilize c-fos as a sensor for ERK signaling duration despite the fact that this transcription factor is coupled to distinct physiological outcomes, differentiation and proliferation, respectively, in these cell types.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

PC12 cells were kindly provided by Patrick Casey (Duke University, Durham, NC, USA). The MEK1/2 inhibitor, UO126, was purchased from Calbiochem (La Jolla, CA, USA). NGF was purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA). Epidermal growth factor and anti-Flag (M2) antibody were purchased from Sigma (St Louis, MO, USA). Anti-ERK2 (C-14 and D-2) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phosphorylation-specific ERK1/2 antibodies (Thr202/Tyr204; no. 9101-polyclonal) and (no. 9106 L-monoclonal) were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-fos (pSer374) antibodies (no. ST1029) were purchased from Calbiochem (La Jolla, CA, USA). LipofectAMINE 2000 was purchased from Invitrogen (Carlsbad, CA, USA). All other chemical reagents were purchased from Sigma.

Cell culture

PC12 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% horse serum and 5% fetal bovine serum at 37°C in 5% CO2 (70% confluence). Unless otherwise stated, cells were deprived of serum for 16 h before being treated with various reagents for luciferase assays, TransAM™ assays, and western blotting.

Plasmids and transfections

PC12 cells were transiently transfected with LipofectAMINE 2000 as per the manufacturer's instructions. After transfection, cells were serum starved and treated with NGF (100 ng/mL), EGF (50 ng/mL), and UO126 (20 μm), unless otherwise stated. Mouse c-fos cDNA (clone ID/ATCC no. 3439554) was purchased from the IMAGE Consortium/ATCC (Manassas, VA, USA). c-fos was amplified by PCR using oligonucleotide primers and cloned with specific restriction enzyme sites (EcoR1/Xba). The resulting fragment was cloned into pcDNA3 (Invitrogen) containing a Flag epitope (Flag-Fos). The c-fos mutations were engineered into Flag-Fos by PCR site directed mutagenesis. All plasmids were verified by sequencing. The CMV promoter was used to drive expression of the Flag-Fos. To generate Gal4-Fos constructs, full-length c-fos and mutants were subcloned into a pcDNA3 vector encoding the DNA binding domain of the yeast transcription factor Gal4. A Gal4-regulated luciferase reporter (Gal4-luciferase) was provided by Richard Maurer (Oregon Health Sciences University). For western blot experiments, the following amounts of DNA were used: 2 μg of Flag-c-fos and 2 μg Flag-GFP, with pcDNA3 (vector) to make total amounts of transfected DNA equal. For luciferase assays, the following amounts of DNA were used: 250 ng of Transin-luciferase, 500 ng AP-1 luciferase (Stratagene, La Jolla, CA, USA), 20 ng pRL-Null (Promega, Madison, WI, USA), 500 ng of Gal4-c-fos plasmids, and 500 ng vector. For TransAM™ assays, 10-cm plates were transfected with 10 μg of CMV-Flag-Fos plasmids or 10 μg of vector.

AP-1-luciferase and transin-luciferase reporter gene assays

PC12 cells were plated onto 24-well plates and transfected the following day as indicated. Cells were serum starved and pretreated with UO126 or dimethylsulfoxide (DMSO) for 20 min, then treated with growth factors for 6 h. Cells were lysed in luciferase lysis buffer (1% triton-X, 110 mm K2HPO4, 15 mm KH2PO4, pH 7.8) and equal amounts of protein were assayed for luciferase enzyme activity in an Autolumat LB953 luminometer (Berthold, Bundoora, Australia). Luciferin (0.15 mm; Sigma) in water was used as a substrate along with 5 mm ATP, 15 mm MgSO4, 25 mm gly-gly to drive the reaction. Data are expressed as relative light units (RLUs).

Gal4-luciferase reporter gene assays

PC12 cells were plated onto 24-well plates and transfected the following day as indicated. Cells were serum starved and treated with NGF for 6 h. Cells were lysed in Passive Lysis Buffer (Promega) and the Dual-Luciferase Reporter Assay System (Promega) was used to determine luciferase activity as per the manufacturer's instructions. Luciferase values were measured in a Veritas Microplate Luminometer (Turner Biosystems, Sunnyvale, CA, USA). Equal volumes of samples were analyzed and both firefly (Photinus pyralis) and sea pansy (Renilla reniformis) luciferase were measured sequentially from the same. Final values are taken as a ratio of the firefly readings (experimental) over the sea pansy readings (internal control), and expressed as arbitrary units (AUs). Unless otherwise stated, each experiment was performed three times with four replicates each and all values normalized to the values of the NGF-treated wild-type Gal4-c-fos (Gal4-WT) samples.

TransAM™ DNA binding assays

To determine DNA binding of Flag-c-fos and mutants, the AP-1 Transcription Factor Assay Kit (Active Motif, Carlsbad, CA, USA) was used with minor modifications to the manufacturer's protocol. PC12 cells were plated onto 10-cm plates and transfected the following day as indicated. Cells were treated with NGF for 2 h and nuclear extracts were prepared using the Nuclear Extract Kit (Active Motif) as per the manufacturer's instructions. Protein concentrations were determined using the Coomassie Plus Bradford Assay kit (Pierce, Rockford, IL, USA). Nuclear extract (20 μg) was added to the matrix containing a TPA-response element (TRE) with the 5′-TGAC/GTCA-3′ sequence. The primary antibody used in this ELISA-based assay was anti-Flag (M2) antibody (Sigma) in place of the manufacturer's primary antibody. Accordingly, an anti-mouse horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences, Buckinghamshire, UK) was used in place of the manufacturer's secondary antibody. Absorbances were read on a Labsystems Multiskan RC Microplate Reader (Fisher Scientific, Rochester, NY, USA) as instructed. Data are generated from an average of three individual experiments normalized to NGF-treated wild-type Flag-c-fos samples (Fos-WT; set at unity).

Lambda protein phosphatase experiments

PC12 cells were plated onto 6-well plates and transfected as indicated. Cells were pretreated with UO126 or vehicle (DMSO) for 20 min. Cells were treated with NGF for 2 h and then lysed in low-stringency lysis buffer [1 × phosphate-buffered saline (PBS), 0.1% NP40, 50 mmβ-glycerolphosphate, 10 mm NaF, 1 mm sodium orthovanadate, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mm phenylmethylsulfonyl fluoride]. Reactions (20 μL) were prepared with equal volumes of lysate with lambda protein phosphatase (New England Biolabs, Beverly, MA, USA) as per the manufacturer's instructions. Briefly, reactions were incubated at 30°C for 20 min and laemmli buffer was added to stop the reactions. Some samples received water in place of phosphatase (DMSO and UO126 samples). Western blotting was then performed as described and Flag (M2) antibody was used to examine changes in the mobility shifts of Flag-Fos.

Western blotting

Unless otherwise stated, cells were deprived of serum for 16 h prior to treatment. Cells were lysed in RIPA buffer (1% triton-X 100, 1% sodium deoxycholate, 0.2% sodium dodecyl sulfate, 125 mm NaCl, 50 mm Tris pH 8.0, 10% glycerol, 1 mm EDTA, 25 mmβ-glycerolphosphate, 25 mm NaF, 1 mm sodium orthovanadate, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mm phenylmethylsulfonyl fluoride) and equal amounts of lysate were sonicated three times in a bath sonicator (S-3000; Misonix Inc., Farmingdale, NY, USA) at level 6 for 30 s. Laemmli buffer was then added to the lysates and they were boiled for 2 min. Proteins were resolved by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and two blotting techniques were employed. In one, proteins were transferred onto polyvinylidine difluoride membranes and blocked in 5% milk diluted in PBS + 0.1% Tween-20 (PBST). Membranes were probed with primary antibodies in PBST as per the manufacturer's instructions. Horseradish peroxidase-conjugated secondary antibodies were used to detect proteins by enhanced chemiluminescence (Western Lightning; PerkinElmer Life Sciences, Boston, MA, USA). In the second method, proteins were transferred onto nitrocellulose (Protran, Schleicher and Schuell, Keene, NH, USA) and blocked in Odyssey™ blocking buffer (LI-COR Biosciences Inc., Lincoln, NE, USA). Primary antibodies were diluted in Odyssey blocking buffer 1 : 1 with PBST, as per the manufacturer's instructions. The appropriate fluorescently labeled secondary antibodies were diluted in Odyssey blocking buffer 1 : 1 with PBST, 0.01% SDS. Membranes were scanned with the Odyssey Infrared Imaging System (LI-COR Biosciences Inc.).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

ERKs are required for AP-1-dependent gene expression by NGF

NGF and EGF are well-known activators of ERKs in PC12 cells (Vaudry et al. 2002; Horgan and Stork 2003). However, these growth factors activate ERKs with very different kinetics. For comparison purposes, we chose concentrations of each growth factor that gave maximal ERK activation at 5 min, a time point of activation shared by both NGF and EGF, and used those concentrations for the duration of this study (Figure S1). One target of ERKs is the transcription factor c-fos, which participates in the activation of AP-1 promoter elements. Using AP-1 luciferase as a reporter of AP-1-dependent gene expression, we show that NGF, when compared with EGF, caused a more robust activation of AP-1-luciferase in PC12 cells. Although both growth factors had a statistically significant increase in AP-1-dependent gene expression, the effect elicited by NGF was greater than twice that of EGF. Both effects were dependent on activation of the ERK cascade as they were blocked by UO126, an inhibitor of the ERK kinase MEK (Fig. 1a). The ERK dependence of growth factor's actions on AP-1 is reflected in the induction of c-fos-response genes. One well-studied gene encodes the metalloprotease transin, which contains well-defined AP-1 sites within its promoter (Machida et al. 1991). Both NGF and EGF stimulated expression of the transin promoter linked to luciferase. Although both effects were statistically significant, NGF activation of the transin promoter was approximately five times greater than that of EGF. Activation of the transin promoter by both NGF and EGF was blocked by UO126 (Fig. 1b). These data suggest that the differences between NGF and EGF may reflect differences in their ability to activate ERKs.

image

Figure 1.  NGF-specific activation of AP-1-dependent gene expression requires ERKs in PC12 cells. (a) PC12 cells were transfected with an AP-1-luciferase plasmid (Stratagene). Cells were pretreated with UO126 or vehicle (DMSO) for 20 min prior to simulation with NGF or EGF (6 h). Lysates were harvested for luciferase assay as described in Materials and methods. Luciferase activity is shown as relative light units (RLUs). The data are means ± SE (n = 4). Asterisks indicate significant differences compared with vehicle control conditions. The statistical significance was calculated by a one-tailed unpaired Student's t-test; *p = 0.0000045, **p = 0.00026. (b) PC12 cells were transfected with a Transin-luciferase plasmid. Cells were pretreated with UO126 or vehicle (DMSO) for 20 min prior to simulation with NGF or EGF (6 h). Lysates were harvested for luciferase assay as described in Materials and methods. The data are means ± SE (n = 4). Asterisks indicate significant differences compared with vehicle control conditions. The statistical significance was calculated by a one-tailed unpaired Student's t-test; *p = 0.00024, **p = 0.00529.

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NGF induction of c-fos protein requires sustained activation of ERKs

One of the hallmarks of c-fos induction in fibroblast cells is the absence of c-fos protein in unstimulated cells (Muller et al. 1984). The more robust induction of AP-1 and the transin promoter by NGF, versus EGF, in PC12 cells may reflect differences in the levels of c-fos protein induced by these growth factors. We examined whether treatment with either NGF or EGF could result in the accumulation of c-fos protein in PC12 cells. NGF triggered a sustained activation of ERKs that remained readily detectable for 120 min (Fig. 2a, middle panel), and was still detectable at 6 h (Figure S2). c-fos protein was undetectable in resting PC12 cells and was induced within 60 min of NGF treatment and remained detectable at 120 min (Fig. 2a, top panel). UO126 dramatically reduced the level of c-fos protein induced by NGF (Fig. 2b, top panel). In contrast to NGF, EGF activation of ERKs in PC12 cells was robust but transient, returning close to baseline levels after 10 min (Fig. 2a, middle panel), and remained at basal levels for at least 6 h (Figure S2). This transient activation was much less efficient at inducing detectable levels of c-fos protein (Fig. 2a, top panel).

image

Figure 2.  NGF stimulation of c-fos protein levels requires MEK. (a) PC12 cells were treated with NGF and EGF and the lysates were resolved by SDS–PAGE. Induction of c-fos protein was detected by SDS–PAGE at the indicated time points (top panel). ERK1/2 activation, measured by phospho-ERK1/2 antibody (pERK Ab), is shown in the middle panel. Equal loading of protein was demonstrated by measuring levels of ERK2 (bottom panel). (b) Cells were pretreated with UO126 or vehicle (DMSO) for 20 min prior to stimulation with NGF. Cells were harvested at the indicated times and c-fos protein levels are shown in the top panel. Activation of ERK1/2 was measured using pERK Ab (middle panel). Equal loading of protein was demonstrated by measuring levels of ERK1/2 (bottom panel). (c) PC12 cells were treated with UO126 before NGF application (−10′) and after NGF application (+20′ and +50′). c-fos protein levels were measured using c-fos antibodies (first panel). Phosphorylation of c-fos on residue Ser374 is measured using anti-Fos (pSer374) antibodies (second panel). Activation of ERK1/2 was measured using pERK Ab (third panel). Equal loading of protein was demonstrated by measuring levels of ERK2 (fourth panel).

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Sustained activation of ERKs has recently been proposed to mediate c-fos protein stabilization (Murphy et al. 2002). To determine whether sustained activation of ERKs is required for NGF's ability to accumulate detectable levels of the c-fos protein, we incubated cells with UO126 at various time points after NGF treatment. Pretreatment of cells with UO126 (−10′) inhibited NGF-dependent increases in c-fos protein levels (Fig. 2b, top panel). Interestingly, inhibition of ERKs at times (+20′, +50′) subsequent to NGF treatment also causes a marked inhibition of c-fos protein levels (Fig. 2c, first panel). In fibroblasts, Ser374 phosphorylation has been shown to be ERK dependent and involved in c-fos protein stabilization (Chen et al. 1996). UO126 application causes inhibition of NGF-dependent Ser374 phosphorylation in PC12 cells (Fig. 2c, second panel). These data suggest that sustained ERK signaling enables NGF to stimulate and maintain c-fos protein levels and phosphorylation.

NGF stabilization of c-fos requires ERKs

ERK stimulates the induction of c-fos protein by both transcriptional and post-translational mechanisms. To specifically examine these post-transcriptional effects, independent of transcriptional effects, we examined the expression of an epitope tagged c-fos construct (Flag-Fos) under the control of the CMV promoter. Initial experiments using Flag-GFP under control of the CMV promoter demonstrated that NGF did not regulate this promoter as shown by equal levels of Flag-GFP protein (Fig. 3a, top panel). In addition, UO126 had no effect on basal Flag-GFP levels (data not shown). In PC12 cells, low levels of Flag-Fos were detectable in the absence of stimulation as a result of basal activation of the CMV promoter. NGF treatment caused an increase in Flag-Fos protein levels that was evident at 2 h (Fig. 3b, top panel). In addition, NGF induced mobility shifts previously associated with hyperphosphorylation of c-fos (Monje et al. 2003). The ERK-dependence of these mobility shifts was confirmed by pretreating cells with UO126, which resulted in the loss of all but the lowest (fastest) migrating band (Fig. 3c, top panel). The phosphorylation dependence of these shifts was confirmed by their absence upon treatment of lysates with lambda phosphatase (Fig. 3c, top panel). In contrast, EGF stimulation had a minor, albeit significant, effect on stabilization and mobility shift of Flag-Fos (Fig. 3b, top panel). Flag-GFP expression levels were constant and served as an internal control (Fig. 3b, bottom panel). Thus, we focused on NGF's regulation of c-fos in the remainder of the study.

image

Figure 3.  Sustained ERK1/2 activation regulates Flag-c-fos phosphorylation and protein levels. (a) PC12 cells were transfected with equal amounts of Flag-GFP plasmids under the control of the CMV promoter. Cells were treated with NGF for 4 h and lysed at the indicated time points. Vector (V) DNA was used as a negative control. Levels of Flag-GFP were determined using anti-Flag antibodies (top panel). Levels of ERK1/2 are provided to demonstrate equal loading of total protein (bottom panel). (b) PC12 cells were transfected with both Flag-c-fos (Flag-Fos) and Flag-GFP and stimulated with NGF or EGF for 2 h. Western blotting was performed with anti-Flag antibodies to detect levels of both Flag-Fos (top panel) and Flag-GFP (bottom panel). (c) PC12 cells were transfected with Flag-Fos and pretreated with UO126 or vehicle for 20 min. Cells were treated with NGF for 2 h. Lambda phosphatase reactions were then performed on lysates as described in Materials and methods. Flag-Fos protein levels and mobility shifts were measured using Flag antibodies (top panel). Levels of ERK1/2 are provided to demonstrate equal loading of total protein (bottom panel).

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Thr325 and Thr331 are required for c-fos activation by NGF

In fibroblasts, Thr325 and Thr331 have been shown to be important for c-fos function (Murphy et al. 2002). We examined whether phosphorylation of these residues was responsible for any of the mobility shifts of Flag-Fos induced by NGF. We mutated both residues to alanine, both together and as single mutations, to inhibit potential phosphorylation at these sites (Flag-Fos-T325A/T331A, Flag-Fos-T331A, and Flag-Fos-T325A referred herein as T325A/T331A, T331A, and T325A, respectively) and compared mobility shifts of wild-type Flag-Fos (WT) with this panel of mutants in response to stimulation by NGF. The band with the highest (slowest) mobility was lost in the T325A/T331A mutant, demonstrating that phosphorylation of these residues are required for the conformational change in the protein associated with this mobility (Fig. 4a, first panel). However, NGF did increase the protein levels of the mutant. Moreover, it also induced an intermediate shift. This suggests that other sites can contribute to the stability of c-fos and produce a protein of intermediate mobility. Both T331A and T325A had similar mobility shifts and protein levels as WT, demonstrating that phosphorylation of either Thr325 or Thr331 is sufficient for migration at the slowest mobility. Flag-GFP levels are shown as an internal control (Fig. 4a, second panel). Activation of ERK1/2 is shown (Fig. 4a, third panel), as well as total levels of ERK2 (Fig. 4a, fourth panel).

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Figure 4.  Thr325 and Thr331 are required for c-fos transactivation by NGF. (a) PC12 cells were transfected with Flag-c-fos wild type (WT), Flag-c-fos T331A (T331A), Flag-c-fos T325A (T325A), Flag-c-fos T325A/T331A (T325A/T331A), and Flag-GFP. Cells were stimulated with NGF for 2 h and western blotting was performed with anti-Flag antibodies to detect levels of Flag-Fos (first panel) and Flag-GFP (second panel). Activation of ERK1/2 was measured using pERK Ab (third panel). Equal loading of protein was demonstrated by measuring levels of ERK2 (fourth panel). (b) PC12 cells were transfected with Gal4-luciferase, pRL-Null (Renilla control), Gal4-WT, Gal4-T331A, Gal4-T325A, and Gal4-T325A/T331A. Cells were treated with NGF for 6 h, harvested, and luciferase activity was measured as described in Materials and methods. The data presented are an average of three independent experiments with four replicates each. The data are means ± SE. All values were then normalized to NGF-treated Gal4-WT (set at unity). Luciferase activity is measured as arbitrary units representing the ratio of firefly light units/Renilla light units.

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We then examined whether these phosphorylations were responsible for c-fos activation. Gal4-Fos fusion proteins are established tools to examine transactivation of c-fos (Monje et al. 2003). We constructed Gal4-Fos fusion proteins encoding full-length wild-type c-fos (Gal4-WT) full-length T331A (Gal4-T331A), full-length T325A (Gal4-T325A), and full-length T325A/T331A (Gal4-T325A/T331A) and measured transactivation of a Gal4-luciferase reporter construct as reported (Monje et al. 2003). In this manner, luciferase activity will only reflect activation by transfected c-fos constructs, with minimal interference from endogenous c-fos. In this assay, NGF induced strong transactivation of Gal4-WT but was unable to activate the threonine double mutant (Gal4-T325A/T331A; Fig. 4b). Gal4-T331A has about 40% of the activity of Gal4-WT in response to NGF, while Gal4-T325A activation is barely over basal levels (Fig. 4b). The data suggest that both Thr325 and Thr331 are required for full activation of c-fos by NGF in PC12 cells, although Thr325 seems to play a greater role.

c-fos contains an ERK binding site known as a DEF domain (docking site for ERK, F/Y-X-F/Y-P; Dimitri et al. 2005). Previous reports in fibroblasts have suggested that binding of ERK to the DEF domain is required for subsequent phosphorylation of Thr325 residue and c-fos transactivation (Murphy et al. 2002). ERK binding can be inhibited by mutating the functional DEF domain (F343TYP) to A343TAP. Using this mutant (FosDEF), we compared its mobility with that of FosWT. In response to NGF, FosDEF was shifted to high mobilities, although the highest mobility seen with FosWT was not apparent (Fig. 5a). This is consistent with the reported loss of Thr325 phosphorylation in DEF mutants (Murphy et al. 2002). We then examined if DEF-dependent phosphorylations were required for c-fos activation using the Gal4-luciferase reporter gene assay. Interestingly, like T325A, activation of the DEF mutant (Gal4-DEF) was drastically reduced in response to NGF when compared with wild-type (Gal4-WT; Fig. 5b). Thus, the DEF domain is required for full activation of c-fos by NGF in PC12 cells through its participation in phosphorylation of downstream sites.

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Figure 5.  The DEF domain of c-fos is required for maximal phosphorylation and transactivation by NGF. (a) PC12 cells were transfected with Flag-Fos-WT, Flag-FosDEF, and Flag-GFP as indicated. Cells were stimulated with NGF for 2 h and western blotting was performed. Anti-Flag antibody was used to detect levels of FosWT and FosDEF and Flag-GFP. (b) PC12 cells were transfected with Gal4-luciferase, pRL-Null, Gal4-WT, and Gal4-DEF. Cells were treated with NGF for 6 h, harvested, and luciferase activity was measured as described in Materials and methods. The data presented are an average of three independent experiments with four replicates each. The data are means ± SE. All values were then normalized to NGF treated Gal4-WT (set at unity). Luciferase activity is measured as arbitrary units representing the ratio of firefly light units/Renilla light units.

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Priming sites mediate ERK-dependent stability

To further examine which phosphorylations are required for the intermediate shift, we treated cells transfected with T325A/T331A with UO126. Treatment with UO126 inhibited the intermediate mobility of T325A/T331A induced by NGF (Fig. 6a). This suggests that additional ERK-dependent phosphorylations are also induced by NGF in PC12 cells. Two ERK-dependent phosphorylation sites, Ser362 and Ser374, have been shown to be important for stabilization and c-fos-dependent transformation in fibroblasts (Okazaki and Sagata 1995; Chen et al. 1996; Murphy et al. 2002). These sites do not require ERK docking to the DEF domain and have been termed ‘priming’ sites for subsequent phosphorylations on Thr325 and Thr331 (Murphy et al. 2002). We mutated these serine residues to alanine (FosAA) and compared mobility shifts and stabilization with FosWT. FosAA was unable to undergo any mobility shift and protein levels were poorly stabilized by NGF treatment (Fig. 6b, first panel). Activation and total levels of ERK1/2 are shown (Fig. 6b, second and third panels, respectively). In order to compare DNA binding of FosAA with FosWT, we used a modified ELISA-based assay (TransAM™) that measures Flag-Fos binding to an AP-1 consensus sequence. As expected, FosAA bound to an AP-1 target sequence poorly, compared with FosWT upon NGF stimulation, consistent with the modest effect of NGF on protein stabilization of this mutant (Fig. 6c).

image

Figure 6.  Ser362 and Ser374 are required for c-fos stabilization by NGF. (a) PC12 cells were transfected with T325A/T331A as indicated. Cells were pretreated with vehicle (DMSO) or UO126 for 20 min prior to stimulation with NGF for 2 h. Western blotting was performed and Flag-Fos protein levels and mobility shifts were measured using anti-Flag antibodies (top panel). ERK1/2 activation and inhibition, measured by pERK Ab, is shown in the middle panel. Levels of ERK1/2 are provided to demonstrate equal loading of total protein (bottom panel). (b) As indicated, PC12 cells were transfected with FosWT and Flag-c-fos-S362A/S374A (FosAA). Cells were either left untreated or stimulated with NGF for 2 h. Western blotting was performed using anti-Flag antibodies to detect levels of Flag-Fos (top panel). Activation of ERK1/2 was measured using pERK Ab (middle panel). Equal loading of protein was demonstrated by measuring levels of ERK1/2 (bottom panel). (c) As indicated, PC12 cells were transfected with FosWT or FosAA. Cells were treated with NGF for 2 h and nuclear extracts were isolated as described in Materials and methods. DNA binding was measured using the TransAM™ AP-1 Transcription Factor Assay Kit (Active Motif) as described in Materials and methods. The data presented are an average of three independent experiments. The data are means ± SE. All values were then normalized to NGF-treated FosWT (set at unity).

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In addition, we mutated both Ser362 and Ser374 to aspartic acids (FosDD) to mimic phosphorylation at these sites, and the effect on Flag-Fos protein levels was examined. FosDD was stable basally and migrated at an intermediate mobility (Fig. 7a, top panel). Stimulation with NGF caused FosDD to migrate at the highest mobility, similar to FosWT. FosDD, in cells treated with UO126, did not show this highest mobility, but displayed intermediate mobilities seen in untreated cells. This demonstrates that these additional phosphorylations are indeed ERK-dependent (Fig. 7a, top panel). Taken together, the data suggest that mimicking phosphorylation of the two priming sites is sufficient to mimic stabilization of c-fos by NGF. Moreover, the intermediate mobility seen basally in FosDD resembles the intermediate mobility seen after NGF treatment of the T325A/T331A mutant, suggesting that phosphorylation of these priming sites represents an intermediate state of c-fos phosphorylation prior to full activation achieved by subsequent phosphorylation of Thr325 and Thr331.

image

Figure 7.  Mimicking phosphorylation on Ser374 produces mutant proteins that are basally stable. The designations for the mutants are listed in Table 1. (a) PC12 cells were transfected with FosWT, FosDD, and Flag-GFP as indicated. Cells were pretreated with vehicle (DMSO) or UO126 for 20 min prior to stimulation with NGF (+) for 2 h. Western blotting was performed using anti-Flag antibodies to detect levels of Flag-Fos (top panel) and Flag-GFP (bottom panel). (b) PC12 cells were transfected with FosWT, FosSD, and FosAD. Cells were left untreated (–) or treated with NGF for 2 h. (+). Total levels of transfected proteins were examined using anti-Flag antibodies (top panel). ERK 1/2 levels were measured to confirm equal protein loading (bottom panel). (c) PC12 were transfected with FosWT or FosSA, and cells were left untreated (–) or treated with NGF for 2 h (+). Western blotting was performed using anti-Flag antibodies to detect levels of Flag-Fos (first panel) and Flag-GFP (fourth panel). Activation of ERK1/2 was measured using pERK Ab (second panel). Equal loading of protein was demonstrated by measuring levels of ERK2 (third panel). (d) PC12 cells were transfected with FosWT, FosDS, and FosDA, and cells were left untreated (–) or treated with NGF for 2 h. (+). Total levels of transfected proteins were examined using anti-Flag antibodies (top panel). ERK 1/2 levels were measured to confirm equal protein loading (bottom panel).

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We then examined the relative contribution of each priming site to both stabilization and to migration at the intermediate mobility. Table 1 lists the mutants at Ser362 and Ser374 that were analyzed. To determine whether mimicking phosphorylation of Ser374 provided a stabilizing signal that was independent of the status of Ser362, we examined the mutants FosSD and FosAD (Fig. 7b, top panel). Both mutants were basally stable; demonstrating that by mimicking phosphorylation of Ser374 stabilized c-fos even in the absence of phosphorylation of Ser362. NGF induced a shift of the majority of FosSD, but only a small fraction of FosAD, consistent with the participation of phosphorylation of Ser362 in the shift. In order to directly test whether Ser374 is required for stabilization of c-fos by NGF, we mutated Ser374 to alanine to inhibit phosphorylation at this site (FosSA). In response to NGF, FosSA is much less stable than FosWT, although its mobility shifts are unchanged (Fig. 7c, first panel). Taken together, these data support a model where phosphorylation of Ser374 appears to be primarily responsible for the stability. Phosphorylation of Ser362 contributes significantly to the intermediate mobility shift.

Table 1.   Analysis of Ser362 and Ser374 Flag-c-fos mutants
Flag-c-fos constructResidue 362Residue 374
  1. This table provides a key to determine the identity of Flag-c-fos mutants corresponding to Ser362 and Ser374. FosWT (wild type), FosAA (S362A, S374A), FosSA (S374A), FosDD (S362D, S374D), FosDS (S362D), FosSD (S374D), FosAD (S362A, S374D), and FosDA (S362D, S374A). A, alanine; D, aspartate; S, serine.

FosWTSS
FosAAAA
FosSASA
FosDDDD
FosDSDS
FosSDSD
FosADAD
FosDADA

To determine whether mimicking phosphorylation of Ser362 could induce a mobility shift independently of Ser374, we compared the mutants FosDS and FosDA (Fig. 7d, top panel). As expected, NGF increased the stability of only FosDS, but not FosDA. In both mutants, the presence of an aspartate at position Ser362 produced a c-fos protein displaying an intermediate mobility in the absence of stimulation. Upon NGF stimulation, the migration of both mutants was further retarded to the highest shifted forms. Together these data strongly support the role of pSer374 in NGF induced c-fos stability, and suggest that priming of additional phosphorylations can occur in the absence of pSer374. We propose a model whereby c-fos is induced at the transcriptional level by growth factor stimulation (Fig. 8). Newly translated c-fos proteins undergo a series of phosphorylations that allows c-fos to activate AP-1-dependent genes. Firstly, phosphorylation of Ser374 stabilizes c-fos, allowing protein levels to accumulate in the cell. Ser362, another ERK-dependent site, is phosphorylated, which changes the conformational structure of the protein, seen as the intermediate mobility shift. Presumably, these two ‘priming’ phosphorylations allow for binding of ERKs to the DEF domain of c-fos. Finally, phosphorylation of Thr325 and Thr331, and possibly other sites (Thr232), induce c-fos transactivation. This sequence of phosphorylations provides a mechanism of how sustained activation of ERKs by NGF is required for maximal activity of c-fos.

image

Figure 8.  Proposed model of NGF-dependent c-fos transactivation. c-fos is basally maintained in an unstable state. Maximal stabilization by NGF is achieved by the ERK-dependent phosphorylation of Ser374, which stabilizes c-fos (Chen et al. 1993), possibly by blocking PEST sequences within this region (Ferrara et al. 2003). The phosphorylation of Ser362, which is thought to occur via the ERK-dependent kinase RSK, contributes to the intermediate mobility shift of c-fos. ERK binding to the DEF domain is required for priming of additional phosphorylations including Thr325 and possibly Thr331 (Murphy et al. 2002). Thr232, another site involved in c-fos transactivation, is phosphorylated by an unknown kinase and recent evidence suggests that it may not be ERK2 (Monje et al. 2003). S, serine; T, threonine.

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c-fos requires constant ERK activity to maintain a hyperphosphorylated state

We propose that sustained activation of ERKs is required to induce the sequence of phosphorylations that are required for maximal activation of c-fos protein. We speculated that EGF might be able to fully activate FosDD as it is already primed and stabilized. Indeed, transient ERK activation by EGF induced a transient mobility shift in both FosWT and FosDD. However, although FosDD was more stable than FosWT at all time points, the shift rapidly returned to basal levels (Fig. 9a, top panel) as ERK levels returned to baseline (bottom panel). This transient effect was insufficient to activate c-fos as seen by the inability of EGF to activate Gal4-DD (Fig. 9b). Thus, although FosDD is basally stable, sustained phosphorylation is still required for maximal c-fos function.

image

Figure 9.  Maximal phosphorylation of c-fos is dependent on sustained ERK activation. (a) PC12 cells were transfected with FosWT, FosDD, and Flag-GFP. Cells were treated with EGF for the indicated time points and western blotting was performed on the lysates. Total levels of Flag-c-fos proteins and Flag-GFP were examined using anti-Flag antibodies (top panel). pERK Abs show activation of ERK1/2 (middle panel). ERK2 levels were measured to confirm equal protein loading (bottom panel). (b) PC12 cells were transfected with Gal4-luciferase, pRL-Null, Gal4-WT, and Gal4-c-fos-S362D/S374D (Gal4-DD). Cells were treated with NGF for 6 h, harvested, and luciferase activity was measured as described in Materials and methods. The data presented are from a single representative experiment. The data are means ± SE (n = 4). Luciferase activity is measured as arbitrary units, which equals the ratio of firefly light units/Renilla light units.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The distinct actions of NGF and EGF on PC12 cells have provided a paradigm for the role of the duration of ERK activation in distinguishing the actions of these growth factors (Chao 1992; Traverse et al. 1992; Cowley et al. 1994; Traverse et al. 1994; Marshall 1995; Vician et al. 1997; Kao et al. 2001; Vaudry et al. 2002). The ability of NGF to induce sustained activation of ERKs in these cells is required for NGF to differentiate these cells (Cowley et al. 1994; Marshall 1995). Sustained ERK activation by NGF permits nuclear localization of ERKs to promote a program of NGF-dependent transcription (Vician et al. 1997) through the induction of ‘immediate-early genes’ including the transcription factor c-fos (Kruijer et al. 1985; Milbrandt 1986; Kujubu et al. 1987; D'Arcangelo and Halegoua 1993; Groot et al. 2000). That we do not observe complete inhibition of c-fos stabilization by UO126 pretreatment may represent an ERK-independent effect. Indeed, other kinases have been implicated in phosphorylation of c-fos (Tratner et al. 1992; Tanos et al. 2005). Following NGF stimulation, c-fos and other immediate-early genes are rapidly induced to initiate a second wave of gene expression that contributes to this differentiated phenotype (Kalman et al. 1990; Ginty et al. 1992; deSouza et al. 1995; Nordstrom et al. 1995; Vician et al. 1997). For one of these immediate early genes, Egr-1, a direct role in neuronal differentiation by NGF has been established (Harada et al. 2001). A recent report has demonstrated that c-fos is also required to initiate the transcriptional program for differentiation in PC12 cells (Gil et al. 2004). In addition, it has also been reported that c-fos may also have a role in neurite outgrowth through its action on membrane biosynthesis (Borioli et al. 2004, 2005).

We show in PC12 cells that NGF induces the stability and transactivation of c-fos. We show that this phenomenon is dependent on the sustained activation of ERKs and is blocked by pharmacological inhibitors of ERK activation. This establishes that c-fos is a sensor for sustained ERK activation in PC12 cells. This is similar to the model proposed for c-fos activation by PDGF in fibroblasts (Murphy et al. 2002). Importantly, PDGF is a proliferative agent in these cells. Therefore, our study demonstrates that the requirement of sustained ERK activation for c-fos function is independent of the physiological outcome of c-fos activation. Recently, studies in fibroblasts have demonstrated that c-myc, Egr-1, Fra-1, Fra-2, and c-jun are also targets for activation by ERKs (Murphy et al. 2004). Indeed, a recent report demonstrates that sustained ERK1/2 activation is required for an AP-1-dependent mitogenic signal by growth factors in fibroblasts (Yamamoto et al. 2006). It is likely that some of these proteins also respond to sustained activation of ERKs by NGF to promote the induction of NGF-responsive genes in neuronal cells (Pap and Szeberenyi 1998; Cosgaya and Aranda 1999; Riccio et al. 1999; Groot et al. 2000; Boss et al. 2001).

We were able to observe a slight, but significant, increase in c-fos transactivation and protein stability by EGF. This effect of EGF can partially be attributed to increased basal levels of transfected Flag-Fos under control of the CMV promoter, which allows transient ERK1/2 activation to immediately act on pre-made c-fos protein. However, small increases of EGF in c-fos transactivation are unable to reach the threshold for induction of differentiation, demonstrating the requirement for sustained ERK activation to initiate this paradigm.

Several reports have examined residues in c-fos that are important for transactivation in fibroblasts (Murphy et al. 2002; Monje et al. 2003; Tanos et al. 2005). In this study, we show that both Thr325 and Thr331 are required for maximal NGF-dependent activation of c-fos in PC12 cells. Our evidence suggests that both residues contribute to c-fos transactivation, although inhibition of Thr325 phosphorylation reduces transactivation more strongly. However, phosphorylation of these residues has a synergistic effect. This may explain why both residues are required for migration of the slowest mobility (top band). Blenis and colleagues showed a robust phosphorylation of Thr325 upon PDGF treatment in non-neuronal cells. This phosphorylation was also dependent on sustained ERK activation and an intact DEF domain. However, when they mutated these same residues, they saw a very modest effect on basal AP-1 activity. Although this difference in results could be cell-type specific, it is more likely as a result of differences in basal versus stimulated c-fos activity. Our data confirm those of Blenis and co-workers that demonstrated the importance of the DEF domain in promoting ERK-dependent phosphorylations critical for c-fos transactivation. At this time, we cannot rule out the possibility that this domain directs the ERK-dependent phosphorylation of additional target proteins.

c-fos is much less potent than its viral counterpart v-fos in inducing cellular transformation, despite sharing extensive homology within the DNA binding and transactivation domains (Cohen and Curran 1989; Piechaczyk and Blanchard 1994; Jotte and Holt 1996). One difference is the presence of C-terminal sequences unique to c-fos (Ofir et al. 1990). c-fos requires growth factor stimulation to become fully activated, in part because growth factors stimulate ERK-dependent phosphorylations within the C-terminal domain (Barber et al. 1987; Chen et al. 1993). ERK-dependent phosphorylations at Ser362 and Ser374 have been implicated in transformation (Okazaki and Sagata 1995; Chen et al. 1996), and transactivation (Murphy et al. 2002; Monje et al. 2003), in part by regulating stability (Chen et al. 1996; Ferrara et al. 2003). Moreover, phosphorylations at these sites are thought to exert a priming function that directs the hyperphosphorylation of c-fos by ERK and other kinases (i.e. Thr325 and Thr331; Murphy et al. 2002). Mutations preventing phosphorylation at both sites (FosAA) block all of these effects (Okazaki and Sagata 1995; Chen et al. 1996; Monje et al. 2003). In contrast, phosphomimetic mutations introduced at both sites (FosDD) enhance stability and transformation (Chen et al. 1996). We show here that NGF also utilizes these sites to regulate c-fos stability in PC12 cells.

It has been assumed that phosphorylation at both sites functions similarly to enhance c-fos stability and transformation, largely because previous studies have examined c-fos mutated simultaneously at both sites. By examining a more complete series of mutants, we demonstrate that phosphorylations at these two serine sites contribute to the function of c-fos in distinct ways. Phosphorylation of Ser374 is required for the stabilizing effects of NGF stimulation, as evidenced by the finding that inhibition of Ser374 phosphorylation by UO126 correlates with decreased stability of c-fos protein. This can be shown by the absence of NGF-induced stabilization in Fos mutants lacking this serine (FosSA, FosDA and FosAA). Phosphorylation at this site appears to be sufficient for stabilization as phosphomimetic mutations at residue Ser374 produce c-fos mutants (FosSD and Fos AD) that are stable even under resting conditions. Phosphorylation of Ser362 appears to contribute significantly to the intermediate mobility shift, even in the absence of phosphorylation of Ser374 (FosDA). This likely reflects the contribution of Ser362 in priming additional phosphorylations. The exact mechanism by which phosphorylation of Ser362 promotes subsequent phosphorylations is not known. It is possible that this is achieved in part by promoting ERK binding to the DEF domain in c-fos that has been proposed to contribute to the hyperphosphorylation and shift. This model is illustrated in Fig. 8.

The requirement for sustained ERK activation was seen not only in the activation of wild-type c-fos proteins but also in c-fos mutants in which priming sites were replaced with aspartates (FosDD). FosDD is basally stable and no longer requires ERK-dependent phosphorylations at the ‘priming’ sites. Even in this mutant, transient ERK activation is not sufficient to induce c-fos activation. The inability of EGF to activate the stable mutant FosDD suggests that sustained activation of ERKs is required to maintain the phosphorylation status of the activating threonines (Thr325 and Thr331). It is likely that these sites are tightly regulated by active phosphatases. This ensures that c-fos requires sustained ERK activation at multiple levels of its regulation.

In summary, the ability of NGF to stimulate c-fos function requires sustained ERK activation. This requirement is as a result of two ERK-dependent phosphorylation sites within the C-terminus (Ser362 and Ser374) that have been proposed to confer stability on nascent c-fos protein and also serve as priming sites for subsequent phosphorylations within the TAD region. We show that phosphorylation of Ser374 is required for stabilization. As previously suggested, phosphorylation of this site may interfere with the well-characterized degradation sequence surrounding this site (Okazaki and Sagata 1995; Ferrara et al. 2003). Phosphorylation of Ser362 is critical in inducing the intermediate mobility shift, and, with some contribution of phosphorylation of Ser374, enables c-fos to be targeted for additional ERK-dependent phosphorylations. These additional phosphorylations, specifically Thr325 and Thr331, are required for c-fos transactivation in this model. Moreover, an intact ERK binding site is also needed for stimulated c-fos activity, presumably for these ‘activating’ phosphorylations. Because c-fos requires multiple ERK-dependent phosphorylations to be stabilized, shifted, and hyperphosphorylated, c-fos is strictly dependent on sustained ERK activation for stability and function. This requirement for sustained ERK activation serves as a paradigm for the distinct actions of NGF versus EGF on c-fos-dependent PC12 gene expression.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We would like to thank Patrick Casey for providing us with PC12 cells. We would like to thank Kirstin Labudda for expert technical assistance. This work was supported by the National Institute of Health grants MH072004 (MJP) and CA72971 (PS).

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  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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