Co-regulation of the mitogen-activated protein kinase, extracellular signal-regulated kinase 1, and the 90-kDa ribosomal S6 kinase in PC12 cells. Distinct effects of the neurotrophic factor, nerve growth factor, and the mitogenic factor, epidermal growth factor.

T. Nguyen, J Scimeca, C. Filloux, P. Peraldi, J. Carpentier, E. van Obberghen

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T. Nguyen, J Scimeca, C. Filloux, P. Peraldi, J. Carpentier, et al.. Co-regulation of the mitogen-activated protein kinase, extracellular signal-regulated kinase 1, and the 90-kDa ribosomal S6 kinase in PC12 cells. Distinct effects of the neurotrophic factor, nerve growth factor, and the mitogenic factor, epidermal growth factor.. Journal of Biological Chemistry, 1993, 268 (13), pp.9803-10. hal-02109561

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# Co-regulation of the Mitogen-activated Protein Kinase, Extracellular Signal-regulated Kinase 1, and the 90-kDa Ribosomal S6 Kinase in PC12 Cells

DISTINCT EFFECTS OF THE NEUROTROPHIC FACTOR, NERVE GROWTH FACTOR, AND THE MITOGENIC FACTOR, EPIDERMAL GROWTH FACTOR*

(Received for publication, August 17, 1992, and in revised form, January 8, 1993)

Tien T. Nguyen‡, Jean-Claude Scimeca§, Chantal Filloux, Pascal Peraldi, Jean-Louis Carpentier¶||, and Emmanuel Van Obberghen**

From the Institut National de la Santé et de la Recherche Médicale U145, Faculté de Médecine, 06107 Nice Cédex 2, France and the ¶Département de Morphologie, Centre Médical Universitaire, 1211 Genève 4, Switzerland

We recently characterized the association of the 44-kDa mitogen-activated protein kinase, also known as extracellular-regulated kinase 1 (ERK1), with the 90-kDa ribosomal S6 kinase (pp90<sup>rsk</sup>), one of its putative substrates in intact PC12 cells. Using antibodies to ERK1 that precipitate a functional ERK1·pp90<sup>rsk</sup> phosphoprotein complex, we demonstrate here the regulation of both kinases by various stimuli. In mouse fibroblasts expressing human insulin receptors, insulin and vanadate swiftly stimulated ERK1 activity within 5 min. While the hormonal effect was short-lived, vanadate led to a first peak followed by a progressively increasing second phase. In PC12 cells, epidermal growth factor, which is a growth promoting factor, provokes a rapid but evanescent activation of ERK1. In contrast, nerve growth factor (NGF), which acts as a neuronal differentiation factor for PC12 cells, induced a swift monophasic response followed by a sustained second phase. This strikingly different pattern of ERK1 stimulation by NGF and epidermal growth factor was associated to a contrasting effect on ERK1 cellular translocation. Thus, NGF induced a nuclear translocation of ERK1, while epidermal growth factor was without noticeable effect on ERK1 localization. In both cell systems all effectors tested stimulated ERK1 phosphorylation on both threonine and tyrosine residues in an 1:1 ratio. During ERK1 inactivation, phosphothreonine and phosphotyrosine were dephosphorylated in a similar fashion. Concurrent with ERK1 activation was the *de novo* appearance of phosphothreonine and an increase in phosphoserine on pp90<sup>rsk</sup>. The pp90<sup>rsk</sup> phosphothreonine content paralleled the ERK1

activity more closely than the phosphoserine level. These results provide compelling evidence that in fibroblasts and PC12 cells ERK1 plays a direct role in the phosphorylation of pp90<sup>rsk</sup> and that pp90<sup>rsk</sup> represents a physiologically relevant substrate of extracellular-regulated kinases. Finally, we would like to suggest that the differentiating action of NGF in PC12 cells might be due, at least in part, to the conjunction of its sustained and robust stimulation of ERK1 and pp90<sup>rsk</sup>, and of its induction of ERK1 nuclear translocation.

Ribosomal protein S6 is a component of the eukaryotic 40 S ribosomal subunit that becomes phosphorylated on multiple serine residues in response to a variety of growth promoting agents and mitogens, including insulin, epidermal growth factor (EGF),<fnref n="1" /> platelet-derived growth factor, fibroblast growth factor, and transforming proteins of many oncogenic viruses (reviewed in Erikson (1991) and Sturgill and Wu (1991)). S6 phosphorylation has been correlated with growth and increased protein synthesis (Duncan and McConkey, 1982; Thomas *et al.*, 1982). Efforts to identify growth factor-activated S6 kinases have led to the cloning of two distinct families of 70-kDa (pp70<sup>S6K</sup>) (Banerjee *et al.*, 1990; Kozma *et al.*, 1990) and 90-kDa (pp90<sup>rsk</sup>) (Jones *et al.*, 1988; Alcorta *et al.*, 1989) ribosomal S6 serine/threonine kinases. Both enzymes are activated by serine/threonine phosphorylation and are inactivated by protein phosphatase 1 and 2A (Ballou *et al.*, 1988; Price *et al.*, 1990). Moreover, neither enzyme contains phosphotyrosine. Hence, the existence of activating serine/threonine-specific kinases in upstream pathways have been implicated in regulation of both types of S6 kinases. Recent studies suggest the involvement of the extracellular-regulated kinase (erk)-encoded serine/threonine protein kinases family, also known as mitogen-activated protein (MAP) kinase, in the regulation of the pp90<sup>rsk</sup> activity as potential candidates for S6 kinase kinases (Sturgill *et al.*, 1988; Ahn and Krebs, 1990; Chung *et al.*, 1991b). Members of the erk-encoded kinase family are distinguished by their ability to phosphorylate microtubule-associated protein 2, myelin basic protein (MBP), and their rapid activation by various extracellular stimuli (Boulton *et al.* (1991), reviewed in Crews

* This work was supported in part by the Institut National de la Santé et de la Recherche Médicale, the Université de Nice-Sophia-Antipolis, Association pour la Recherche contre le Cancer Grant 6760, the Ligue Nationale Française contre le Cancer, Fédération des Comités Départementaux, Comité Départemental du Var. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "*adver- tument*" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

‡ Recipient of a fellowship from the Medical Research Council of Canada.

§ Recipient of a fellowship from the Ligue Nationale Française contre le Cancer, Fédération des Comités Départementaux, Comité Départemental du Var.

|| Supported by Grant 31-34093.92 from the Swiss National Science Foundation (Geneva) and the Juvenile Diabetes Foundation Int.

** To whom correspondence should be addressed: INSERM U145, Faculté de Médecine, Ave. de Valombrose, 06107 Nice Cédex 2, France. Tel.: 93-85-16-54; Fax: 93-92-07-13.

<footnote n="1">¹ The abbreviations used are: EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; FCS, fetal calf serum; MAP, mitogen-activated protein kinase; MBP, myelin basic protein; NGF, nerve growth factor; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.</footnote>
et al. (1992) and Thomas (1992)). It has been shown that the insulin-stimulated MAP kinase can phosphorylate and reactivate the 92-kDa phosphatase-treated *Xenopus* S6 kinase II *in vitro*, with the ensuing appearance of phosphothreonine (Sturgill et al., 1988). Since *Xenopus* S6 kinase II autophosphorylated exclusively on serine residues, at least one MAP kinase phosphorylation site in S6 kinase II is a threonine residue. Favoring the role of MAP kinase in phosphorylating S6 kinase *in vivo* is the fact that MAP kinase activity reaches a peak of activation prior to S6 kinase activity in EGF-stimulated Swiss 3T3 fibroblasts (Ahn et al., 1990). *In vitro* phosphorylation and partial reactivation of S6 kinase activity by MAP kinase can be demonstrated in partially purified and reconstituted fractions from these cells (Ahn and Krebs, 1990; Ahn et al., 1990). Moreover, it has been shown that Swiss 3T3 ribosomal S6 kinase kinase phosphorylates and activates recombinant pp90^rsk *in vitro* (Chung et al., 1991a, 1991b). Taken together, these studies suggest a role for MAP kinase as a ribosomal S6 kinase kinase.

Using murine fibroblasts expressing human insulin receptor, we have demonstrated that the 44-kDa MAP kinase, ERK1, exhibits *in vitro* phosphorylation on tyrosine and threonine residues, suggesting an autophosphorylating activity and/or copurification of a MAP kinase kinase with dual specificity or two MAP kinase kinases (Scimeca et al., 1991). This dual phosphorylation is accompanied by the activation of ERK1 as determined by MBP phosphorylation in eluates of anti-ERK1 immunoprecipitates. In search for ERK-associated proteins, we next demonstrated the association of pp90^rsk, a mammalian homologue of *Xenopus* S6 kinase II, with ERK1 in anti-ERK1 immunoprecipitates from both PC12 cells and fibroblasts expressing human insulin receptors (Scimeca et al., 1992). The coprecipitated pp90^rsk is phosphorylated *in vitro* on serine and threonine residues. Following separation by SDS-PAGE and subsequent protein renaturation *in situ*, the pp90^rsk band autophosphorylated solely on serine residues. These results suggest that the pp90^rsk threonine phosphorylation would come from the copurifying ERK1. We now report on the regulation of both ERK1 and pp90^rsk phosphorylation states by various extracellular stimuli in two cell lines, undifferentiated PC12 cells and fibroblasts. In the PC12 cells a sharp contrast was observed in the activation pattern of ERK1 and pp90^rsk after stimulation by the differentiative factor, NGF, compared to after stimulation by the mitogenic factor, EGF. In fibroblasts expressing human insulin receptors both vanadate and insulin stimulated ERK1 and pp90^rsk phosphorylation in a biphasic fashion, vanadate leading to a more pronounced second phase. Taken together, our results provide compelling evidence that pp90^rsk and ERK are coregulated, probably as a result of a direct phosphorylation of pp90^rsk by ERK1. We also present biochemical and morphological data on ERK1 regulation in PC12 cells which might explain, at least in part, the effect of NGF on neuronal differentiation.

## EXPERIMENTAL PROCEDURES

**Materials**—The keyhole limpet hemocyanin-coupled 12-amino acid carboxyl-terminal of ERK1-(356-367) (TARFQPGAPEAP), synthesized by Neosystem (Strasbourg, France), was used to produce rabbit polyclonal antisera as previously described (Scimeca et al., 1991). Sodium orthovanadate, bovine serum albumin (type 7030) for cell culture, EGF, leupeptin, phenylmethylsulfonyl fluoride, MBP from bovine brain, protein A-Sepharose, Triton X-100, and ninhydrin were purchased from Sigma. Immobilon polyvinylidene difluoride membrane was from Millipore (Bedford, MA). Nerve growth factor (NGF) and the rat pheochromocytoma PC12 cell line were kindly provided to us by Dr. P. Kitabgi (Nice-Sophia Antipolis, France). [γ-³²P]ATP, [³²P]orthophosphate, Dulbecco's modified Eagle's medium, and

RPMI without phosphate were purchased from ICN (Costa Mesa, CA).

**Cell Culture**—NIH 3T3 cells (clone HIR 3.5) transfected with an expression plasmid encoding the human insulin receptor were kindly provided to us by Dr. Whittaker (Stony Brook, NY) (Whittaker et al., 1987). Expressing approximately 6 × 10⁶ receptors/cell, these HIR cells exhibit several typical insulin-induced biological responses (Hofmann et al., 1989). Mouse fibroblast HIR cells were cultured to confluence in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum (FCS), 50 units/ml penicillin, and 50 µg/ml streptomycin. Undifferentiated rat pheochromocytoma PC12 cells were maintained in RPMI medium supplemented with 10% FCS to near confluency. Cells were plated at 2.5 × 10⁴ cells/cm² in a 12-well plate and in a 100-mm Petri dish for MBP kinase assay and ³²P labeling, respectively. For MBP kinase assay, each time point was assayed in triplicate wells and results are expressed as mean ± S.E. HIR and PC12 cells were starved, respectively, in Dulbecco's modified Eagle's medium or RPMI containing 0.2% (w/v) bovine serum albu- min for 18 or 1 h prior to the addition of various effectors.

**Cell Stimulation of ERK1 Activity and ERK1 Immunoprecipitation**—Following the starvation period, the same amount of HIR or PC12 cells were incubated for various times in the presence of the following effectors: 0.1 µM insulin, 100 ng/ml NGF, 0.1 µM EGF, 10% (v/v) FCS, 1 mM vanadate. Cell monolayers were washed twice in ice-cold phosphate-buffered saline (PBS) containing 140 mM NaCl, 3 mM KCl, 6 mM Na₂HPO₄, 1 mM KH₂PO₄, pH 7.4, and once in 50 mM Hepes, pH 7.5, 150 mM NaCl, 10 mM EDTA, 10 mM Na₄P₂O₇, 2 mM Na₃VO₄, 100 mM NaF, 100 units/ml aprotinin, 20 µM leupeptin, and 0.2 mg/ml phenylmethylsulfonyl fluoride (buffer A). Cell mono- layers were then solubilized for 15 min in buffer A supplemented with 1% (v/v) Triton X-100. The solubilized monolayers were clarified by centrifugation at 18,000 × g for 15 min and then incubated for 90 min with ERK1 antibodies preadsorbed on protein A-Sepharose beads.

**³²P Labeling in Intact Cells**—After the serum-depletion period, the same amount of cells were washed twice in culture medium without phosphate and incubated for 3 h in this medium enriched with [³²P] orthophosphate (0.5 mCi/ml). Effectors were added at the end of the labeling period. Following the indicated incubation times, the mono- layer was then washed 3 times in ice-cold PBS and incubated for a further 15 min in the solubilization buffer. The cell extracts were then clarified by centrifugation and incubated with antibodies to ERK1. The protein A-Sepharose beads were washed alternatively for 6 times each in the solubilization buffer and a buffer consisting of 50 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10% (v/v) glycerol, 0.2 mM Na₃VO₄, 100 units/ml aprotinin, 20 µM leupeptin, and 0.2 mg/ml phenylmethylsulfonyl fluoride (HNTG buffer). Laem- mli buffer (3% SDS) was added to the beads which were then boiled for 5 min, and solubilized proteins were submitted to SDS-PAGE under reducing conditions on a 10% acrylamide resolving gel.

**MBP Phosphotransferase Assay**—At the end of the immunoprecipitation period, the beads were submitted to 6 alternate washes in solubilization and HNTG buffers. Bound proteins were then eluted at 4 °C for 30 min in MAP kinase buffer consisting of 25 mM Hepes, pH 7.5, 25 mM NaCl, 40 mM *p*-nitrophenyl phosphate, 2 mM EGTA, 1 mM dithiothreitol, 10% glycerol, 100 units/ml aprotinin, 20 µM leupeptin, 0.2 mg/ml phenylmethylsulfonyl fluoride, and the peptide antigen (10 µM). MBP phosphorylation was performed for 40 min at room temperature (linear assay condition) in the presence of 150 µg/ml MBP, 10 mM magnesium acetate, and [γ-³²P]ATP (5 µM, 33 Ci/mmol). The phosphorylation reaction was stopped by spotting pieces of Whatman P81 paper which were then dropped in 10% (w/v) trichloroacetic acid solution. The papers were washed 3 times in 5% trichloroacetic acid solution containing 1% (w/v) Na₄P₂O₇, rinsed in ethanol, air dried, and counted in the presence of scintillation mix- ture.

**Phosphoamino Acid Analysis**—The phosphoamino acid analysis of *in vivo* ³²P-labeled proteins was performed according to the method of Kamps and Sefton (1989). Briefly, following electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane over 3 h in 12 mM Tris, pH 8.3, 96 mM glycine, 20% (v/v) methanol, and 0.05% (w/v) SDS. Membrane segments corresponding to the phosphoproteins of interest were cut off, wet in methanol, washed in water, and then submitted to hydrolysis in 5.7 M HCl for 90 min at 110 °C. Extended acid hydrolysis up to 4 h did not alter the relative phosphoamino acid contents. The hydrolysates were dried under reduced pressure and the released phosphoamino acids were resuspended in electrophoresis buffer consisting of water:acetic acid:pyridine (945:50:5), pH 3.5, containing 0.2 mg/ml each of phos-
phosserine, phosphothreonine, and phosphotyrosine. The phosphoamino acids were then separated by high voltage thin layer chromatography (TLC) on cellulose glass plates (Merck, Darmstadt, Germany) for 90 min at 960 V, visualized by ninhydrin staining, and exposed to Kodak X-AR5 film at -80 °C for 2 h to 14 days.

**Immunofluorescence**—To localize the MAP kinase, ERK1, PC12 cells grown on coverslips were fixed and permeabilized by acetone at -80 °C. Briefly, cells were washed five times in 0.1 M PBS, pH 7.4, at 4 °C, covered with acetone at -80 °C, and incubated at -20 °C for 3 min. Then the acetone was removed, the cells were dried for 5 min, and PBS was added at 4 °C. Cells were washed with PBS at 22 °C before incubation for 2 h at the same temperature with PBS containing antibodies to ERK1 at a dilution of 1:200. Cells were washed in PBS prior to incubation with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG at a dilution of 1:200 for 1 h at 22 °C, rinsed in PBS, and counterstained with Evans blue before examination in a Zeiss Axiophot fluorescence microscope. Control experiments included incubation in the presence of second antibody alone or incubation in the presence of an irrelevant first antibody.

## RESULTS

**Kinetics of ERK1 Activation in PC12 and HIR Cells**—We first studied the kinetics of the activation of ERK1 isolated from the rat phaeochromocytoma PC12 cell line using MBP as an exogenous substrate. Cell extracts from serum-depleted PC12 cells stimulated with 0.1 µM EGF, 100 ng/ml NGF, or 1 mM vanadate were immunoprecipitated with antibodies to ERK1. The immunoaffinity purified eluates were then used to phosphorylate MBP *in vitro* (Fig. 1). EGF (0.1 µM) promoted a swift activation of ERK1 within 2.5 min. This activation was followed by a return to near basal levels by 30 min, a low level that was maintained up to 3 h. Vanadate (1 mM) produced a slower onset of ERK1 activation which maximized by 15 min and which remained stable up to 3 h. Interestingly, NGF (100 ng/ml), a differentiating factor for these cells, promoted a sustained activation of ERK1. The early phase of ERK1 activation was rapidly reached within 15 min. This was followed by a slight transient decline by 30 min and then a late robust phase of ERK1 activation that was sustained up to 3 h.

We next studied the time course of ERK1 activity in HIR fibroblasts (Fig. 2). Insulin as well as vanadate rapidly activated ERK1. The ERK1 activity swiftly peaked within 5 min, followed by a slight transient decline between 10 and 20 min. In both insulin- and vanadate-treated cells, we also observed a broad weaker ERK1 activation phase between 30 and 60 min, which is enhanced by vanadate treatment. In insulin-treated cells, the ERK1 activity returned to near basal levels between 60 and 180 min. In contrast, vanadate produced a

<figure><img src="image_2.png" /><figcaption>**FIG. 1.** Time course of ERK1 activation in PC12 cells. Serum-depleted PC12 cells were incubated for the indicated times in the presence of 100 ng/ml NGF, 0.1 µM EGF, and 1 mM vanadate. The peptide antigen eluates of anti-ERK1 immunoprecipitates were used to phosphorylate exogenously added MBP as described under "Experimental Procedures." Results are expressed as means of triplicate well determinations from a representative experiment.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>**FIG. 2.** Time course of ERK1 activation in HIR cells. Serum-depleted HIR cells were incubated for the indicated times in the presence of 0.1 µM insulin and 1 mM vanadate. Detergent cell extracts were submitted to immunoprecipitation by antibodies to ERK1 and the peptide antigen eluates were used to phosphorylate MBP *in vitro* in the presence of [γ-³²P]ATP as described under "Experimental Procedures." Phosphorylated MBP was precipitated on paper in trichloroacetic acid solution and assessed by liquid scintillation counting. Results are expressed as means of triplicate well determinations from a representative experiment.</figcaption></figure>

sustained elevation of ERK1 activity up to 180 min in addition to the early peak of activity.

*In Vivo* Phosphorylation of ERK1 and pp90<sup>rsk</sup> in PC12 Cells—We next looked at the phosphorylation states of ERK1 and its associated pp90<sup>rsk</sup> at various levels of ERK1 activity. To do so, PC12 cells were ³²P-labeled and treated with various stimuli. After solubilization of the cells, proteins were subjected to immunoprecipitation with nonimmune serum (NI) or antibodies to ERK1 (I), and the immunoprecipitates were submitted to SDS-PAGE analysis (Fig. 3). Antibodies to ERK1 precipitated two phosphoproteins of 44 and 90 kDa, which were not seen with the nonimmune serum. These two proteins have previously been identified as ERK1 and pp90<sup>rsk</sup>, respectively (Scimeca *et al.*, 1991, 1992). NGF induced a robust increase in the phosphorylation of ERK1 at 15 min relative to basal phosphorylation. At 50 min, the phosphate incorporation into ERK1 was still elevated relative to basal levels. In parallel, the phosphate incorporation into pp90<sup>rsk</sup> peaked at 15 min and then decreased slightly by 50 min. In contrast, vanadate produced a scanty increase in ERK1 phosphorylation relative to basal levels at both 15 and 50 min and no significant change in the phosphorylation state of pp90<sup>rsk</sup>. When PC12 cells were treated with EGF, there was a strong phosphate incorporation into ERK1 within 5 min which then substantially decreased, but remained greater than basal phosphorylation by 45 min. Meanwhile, the pp90<sup>rsk</sup> phosphorylation level was also maximal at 5 min, and at 45 min the EGF induced phosphorylation of the 90-kDa protein was not different from basal phosphorylation. In contrast to vanadate-treated PC12 cells, both NGF and EGF provoked a tremendous ³²P incorporation into the ERK1 protein doublet. The slower SDS-PAGE migrating entity would be consistent with the hyperphosphorylated ERK1 subpopulation.

In order to compare the effects of the various stimuli on the ERK1 phosphorylation states, only the lower molecular mass species (44 kDa) of the ERK1 protein doublet were submitted to acid hydrolysis. Phosphoamino acid composition of ERK1 stimulated by NGF revealed a strong dual phosphorylation of threonine and tyrosine residues at 15 min, the extent of which was diminished by 50 min (Fig. 4A). Vanadate produced a small ERK1 phosphorylation on both threonine and tyrosine residues at 15 and 50 min, with the phosphorysine level slightly greater than that of phosphothreonine. The level of EKR1 threonine and tyrosine phosphorylation
<figure><img src="image_4.png" /><figcaption>FIG. 3. ³²P labeling of PC12 cells: immunoprecipitation with antipeptide to ERK1. Near confluent PC12 cells were ³²P-loaded for 3 h in RPMI without phosphate containing 0.2% bovine serum albumin and 100 ng/ml NGF, 1 mM vanadate, 0.1 µM EGF, or buffer were added for the indicated times. Phosphoproteins were immunoprecipitated by nonimmune serum (NI) or by antibodies to ERK1 (I), eluted in Laemmli buffer, analyzed on a 10% resolving gel, and transferred to a polyvinylidene difluoride membrane. A 2-h autoradiograph exposure of the dried membrane from two representative experiments is shown.</figcaption></figure>

increased to a maximum within 5 min of EGF treatment and returned to near basal levels by 45 min. With the exception of vanadate-treated cells, there was a 1:1 ratio of phosphate incorporation into threonine and tyrosine residues of ERK1. The ERK1 phosphoserine level was not significantly affected by various stimuli at any time point studied.

The associated pp90<sup>rsk</sup> coprecipitating with ERK1 was phosphorylated mainly on serine residues in the basal condition (Fig. 4B). At 15 min, NGF provoked a strong phosphorylation on both serine and threonine residues, followed by a parallel decrease up to 50 min. Vanadate did not produce any detectable increment in both phosphoserine and phosphothreonine levels of the 90-kDa protein at 15 and 50 min. EGF induced a modest increase in pp90<sup>rsk</sup> phosphothreonine content and to a lesser extent, the phosphoserine level at 5 min. By 45 min of EGF treatment, the pp90<sup>rsk</sup> phosphothreonine level was undetectable while the phosphoserine content sparely diminished. Autoradiography of the pp90<sup>rsk</sup> phospho-amino acid analysis plates for up to 2 weeks did not reveal any phosphate incorporation into tyrosine residues. Thus NGF, EGF, and vanadate all stimulated the dual threonine and tyrosine phosphorylation of ERK1 as well as the threonine and serine phosphorylation of pp90<sup>rsk</sup> in PC12 cells.

*In Vivo Phosphorylation of ERK1 and pp90<sup>rsk</sup> in HIR Cells—* Serum-deprived HIR cells were also loaded with [³²P]orthophosphate and then treated with various ligands for selected periods of time. The MAP kinase ERK1 as well as the associated pp90<sup>rsk</sup> were isolated from cell extracts by immunoprecipitation with antibodies to ERK1. As illustrated in Fig. 5, insulin provoked a marked phosphate incorporation into ERK1 (44 kDa) by 5 min, corresponding to the peak of MBP kinase activity. This was followed by a diminution of the ERK1 phosphorylation state by 20 min, a situation which was slightly attenuated by 3 h. Meanwhile the ³²P incorporation into pp90<sup>rsk</sup> (90 kDa) also peaked at 5 min, diminished by 20 min, and then was reaugmented by 180 min of insulin treatment. Vanadate produced a phosphate incorporation into

ERK1 which was progressively greater as the exposure time was increased from 5 to 20 and 180 min. In agreement with the ERK1 phosphorylation state, vanadate induced a gradual increase in pp90<sup>rsk</sup> ³²P labeling over the same period.

We next performed phosphoamino acid analysis in order to investigate which amino acids were modified along this time course. As shown in Fig. 6A, within 5 min insulin stimulated the incorporation of phosphate into ERK1 on both threonine and tyrosine residues. This dual phosphorylation was diminished gradually over time by 20 and 180 min, but remained elevated as compared to the basal condition. Vanadate also stimulated a dual phosphorylation of ERK1 on threonine and tyrosine residues which increased gradually from 5 to 180 min. In insulin- and vanadate-treated HIR cells, there was a consistent 1:1 ratio of phosphothreonine to phosphotyrosine in ERK1 at all of the selected periods of time. The phosphoserine level of ERK1 was not affected by any of the stimuli at any time point studied.

Concerning the pp90<sup>rsk</sup> phosphorylation state, it can be seen on Fig. 6B that the coprecipitated 90-kDa protein was phosphorylated solely on serine residues in the basal condition. Within 5 min, insulin induced a de novo appearance of phosphothreonine concomitant with an increased phosphorylation on serine residues. By 20 and 180 min, both the phosphoserine and phosphothreonine levels were diminished, although phosphoserine to a lesser extent. Vanadate also stimulated both threonine and serine phosphorylation of pp90<sup>rsk</sup> at 5, 20, and 180 min, although phosphoserine to a lesser extent. However, none of these agents produced a detectable phosphotyrosine incorporation into pp90<sup>rsk</sup> even after 2 weeks of autoradiography. Thus, insulin and vanadate produced a parallel increase in the threonine and tyrosine phosphorylation of ERK1 as well as the simultaneous threonine and serine phosphorylation of pp90<sup>rsk</sup>.

*Localization of ERK1 in PC12 Cells by Immunofluorescence—* When PC12 cells cultured for 48 h in the absence of serum and any growth factor were fixed and permeabilized in
FIG. 4. Phosphoamino acid analysis of ERK1 and pp90<sup>rsk</sup> phosphorylated in PC12 cells. Membrane segments containing ERK1 (44 kDa) and pp90<sup>rsk</sup> (90 kDa) phosphoproteins from Fig. 3 were excised and submitted to acid hydrolysis for 90 min at 110 °C. The amount of released phosphoamino acids separated by TLC were, respectively, 90% for effector-stimulated ERK1, NGF- and vanadate-stimulated pp90<sup>rsk</sup>, and 20% for EGF-stimulated pp90<sup>rsk</sup>. The ERK1 (A) and pp90<sup>rsk</sup> (B) phospho-amino acid plates were autoradiographed for 18 and 48 h, respectively.

<figure><img src="image_5.png" /></figure>

acetone at -80 °C and then incubated with antibodies to ERK1 and stained with fluorescence isothiocyanate-conjugated goat anti-rabbit IgG, the immunofluorescence reaction was concentrated in the cytoplasm and only a weak reaction was detected in the nucleus (Fig. 7, A and B). By contrast, if the fixation step was preceded by a 5- or 45-min incubation at 37 °C with 100 ng/ml NGF, clear immunofluorescence reaction was evident in the nucleus (Fig. 7, C and E). When cells were incubated for the same time periods with 0.1 µM EGF, the immunofluorescence reaction pattern was superposable to that observed in control conditions (Fig. 7, D and F).

DISCUSSION

Taking advantage of the fact that our antipeptides to ERK1 precipitated a functional ERK1·pp90<sup>rsk</sup> phosphoprotein complex (Scimeca *et al.*, 1992), we investigated the regulation of both molecular species by various extracellular stimuli in PC12 cells and in intact fibroblasts expressing human insulin receptors. We first studied the activation kinetics of ERK1 in the eluates of anti-ERK1 immunoprecipitates using MBP as exogenous substrate. In serum-deprived HIR fibroblasts, insulin and vanadate rapidly stimulated the early phase of ERK1 activity within 5 min (Fig. 2). While the ERK1 activity
<figure><img src="image_6.png" /></figure>

FIG. 5. ³²P labeling of HIR cells: immunoprecipitation with antipeptide to ERK1. Confluent HIR cells growing in 100-mm Petri dishes were starved overnight, and the ³²P labeling was performed as described under "Experimental Procedures." 0.1 µM insulin and 1 mM vanadate were added at the indicated times and the solubilized proteins were submitted to precipitation by antibodies to ERK1. Following washes, phosphoproteins were eluted in Laemmli buffer and analyzed by SDS-PAGE on a 10% acrylamide resolving gel under reducing conditions. Phosphoproteins were then transferred to a polyvinylidene difluoride membrane and a 2-h autoradiograph exposure of the dried membrane from a representative experiment is shown.

returned to a much lower second phase in insulin-treated cells, vanadate produced a persistently increasing late phase ERK1 activation up to 180 min. As vanadate is an *in vitro* (Swarup *et al.*, 1982) and *in vivo* (Kharlund, 1985) inhibitor of tyrosine phosphatase activity, the first phase of MBP kinase activity might reflect the action of vanadate at the insulin receptor level. Thus by inhibiting the dephosphorylation of tyrosines of the expressed insulin receptors, vanadate would provoke a stimulation of the insulin receptor kinase activity, mimicking the hormone's action on its receptors. The late sustained activation of MBP kinase could be attributed to vanadate inhibiting cellular tyrosine phosphatases involved in the deactivation of ERK1 and expressed insulin receptors. Indeed, in fibroblasts not transfected with insulin receptors, vanadate induced a weak ERK1 activation which was maximal by 30 min and remained stable up to 3 h (data not shown). Neither the early peak of ERK1 activity nor the late phase increase were observed. In PC12 cells, NGF, EGF, and vanadate also activated ERK1 (Fig. 1). Vanadate produced a weak activation of ERK1 which reached a maximum by 15 min and which remained stable thereafter, similar to that seen in nontransfected fibroblasts (data not shown). NGF potently stimulated ERK1 activity, which attained a maximum at 15 min. There was another ERK1 activation at 30-60 min, and activity was still elevated at 180 min. Both the transient ERK1 deactivation at 30 min and the sustained late activation phase of ERK1 by NGF in PC12 cells corroborated with some previous studies (Tsao and Greene, 1991; Robbins and Cobb, 1992). However, a fast declining late phase of ERK1 activity has been observed in other studies (Gotoh *et al.*, 1990; Miyasaka *et al.*, 1990). Such discrepancies might be due to variations in experimental protocols and distinct clonal PC12 cell lines. In contrast to NGF, EGF provoked only a rapid, transient activation of ERK1 activity which was maximal by 2.5 min and returned to near basal levels by 30 min.

<figure><img src="image_7.png" /></figure>

FIG. 6. Phosphoamino acid analysis of ERK1 and pp90<sup>rsk</sup> phosphorylated in HIR cells. Membrane segments containing *in vivo* phosphorylated ERK1 (44 kDa) and pp90<sup>rsk</sup> (90 kDa) from Fig. 5 were excised and submitted to hydrolysis in 5.7 M HCl for 90 min at 110 °C. Ninety percent of both ERK1 and pp90<sup>rsk</sup> released phosphoamino acids were separated by TLC. The ERK1 (A) and pp90<sup>rsk</sup> (B) phosphoamino acid plates were autoradiographed for 18 and 48 h, respectively.

We have ³²P-loaded HIR and PC12 cells in order to investigate the regulation of the phosphorylation states of both ERK1 and the coprecipitating pp90rsk protein. All the stimuli tested, insulin, NGF, EGF, and vanadate, augmented the phosphorylation of ERK1 on both threonine and tyrosine residues in an 1:1 ratio (Figs. 4A and 6A). The increase in the phosphothreonine and phosphotyrosine content of ERK1 was in agreement with its activation as measured via MBP phosphorylation. There was also a simultaneous decrease in the
FIG. 7. Immunofluorescence staining of PC12 cells with antibody to ERK1. Cells were cultured in the absence of FCS or any growth factor for 48 h and fixed (A and B) or further incubated for 5 or 45 min at 37 °C in the presence of 100 ng/ml NGF (C and E) or 0.1 µM EGF (D and F). In the control conditions and in the presence of EGF, the cell cytoplasms show a fluorescent reaction, while the nuclei were poorly labeled. In contrast, after 5 and 45 min of exposure to NGF an increased labeling of the nucleus was observed. A-D, × 6000; E and F, × 3800.

<figure><img src="image_8.png" /></figure>

labeling of both threonine and tyrosine residues that followed closely the inactivation of MBP kinase activity. The results further strengthen the notion that both threonine and tyrosine phosphorylation are required for ERK phosphotransferase activity (Ray and Sturgill, 1988; Anderson *et al.*, 1990). It is of interest that neither phosphoamino acid was preferentially dephosphorylated. Such concomitant dephosphorylation of both threonine and tyrosine residues suggests that the regulation of ERK1 kinase activity involves both cellular serine/threonine and tyrosine phosphatases or a phosphatase activity with specificity for both residues. Alternatively, the regulation of ERK1 activity could lie further upstream through the existence of a serine/threonine phosphatase activity that acts on the recently identified MAP kinase kinase (Gomez and Cohen, 1991; Nakielny *et al.*, 1992).

The phosphorylation of pp90<sup>rsk</sup> was in good correlation with the phosphorylation and activation of ERK1. Following effector addition, there was a *de novo* appearance of phosphothreonine on pp90<sup>rsk</sup> concomitant with an increased phosphate incorporation on serine residues (Figs. 4B and 6B). We have recently shown that the coprecipitating pp90<sup>rsk</sup> undergoes *in vitro* phosphorylation on both serine and threonine residues. Following electrophoretic separation and *in situ* renaturation kinase assay, the 90-kDa protein becomes phosphorylated only on serine residues. Hence pp90<sup>rsk</sup> serine phosphorylation is likely to be due at least in part to autophosphorylation, while the threonine phosphorylation would come from the copurifying ERK1 (Scimeca *et al.*, 1992). It is worth noting that reports from P. Cohen's laboratory have described an insulin-stimulated protein kinase-1 which is closely related, if not identical, to the *Xenopus* S6 kinase II and which is thought to play a key role in the insulin regulation of glycogen metabolism in skeletal muscle (Dent *et al.*, 1990; Lavoinne *et al.*, 1991). The relationship between insulin-stimulated protein kinase-1 and our observed ERK1-associated pp90<sup>rsk</sup> remains to be established. In general, the pp90<sup>rsk</sup> phosphothreonine content paralleled the ERK1 activity more closely than

the phosphoserine level. Taking into account the close relationship between ERK1 and pp90<sup>rsk</sup> phosphorylation states, our results provide evidence of a direct interaction and phosphorylation of pp90<sup>rsk</sup> by ERK1. These results are in agreement with previous studies showing that MAP kinase can phosphorylate and partially activate pp90<sup>rsk</sup> *in vitro* with ensuing appearance of phosphothreonine (Sturgill *et al.*, 1988; Chung *et al.*, 1991b). Indeed, we observed Kemptide (LRRASLG) phosphorylation as a reflection of pp90<sup>rsk</sup> activation in our anti-ERK1 immunoprecipitates following cell stimulation (data not shown). This suggests that the phosphorylation of pp90<sup>rsk</sup> by ERK1 in intact cells stimulated its Kemptide phosphotransferase activity. Peptide maps of pp90<sup>rsk</sup> immunoprecipitated from <sup>32</sup>P-labeled cells were similar but more complex than those obtained from pp90<sup>rsk</sup> phosphorylated *in vitro* by *erk*-encoded kinases (Chung *et al.*, 1991a). Taken together, a change in pp90<sup>rsk</sup> activity as a result of phosphorylation by ERK and the presence of common phosphorylation sites *in vitro* as *in vivo* support the notion that these two enzymes participate in a phosphorylation cascade. In contrast, the regulation of the pp70<sup>S6K</sup> appears to be less straightforward. Some studies have suggested that pp70<sup>S6K</sup> and MAP kinase may be controlled by different regulatory pathways (Ahn and Krebs, 1990; Ballou *et al.*, 1991). However, more recent studies showed a parallel bi-phasic activation of both ERK1 and pp70<sup>S6K</sup>, with MBP kinase activity preceding S6 kinase activity (Kahan *et al.*, 1992). Although ERK1 might not activate pp70<sup>S6K</sup> directly, it remains possible that both kinases could be controlled by common regulatory elements.

The key observations of the present report are the strikingly different effects of NGF and EGF on ERK1 activation and intracellular fate. While NGF and EGF elicit an impressively long list of common early responses in PC12 cells, they clearly induce distinct final effects on cell destiny, i.e. growth arrest followed by neurite outgrowth for NGF, and cell division without cell differentiation for EGF (Huff *et al.*, 1981; Chao,
1992). We show here that both polypeptides regulate ERK1 and pp90rk in a coordinate but clearly ligand-specific fashion. To be specific, EGF evokes only a monophasic short-lived activation/phosphorylation of ERK1, and phosphorylation of pp90rk, while NGF induces a sustained and robust phase of similar modifications of both ERK1 and pp90rk. In addition, NGF provokes the translocation to the nucleus of ERK1, while EGF is apparently without detectable effect on intra-cellular ERK1 distribution. Very recent studies have shown that the inhibition of cell cycle progression and cell growth seen with rapamycin are associated with an inhibition of growth factor-stimulated activity of pp70s6k (Chung et al., 1992; Kuo et al., 1992; Price et al., 1992). In contrast, the pp90rk and MAP kinase are unaffected. These data have led the authors to conclude that rapamycin interferes with a signal transduction step that is unnecessary for activation of pp90rk or MAP kinase, but that is an absolute requisite for the activation of pp70s6k and mitogenesis. The lack of effect of rapamycin on pp90rk concurrent with the block of growth factor-dependent cell growth could suggest that the MAP kinase-pp90rk "couple" might be redundant for the control of growth regulation of mammalian cells, but that it plays a determining role in other major cell functions, such as the regulation of glycogen metabolism (Dent et al., 1990) and of cell differentiation. As shown here both NGF and EGF stimulate the phosphorylation cascade involving ERK1 and pp90rk, but with distinctive profiles. For PC12 cells the mitogenic events induced by EGF could be different from the neurotrophic signals generated by NGF. Alternatively, the mitogenic program versus the differentiative program could be generated by the same signals, but occurring with a duration and intensity which is characteristic for a growth factor (e.g. EGF) versus a differentiating factor (e.g. NGF). To be specific, we would like to suggest that the differentiating action of NGF in PC12 cells could be due, at least in part, to the sustained and coordinate activation of ERK1 and pp90rk coupled to the nuclear translocation of ERK1. Along the same lines of thought the failure of EGF to induce both phenomena, i.e. sustained activation of ERK1/pp90rk and nuclear translocation of ERK1, could explain why EGF does not lead to differentiation of PC12 cells.

In summary, the results reported here provide compelling evidence that the ERK1·pp90rk phosphoprotein complex is functionally regulated in a coordinate manner in intact fibroblasts and PC12 cells, and that pp90rk is activated *in vivo* as a result of direct phosphorylation by ERK1. Hence the *in vivo* association of pp90rk with ERK1 (Scimeca et al., 1992) together with the present report on the ERK1-pp90rk phosphorylation cascade in the same cells further strengthen the notion that pp90rk represents a physiologically relevant substrate of the extracellular-regulated kinases. Finally, based on the distinctive actions of NGF *versus* EGF on ERK1-pp90rk activation, and on ERK1 nuclear translocation, we would like to suggest that for PC12 cells the differentiative *versus* proliferative program results from the same signaling events, but which have a vigor and duration specific for a differentiating *versus* a growth promoting factor.

**Acknowledgments**—We thank Drs. R. Ballotti, V. Baron, S. Hey-

drick, and S. Tartare for reviewing the manuscript and for constructive comments. J. Duch, L. Capolongo, and G. Visciano are acknowledged, respectively, for secretarial assistance and illustrative work. The generous gift of HIR cells by Dr. J. Whittaker (Stony Brook, NY) and of NGF and the PC12 cell line by Dr. P. Kitabgi (Nice-Sophia-Antipolis, France) are gratefully acknowledged.

**Addendum**—While our manuscript was under review, Qiu and Green (1992) and Traverse *et al.* (1992) reported results similar to ours as far as the effect of EGF and NGF on ERK activity is concerned and reached similar conclusions concerning PC12 cell neuronal differentiation.

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