Activation and Protein Kinase C-Dependent Nuclear Accumulation of ERK in Differentiating Human Neuroblastoma Cells

Experimental Cell Research 256, 454 – 467 (2000) doi:10.1006/excr.2000.4843, available online at http://www.idealibrary.com on

Activation and Protein Kinase C-Dependent Nuclear Accumulation of ERK in Differentiating Human Neuroblastoma Cells Anna-Karin Olsson, Karin Vadhammar, and Eewa Nånberg 1 Department of Genetics and Pathology, The Rubeck Laboratory, Uppsala University, S-751 85 Uppsala, Sweden

The human neuroblastoma cell line SH-SY5Y is a well characterized model for sympathetic neuronal differentiation in vitro. Several differentiation protocols exist, one of which, the addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of serum, has been thoroughly studied. Wild-type SH-SY5Y cells are unresponsive to nerve growth factor (NGF), but cells transfected with the high-affinity NGF receptor TrkA (SH-SY5Y/TrkA) differentiate in response to NGF. In the present study, we have addressed the existence of a differentiation-specific mode of activation and subcellular distribution of the extracellular signal-regulated kinases ERK1 and ERK2 in SH-SY5Y/wt and SH-SY5Y/TrkA. Both TPA and NGF induced a sustained activation and nuclear accumulation of ERK that was accompanied by transactivation of a serum response element (SRE)-driven reporter and of the c-fos gene. However, activation and nuclear accumulation of ERK were not sufficient to induce neuronal differentiation in SH-SY5Y, as demonstrated by the response to TPA in serum-free cultures. Nuclear accumulation but not activation of ERK was demonstrated to require active protein kinase C (PKC). The effect of specific PKC inhibitors on subcellular distribution of ERK and ERK-dependent transcription suggests a functional role for PKC in the regulation of nuclear ERK activity in SH-SY5Y neuroblastoma cells. © 2000 Academic Press Key Words: neuroblastoma; differentiation; extracellular signal-regulated kinase; protein kinase C; NGF.

INTRODUCTION

During the development of the peripheral nervous system, immature neuroblasts undergo differentiation and acquire a sympathetic neuronal phenotype as a consequence of sequential exposure to growth factors and neurotrophins and interaction with extracellular matrix components [1, 2]. During this process, the cells become growth arrested and develop extended axonal 1 To whom correspondence and reprint requests should be addressed. Fax: 46-18-55 89 31. E-mail: [email protected].

0014-4827/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

processes and express neuronal marker genes. A commonly used in vitro model system for sympathetic neuronal differentiation is the rat phaeochromocytoma cell line, PC12 [3]. These cells express the high-affinity NGF receptor TrkA and respond to NGF by expression of differentiation-specific genes, neurite formation, and excitability [4, 5]. The monomeric GTP-binding protein p21Ras plays an important role during differentiation in PC12 cells. Introduction of constitutively active Val12Ras promotes neurite formation [6] while the dominant inhibitory mutant form, N17Ras, prevents NGF-stimulated neuritogenesis [7]. An important role of the Ras effector Raf and downstream activation of the MEK (MAPK/ERK kinase) and ERK kinases have also been demonstrated by transfection studies [8 –11]. A sustained activation of the mitogen-activated protein kinases (MAPK) ERK1 and ERK2 accompanied by nuclear translocation of active ERK promotes the differentiation response, while a transient activation stimulates proliferation in PC12 cells [12–15; for review, see 16]. Cell lines established from tumors originating from the nervous system and with a retained capacity to undergo differentiation in vitro provide useful models for studying the signal transduction involved in neuronal differentiation at a biochemical level. We have addressed the issue of ERK activity and subcellular distribution of active ERK in a well-characterized human in vitro model for neuronal differentiation, the neuroblastoma cell line SH-SY5Y. Neuroblastomas are pediatric tumors originating from immature neuroblasts in the developing peripheral nervous system. The SHSY5Y cell line was established from a highly malignant tumor with no N-myc amplification [17]. Amplification of the N-myc gene is found in a subset of highly malignant neuroblastomas and is correlated to a poor prognosis. Ras mutations have so far not been found in neuroblastomas, but high expression of normal Ras has been suggested to correlate with good prognosis in patients lacking N-myc amplification [18]. High expression of TrkA indicates a higher degree of differentiation and also correlates with a more favorable prognosis [18, 19]. Thus it is likely that signaling through a

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growth factor receptor to Ras and downstream targets may be of importance for the in vivo development of neuroblastomas possibly by promoting a higher degree of differentiation. SH-SY5Y cells undergo sympathetic neuronal differentiation when treated with low concentrations of the phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA), in the presence of serum [20, 21] or defined growth factors such as bFGF, IGF-1, PDGFAA, and PDGF-BB [22–24]. TPA added alone under serum-free conditions does not promote a differentiation response [22]. Wild-type SH-SY5Y cells (SH-SY5Y/ wt) express low levels of TrkA but show a very poor trophic and differentiation response to NGF [25]. Stable and transient transfection with full-length human trkA cDNA (SH-SY5Y/TrkA) renders the cells to respond to NGF with formation of extended neurites with growth cones and varicosities and expression of neuronal marker genes such as growth-associated protein-43 (GAP-43) and neuropeptide tyrosine (NPY) [25, 26]. Despite the tumor origin, SH-SY5Y/TrkA cells undergo progressive growth arrest during TPA- and NGF-induced differentiation. We have previously reported that induction of differentiation in SH-SY5Y cells is concomitant with a sustained increased activity of protein kinase C (PKC) [24, 27, 28]. SH-SY5Y expresses at least six isoforms of PKC (␣, ␤, ␦, ⑀, ␨, and ␮), but particularly isoforms of the novel type, PKC-⑀ and possibly PKC-␦, seem to be crucial for transcriptional activation and formation and maintenance of functional growth cones in differentiating SH-SY5Y [29 –31]. In the present paper, we have addressed the existence of a differentiation-specific mode of activation and subcellular distribution of ERK in SH-SY5Y/wt and SH-SY5Y/TrkA cells. We have used two defined differentiation protocols, NGF and TPA, in the presence of serum. Both protocols induced a sustained activation and nuclear accumulation of ERK compared to the effect of mitogenic treatment (PDGF-BB). However, the data showed that sustained activation and nuclear accumulation of ERK were insufficient to initiate differentiation, demonstrating the requirement for additional signals in these cells. Interestingly, while NGF-induced activation of ERK in the cytosol was insensitive to a specific PKC inhibitor, nuclear accumulation of ERK and ERK-dependent transcription in NGF-treated cells were demonstrated to require active PKC. MATERIALS AND METHODS Cell culture and reagents. SH-SY5Y wild-type cells (SH-SY5Y/ wt) [17] and SH-SY5Y stably transfected with human trkA cDNA (SH-SY5Y/TrkA) [25] were cultured in Eagle’s minimum essential medium supplemented with 10% fetal calf serum (FCS) (GIBCO), 100 IU/ml penicillin, and 50 ␮g/ml streptomycin. Prior to the experiments, cells were washed twice in RPMI 1640 and kept in serumfree SHTE medium (RPMI 1640, 30 nM selenium, 10 nM hydrocor-

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tisone, 10 nM ␤-estradiol, 30 ␮g/ml transferrin) for 24 h. Transient transfections were carried out in regular culture medium using the Ca-phosphate precipitation method according to Protocols and Application Guide, Promega. TPA was purchased from Sigma, NGF from Promega, and PDGF-BB kindly provided by Dr. C-H. Heldin, The Ludwig Institute for Cancer Research, Uppsala, Sweden. The protein kinase C inhibitors GF109203X [32] used at 2 and 4 ␮M and Ro-32-0432 [33] used at 4 ␮M, and the MEK inhibitor PD98059 [34] used at 75 ␮M were all purchased from Calbiochem. The inhibitors were dissolved in dimethyl sulfoxide. Analysis of cell morphology. SH-SY5Y/wt cells were seeded sparsely on 100-mm dishes in regular culture medium and allowed to attach to the dish for about 36 h. Thereafter the indicated cultures were transferred to SHTE and the rest received fresh regular culture medium. TPA (16 nM) was added to both serum-free and serumcontaining cultures. After 4 days the cultures were photographed in an Olympus IMT-2 phase-contrast microscope at 20⫻ magnification. Morphologically differentiated cells were defined as cells with neurites with a length equal to or greater than the diameter of two cell bodies. The percentage of positive cells in a given culture was obtained by counting all cells in a defined area and scoring the neurite length. Analysis of phosphorylated ERK and ERK activity. For analysis of phosphorylated ERK (phospho-ERK), serum-starved cells were treated and lysed in 0.3 ml of 0.5% Triton X-100, 0.5% sodiumdesoxycholate (Na-doc), 20 mM Tris-HCl, pH 7.5, 10 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 1 mM benzamidine, 1 mM Na 3VO 4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 ␮g/ml leupeptin, and 10 ␮g/ml aprotinin. Total cell lysates were separated by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), transferred to Hybond C membrane (Amersham Pharmacia Biotech), and immunoblotted with an anti-phospho-ERK antibody (New England BioLabs. Inc.) diluted 1:1000 in TBS-T (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20). Immunoreactivity was visualized by incubation with an anti-rabbitHRP antibody and ECL reagents according to the manufacturer’s protocol (Amersham Pharmacia Biotech). The membranes were subsequently stripped and reprobed with an anti-ERK antibody (sc94; Santa Cruz Biotechnology Inc.) for loading of the total amount of ERK1 and ERK2. The intensity of the phospho-ERK and ERK signal was determined by computer scanning of the original ECL films and quantification with the software MacBAS v.2.2 (Fuji Inc., Japan). For analysis of ERK activity the cells were lysed in 1% Triton X-100, 0.5% Na-doc, 20 mM (N-[2-hydroxyethyl]piperazine-N⬘-[2ethanesulfonic acid] (Hepes), pH 8.0, 50 mM NaF, 20 mM Na 2H 2P 2O 7, 10 mM ethylene glycol-bis(␤-aminoethyl ether) N,N,N⬘,N⬘-tetraacetic acid (EGTA), 5 mM MgCl 2, 1 mM dithiothreitol (DTT), 20 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, 100 ␮M Na 3VO 4, 1 mM PMSF. ERK protein was immunoprecipitated using an antiERK2 antiserum generated against a C-terminal ERK2 peptide [35], kindly provided by Dr. Lars Ro¨nnstrand, The Ludwig Institute for Cancer Research, Uppsala, Sweden, and protein A-Sepharose (Amersham Pharmacia Biotech). The kinase activity in the precipitates was measured by in vitro phosphorylation of 10 ␮g myelin basic protein (MBP) for 15 min at 30°C in 40 ␮l of a reaction mixture containing 20 mM MgCl 2, 2 mM MnCl 2, 20 mM Hepes, pH 8.0, 1 mM DTT, 20 ␮M ATP, and 3 ␮Ci [␥- 32P]ATP. The kinase reaction was terminated by the addition of 20 ␮l of 3X Laemmli sample buffer [36]. The proteins were resolved on 15% SDS-PAGE and the position of MBP was determined by Coomassie blue staining. Quantification was done by phosphorimage analysis (Bas 2000, Fuji Inc.). Subcellular distribution of ERK1 and ERK2. SH-SY5Y/TrkA cells seeded on coverslips were starved as described above. After treatment with the indicated additions, the cells were fixated and permeabilized in 100% methanol at ⫺20°C for 7 min. The coverslips were rehydrated and blocked with 2% bovine serum albumine (BSA) in phosphate-buffered saline (PBS) before incubation with an anti-

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ERK antibody (sc94 from Santa Cruz Biotechnology Inc.) and a FITC-labeled secondary anti-rabbit IgG (F0205, Dako). The coverslips were subsequently stained with Dapi reagent to visualize the nuclei and mounted using Vectashield (Vector Laboratories). Subcellular distribution of active ERK. SH-SY5Y/TrkA cells were cultured and starved as described above for staining of total ERK. After stimulation the cells were fixated in cold 3% paraformaldehyde and the coverslips blocked with 5.5% horse serum in TBS-T-X100 (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). The coverslips were subsequently incubated with the anti-phospho-ERK antibody (New England BioLabs, Inc.), followed by a mouse antirabbit antibody (M0737, Dako) and a rabbit anti-mouse antibody (Z259, Dako) and finally with mouse APAAP (alkaline phosphatase and mouse monoclonal anti-alkaline phosphatase; D0651, Dako). The slides were developed in substrate solution containing Fast Red (Sigma) and mounted using Immunomount (Shandon No. 9990402). Quantification of nuclear staining of ERK. Semiquantitative analysis of the staining was performed using a software package, Signifier, from Diascan AB in Uppsala, Sweden. The slides were viewed in a standard Olympus BH-10 optical microscope with an attached Sony DXC-151 colorvideo camera, and the images were transferred to a Silicon Graphics Indy workstation for quantification. Alternatively, the slides were photographed and the negative film images scanned and analyzed as above. The areas of more than 100 nuclei from each treatment were marked and the relative intensity in staining was calculated by the software. Analysis of SRE-dependent transcription. SH-SY5Y/TrkA cells seeded in routine culture medium in 10-cm dishes were transfected for 12–16 h with 15 ␮g of the vector p3TDlux [37] that contains three repeats of the SRE sequence from the human c-fos promoter (kindly provided by Dr. Joan Massague´, Memorial Sloan-Kettering Cancer Center, New York) and 5 ␮g of a constitutively active ␤-actin promoter-driven lacZ plasmid using the calcium-phosphate method. Thereafter the transfected cells were washed and transferred to serum-free or regular culture medium. When serum-free cultures were included in the experiment, at least 18 h were allowed to pass before the cells were stimulated for 3 h and then harvested in 25 mM Tris-HCl, pH 7.8, 2 mM EDTA, 10% glycerol, 1% Triton X-100, and 2 mM DTT. The luciferase activity was assayed using luciferase assay reagent (Promega) and the ␤-galactosidase activity with Galacton and Emerald (Tropix). The luciferase activity in each sample was correlated to transfection efficacy measured as ␤-galactosidase activity. Northern blot analysis. RNA was prepared according to Chomczynski and Sacchi [38]. The amount of 15 ␮g of total RNA was loaded per sample and separated by formaldehyde gel electrophoresis according to Sambrook et al. [36]. The RNA was blotted onto Hybond C extranitrocellulose membranes and hybridized with the following cDNA probes labeled with [ 32P] by the mega prime method (Amersham Pharmacia Biotech) according to the manufacturer’s protocol: human c-fos [39] and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [40]. The Hybond C membrane was dehybridized in 50% formamide, 0.1% SDS, and 0.1X SSC (30 mM sodium citrate, 300 mM NaCl) for 30 min at 65°C between hybridization with the two probes. The filters were exposed and quantified by phosphorimage analysis as above.

phorbol ester TPA in the presence of 10% FCS [20, 21, 41]. The differentiating cells express neuronal marker genes and extend neurites with varicosities and growth cones, a response that is well pronounced after 4 days of treatment [41]. The differentiation response requires the presence of serum or defined growth factors and is not induced by TPA alone [22, 41]. We have now measured the amplitude and duration of TPA-induced phosphorylation of ERK under serum-free and serumcontaining conditions to investigate if there was a correlation between the activation of ERK and the differentiation response. Immunoblotting was done with an antibody specifically recognizing active phosphorylated ERK1 and ERK2 and subsequent incubation of the membranes with an antibody recognizing total ERK1 and ERK2 for standardization of the data by comparison of the relative intensity of the phospho-ERK signal with the total amount of ERK in each sample. The results showed that TPA induced a potent and longlasting (24 h) phosphorylation of ERK in both serumfree and serum-containing cultures (Fig. 1A). The upper and lower band in each panel represents the 44kDa ERK1 and 42-kDa ERK2, respectively. The serum-free cultures showed a lower basal level of phosphorylated ERK compared with serum-containing cultures, but the relative amount of phosphorylated ERK after TPA treatment reached similar levels in serumfree and serum-containing cultures. Also, the duration of the response was similar under both conditions. The morphology of cultures treated with TPA in the absence or presence of serum is shown in Fig. 1B. We have defined a morphologically differentiated cell as having a neurite length greater than or equal to the diameter of two cell bodies. Quantification of the relative number of neurite-bearing cells in cultures treated with TPA in the absence or presence of serum for 4 days (Table 1) demonstrated that TPA in the presence of serum induced a ninefold increase in the number of morphologically differentiated cells, while TPA had no effect under serum-deprived conditions. These results suggested that although cultures treated with TPA alone showed a strong and sustained phosphorylation of ERK, this ERK response was not in itself sufficient for induction of the sympathetic differentiation response in these cells. The data were further supported by our findings that the specific MEK inhibitor PD98059 had no effect on TPA-induced neurite formation (Olsson and Nånberg, manuscript in preparation).

RESULTS

Phorbol Ester-Induced Phosphorylation of ERK in SH-SY5Y Cells The neuroblastoma cell line SH-SY5Y undergoes differentiation and acquires a sympathetic phenotype when treated with a low (16 nM) concentration of the

Phosphorylation of ERK and ERK Activity in SH-SY5Y/TrkA Cells Previous reports have described that NGF added to PC12 cells induces a sustained increase in ERK activity that is a necessary event for NGF-dependent development of a sympathetic phenotype [9, 13, 16,

ACTIVATION OF ERK IN NEUROBLASTOMA CELLS

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FIG. 1. Effect of TPA treatment on ERK activity and neurite formation in the absence and presence of serum. (A) Cultures of SH-SY5Y/wt cells were treated with 16 nM TPA in the absence (⫺FCS) or presence (⫹FCS) of serum for the indicated lengths of time. Total lysates were prepared and resolved by 10% SDS-PAGE, immunoblotted with an anti-phospho-ERK antibody (P-ERK), and visualized by ECL detection. The filters were subsequently reprobed with an anti-ERK antibody (ERK). The intensity of the phospho-ERK signal was correlated to the signal from the total amount of ERK and calculated as fold values compared to serum-free control at 0.5 h. The values are given beneath each lane. The figure shows one representative experiment out of three. (B) Cultures of SH-SY5Y/wt were treated under the same conditions as in A and their morphology was monitored during several days. The micrographs show the morphology after 4 days of treatment in one representative experiment out of three.

42, 43]. To better understand the role of ERK activity during sympathetic differentiation of neuroblastoma cells, we wanted to use two distinct differentiation protocols that both stimulate neurite formation, gene expresssion, and growth arrest, and compare the TABLE 1 TPA-Induced Neurite Formation in SH-SY5Y/TrkA in the Absence and Presence of Serum Condition

No FCS

10% FCS

Control 16 nM TPA

16% (n ⫽ 140) 21% (n ⫽ 173)

8% (n ⫽ 173) 66% (n ⫽ 202)

Note. The number of neurite-bearing cells was analyzed from micrographs of cultures treated with 16 nM TPA for 4 days in the absence or presence of 10% FCS as in Fig. 1B. A neurite was defined as a process with a length equal to or exceeding the diameter of two cell bodies. The total number of cells analyzed under each condition is indicated within parentheses.

effect on ERK activity. TPA is biologically stable and even a low dose of 16 nM induces a sustained activation of PKC [27] that may cause a forced sustained ERK activity compared with the effect of a metabolized differentiation-promoting ligand such as NGF or a growth factor such as PDGF. To allow studies of TPA- and NGF-induced ERK signaling in the same cells, we used SH-SY5Y cells stably transfected to express full-length human NGF-receptor TrkA (SHSY5Y/TrkA) [25]. In contrast to the wild-type SHSY5Y cells, SH-SY5Y/TrkA are NGF responsive and develop a differentiated phenotype upon treatment with 100 ng/ml NGF [25]. The SH-SY5Y/TrkA have retained their responsivenes to TPA and requirement for serum factors for a full differentiation response. Total lysates were analyzed by immunoblotting with the antibody specifically recognizing active phosphorylated ERK1 and ERK2 (Figs. 2A–C, upper part). The cells were treated using the defined dif-

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but also of ERK1, compared to unstimulated cells (Fig. 2A). After 4 h (Fig. 2B), NGF was the strongest activator of phosphorylation of ERK and this was also seen after 24 h (Fig. 2C). PDGF and TPA had similar effects both with respect to amplitude and duration of the increased amounts of phospho-ERK inducing a slowly declining response that remained for 24 h of incubation (Fig. 2). The similar effects of TPA in the absence and presence of serum were in good agreement with the results obtained from SHSY5Y wild-type cells (Fig. 2 and Fig. 1A). Total amount and loading of ERK1 and ERK2 were again estimated by incubation of the membranes with an anti-ERK-antibody (Figs. 2A–C, lower part). To measure ERK kinase activity, we immunoprecipitated ERK protein from treated cultures using an antiERK2 antiserum [35] and measured the kinase activity as in vitro phosphorylation of myelin basic protein (Table 2). In agreement with the phospho-ERK immunoblots, NGF gave a strong and sustained kinase activation measured over 24 h. Even after 48 h, this stimulatory effect of NGF was seen, while the other protocols were without effect at this late time point (data not shown). PDGF-BB stimulated an early transient kinase activity, which after 2 h had declined and reached a plateau corresponding to 30% of the response seen at 30 min. TPA, in the absence or presence of serum, caused an equally potent initial ERK activity, which remained for 4 h but was largely gone after 24 h. TABLE 2 Immunoprecipitated ERK2 Activity from SH-SY5Y/TrkA Cells Measured as in Vitro Phosphorylation of Myelin Basic Protein Condition

FIG. 2. Growth factor- and TPA-induced phosphorylation of ERK. Serum-starved cultures of SH-SY5Y/TrkA cells were incubated with 100 ng/ml NGF, 16 nM TPA ⫹/⫺ 10% FCS, 10 ng/ml PDGF-BB, or no addition (⫺) for 30 min (A), 4 h (B), or 24 h (C). Total lysates were prepared and resolved by 10% SDS-PAGE, immunoblotted with an anti-phospho-ERK antibody (P-ERK), and visualized by ECL detection. The filters were subsequently reprobed with an anti-ERK antibody (ERK), and the intensity of the phospho-ERK signal was analyzed as in Fig. 1A.

ferentiation protocols, 100 ng/ml NGF or 16 nM TPA in the presence of 10% FCS (TPA/FCS), or with the mitogen PDGF-BB (10 ng/ml). NGF and PDGF-BB were used under serum-free conditions and TPA in the absence of FCS (TPA) was also included. After 30 min of stimulation, all four treatments induced a significant phosphorylation of particularly ERK2,

30 min

2h

4h

24 h

Control

Experiment 1 Experiment 2 Mean

1.0 1.0 1.0

1.0 1.0 1.0

1.0 1.0 1.0

1.0 1.0 1.0

NGF

Experiment 1 Experiment 2 Mean

6.9 5.5 6.2

5.2 4.2 4.7

5.5 7.1 6.3

3.8 5.3 4.6

TPA/FCS

Experiment 1 Experiment 2 Mean

5.2 5.5 5.4

5.0 2.5 3.8

4.0 4.1 4.0

1.3 2.1 1.7

TPA

Experiment 1 Experiment 2 Mean

3.2 5.8 4.5

4.9 4.9 4.9

4.3 4.3 4.3

2.2 1.9 2.0

PDGF-BB

Experiment 1 Experiment 2 Mean

5.1 5.2 5.2

3.1 2.6 2.8

1.8 2.3 2.0

2.3 3.0 2.6

Note. Kinase activity was quantified by phosphorimage analysis and the pixel value in each control sample was set to 1.0 and the other results from the same time point were calculated as fold of the control value.

ACTIVATION OF ERK IN NEUROBLASTOMA CELLS

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FIG. 3. Growth factor- and TPA-induced nuclear accumulation of ERK1 and ERK2. Serum-starved SH-SY5Y/TrkA cells seeded on coverslips were preincubated with vehicle (the two columns to the left) or with the PKC inhibitor GF109203X (the two columns to the right) for 30 min prior to treatment with 16 nM TPA, 16 nM TPA ⫹ 10% FCS, 100 ng/ml NGF, 10 ng/ml PDGF-BB, or no addition (ctrl) for 4 h. The fixated and permeabilized cells were incubated with an anti-ERK antibody (sc94) followed by a FITC-labeled anti-rabbit IgG antibody (anti-ERK). Dapi staining was performed to visualize the nuclei (Dapi). The micrographs depict the result from one representative experiment out of several.

Growth Factor- and TPA-Induced Subcellular Redistribution of ERK Based on data especially from PC12 cells, it has been suggested that nuclear translocation of active ERK and subsequent transcriptional activation, rather than the duration of total kinase activity, are the ERK-related events important for promotion of a neuronal differentiation response. To evaluate a possible differentiationspecific pattern in the subcellular distribution of ERK in SH-SY5Y/TrkA cells, we did immunocytochemical staining for total ERK on cultures treated with the same additions as in Fig. 2. The immunoreactivity was visualized using a FITC-coupled secondary antibody and the localization of the nuclei was visualized by counterstaining with Dapi reagent. The Dapi staining also illustrated the relatively large proportion of the cell body that is occupied by the nucleus in these cells. The specificity of the fluorescence signal was verified by staining of cells with peptide-blocked primary antibody or no primary antibody (data not shown). Unstimulated SH-SY5Y/TrkA cells showed strong immunoreactivity for ERK in the cytoplasm and in the short spikes and immature neurites (Fig. 3, left panel). Ad-

dition of TPA in the absence or presence of serum caused a rapidly formed pattern of granulated staining of ERK all over the cells as well as in the nuclei. This effect could be seen already after 30 min but was even more pronounced after 2 and 4 h. Figure 3, left panel, shows cells treated with TPA for 4 h. Addition of serum caused a change in cell morphology with a rounding up and clustering of the cells that severely hampered a more detailed analysis of the subcellular distribution of ERK under those conditions (Fig. 3, left panel, TPA/ FCS). The rounded cells showed a layer of cytoplasm with immunoreactive ERK superimposed over the nucleus while the cells in serum-free cultures retained a more flattened morphology with better resolution of staining in the area of the nuclei. Addition of serum alone did not cause any apparent translocation of ERK into the nuclear compartment but strong immunoreactive signal from the clustered superimposed cells (data not shown). NGF induced granulated staining of ERK in the nuclei with a similar time course as TPA (4 h illustrated in Fig. 3, left panel, NGF). PDGF-BB on the other hand failed to cause any detectable increase in nuclear ERK at any time point measured between 30

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min and 4 h (Fig. 3 and data not shown). Semiquantitative analysis (see Materials and Methods) of the nuclear staining intensity after each treatment revealed that TPA, TPA/FCS, and NGF, but not PDGF-BB, induced more nuclear ERK than that seen in control cells (arbitrary units: control, 42; TPA, 87; TPA/FCS, 74; NGF, 79; PDGF-BB, 38). Identical results were obtained using two different antibodies recognizing both ERK1 and ERK2 (sc93, data not shown and sc94 in Fig. 3) and the anti-ERK2 antiserum used for the kinase activity measurements described above (data not shown). Different methods for fixation of the cells have also been tested with the same result. All together, the ERK staining has been performed 50 – 60 times and consistently demonstrated that a large proportion of the ERK proteins remains in the cytosol even in the stimulated cells. Thus, TPA with and without serum as well as NGF induced a nuclear accumulation of ERK that was detectable after 30 min and stayed at a steady maximal level between 2 and 4 h. ERK-Dependent Transcriptional Activity An established target for active ERK in the nucleus is phosphorylation of the transcription factor Elk-1. Phosphorylated Elk-1 in complex with serum response factor (SRF) binds to and activates SRE in the c-fos promoter and in other promoters. To analyze if the

FIG. 4. Growth factor- and TPA-induced SRE-driven transcription in the absence and presence of serum. Cultures of SH-SY5Y/ TrkA were transiently transfected with a SRE-driven luciferase reporter (p3TD-lux). After the transfection, half of the cultures were put in serum-free medium (⫺FCS) and the rest in regular culture medium (⫹FCS). 18 h later the cells were treated for 3 h with 16 nM TPA, 100 ng/ml NGF, 10 ng/ml PDGF-BB, or no addition (ctrl) before harvest and analysis of luciferase activity. Each bar represents the mean value ⫹/⫺ SEM of the luciferase activity in three independent experiments, correlated to transfection efficacy measured as transcription from a constitutively active ␤-galactosidase-encoding vector.

FIG. 5. Effect of PKC inhibition on NGF- and TPA-induced SREdriven transcription. SH-SY5Y/TrkA cells transiently transfected with the SRE-driven luciferase reporter p3TD-lux were cultured in the presence of serum and preincubated with vehicle or one of the two PKC inhibitors GF109203X or Ro-32-0432 or the MEK inhibitor PD98059. Then 16 nM TPA (gray bars), 100 ng/ml NGF (black bars), or no addition (open bars) was added and the cells were harvested after 3 h. Luciferase activity was correlated to transfection efficacy measured as transcription from a constitutively active ␤-galactosidase-encoding vector. The figure represents mean values ⫹/⫺ SEM from three independent experiments.

data on nuclear accumulation of ERK presented in Fig. 3 reflected increased amounts of functionally active ERK in the nuclei, we measured transcriptional activation of a reporter construct encoding a luciferase gene under the control of 3X SRE from the human c-fos-promoter, p3TD-lux [37]. This reporter system gives a more specific readout for nuclear MAP kinase activity than measurement of c-fos mRNA, since c-fos transcripts also can be induced by ERK-independent pathways [44 – 46]. Cultures of SH-SY5Y/TrkA cells were transiently transfected with p3TD-lux and subsequently serum-starved or cultured in ordinary medium before incubation with TPA, NGF, or PDGF-BB for 3 h prior to harvest of cell lysates and measurement of luciferase activity. To compensate for differences in transfection efficacy and basal transcriptional activity, the cultures were cotransfected with a constitutively active ␤-galactosidase vector (␤-actin lacZ). Both TPA and NGF induced an increase in transcriptional activity of the SRE reporter. TPA was more potent than NGF while PDGF-BB had no effect (Fig. 4). This was seen in both the absence and the presence of serum, with the exception that TPA was even more potent in the absence of serum. Both TPA- and NGF-induced transactivation of p3TD-lux was abolished by pretreatment with PD98059, demonstrating that the response was mediated by MEK-dependent activation of ERK, and not by other MAP kinases under the given conditions (Fig. 5). The ERK-dependent transactivation of p3TD-lux (Figs. 4 and 5) corroborated the immunocytochemical staining data (Fig. 3) and demonstrated that both TPA and NGF induced a nuclear ERK activ-

ACTIVATION OF ERK IN NEUROBLASTOMA CELLS

FIG. 6. Effect of PKC inhibition on activation of ERK. Cultures of serum-starved SH-SY5Y/TrkA cells were incubated for 2 h with 16 nM TPA, 100 ng/ml NGF, or no addition (ctrl), in the presence of the PKC inhibitor GF109203X (⫹) or vehicle (⫺) and analyzed for ERK activity measured as in vitro phosphorylation of MBP. Quantification was done by phosphorimage analysis. The values represent means from two independent experiments.

ity in these neuroblastoma cells while PDGF-BB failed to cause nuclear accumulation of active ERK. The stronger potency of TPA compared with NGF on SREdriven transcription (Figs. 4 and 5) was also true for phosphorylation of Elk-1 analyzed in total lysates of SH-SY5Y/TrkA cultures treated with TPA and NGF for 2 h in the absence or presence of serum (data not shown). Role of Protein Kinase C for Activation of ERK in SH-SY5Y/TrkA Cells The data in Figs. 4 and 5 showed that TPA was a more potent activator of ERK-dependent transcription compared with NGF. This could reflect a more potent activation of ERK and/or a more efficient nuclear accumulation of active ERK in TPA-treated cells. To evaluate the relative potency of TPA and NGF on activation of ERK versus nuclear accumulation of active kinase and subsequent transactivation, we compared the effect of NGF and TPA on ERK-dependent MBP phosphorylation, immunocytochemical staining for active ERK, and SRE-dependent transactivation. As shown in Fig. 6, in vitro kinase measurements of ERK activity demonstrated that TPA and NGF were equally potent stimulating total ERK activity in serum-starved SH-SY5Y/TrkA cells (see also Table 2). Thus, activation of PKC by TPA could feed into the Ras-ERK pathway as efficiently as NGF-dependent signaling via TrkA also in the absence of serum factors. We addressed the role of PKC for NGF-induced ERK activity in cultures treated with NGF in the presence of the specific PKC-inhibitor GF109203X or vehicle. GF109203X did not attenuate, but rather enhanced,

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the NGF-induced activation of ERK, while the effect of TPA was inhibited to 85% (Fig. 6). Preincubation with GF109203X did also block TPA-induced phosphorylation of ERK but had no effect on NGF-induced phosphorylation as analyzed by immunoblotting with the anti-phospho-ERK-specific antibody (data not shown). Treatment with GF109203X for up to 24 h had no effect on the total amount of immunoreactive ERK1 and ERK2 (data not shown). Thus, we conclude that although both TPA and NGF efficiently caused activation of ERK, the NGF-induced ERK activity was not prevented by the PKC inhibitor. To evaluate the relative efficiency of NGF and TPA on nuclear accumulation of active ERK we did immunocytochemical staining using the specific anti-phospho-ERK antibody to avoid staining of both active and inactive ERK1 and ERK2 as in Fig. 3. For technical reasons, we were unable to use any fluorochrome-coupled secondary antibody in combination with this primary antibody and therefore had to use a coupled enzyme reaction. Hence, no counterstaining with Dapi was performed in this series of experiments. Unstimulated cells showed very low background of staining while both TPA and NGF induced strong anti-phosphoERK reactivity after 2 h (Fig. 7A). Both TPA- and NGF-treated cells showed staining in the cytoplasm, the perinuclear compartment and in the nuclei. However, nuclear staining was stronger in the TPA-treated cells. The specificity of the immunoreactivity was demonstrated by the very low level of staining in cultures pretreated with the MEK inhibitor PD98059 prior to addition of TPA and NGF (Fig. 7A, right panel). Cultures incubated without primary antibody completely lacked staining (data not shown). Semiquantitative image analysis of 100 nuclei from each treatment revealed that TPA induced 200% and NGF 50% more nuclear staining compared to control cells (arbitrary units: control, 14; TPA, 41; NGF, 21). Analysis of cultures treated with vehicle, TPA, and NGF, respectively, for 24 h (Fig. 7B) demonstrated increased staining in the cytoplasm of NGF, but not to the same extent in TPA-treated cultures, which was in good agreement with the measurements of ERK activity by in vitro phosphorylation of MBP (Table 2). However, quantification of the coverslips in Fig. 7B revealed no significant difference in nuclear staining between TPA- and NGF-treated cultures (arbitrary units: control, 16; TPA, 24; NGF, 23). Role of Protein Kinase C for Nuclear Accumulation of Active ERK We have previously published data demonstrating a requirement for functional PKC for the development of a differentiated neuronal phenotype in SH-SY5Y/wt and SH-SY5Y/TrkA cells. Inhibition of PKC by treat-

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FIG. 7. PKC-dependent nuclear accumulation of phosphorylated ERK. (A) Serum-starved SH-SY5Y/TrkA cells seeded on coverslips were preincubated with vehicle, GF109203X, or PD98059 for 30 min prior to the addition of 16 nM TPA, 100 ng/ml NGF, or no addition (control) for 2 h. The fixated and permeabilized cells were incubated with an anti-phospho-ERK antibody and the immunoreactivity was visualized using APAAP enzyme reaction with Fast Red as substrate. (B) Cultures of serum-starved SH-SY5Y/TrkA were treated with 16 nM TPA, 100 ng/ml NGF, or no addition for 24 h and analyzed as in A. The micrographs depict the result from one representative experiment repeated four times.

ment with the specific inhibitor GF109203X prevents formation and maintenance of functional growth cones and elongated neurites as well as transcriptional activation of the GAP-43 and NPY genes in cultures of SH-SY5Y/wt differentiated by treatment with TPA/ FCS [29]. Also NGF-dependent formation of neurites requires functional PKC, while the activation of neuronal marker genes is less dependent on PKC (30% inhibition) [29]. Analysis of the subcellular distribution of ERK after preincubation with GF109203X revealed that the PKC inhibitor did not only prevent TPA-induced activation and nuclear accumulation of ERK, but also NGF-induced nuclear accumulation of active kinase (Figs. 3 and 7A). Identical results were obtained using another specific PKC inhibitor, Ro-32-0432 (data not shown). Semiquantitative analysis of the nuclear staining (as described above) in NGF ⫹ GF109203X-treated and control cells in Fig. 7A showed that addition of GF109203X reduced the nuclear staining to background levels (arbitrary units: NGF ⫹ GF109203X, 13;

control, 14). Qualitatively similar results were obtained from semiquantitative analysis of cells fixated and stained as in Fig. 3 (arbitrary units: TPA ⫹ GF109203X, 30; TPA/FCS ⫹ GF109203X, 31; NGF ⫹ GF109203X, 29; control, 32). However, the cytoplasmic staining in NGF-treated cells was unaffected by the PKC inhibitor. We cannot exclude that some of the staining in the nuclear area of the fixated cells in Fig. 7 originates from active ERK in a cytoplasmic layer superimposed over the nucleus. But the very low degree of staining of the corresponding area in cells treated with both GF109203X and NGF speaks against this being a significant component. These results were further supported by analysis of SRE-dependent transcription. GF109203X prevented both TPA- and NGFinduced transcription from the SRE reporter. Identical results were obtained with Ro-32-0432 (Fig. 5). Thus, we conclude that the NGF-induced activation of ERK was insensitive to the PKC inhibitor, but nuclear translocation of active ERK and ERK-dependent transcription required functional PKC in NGF-treated cells. Both NGF and TPA induce expression of c-fos in SH-SY5Y/TrkA [25] and SH-SY5Y/wt [47], respectively. Since activation via SRE is important for regulation of c-fos transactivation, we analyzed to what extent inhibition of PKC affected induction of c-fos in response to TPA and NGF. As shown in Fig. 8, NGFinduced c-fos transcript was reduced to 40% by the PKC inhibitor, while the response to TPA was fully blocked as expected. Further experiments will be required to analyze the contribution of PKC-dependent

FIG. 8. PKC-dependent induction of c-fos transcript. Cultures of serum-starved SH-SY5Y/TrkA were stimulated with 16 nM TPA, 100 ng/ml NGF, or no addition (ctrl) for 1 h, in the presence (⫹) or absence (⫺) of the PKC inhibitor GF109203X. Total RNA was prepared and analyzed for the amount of c-fos transcript by Northern blotting. The c-fos transcript was quantified by phosphorimage analysis and correlated to the GAPDH mRNA signal that was used as an internal standard for the amount of loaded RNA. The figure represents mean values ⫹/⫺ SEM from three independent experiments.

ACTIVATION OF ERK IN NEUROBLASTOMA CELLS

nuclear accumulation of ERK for induction of c-fos and other genes in more detail, but the available results open up the possibility that regulation of subcellular distribution of ERK is one component in the reported PKC dependency of sympathetic differentiation of the SH-SY5Y cells. DISCUSSION

Our results presented here show that two established protocols that induce SH-SY5Y/TrkA neuroblastoma cells to undergo sympathetic differentiation, NGF and TPA in the presence of serum, stimulated an increase in ERK activity that had reached a plateau after 30 min and remained elevated for several hours. This increase in total ERK activity was accompanied by accumulation of active kinase in the nuclei. In contrast, PDGF-BB that promotes proliferation and survival of SH-SY5Y cells [23] induced a rapid and transient activation but no nuclear accumulation of ERK. Addition of TPA alone in serum-free cultures induces a low and sustained activation of PKC in SH-SY5Y [24], but no differentiation with respect to neurite formation has been recorded (Fig. 1 and [22, 41]). In addition, a very limited transactivation of neuronal marker genes was demonstrated under these conditions [22]. To promote a full differentiation response, TPA must be added in combination with serum or defined growth factors [41]. However, the data presented here demonstrated that TPA-induced ERK activity was identical with respect to amplitude and duration and nuclear accumulation when we compared serum-depleted and serum-containing cultures of SH-SY5Y/wt. We interpret these results so that a sustained activation and/or nuclear accumulation of ERK was not sufficient for, but may be necessary for, induction of a full neuronal differentiation in SH-SY5Y cells. For a differentiation response to take place, additional signals induced by serum or growth factors activating protein-tyrosine kinase receptors are required. Several authors have demonstrated formation of neurites in PC12 cells transfected with active Ras [6], Raf-1 [8], MEK [9, 10], or ERK [10, 11] and reported that inhibition of MEK by PD98059 prevents NGF-dependent neurite formation [48], but contradictory results have also been reported with respect to the differentiation promoting capacity of ERK [49, 50]. A direct comparison between our findings and data on PC12 cells expressing active and inactive mutants of MEK is complicated by the presence of serum factors in the PC12 study [9], but the existing data suggest distinct roles of ERK in differentiating PC12 and SH-SY5Y cells. Expression of active Ras induces neurite formation in SH-SY5Y/wt cells (Olsson and Nånberg, to be published) and we are in the progress of identifying the downstream effector pathway(s) for this, as well as the functional role of

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ERK for neuritogenesis and other differentiationlinked events in neuroblastoma cells. At present we conclude that nuclear accumulation of active ERK may well be necessary for induction of a sympathetic differentiation response in SH-SY5Y cells, but concomitant activation of additional signals is also required. What are the targets that constitute a functional coupling between a sustained ERK activity and the accompanying nuclear accumulation of active kinase that contribute to a differentiation response? ERK phosphorylates a number of substrates with very different cellular functions. It activates the cytoplasmic target molecule phospholipase A 2 by phosphorylation on serine 505 and this leads to release of the important metabolite arachidonic acid in agonist-stimulated cells [51]. Another important cytosolic target molecule is RSK [52]. In the nucleus, ERK phosphorylates the transcription factor c-Myc, generating a specific ubiquitination site and thus promoting degradation of the Myc protein [53, 54]. This makes N-Myc a potential target for ERK-induced growth inhibitory effects in neuroblastoma cells. A better characterized nuclear target is the transcription factor Elk-1 that can be phosphorylated and activated by the MAP kinases ERK, p38, and JNK (c-Jun amino-terminal kinase) in response to a large selection of cellular stimuli [55–57]. It was therefore an interesting observation that both NGF- and TPA-induced SRE reporter activity was completely blocked by the specific MEK inhibitor PD98059, a result that indicated that signaling via MEK and ERK is the predominant mechanism for SRE-driven transcription in the differentiating SH-SY5Y/TrkA cells. Raf and ERK-dependent activation of Elk-1/SRF induces transactivation of the genes encoding c-fos and the CDK inhibitor p21 cip1 [58]. Regulation of both these genes is of great interest in neuroblastoma cells. TPA in the presence of serum causes a biphasic prolonged activation of c-fos and c-jun in SH-SY5Y cells [59] with a peak around 1 h and a second weaker response between 8 and 96 h. This has so far only been seen in cultures of SH-SY5Y undergoing sympathetic differentiation and not in growth-stimulated cultures. p21 cip1 mediates Ras- and Raf-induced cell cycle arrest [58] and one report has suggested that expression of p21 cip1 in neuroblastoma cells may promote NGF sensitivity and differentiation by reducing cell cycle activity and is necessary for survival of differentiating cells [60]. We are currently investigating the role of nuclear ERK activity for transcriptional activation of c-fos and other neuronal marker genes in neuroblastoma cells. The cellular response to increased ERK activity is dependent on the amplitude and duration of the activating signal and induction of inactivating specific phosphatases (MKPs) [61– 64], but equally important is the subcellular localization of active ERK determining the interaction with its specific target substrates.

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Our here reported finding that PKC can interfere with ERK-dependent signaling by regulation of the amount of nuclear ERK is therefore potentially very important. It also suggests that PKC can not only feed into the ERK pathway by activation of Raf [65– 69] and possibly even MEK [70, 71] but also regulate the nuclear distribution of active ERK, a finding that adds even more complexity to the ERK pathway. Although SH-SY5Y/ TrkA cells express at least PKC-␣, -␤, -␦, -⑀, -␨, and -␮, particularly PKC-␦ and PKC-⑀ are strong candidates mediating the reported PKC dependency for gene induction and neurite formation in differentiating SHSY5Y/wt and SH-SY5Y/TrkA cells [29 –31]. The PKC inhibitors used in the present report have high affinity for the classic forms of PKC (␣, ␤, and ␥) and inhibit PKC-⑀ with IC 50 values in the 100 nM range. GF109203X inhibits PKC-␦ (IC 50 ⫽ 210 nM) while Ro32-0432 has no reported effect on that isoform. GF109203X also has a low affinity for PKC-␨ (IC 50 ⫽ 5.8 ␮M). The fact that both GF109203X and Ro-320432 efficiently blocked NGF-induced nuclear accumulation of active ERK (Figs. 3 and 7A and data not shown) as well as SRE-dependent transcription (Fig. 5) suggested that either classic isoforms and/or PKC-⑀ was important for NGF-induced nuclear accumulation of ERK in SH-SY5Y/TrkA cells. More conclusive data on the role of different PKC-isoforms will require further analysis with, e.g., mutated PKC and antisense constructs. PKC is known to have regulatory effects on the actin cytoskeleton and the microtubuli [72], a structure to which ERK associates [73]. A simple explanation for our presented data would be that inhibition of both basal and NGF-dependent PKC activity leads to perturbed subcellular distribution of ERK via effects on the microtubuli. But the results showed that ERK was enriched in the perinuclear region in NGF-stimulated cells, compared with control cells in which ERK was evenly distributed in the cytoplasm and in the short neurites (Fig. 7). This speaks against microtubuli-related effects as the only explanation for PKC-dependent nuclear translocation of ERK. The mechanisms determining nuclear translocation and accumulation of ERK are unclear. If nuclear translocation of active ERK was the result of increased amounts of active ERK in the cytoplasm exclusively, one would expect an initial transient increase in nuclear ERK after addition of, e.g., PDGF-BB to responsive cells. This is the case in NIH3T3 fibroblasts [74] but as reported here, not in SH-SY5Y/TrkA cells, although PDGF-BB induced a potent transient activation of total ERK. A similar situation has been described for PC12 cells stimulated with EGF [16]. Increased amounts of phosphorylated active ERK in the nucleus may be the result of increased nuclear import or attenuated nuclear export of ERK or sup-

pressed phosphatase activity. A recent paper has described how ERK2 forms homodimers in a phosphorylation-dependent way and that these dimers accumulate in the nucleus [75]. In preliminary experiments, we have failed to detect any effects of inhibition of PKC on complex formation of ERK with other proteins or on migration of ERK at higher molecular weights in lysates prepared and analyzed under native conditions. Possible mechanisms whereby PKC can interfere with nuclear accumulation of ERK would be by facilitating nuclear import, by stabilizing ERK dimers, by affecting MEK-ERK complex formation, and by counteracting dephosphorylation of ERK or its export from the nucleus. This will hopefully be elucidated in the future. PKC-dependent effects both in the cytosol and in the nucleus must be taken into account since several isoforms of PKC have been shown to translocate into the nucleus [76 – 80]. Although the numerical values obtained from the experimental protocols cannot be directly compared, it was striking that TPA consistently was the more potent activator of nuclear ERK signaling compared with NGF. Our findings that TPA stimulated both more active ERK to accumulate in the nucleus (Fig. 7), was a stronger inducer of PD98059-sensitive transactivation of the SRE reporter gene compared to NGF (Figs. 4 and 5), and gave a stronger induction of c-fos transcript (Fig. 8), although TPA and NGF were equally potent stimulating total ERK activity (Fig. 6), may be interpreted so that concomitant strong activation of PKC and ERK promoted more ERK to accumulate within the nucleus. If stimulation of PKC activity is required for all full nuclear response to ERK, a potent activator of ERK with very limited or no stimulatory capacity with respect to cellular PKC activity would have lesser effects on ERK-dependent transcription via Elk-1/SRE but promote signaling to cytosolic ERK target molecules. In agreement with this hypothesis, NGF had a much weaker effect compared with 16 nM TPA on PKC activity measured as in vivo phosphorylation of MARCKS and translocation of PKC-␣, -␦, and -⑀ to the particulate fraction in stimulated SH-SY5Y/TrkA cells (So¨derholm, Olsson, Lavenius, Ro¨nnstrand, and Nånberg, manuscript in preparation.). Thus in SHSY5Y cells a potent and prolonged ERK activation seems to be required for nuclear accumulation of ERK, but additional signals such as PKC also determine the amount of nuclear ERK. The finding that inhibition of PKC reduced NGF-induced expression of c-fos with 60% implies that a PKC-dependent mechanism regulating nuclear activity of ERK can be an important factor during differentiation of these neuroblastoma cells and expression of AP1-induced genes required for differentiation. Future studies will shed light on the molecular mechanisms behind PKC-dependent nuclear ERK activity.

ACTIVATION OF ERK IN NEUROBLASTOMA CELLS We thank Dr. Lars Ro¨nnstrand for generously providing the antiERK-2 antibody and Dr. Joan Massague´ for the p3TD-lux plasmid. We would also like to thank Ulrika Larsson for helpful advice concerning the phospho-ERK stainings and Kenneth Wester for access to and help with the quantification software. We are grateful to Dr. Pa¨r Gerwins for valuable comments on the manuscript. This study was supported by grants from The Swedish Cancer Society, The Swedish Childrens Cancer Society, and Go¨ran Gustavssons stiftelse.

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