Role of the subcellular localization of ALK tyrosine kinase domain in neuronal differentiation of PC12 cells

Jean Y. Gouzi, Christel Moog-Lutz, Marc Vigny* and Nicole Brunet-de Carvalho

INSERM, U706, Institut du Fer à Moulin, 17 rue du Fer à Moulin; and UPMC, 4 Place Jussein, Paris, F-75005, France

* Author for correspondence (e-mail: vigny{at}fer-a-moulin.inserm.fr)

Accepted 15 September 2005


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Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase essentially and transiently expressed in specific areas of the developing central and peripheral nervous systems. We previously demonstrated that a membrane-bound and constitutively active form of the ALK protein tyrosine kinase (PTK) domain induced the neuron-like differentiation of PC12 cells through specific activation of the mitogen-activated protein kinase (MAP kinase) pathway. Its PTK domain had been originally identified in a nucleo-cytosolic and constitutively active transforming protein, NPM-ALK. Downstream targets involved in oncogenic proliferation and survival processes have been proposed to include phospholipase C{gamma} (PLC{gamma}), phosphoinositide 3-kinase (PI 3-kinase)/AKT, STAT 3/5 and Src. We therefore postulated that activation of specific signaling pathways leading to differentiation or proliferation can be differently controlled depending on the subcellular localization of ALK PTK domain. To increase knowledge of its physiological role in the nervous system, we focused in the present study on the influence of its subcellular localization on neuronal differentiation. To achieve this goal, we characterized biological responses and transduction pathways in PC12 cells elicited by various constructs encoding membrane-bound (through transmembrane or myristyl sequences) or cytosolic ALK-derived proteins. In order to control the activation of their PTK domain, we used an inducible dimerization system. Here, we demonstrate that membrane attachment of the ALK PTK domain, in PC12 cells, is crucial for initiation of neurite outgrowth and proliferation arrest through a decrease of DNA synthesis. Furthermore, we show that this differentiation process relies on specific and sustained activation of ERK 1/2 proteins. By contrast, activation of the cytosolic form of this domain fails to induce MAP kinase activation and cell differentiation but promotes a PI 3-kinase/AKT-dependant PC12 cell proliferation. These data indicate that subcellular localization of the ALK PTK domain was a determinant for the control and specificity of downstream transduction cascades and was crucial for deciding the fate to which the neuronal cell will be committed.

Key words: ALK, FK506-binding protein, PC12 cells, MAP kinase, Neurite outgrowth, Cell proliferation


    Introduction
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 Introduction
 Materials and Methods
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During development of the nervous system, a wide range of cellular processes such as proliferation, survival, differentiation, synaptogenesis and maturation are driven by multiple extracellular growth factors known as neurotrophic factors. Their effects are usually mediated by membrane receptors belonging to the receptor with tyrosine kinase activity (RTK) family (Blume-Jensen and Hunter, 2001Go; Manning et al., 2002Go). Generally, binding of the ligand to the RTK extracellular region leads to receptor dimerization followed by activation of its intracellular PTK domain by conformational changes of the catalytic loop associated with phosphorylation of key tyrosine residues (Schlessinger, 2000Go). Phosphorylated tyrosine residues can bind different adaptor substrate proteins that subsequently trigger various signaling cascades such as those of MAP kinase, PI 3-kinase or PLC{gamma}. The nature and degree of activation of these pathways largely depends on the involved RTK and on the cell type in which it is expressed. This variability could explain the broad range of subsequent neural cellular responses (Bibel and Barde, 2000Go; Huang and Reichardt, 2001Go; Huang and Reichardt, 2003Go; Kaplan and Miller, 2000Go; Schlessinger, 2000Go). The establishment of the rat pheochromocytoma cell line PC12 has considerably facilitated the study of action of some neurotrophic factors (Greene and Tischler, 1976Go). These cells can either differentiate from chromaffin-like cells to sympathetic neuron-like cells when treated with nerve growth factor (NGF) or proliferate upon epidermal growth factor (EGF) stimulation (Huff et al., 1981Go), although the corresponding receptors all belong to the RTK family.

Anaplastic lymphoma kinase (ALK) is a novel RTK belonging to the insulin receptor subfamily. ALK has been characterized in humans, mouse and, more recently, in Drosophila (Iwahara et al., 1997Go; Loren et al., 2001Go; Morris et al., 1997Go). ALK possesses a classical structure shared with other RTKs: a large extracellular domain, a single transmembrane domain and an intracellular domain containing the tyrosine kinase catalytic site. Recently, pleiotrophin (PTN) and midkine (MK) have been proposed as potential ligands of ALK (Stoica et al., 2001Go; Stoica et al., 2002Go), although several studies do not confirm this hypothesis (Dirks et al., 2002Go; Miyake et al., 2002Go; Moog-Lutz et al., 2005Go; Motegi et al., 2004Go). However, a recent report suggests that a truncated form of PTN (and not the full-length PTN) could activate ALK (Lu et al., 2005Go). In addition, the protein Jelly Belly (Jeb) has been identified as the ligand of Drosophila ALK (Englund et al., 2003Go; Lee et al., 2003Go) and Jeb is distinct from the Drosophila homologs of PTN and MK, Miple 1 and 2 (Lee et al., 2003Go). Finally, no obvious vertebrate homolog has been identified for Jeb in the sequence database.

In situ hybridization and northern blot studies in mammals revealed that alk transcripts were essentially and transiently expressed in specific regions of the developing central and peripheral nervous systems (Iwahara et al., 1997Go; Morris et al., 1997Go). In Drosophila, regulated DAlk mRNA and DAlk protein expressions were also described in the developing brain and ventral nerve cord (Loren et al., 2001Go). This distribution strongly suggested that ALK could play an important role in the normal development and function of the nervous system. In agreement with this hypothesis, we previously demonstrated that a membrane-bound and constitutively active form of the ALK PTK domain (a Fc-ALK chimera in which a Fc fragment of mouse IgG is substituted with the ALK extracellular domain) induced the neuron-like differentiation of PC12 cells (Souttou et al., 2001Go). Analysis of the signaling pathways involved in this process pointed to an essential role of the MAP kinase cascade. Recently, we and others (Moog-Lutz et al., 2005Go; Motegi et al., 2004Go) reported that ALK activation triggered by specific monoclonal antibodies promoted neurite outgrowth of neuronal cell lines through activation of this pathway.

The intracellular domain of ALK had been previously identified in some anaplastic large cell lymphomas (ALCL) resulting from abnormal chromosomal translocations that lead to the expression of oncogenic fusion kinases (reviewed by Pulford et al., 2004Go). The most characteristic is the nucleo-cytosolic NPM-ALK protein, which contains the complete ALK intracellular domain fused to the N-terminal part of nucleophosmin [NPM (Morris et al., 1994Go)]. NPM oligomerization leads to the constitutive activation of the ALK kinase domain (Bischof et al., 1997Go) and PLC{gamma}, PI 3-kinase/AKT, STAT 3/5 and Src have been proposed as downstream targets of NPM-ALK involved in oncogenic proliferation and survival processes. It had also been reported that cytoplasmic localization, but not nuclear localization, of NPM-ALK was required for its transforming activity (Bai et al., 1998Go; Bai et al., 2000Go; Bischof et al., 1997Go; Fujimoto et al., 1996Go; Nieborowska-Skorska et al., 2001Go; Slupianek et al., 2001Go; Zamo et al., 2002Go).

On the basis of the above-mentioned previous results, we postulated that activation of specific signaling pathways leading to differentiation or proliferation can be differently controlled depending on the subcellular localization of the ALK kinase domain. It is noteworthy that subcellular localization of a PTK domain as a factor that determines the signal transduction specificity has been poorly documented so far and could have a profound impact on cellular processes (Schlessinger, 2000Go). Here, taking account of the cellular expression of ALK during development, we particularly studied the role of the subcellular localization of its PTK domain in neuronal differentiation. To achieve this goal, we expressed different constructs encoding membrane-bound or cytosolic ALK-derived proteins in PC12 cells. In order to control the activation of their intracellular PTK domain, we used an inducible dimerization system. Here, we show that membrane attachment of the ALK PTK domain is crucial for initiation of neurite outgrowth and proliferation arrest of P12 cells through a decrease of DNA synthesis. Furthermore, we show that this differentiation process relies on specific and sustained activation of the MAP kinase pathway. By contrast, activation of the cytosolic form of this domain fails to induce MAP kinase activation and cell differentiation but promotes a PI 3-kinase/AKT-dependent PC12 cell proliferation.


    Materials and Methods
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Plasmid constructions
The pC4M-Fv2E plasmid (ARIAD Pharmaceuticals) encodes a protein that we named myr-FH (Fig. 1B) composed of an N-terminal myristoylation signal, two tandem Fv domains (derived from the FKBP domain) followed by a C-terminal hemagglutinin (HA) tag. The cDNA encoding the ALK intracellular domain (1058-1620 aa) was cloned by PCR amplification of the human ALK cDNA plasmid (Souttou et al., 2001Go) using the primers forward 5'-GGCCTCTAGAGTGTACCGCCGGAAGCACCAG-3' and reverse 5'-CCGGTCTAGAGGGCCCAGGCTGGTTCATGCT-3', then sequenced and subcloned into the XbaI site of the pC4M-Fv2E vector to make a construct expressing the myrIA-FH fusion protein (Fig. 1B). To construct the pC4-Fv2E vector, we inserted the XbaI-XhoI Fv fragment of the pC4M-Fv2E plasmid into the XbaI site of the pC4-Fv1E plasmid (ARIAD Pharmaceuticals). The cDNA coding for the ALK intracellular domain was cloned into the XbaI site of this vector to make a construct expressing the cytoIA-FH fusion protein (Fig. 1B). We performed directed mutagenesis on the ALK cDNA to create a SmaI site in order to remove the extracellular domain of the protein (42-1026 aa) and a XbaI site (replacing the stop codon). This cDNA was sequenced and inserted into the EcoRI-XbaI sites of the pC4M-Fv2E vector, yielding expression of the tmbIA-FH protein (Fig. 1B). By an exchange of KpnI fragments between this mutated cDNA and the wild-type ALK cDNA (821-5619 bp), we constructed the vector expressing the ALK-FH protein (Fig. 1B). A construct encoding a kinase-defective form of the full-length human ALK, in which the invariant lysine residue located in the ATP-binding portion of the catalytic domain was changed to arginine, was previously inserted in the mammalian expression vector pcDNA 3.1 (Souttou et al., 2001Go). From this construct, we generated the different constructs encoding the kinase-defective forms of ALK-FH, myrIA-FH and cytoIA-FH proteins. In addition, inserts encoding cytoIA-FH and myrIA-FH were subcloned into the pCBC vector (Moog-Lutz et al., 2005Go) using directed mutagenesis and constructs were verified by DNA sequencing.



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Fig. 1. ALK-FH-derived proteins and the inducible dimerization system. (A) Schematic representation of the induced dimerization of ALK-FH protein by the synthetic lipophylic and bifunctional dimerizer AP20187. (B) Schematic representation of the full-length ALK-FH protein and its intracellular domain as membrane-bound (transmembrane, tmbIA-FH; or myristoylated, myrIA-FH) or cytosolic (cytoIA-FH) proteins. ECD, extracellular domain; TM, transmembrane domain; PTK, protein tyrosine kinase domain; 2x FKBPv, tandem copies of modified form of the intracellular FK506-binding protein; HA, hemagglutinin tag; myr, myristoylation sequence from Src.

 
Cell culture and transfection
Rat pheochromocytoma (PC12) cells (kindly provided by T. Galli, Institut Jacques Monod, Paris, France) were grown in a complete medium (RPMI 1640; Gibco Life technologies) supplemented with 10% horse serum (HS) and 5% fetal calf serum (FCS) at 37°C in an atmosphere containing 5% CO2. Cells were cultured in flasks or plastic dishes coated with collagen (1 µg/cm2). PC12 cells were electroporated as previously described (Moog-Lutz et al., 2005Go) and immediately transferred to fresh complete medium for 1 day. Then, unless otherwise specified, cells were serum starved in a low-serum medium (1% HS without FCS) for 16 hours before the addition of the dimerizer. For experiments with various kinase inhibitors, cells were pre-treated with the agents for 30-60 minutes before the addition of the dimerizer.

Human 293 embryonic kidney (HEK 293) cells were cultivated and transfected using the calcium phosphate precipitation method. Cells stably expressing cytoIA-FH and myrIA-FH were established using pCBC vectors as previously described (Moog-Lutz et al., 2005Go).

Reagents and antibodies
The dimerizer (AP20187) was obtained from ARIAD Pharmaceuticals. U0126 was purchased from Cell Signaling Technology. LY294002 and wortmannin were from Calbiochem. 5-Bromodeoxyuridine (BrdU) and mouse anti-BrdU FITC-conjugated antibody were from Roche Diagnostics. Antibodies and their sources were as follows: mouse monoclonal anti-HA-tag antibody (12CA5; generous gift of Y. Frobert, CEA, Saclay, France); rat anti-HA-tag antibody (3F10; Roche Diagnostics); mouse anti-PY antibody (4G10) and rabbit anti-ERK antibody (Upstate Biotechnology); mouse anti-P-ERK antibody (Sigma); rabbit anti-AKT phosphoserine-473 antibody and anti-AKT antibody (Cell Signaling Technology); mouse anti-transferrin receptor antibody (H 68.4; Zymed); and rabbit anti-stathmin antibody (STC3; kindly provided by A. Sobel, INSERM U706, Paris, France).

Immunocytochemistry and confocal microscopy
PC12 cells transiently transfected with the different constructs were grown on collagen-coated plastic dishes for 2 days in a complete medium, fixed for 15 minutes at room temperature with pre-warmed 2% formaldhehyde/30 mM sucrose and washed three times with PBS. Cells were then permeabilized in 0.5% PBS-Triton X-100 for 5 minutes and washed with 0.1 M PBS-glycine for 15 minutes. After 1 hour of blocking in PBS containing 1.5% BSA, cells were incubated in the same buffer with monoclonal mouse anti-HA-tag (12CA5) antibody (0.9 µg/ml) to visualize cells expressing ALK-FH-derived proteins. The cells were then washed five times with PBS before and after incubation with anti-mouse IgG Alexa Fluor 488-conjugated secondary antibody (Molecular Probes). In order to visualize nuclei, cells were incubated with 25 nM Sytox Orange nucleic acid stain (Molecular Probes) for 2 minutes and washed with water. The cells were then mounted in Mowiol 4-88 (Calbiochem). Confocal laser microscopy was performed using a TCS SP2 confocal microscope (Leica). Images were assembled using Adobe Photoshop software.

Subcellular fractionation
Electroporated PC12 cells grown for two days were rapidly washed with cold PBS and resuspended in 200 µl hypotonic buffer [10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM EGTA, 25 mM ß-glycerol phosphate, 5 mM sodium fluoride, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate and protease inhibitor mixture `complete' (Roche)]. Cells were disrupted by three cycles of freezing followed by thawing, after each of which a 25-gauge needle was used to disrupt the material further. Whole-cell lysates were spun at 800 g for 10 minutes at 4°C to remove cell nuclei and crude debris, and the supernatants were submitted to ultracentrifugation at 100,000 g for 45 minutes at 4°C in a TLA 100.1 rotor (Optima Beckman) to generate supernatant (cytosol) and pellet (membrane) fractions. Membrane fractions were then resuspended in a RIPA buffer and clarified by centrifugation at 21,000 g for 10 minutes at 4°C.

Neurite outgrowth assay
Electroporated PC12 cells were treated at day 2 with the dimerizer. At day 4, cells were fixed and processed for immunofluorescence as described above. Cells were first incubated with monoclonal mouse anti-HA-tag (12CA5) antibody (0.9 µg/ml) and then with anti-mouse IgG FITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) to visualize cells expressing ALK-FH-derived proteins. Then cells were mounted in Mowiol 4-88 supplemented with Hoechst 33258 nucleic acid stain (0.5 µg/ml, Molecular Probes) in order to visualize nuclei. Conventional fluorescence microscopy was performed on a Leica microscope equipped with a MicroMax CCD camera (Princeton Instruments). Images were assembled using Adobe Photoshop software. The Metamorph software was used (Roper Scientific) for quantification, and 100 transfected cells were counted and cells bearing neurites longer than twice the diameter of the cell body were scored as differentiated. The experiments were performed in triplicate.

BrdU-incorporation assay
Electroporated PC12 cells were treated with the dimerizer for only 12 hours; 6 hours after dimerizer application, cells were incubated with BrdU (10 µM) for 6 hours, then fixed and processed for immunofluorescence as described above. After DNA denaturation with 2N HCl for 30 minutes and neutralization with a borate buffer (0.1 M pH 8.5; three washes), cells were first incubated with mouse anti-BrdU FITC-conjugated (2 µg/ml) and rat anti-HA-tag (3F10; 0.5 µg/ml) antibodies and then with anti-rat IgG TRITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) to visualize cells expressing ALK-FH-derived proteins and BrdU-positive cells. The cells were then mounted and conventional fluorescence microscopy and quantification were performed as described above. 100 transfected cells were counted and cells with positive BrdU staining were scored as having undergone DNA replication during the time of labeling. The experiments were performed in triplicate.

Cell lysates and immunoblotting analysis
Cell extracts were prepared by lysing the cells in a RIPA buffer [10 mM NaPi buffer, pH 7.8, 60 mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, 10% glycerol, 25 mM ß-glycerol phosphate, 50 mM sodium fluoride, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate and protease inhibitor mixture `complete' (Roche)] and analysed by immunoblotting as previously described (Moog-Lutz et al., 2005Go). Bound proteins were visualized using the ECL system (Amersham Bioscience) or the Odyssey Imaging System (LI-COR bioscience). This latter was also used for quantification.


    Results
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 Materials and Methods
 Results
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 References
 
Constructs encoding ALK-derived proteins
We used an inducible dimerization system recently applied to the study of RTKs such as the platelet-derived growth factor (PDGF) and insulin receptors (Yang et al., 1998Go) or the hepatocyte growth factor (HGF) receptor (Boccaccio et al., 2002Go). This method is based on the use of a small, synthetic, lipophylic and bifunctional ligand known as the dimerizer (AP20187). The dimerizer binds in trans with high affinity to a modified form of the intracellular FK506-binding protein (FKBPv) and has no ability to bind endogenous FKBP. It allows the dimerization of intracellular functional domains of various proteins that have been fused to the FKBP modules (Clackson et al., 1998Go) as represented in Fig. 1A. Thus, we engineered several plasmid constructs encoding chimeric proteins in which the C-terminus of the human ALK intracellular domain was fused to two tandem copies of FKBPv (Fig. 1B) and a HA tag. These constructs allowed the expression of the full-length receptor (ALK-FH), of the entire ALK intracellular domain linked to the membrane either through the ALK transmembrane sequence (tmbIA-FH) or the Src myristoyl sequence (myrIA-FH) and finally of the complete ALK intracellular domain without any membrane attachment sequence (cytoIA-FH). Several control constructs were also generated. One (myr-FH) encodes the two FKBPv modules bound to the membrane after deletion of the entire ALK intracellular domain from the myrIA-FH construct. The other corresponds to kinase-defective mutants of the various ALK constructs (schematic representations not shown) in which the invariant lysine residue located in the ATP-binding site of the catalytic domain was changed to arginine (see Materials and Methods). This point mutation has been previously shown to inhibit completely the transforming capability of the NPM-ALK oncogenic protein (Bischof et al., 1997Go) and the PC12 neurite outgrowth induced by the expression of the constitutive active form of the ALK tyrosine kinase domain (Souttou et al., 2001Go).

Induced kinase activation of full-length ALK resulted in PC12 cell neurite extension and ERK 1/2 activation
To test whether the inducible dimerization system we used was functional with the full-length ALK, we first transfected PC12 cells with the construct encoding ALK-FH and assayed for neurite outgrowth in the presence of increasing doses of dimerizer (0-1 µM). As shown in Fig. 2A, the dimerizer increased the proportion of cells expressing ALK-FH extending neurites in a dose-dependent manner from a basal level of about 20% in the absence of dimerizer to a maximum level of about 50% obtained with the dimerizer at 20 nM. This optimal concentration was used for the subsequent experiments. Cells transfected with the ALK-FH kinase-defective mutant (dALK-FH; Fig. 2B) exhibited a basal level of only 5% of transfected cells bearing neurites, significantly lower compared with the 15-20% obtained with ALK-FH-expressing cells and similar to that observed with control cells transfected with the empty vector (not shown). In this case, addition of the dimerizer did not induce any differentiation. These data indicated that both the basal and induced neurite outgrowth described specifically relied on the kinase activity of the ALK-FH protein and furthermore indicated that the presence of the FKBPv modules and/or the dimerizer did not interfere with neurite extension.



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Fig. 2. Induced kinase activation of the full-length receptor ALK results in neurite extension and ERK 1/2 activation. (A) Dimerizer dose-dependent effect on neurite outgrowth induced by the activation of ALK-FH protein. PC12 cells were electroporated with the ALK-FH construct (25 µg), cultured overnight in a 1% horse serum medium and treated without or with increasing concentrations of dimerizer as indicated for 2 days. Then immunofluorescence assay was performed using the anti-HA-tag antibody (12CA5); 100 transfected cells were counted and cells bearing neurites longer than twice the diameter of the cell body were scored as differentiated. The experiment was performed in triplicate and values are expressed as the mean ± s.e.m. (%). (B) Neurite outgrowth induced by ALK-FH dimerization is a result of its own kinase activity. PC12 cells were electroporated with the indicated constructs (25 µg) and then submitted to the same protocol as described in (A). Cells were treated with a dimerizer concentration of 20 nM. The experiment was performed in triplicate and values are expressed as the mean ± s.e.m. (%). Time course of (C) ALK-FH and (D) ERK 1/2 phosphorylation following dimerizer treatment. ALK-FH-transfected PC12 cells were incubated with the dimerizer (20 nM) for the indicated periods. As controls, non-transfected cells (Mock) or cells transfected with the dALK-FH mutant were treated with the dimerizer during 30 minutes. Cells were lysed in a RIPA buffer as described in the Materials and Methods and the lysates (10 µg) were submitted to western blot analysis using the anti-PY (4G10) or the anti-P-ERK 1/2 antibody (upper panels) and then reprobed with the indicated antibodies (lower panels). The P-ERK 2 densitometry quantification was performed using the Odyssey Imaging System on three independent experiments. Statistical analyses were carried out by Student's t-test (***P<0.005). Asterisk in C indicates a 160 kDa protein that was not revealed by the anti-HA-tag antibody and therefore not related to ALK.

 
To demonstrate that ALK-FH was actually activated we directly assayed its phosphorylation level by western blot analysis of whole-cell lysates after various periods of dimerizer treatment (Fig. 2C). The anti-HA-tag antibody revealed two major species of ALK-FH as previously reported in rat and mouse brain (Iwahara et al., 1997Go; Morris et al., 1997Go) and in HEK 293 or NIH3T3 cells stably transfected with the ALK receptor (Moog-Lutz et al., 2005Go; Motegi et al., 2004Go). In the absence of dimerizer, both species of ALK-FH (220 kDa and 140 kDa) exhibited a basal level of phosphorylation that was strongly increased upon dimerizer treatment to a level that reached a maximum within 5 minutes and then remained constant for at least 30 minutes. No ALK phosphorylation was observed in cells expressing the kinase-defective mutant (dALK-FH), thus confirming that both basal and induced phosphorylation were a result of its intrinsic kinase activity. We then assayed for ERK 1/2 phosphorylation (Fig. 2D) since MAP kinase activation had been previously shown to be essential in PC12 cell differentiation triggered by the expression of the constitutively active form of ALK (Souttou et al., 2001Go). Immunoblotting with anti-phospho-ERK 1/2 antibodies revealed that the ERK 1/2 proteins were activated in the presence of dimerizer, reaching a maximum phosphorylation within 10 minutes. We further showed that their phosphorylation was sustained in a long-lasting manner since it remained elevated for at least 1 hour and even after 24 hours (data not shown). In addition, no ERK 1/2 activation was detected in cells expressing the kinase-defective mutant dALK-FH, which demonstrates that basal and induced ERK activations were dependent on ALK kinase activity. Altogether, these results were in agreement with those obtained previously with the constitutively active Fc-ALK chimera, clearly establishing that the ALK-FH protein dimerized by its intracellular part was functional, and therefore validating the dimerization system we used.

Normalized expression and subcellular localization of ALK-FH-derived proteins
Since the major aim of this study was to analyze the role of the subcellular localization of ALK PTK domain in differentiation and/or proliferation of PC12 cells, we normalized the expression of the different constructs and we analyzed the localization of the different ALK-FH-derived proteins. Thus, PC12 cells were initially transfected by electroporation with equal amounts of the different ALK-derived constructs (25 µg DNA for 5x106 cells). Immunoblotting analysis using the anti-HA-tag antibody revealed that ALK-derived proteins display similar apparent molecular weight and that their expression levels were highly heterogeneous (data not shown). myrIA-FH protein displayed the highest expression level whereas, in comparison, tmbIA-FH was weakly expressed. On the basis of the expression of the tmbIA-FH protein, we performed cell transfections with a range of decreasing concentrations of DNA constructs encoding myrIA-FH and cytoIA-FH proteins, and established those required to obtain approximately identical protein expression levels as shown in Fig. 3A.



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Fig. 3. Cellular expression of ALK-FH-derived proteins: normalization of the expression levels and subcellular localization. (A) Western blot analysis of the expression of ALK-FH-derived proteins after normalization of their expression levels. PC12 cells were electroporated with the indicated quantities of the DNA constructs, in order to obtain comparable levels of protein expression. Cells were lysed after two days in a RIPA buffer as described in the Materials and Methods, and the lysates (10 µg) were submitted to a western blot analysis using the anti-HA-tag (12CA5) monoclonal antibody. (B) Immunofluorescence confocal microscopy analysis of the subcellular localization of ALK-FH-derived proteins revealed with the anti-HA-tag antibody (green) in permeabilized PC12 cells (representative fields are shown). Cell nuclei were labeled with the Sytox Orange nucleic acid stain (red). Bar, 10 µm.

 
The second step was to verify that all these proteins were correctly located in the cell by performing confocal microscopy analysis (Fig. 3B). tmbIA-FH and myrIA-FH proteins exhibited a potent enrichment at the periphery of the transfected cells, suggesting a plasma membrane localization. A much less intense intracellular staining could also be observed, probably corresponding to intracellular membrane compartments. By contrast, the cytoIA-FH protein showed essentially a uniform distribution throughout the cytoplasm and within the cell nucleus. Subcellular fractionation mainly confirmed these results and also revealed that, compared with the pure cytosolic marker stathmin, a minor but significant part of the cytoIA-FH protein was associated with the membrane fraction (Fig. S1, supplementary material).

Membrane attachment of ALK intracellular domain is required for the induction of neurite outgrowth of PC12 cells and MAP kinase activation
We investigated the phenotypic effect induced after two days of dimerizer treatment in cells expressing ALK-FH-derived proteins (Fig. 4A). In the absence of dimerizer, control cells transfected with the myr-FH construct showed essentially a round shape and undifferentiated phenotype. By contrast, only cells transfected with the constructs encoding the membrane-bound proteins (tmbIA-FH and myrIA-FH) exhibited a significant level of basal differentiation (25% and 32% respectively, Fig. 4B), displaying short and thin neurites (Fig. 4A). However, when treated with the dimerizer, the percentage of these cells extending neurites was greatly increased (55% and 75%, respectively) and they displayed a much larger and spread cell body. Moreover, they harbored thick neurite extensions (reaching several folds the cell body size), which could be visible as early as the 12th hour of dimerizer treatment. By contrast, even in the presence of dimerizer, fewer than 10% of the cells expressing the cytosolic form of the ALK intracellular domain (cytoIA-FH) extend neurites (Fig. 4B). Thus, they essentially retained their undifferentiated phenotype. This very weak differentiation level probably resulted from the activation of the tiny cytoplasmic protein fraction associated with the membrane (Fig. S4, supplementary material). Control cells expressing membrane-bound FKBPv modules alone did not trigger any neurite outgrowth.



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Fig. 4. Membrane attachment of the ALK intracellular domain is required to induce neurite extension of PC12 cells and MAP kinase activation. (A) PC12 cells were electroporated with normalized quantities of the indicated constructs, cultured overnight in a 1% horse serum medium and treated with the dimerizer (20 nM) for 2 days. Then cells were fixed, permeabilized and immunofluorescence assay was performed. Cells expressing the ALK-FH-derived proteins were labeled with the anti-HA-tag antibody (12CA5) and cell nuclei were colored with the nucleic acid stain Hoechst 33258. Bar, 100 µm. (B) Quantification of the effect of the induced activation of the different proteins on neurite outgrowth, as indicated in the Materials and Methods. The experiment was performed in triplicate and values are expressed as the mean ± s.e.m. (%). (C) Time course of ERK 1/2 phosphorylation following dimerizer treatment. PC12 cells expressing the indicated proteins were cultured overnight in a 1% horse serum medium and then incubated with the dimerizer (20 nM) for the indicated periods. Cells were lysed in RIPA buffer as described in the Materials and Methods and the lysates (10 µg) were submitted to western blot analysis using the anti-HA-tag or the anti-P-ERK antibody and then reprobed with the anti-PY (4G10) or the anti-ERK antibody.

 
As we previously showed that induced activation of the transmembrane full-length receptor ALK-FH led to PC12 cell differentiation and sustained activation of ERK 1/2 proteins (Fig. 2D), we investigated how activation of the membrane-bound or cytosolic ALK-FH-derived proteins could induce the activation of ERK proteins. We focused our comparative study on the two most closely related proteins: myrIA-FH and cytoIA-FH. Immunoblot analysis of whole-cell extracts faintly revealed a dimerizer-induced phosphorylation of myrIA-FH and cytoIA-FH (Fig. 4C). Taking into account the strong activation of the downstream pathways induced by the dimerizer (see below), one could have expected a stronger increase in the level of tyrosine phosphorylation of ALK-FH-derived proteins as observed for the full-length receptor (Fig. 2C). The use of stable HEK 293 clones expressing the same ALK-derived proteins at a much lower level allowed us to detect a stronger increase in their dimerizer-induced tyrosine phosphorylation level, which was revealed in immunoprecipitation experiments (Fig. S2A, supplementary material). Similar HA immunoprecipitates were also performed using PC12 cell lysates. We did not detect a stronger change in tyrosine phosphorylation in the immunoprecipitates than in the whole-cell lysates. Thus, the basal phosphorylation of these proteins probably resulted from their spontaneous dimerization in the absence of any treatment, as suggested by the absence of phosphorylation in kinase-defective corresponding proteins (Fig. S2B, supplementary material). Note that the levels of basal and induced phosphorylation of the cytoIA-FH protein were slightly lower than that of the myrIA-FH protein. As shown in Fig. 4C, the myrIA-FH protein induced a significant increase of ERK 1/2 phosphorylation upon only 2 minutes of dimerizer treatment, which was sustained in a long-lasting manner as it remained constant after at least 1 hour and even after 24 hours (not shown). By contrast, the expression of the cytosolic protein (cytoIA-FH) resulted in a weak ERK phosphorylation. This much weaker ERK activation could be explained by the lower level of phosphorylation of this protein. To test this hypothesis, we overexpressed the cytoIA-FH protein to obtain a degree of tyrosine phosphorylation similar to that of the myrIA-FH protein. In these conditions, this weak level of ERK activation was identically observed (data not shown). Note that either basal or induced ERK phosphorylations triggered by the myrIA-FH and cytoIA-FH proteins were a result of their intrinsic kinase activities (Fig. S2B, supplementary material). Thus, all these data provided evidence for the first time that membrane attachment of the ALK PTK domain was required for the induction of neurite outgrowth of PC12 cells and for the activation of MAP kinase.

Induced neurite extension of PC12 cells is dependent on ERK 1/2 but not PI 3-kinase
It had previously been shown that MAP kinase but not PI 3-kinase activation was essential to the neuron-like differentiation of PC12 cells expressing the constitutively active Fc-ALK chimera (Souttou et al., 2001Go). We therefore asked whether the sustained ERK 1/2 activation following induced activation of the membrane-bound ALK proteins (Fig. 2B, Fig. 4B) was actually required for the induction of the neurite outgrowth process. Thus, the effect of U0126, one of the selective inhibitors of MEK 1/2 (MAP kinase kinase) proteins (Favata et al., 1998Go) was first tested on neurite outgrowth induced by activation of the full-length ALK-FH protein. As shown in Fig. 5A, and as expected, U0126 treatment led to a progressive inhibition of the induced neurite extension of transfected cells in a dose-dependant manner, reaching a weak level of about 10% of these cells bearing neurites at a concentration of 25 µM of U0126 that we chose for subsequent analysis. This concentration was actually shown to inhibit either the basal or induced ERK phosphorylation in these cells completely (Fig. S3, supplementary material). Cells expressing the membrane-bound proteins tmbIA-FH or myrIA-FH were treated or not with 20 nM of dimerizer in the presence or absence of U0126. As shown in Fig. 5B, the U0126 treatment almost completely abolished both the basal and dimerizer-induced neurite outgrowth, which also reached a weak differentiation level of about 10%. By contrast, wortmannin (Fig. 5C), one of the widely used pharmacological inhibitors of PI 3-kinase activity (Nakanishi et al., 1992Go; Powis et al., 1994Go), did not trigger any neurite outgrowth inhibition when used at the active dose of 50 nM (see below). Thus, the neurite extension process induced by activation of the membrane-bound proteins ALK-FH, tmbIA-FH and myrIA-FH required activation of the MAP kinase pathway but not the PI 3-kinase pathway.



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Fig. 5. Activation of the MAP kinase pathway, but not the PI 3-kinase pathway, is required to induce neurite extension of PC12 cells. (A) MEK inhibitor dose-dependent inhibition of neurite outgrowth induced by the activation of the full-length ALK-FH protein. PC12 cells were electroporated with ALK-FH construct (25 µg), cultured overnight in a 1% horse serum medium and treated with the dimerizer (20 nM) for two days in the presence of increasing concentrations of the MEK inhibitor U0126 dissolved in DMSO. As a control, cells were treated without DMSO (No vehicle). DMSO control (0 µM) showed no adverse effects. (B,C) Inhibition of neurite outgrowth by the MEK inhibitor U0126 but not by the PI 3-kinase inhibitor wortmannin. PC12 cells were electroporated with normalized quantities of the indicated constructs, cultured as described above and treated with the dimerizer (20 nM) in the presence or not of U0126 (25 µM) (B) or wortmannin (50 nM) (C) for two days. All the data (A-C) were obtained following immunofluorescence detection with the anti-HA-tag antibody (12CA5), neurite outgrowth of transfected cells being scored as indicated in the Materials and Methods. The experiments were performed in triplicate and values are expressed as the mean ± s.e.m. (%).

 

The differentiation process induced by activation of the membrane-bound ALK intracellular domain is concomitant with DNA synthesis arrest
It has previously been described (Greene and Tischler, 1976Go; Rudkin et al., 1989Go; van Grunsven et al., 1996aGo; van Grunsven et al., 1996bGo; Yan and Ziff, 1995Go; Yan and Ziff, 1997Go) that, upon NGF treatment, PC12 cells undergo a differentiation process and a proliferation arrest in the G0-G1 phase of the cell cycle by decreasing their proliferation rate and DNA synthesis. In the present study, we show that induced activation of the membrane-bound proteins tmbIA-FH and myrIA-FH leads to neurite outgrowth of PC12 cells (Fig. 4A,B). Therefore, we asked whether activation of these proteins was accompanied by DNA synthesis arrest. This process was monitored through a BrdU-incorporation assay using PC12 cells grown asynchronously in the presence of serum. As shown in Fig. 6, induced activation of tmbIA-FH and myrIA-FH proteins significantly decreased the proportion of transfected cells incorporating BrdU during the time of labeling, revealing an arrest of DNA synthesis. By contrast, cells expressing the myrIA-FH-related kinase-defective mutant (dmyrIA-FH) did not exhibit such an effect. This indicates that this DNA synthesis arrest process was specifically a result of the kinase activity of the membrane-bound ALK-FH-derived proteins. Furthermore, we showed that this process indeed required ERK 1/2 activation since it was abolished by U0126 treatment. In conclusion, our results show for the first time that the induced activation of these membrane-bound proteins leads to differentiation (see above) and concomitantly to an arrest of the continued proliferation of transfected PC12 cells, and that both of these processes are controlled by the ERK pathway.



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Fig. 6. Neurite outgrowth of PC12 cells mediated by ALK-FH-derived protein is accompanied by arrest of DNA synthesis. Cells were electroporated with normalized quantities of the indicated constructs, continually cultured in a normal serum medium (10% horse serum, 5% fetal calf serum) in order to study their normal asynchronous growth and treated with the dimerizer (20 nM) for 12 hours in the presence or not of U0126 (25 µM). At 6 hours after dimerizer application, cells were incubated with BrdU (10 µM) for 6 hours of incorporation. Then immunofluorescence assay was performed using the anti-HA-tag (3F10) and the FITC-conjugated anti-BrdU antibodies; 100 transfected cells were counted and cells with positive BrdU staining were scored as having undergone DNA replication during the time of labeling. The experiment was performed in triplicate and values are expressed as the mean ± s.e.m. (%). Statistical analyses were carried out by Student's t-test (*P<0.05; **P<0.01).

 

Activation of the cytosolic form of the ALK intracellular domain induces DNA synthesis through involvement of the PI 3-kinase/AKT pathway
As the cytosolic form of the ALK intracellular domain was shown to fail to induce any neurite outgrowth of PC12 cells, we looked for its potential functional role. Since the oncogenic and cytoplasmic NPM-ALK protein has been shown to trigger cell transformation and cell growth (Bai et al., 2000Go; Bischof et al., 1997Go; Slupianek et al., 2001Go), we investigated whether activation of the cytoplasmic cytoIA-FH protein could control PC12 cell growth. We therefore monitored DNA synthesis following activation of cytoIA-FH in PC12 cells synchronized in low-serum medium, as usually required to study DNA synthesis induction (Pardee, 1989Go). As shown in Fig. 7A, induced activation of the cytoIA-FH protein significantly increased the proportion of transfected cells incorporating BrdU (from 27% to 37%), revealing an increase of DNA synthesis. By contrast, cells expressing its related kinase-defective mutant (dcytoIA-FH) or the membrane-bound myrIA-FH protein did not exhibit such an effect, indicating that this induced DNA synthesis was specifically a result of the kinase activity of the cytosolic protein and that cytosolic localization was required for this effect. We then analyzed the signal transduction pathways involved in this process. We investigated whether this induced DNA synthesis was PI 3-kinase/AKT dependent, as it has been previously shown for NPM-ALK-induced cell proliferation (Bai et al., 2000Go; Slupianek et al., 2001Go). Thus, we tested the effect of two selective and widely used inhibitors of PI 3-kinase activity, wortmannin and LY294002 (Vlahos et al., 1994Go), on BrdU incorporation induced by dimerizer activation of the cytoIA-FH protein. As shown in Fig. 7B, both PI 3-kinase inhibitors induced progressive inhibition of DNA synthesis of transfected cells in a dose-dependent manner from 35% to about 17% of these cells incorporating BrdU. Note that, in the absence of dimerizer, wortmannin reduced BrdU incorporation to a similar level (Fig. 7C). By contrast and as expected, cells did not show any significant inhibition of BrdU incorporation when treated with 25 µM of the inhibitor of ERK 1/2 activity U0126 (Fig. 7C). Thus, DNA synthesis induced by activation of the cytosolic protein (cytoIA-FH) required activation of PI 3-kinase but not the MAP kinase pathway. As AKT protein is one of the major downstream effectors of PI 3-kinase, we studied the time-course activation of AKT by dimerizer treatment in cells transfected with either the cytoIA-FH or myrIA-FH constructs. As shown in Fig. 7D in cells transfected with the cytoIA-FH construct, AKT phosphorylation was increased after 10 minutes of dimerizer treatment and was maximal after 30 minutes. This significantly induced phosphorylation was completely inhibited in the presence of 50 nM wortmannin (Fig. S4, supplementary material). In good agreement with the preceding results, these data confirm that, following dimerizer stimulation, cells expressing the cytosolic protein actually displayed activation of the PI 3-kinase/AKT pathway, which was required to trigger cell growth through an increase of DNA synthesis.



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Fig. 7. Activation of the cytosolic form of the ALK intracellular domain induces DNA synthesis through the PI 3-kinase/AKT pathway. (A) Induced activation of the cytoIA-FH but not of the myrIA-FH protein leads to DNA synthesis in PC12 cells. Cells were electroporated with normalized quantities of the indicated constructs and were submitted to the same protocol as in Fig. 6 except that they were cultured overnight in a 1% horse serum medium before dimerizer treatment. ***P<0.005 (Student's t-test). (B) PI 3-kinase inhibitor inhibition of DNA synthesis induced by the activation of the cytoIA-FH protein. PC12 cells transiently expressing the cytoIA-FH protein were submitted to the same protocol as in A. They were treated with the dimerizer (20 nM) for 12 hours in the presence of the indicated increasing concentrations of PI 3-kinase inhibitors wortmannin or LY294002 dissolved in DMSO. As a control, cells were treated without DMSO (No vehicle). DMSO controls (0 nM or µM) showed no adverse effects. The experiments were performed in triplicate and values are expressed as the mean ± s.e.m. (%). (C) Induced DNA synthesis of PC12 cells expressing the cytoIA-FH protein is dependent on PI 3-kinase but not on ERK 1/2. Cells transiently expressing the cytoIA-FH protein were submitted to the same protocol as in A. U0126 (25 µM) and/or wortmannin (50 nM) were added to the medium 60 and 30 minutes, respectively, before dimerizer treatment. **P<0.01; ***P<0.005 (Student's t-test). (D) Time course of AKT phosphorylation following dimerizer treatment. PC12 cells transfected with the indicated constructs were cultured overnight in 0% horse serum medium and then incubated with the dimerizer (20 nM) for the indicated periods. Cells were lysed in RIPA buffer as described in the Materials and Methods and the lysates (10 µg) were submitted to western blot analysis using the anti-AKT phosphoserine-473 antibody and then reprobed with the anti-AKT antibody. (E) MAP kinase pathway inhibition revealed a potential activation effect of myrIA-FH protein on DNA synthesis through the PI 3-kinase pathway. Cells transiently expressing the myrIA-FH protein were submitted to the same protocol as in C. **P<0.01 (Student's t-test).

 

It is also noteworthy that in the same treatment conditions cells expressing the membrane-bound protein myrIA-FH (Fig. 7D) also displayed an increase of AKT phosphorylation. Interestingly, we observed that activation of myrIA-FH protein led to a significant increase in BrdU incorporation in the presence of U0126 (Fig. 7E). This increase was abolished by the dual addition of U0126 and wortmannin. These results strongly suggested that the activation of the MAP kinase pathway masked the potential proliferation process that could be triggered by the PI 3-kinase pathway. Thus, activation of the membrane-bound proteins led to the activation of both the MAP kinase and PI 3-kinase/AKT pathways, whereas neurite outgrowth and proliferation arrest processes observed in this case essentially relied on the activation of the MAP kinase cascade.


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The aim of the present report was to provide further elements to the comprehension of the role of ALK in neuronal differentiation and/or proliferation and to analyze the role and mechanisms of action of the subcellular localization of its PTK domain in relation to these processes and their related signaling pathways. Indeed, subcellular localization of a PTK domain as a factor that determines the signal transduction specificity has been poorly documented so far (Schlessinger, 2000Go). In this report, we established for the first time that membrane attachment of the ALK PTK domain is crucial for induction of neurite outgrowth of PC12 cells and their proliferation arrest. We further showed that these two processes resulted from specific and sustained activation of the MAP kinase pathway. By contrast, we demonstrated that activation of the cytosolic form of this domain failed to induce MAP kinase activation and cell differentiation but rather promoted a PI 3-kinase/AKT-dependent cell growth.

We first showed that the dimerizer induced the phosphorylation of the full-length ALK-FH protein and triggered a MAP-kinase-dependent neurite outgrowth of PC12 cells. This indicates that the induced activation of ALK by intracellular dimerization was functional. These data were consistent with previous studies reporting that this pathway was required for the ALK-dependent differentiation of neuron-like cells through dimerization of the Fc-ALK chimera or ALK extracellular domains (Moog-Lutz et al., 2005Go; Motegi et al., 2004Go; Souttou et al., 2001Go). Our data also indicated that MAP-kinase-dependent neurite outgrowth of PC12 cells was not dependent on the presence of the ALK extracellular domain as comparable neurite outgrowth efficiencies were found following activation of full-length ALK-FH or truncated tmbIA-FH proteins. MAP-kinase-dependent neurite outgrowth of PC12 cells was also not dependent on the type of membrane anchoring (transmembrane versus myristoyl). However, some noticeable differences were detected. The myristoylated protein appeared more efficient at promoting both the basal and dimerizer-induced differentiation of PC12 cells. An attractive hypothesis would be that the myrIA-FH protein, like other myristoylated proteins, is concentrated into the lipid rafts of the plasma membrane. This concentration by itself could facilitate the basal or induced dimerization of the protein. In addition, the compartmentalized assembly of MAP kinase signaling proteins within lipid rafts might provide a higher efficiency for the myrIA-FH protein to activate these signaling proteins than for the transmembrane form tmbIA-FH. For instance, several reports pointed out the crucial role of the FRS2 adaptor in the NGF- or FGF-induced differentiation of PC12 cells and established that FRS2 was associated within lipid rafts through myristoyl anchors (Ong et al., 2000Go; Ridyard and Robbins, 2003Go).

The sustained activation of the ERK 1/2 proteins triggered by the membrane-bound form of ALK also deserves comments. This typical kinetics pattern is in agreement with those reported in several previous studies stating that a sustained activation of the MAP kinase pathway induced by various neurotrophic factors such as NGF or FGF was essential to promote PC12 cell differentiation (Marshall, 1995Go). In fact, it was also previously shown in PC12 cells that overexpression of the EGF or insulin receptors in the presence of their respective ligands led to a sustained activation of the MAP kinase cascade and subsequently to neuronal differentiation, whereas only a transient activation leading to cell proliferation has been described when these receptors were expressed at a physiological level (Dikic et al., 1994Go; Traverse et al., 1994Go). This indicates that the level of receptor expression could be crucial for the induction of the MAP kinase cascade and cell differentiation. As in transiently transfected cells, proteins are usually overexpressed, which could explain the observed sustained activation of the MAP kinase pathway induced by the dimerizer. However, our unpublished results obtained with stable HEK 293 clones expressing the same ALK-derived proteins at a much lower level confirmed that ERK proteins also displayed a sustained phosphorylation kinetics following dimerizer treatment (data not shown). In addition, it had also been reported that endogenously expressed ALK receptor in neuroblastoma cells could promote neurite outgrowth through sustained MAP kinase activation (Motegi et al., 2004Go). Altogether, these data suggest that ligand-induced activation of the endogenous ALK receptor in physiological conditions could trigger a sustained activation of the MAP kinase cascade leading to subsequent neuronal differentiation in vivo.

The difference between the membrane-bound and cytosolic proteins in the ERK cascade activation could result from the crucial role played by specific signaling complexes located at the cell membrane. Indeed, activated RTKs could recruit to the membrane different cytosolic adaptors (Sos, Grb2, Shc, etc.) or directly link membrane-anchored docking proteins (FRS2). These protein complexes facilitate subsequent activation of GTPase proteins of the Ras family (Ras, Rap1, etc.) that are also associated to the membrane through lipid anchors (Schlessinger, 2000Go). Thus, these confined protein assemblages bring together signal proteins, and organize and coordinate the function of a large part of the MAP kinase signaling cascade at the membrane. Thus, in contrast to the membrane-bound forms of ALK, the cytosolic form probably failed to activate these membrane-bound protein complexes, resulting in the lack of MAP kinase activation. In this context, it is noteworthy that the activation of the MAP kinase pathway by the NPM-ALK protein or by other cytosolic forms of ALK resulting from various chromosomal translocations had never been reported (reviewed by Pulford et al., 2004Go). Interestingly, the gene encoding moesin (MSN) at Xq11-12 has been recently reported as a new partner of ALK in rare cases of anaplastic large cell lymphoma (ALCL). The fusion protein MSN-ALK (Tort et al., 2001Go) exhibited a distinctive membrane-restricted labeling pattern. This particular membrane localization of an ALK oncogenic form is presumed to reflect association of moesin with cell membrane proteins. Therefore, it would be interesting to know whether this membrane-bound form of ALK is able to activate the MAP kinase pathway.

Here, we cultured PC12 cells using the culture conditions of low-serum medium (1% horse serum, overnight) before dimerizer stimulation. We chose this particular cell culture condition because it had been previously reported that NGF-induced neurite extension occurred more rapidly in PC12 cells arrested in G0 phase than in asynchronously growing cells (Rudkin et al., 1989Go). In addition, synchronized cells are also usually required to study DNA synthesis induction. Indeed, when deprived of serum, cells continue to cycle until they terminate mitosis, at which point they exit into the G0 state (Pardee, 1989Go). Then, they can be reintroduced into the cell cycle by re-addition of growth factor. In agreement with these data, we showed here that, when cultured in low-serum medium, cells expressing the cytosolic ALK-derived form underwent DNA synthesis in the presence of dimerizer, probably reflecting their reintroduction into the cell cycle. Such an effect was also observed with cells cultured in a serum-containing medium, but DNA synthesis induction was less significant. We also demonstrated that this phenomenon mainly required activation of the PI 3-kinase/AKT pathway. This is in full agreement with several previous reports showing that PI 3-kinase/AKT activation was required and sufficient for cell-cycle entry and DNA synthesis (Roche et al., 1994Go; Valius and Kazlauskas, 1993Go) and that this pathway was essential to enhance proliferation of cells expressing the cytosolic NPM-ALK protein (Bai et al., 2000Go; Slupianek et al., 2001Go). By using the engineered cytosolic ALK-derived protein, we essentially reproduced the results obtained in NPM-ALK-positive cells. Thus, our model is to a certain extent a relevant transposition of the oncogenic system.

Induced AKT phosphorylation was also noticeable with cells expressing the membrane-bound myrIA-FH protein. Therefore, PI 3-kinase/AKT activation also occurs when the ALK PTK domain is located at the membrane. Thus, activation of the membrane-bound protein led to activation of both the MAP kinase and PI 3-kinase pathways. Neurite outgrowth and proliferation arrest processes visualized in this case essentially relied on the activation of the ERK pathway. Nevertheless, inhibition of the ERK pathway revealed a potential proliferation effect of the myrIA-FH protein that is controlled by the PI 3-kinase/AKT pathway. These results fit different reports analyzing the NGF effects through the MAP kinase and PI 3-kinase/AKT pathways in PC12 cells (Klesse et al., 1999Go). We showed here for the first time that induced activation of the membrane-bound forms of ALK triggered PC12 cell differentiation and proliferation arrest. In contrast to our results and the NGF effects on PC12 cells, a recent study showed that activation of endogenous ALK expressed in the SK-N-SH neuroblastoma cell line induced both neurite outgrowth and cell proliferation (Motegi et al., 2004Go). A possible explanation is that the level of expression of the ALK receptor in these cells is crucial as previously discussed. Furthermore, as pointed out by these authors, this difference of biological effects might be a result of cell-type specificity. Thus, depending on the levels of activation of the ERK and PI 3-kinase pathways, the cell could either differentiate and/or proliferate. A complete analysis of both effects and inhibition of these two pathways would certainly be informative. The availability of monoclonal antibodies (Moog-Lutz et al., 2005Go) will now allow us to study directly the biological effects and related signaling pathways triggered by the activation of ALK during development, in particular in primary neuronal cell cultures endogenously expressing this receptor.

In conclusion, our results strongly support our initial hypothesis postulating that membrane attachment of the ALK PTK domain could be a determinant for the control and specificity of the downstream transduction cascades. This membrane attachment was crucial for promotion of neuron-like differentiation and cell proliferation arrest of PC12 cells through specific activation of the MAP kinase pathway. In addition, our model allowed for the first time a direct comparison between the full-length membrane-bound receptor and a cytosolic form of its PTK domain that strongly parallels the oncogenic forms of ALK resulting from various chromosomal translocations. Indeed, our data showed that subcellular localization of the ALK PTK domain was crucial for deciding the fate to which the neuronal cell will be committed.


    Acknowledgments
 
This work was supported in part by institutional funding from INSERM and Université Paris 6, as well as by grants from the Association pour la Recherche sur le Cancer (ARC) and Association Française contre les Myopathies (AFM). We thank ARIAD Pharmaceuticals (http://www.ariad.com/regulationkits) for providing vectors and AP20187. We thank J. Gavard, H. Enslen and J. Degoutin for fruitful discussions. We are particularly grateful to A. Sobel for his continual support and helpful comments on the manuscript.


    Footnotes
 
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/119/24/5811/DC1


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

Bai, R. Y., Dieter, P., Peschel, C., Morris, S. W. and Duyster, J. (1998). Nucleophosmin-anaplastic lymphoma kinase of large-cell anaplastic lymphoma is a constitutively active tyrosine kinase that utilizes phospholipase C-gamma to mediate its mitogenicity. Mol. Cell. Biol. 18, 6951-6961.[Abstract/Free Full Text]

Bai, R. Y., Ouyang, T., Miething, C., Morris, S. W., Peschel, C. and Duyster, J. (2000). Nucleophosmin-anaplastic lymphoma kinase associated with anaplastic large-cell lymphoma activates the phosphatidylinositol 3-kinase/Akt antiapoptotic signaling pathway. Blood 96, 4319-4327.[Abstract/Free Full Text]

Bibel, M. and Barde, Y. A. (2000). Neurotrophins: key regulators of cell fate and cell shape in the vertebrate nervous system. Genes Dev. 14, 2919-2937.[Free Full Text]

Bischof, D., Pulford, K., Mason, D. Y. and Morris, S. W. (1997). Role of the nucleophosmin (NPM) portion of the non-Hodgkin's lymphoma-associated NPM-anaplastic lymphoma kinase fusion protein in oncogenesis. Mol. Cell. Biol. 17, 2312-2325.[Abstract]

Blume-Jensen, P. and Hunter, T. (2001). Oncogenic kinase signalling. Nature 411, 355-365.[CrossRef][Medline]

Boccaccio, C., Ando, M. and Comoglio, P. M. (2002). A differentiation switch for genetically modified hepatocytes. FASEB J. 16, 120-122.[Abstract/Free Full Text]

Clackson, T., Yang, W., Rozamus, L. W., Hatada, M., Amara, J. F., Rollins, C. T., Stevenson, L. F., Magari, S. R., Wood, S. A., Courage, N. L. et al. (1998). Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity. Proc. Natl. Acad. Sci. USA 95, 10437-10442.[Abstract/Free Full Text]

Dikic, I., Schlessinger, J. and Lax, I. (1994). PC12 cells overexpressing the insulin receptor undergo insulin-dependent neuronal differentiation. Curr. Biol. 4, 702-708.[CrossRef][Medline]

Dirks, W. G., Fahnrich, S., Lis, Y., Becker, E., MacLeod, R. A. and Drexler, H. G. (2002). Expression and functional analysis of the anaplastic lymphoma kinase (ALK) gene in tumor cell lines. Int. J. Cancer 100, 49-56.[CrossRef][Medline]

Englund, C., Loren, C. E., Grabbe, C., Varshney, G. K., Deleuil, F., Hallberg, B. and Palmer, R. H. (2003). Jeb signals through the Alk receptor tyrosine kinase to drive visceral muscle fusion. Nature 425, 512-516.[CrossRef][Medline]

Favata, M. F., Horiuchi, K. Y., Manos, E. J., Daulerio, A. J., Stradley, D. A., Feeser, W. S., Van Dyk, D. E., Pitts, W. J., Earl, R. A., Hobbs, F. et al. (1998). Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem. 273, 18623-18632.[Abstract/Free Full Text]

Fujimoto, J., Shiota, M., Iwahara, T., Seki, N., Satoh, H., Mori, S. and Yamamoto, T. (1996). Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5). Proc. Natl. Acad. Sci. USA 93, 4181-4186.[Abstract/Free Full Text]

Greene, L. A. and Tischler, A. S. (1976). Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 73, 2424-2428.[Abstract/Free Full Text]

Huang, E. J. and Reichardt, L. F. (2001). Neurotrophins: roles in neuronal development and function. Annu. Rev. Neurosci. 24, 677-736.[CrossRef][Medline]

Huang, E. J. and Reichardt, L. F. (2003). Trk receptors: roles in neuronal signal transduction. Annu. Rev. Biochem. 72, 609-642.[CrossRef][Medline]

Huff, K., End, D. and Guroff, G. (1981). Nerve growth factor-induced alteration in the response of PC12 pheochromocytoma cells to epidermal growth factor. J. Cell Biol. 88, 189-198.[Abstract/Free Full Text]

Iwahara, T., Fujimoto, J., Wen, D., Cupples, R., Bucay, N., Arakawa, T., Mori, S., Ratzkin, B. and Yamamoto, T. (1997). Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system. Oncogene 14, 439-449.[CrossRef][Medline]

Kaplan, D. R. and Miller, F. D. (2000). Neurotrophin signal transduction in the nervous system. Curr. Opin. Neurobiol. 10, 381-391.[CrossRef][Medline]

Klesse, L. J., Meyers, K. A., Marshall, C. J. and Parada, L. F. (1999). Nerve growth factor induces survival and differentiation through two distinct signaling cascades in PC12 cells. Oncogene 18, 2055-2068.[CrossRef][Medline]

Lee, H. H., Norris, A., Weiss, J. B. and Frasch, M. (2003). Jelly belly protein activates the receptor tyrosine kinase Alk to specify visceral muscle pioneers. Nature 425, 507-512.[CrossRef][Medline]

Loren, C. E., Scully, A., Grabbe, C., Edeen, P. T., Thomas, J., McKeown, M., Hunter, T. and Palmer, R. H. (2001). Identification and characterization of DAlk: a novel Drosophila melanogaster RTK which drives ERK activation in vivo. Genes Cells 6, 531-544.[Abstract/Free Full Text]

Lu, K. V., Jong, K. A., Kim, G. Y., Singh, J., Dia, E. Q., Yoshimoto, K., Wang, M. Y., Cloughesy, T. F., Nelson, S. F. and Mischel, P. S. (2005). Differential Induction of Glioblastoma Migration and Growth by Two Forms of Pleiotrophin. J. Biol. Chem. 280, 26953-26964.[Abstract/Free Full Text]

Manning, G., Whyte, D. B., Martinez, R., Hunter, T. and Sudarsanam, S. (2002). The protein kinase complement of the human genome. Science 298, 1912-1934.[Abstract/Free Full Text]

Marshall, C. J. (1995). Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80, 179-185.[CrossRef][Medline]

Miyake, I., Hakomori, Y., Shinohara, A., Gamou, T., Saito, M., Iwamatsu, A. and Sakai, R. (2002). Activation of anaplastic lymphoma kinase is responsible for hyperphosphorylation of ShcC in neuroblastoma cell lines. Oncogene 21, 5823-5834.[CrossRef][Medline]

Moog-Lutz, C., Degoutin, J., Gouzi, J. Y., Frobert, Y., Carvalho, N. B.-d., Bureau, J., Creminon, C. and Vigny, M. (2005). Activation and inhibition of anaplastic lymphoma kinase receptor tyrosine kinase by monoclonal antibodies and absence of agonist activity of pleiotrophin. J. Biol. Chem. 280, 26039-26048.[Abstract/Free Full Text]

Morris, S. W., Kirstein, M. N., Valentine, M. B., Dittmer, K. G., Shapiro, D. N., Saltman, D. L. and Look, A. T. (1994). Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. Science 263, 1281-1284.[Medline]

Morris, S. W., Naeve, C., Mathew, P., James, P. L., Kirstein, M. N., Cui, X. and Witte, D. P. (1997). ALK, the chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin's lymphoma, encodes a novel neural receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK). Oncogene 14, 2175-2188.[CrossRef][Medline]

Motegi, A., Fujimoto, J., Kotani, M., Sakuraba, H. and Yamamoto, T. (2004). ALK receptor tyrosine kinase promotes cell growth and neurite outgrowth. J. Cell Sci. 117, 3319-3329.[Abstract/Free Full Text]

Nakanishi, S., Kakita, S., Takahashi, I., Kawahara, K., Tsukuda, E., Sano, T., Yamada, K., Yoshida, M., Kase, H., Matsuda, Y. et al. (1992). Wortmannin, a microbial product inhibitor of myosin light chain kinase. J. Biol. Chem. 267, 2157-2163.[Abstract/Free Full Text]

Nieborowska-Skorska, M., Slupianek, A., Xue, L., Zhang, Q., Raghunath, P. N., Hoser, G., Wasik, M. A., Morris, S. W. and Skorski, T. (2001). Role of signal transducer and activator of transcription 5 in nucleophosmin/anaplastic lymphoma kinase-mediated malignant transformation of lymphoid cells. Cancer Res. 61, 6517-6523.[Abstract/Free Full Text]

Ong, S. H., Guy, G. R., Hadari, Y. R., Laks, S., Gotoh, N., Schlessinger, J. and Lax, I. (2000). FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors. Mol. Cell. Biol. 20, 979-989.[Abstract/Free Full Text]

Pardee, A. B. (1989). G1 events and regulation of cell proliferation. Science 246, 603-608.[Medline]

Powis, G., Bonjouklian, R., Berggren, M. M., Gallegos, A., Abraham, R., Ashendel, C., Zalkow, L., Matter, W. F., Dodge, J., Grindey, G. et al. (1994). Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase. Cancer Res. 54, 2419-2423.[Abstract]

Pulford, K., Morris, S. W. and Turturro, F. (2004). Anaplastic lymphoma kinase proteins in growth control and cancer. J. Cell Physiol. 199, 330-358.[CrossRef][Medline]

Ridyard, M. S. and Robbins, S. M. (2003). Fibroblast growth factor-2-induced signaling through lipid raft-associated fibroblast growth factor receptor substrate 2 (FRS2). J. Biol. Chem. 278, 13803-13809.[Abstract/Free Full Text]

Roche, S., Koegl, M. and Courtneidge, S. A. (1994). The phosphatidylinositol 3-kinase alpha is required for DNA synthesis induced by some, but not all, growth factors. Proc. Natl. Acad. Sci. USA 91, 9185-9189.[Abstract/Free Full Text]

Rudkin, B. B., Lazarovici, P., Levi, B. Z., Abe, Y., Fujita, K. and Guroff, G. (1989). Cell cycle-specific action of nerve growth factor in PC12 cells: differentiation without proliferation. EMBO J. 8, 3319-3325.[Abstract]

Schlessinger, J. (2000). Cell signaling by receptor tyrosine kinases. Cell 103, 211-225.[CrossRef][Medline]

Slupianek, A., Nieborowska-Skorska, M., Hoser, G., Morrione, A., Majewski, M., Xue, L., Morris, S. W., Wasik, M. A. and Skorski, T. (2001). Role of phosphatidylinositol 3-kinase-Akt pathway in nucleophosmin/anaplastic lymphoma kinase-mediated lymphomagenesis. Cancer Res. 61, 2194-2199.[Abstract/Free Full Text]

Souttou, B., Carvalho, N. B., Raulais, D. and Vigny, M. (2001). Activation of anaplastic lymphoma kinase receptor tyrosine kinase induces neuronal differentiation through the mitogen-activated protein kinase pathway. J. Biol. Chem. 276, 9526-9531.[Abstract/Free Full Text]

Stoica, G. E., Kuo, A., Aigner, A., Sunitha, I., Souttou, B., Malerczyk, C., Caughey, D. J., Wen, D., Karavanov, A., Riegel, A. T. et al. (2001). Identification of anaplastic lymphoma kinase as a receptor for the growth factor pleiotrophin. J. Biol. Chem. 276, 16772-16779.[Abstract/Free Full Text]

Stoica, G. E., Kuo, A., Powers, C., Bowden, E. T., Sale, E. B., Riegel, A. T. and Wellstein, A. (2002). Midkine binds to anaplastic lymphoma kinase (ALK) and acts as a growth factor for different cell types. J. Biol. Chem. 277, 35990-35998.[Abstract/Free Full Text]

Tort, F., Pinyol, M., Pulford, K., Roncador, G., Hernandez, L., Nayach, I., Kluin-Nelemans, H. C., Kluin, P., Touriol, C., Delsol, G. et al. (2001). Molecular characterization of a new ALK translocation involving moesin (MSN-ALK) in anaplastic large cell lymphoma. Lab. Invest. 81, 419-426.[Medline]

Traverse, S., Seedorf, K., Paterson, H., Marshall, C. J., Cohen, P. and Ullrich, A. (1994). EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor. Curr. Biol. 4, 694-701.[CrossRef][Medline]

Valius, M. and Kazlauskas, A. (1993). Phospholipase C-gamma 1 and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor's mitogenic signal. Cell 73, 321-334.[CrossRef][Medline]

van Grunsven, L. A., Billon, N., Savatier, P., Thomas, A., Urdiales, J. L. and Rudkin, B. B. (1996a). Effect of nerve growth factor on the expression of cell cycle regulatory proteins in PC12 cells: dissection of the neurotrophic response from the anti-mitogenic response. Oncogene 12, 1347-1356.[Medline]

van Grunsven, L. A., Thomas, A., Urdiales, J. L., Machenaud, S., Choler, P., Durand, I. and Rudkin, B. B. (1996b). Nerve growth factor-induced accumulation of PC12 cells expressing cyclin D1: evidence for a G1 phase block. Oncogene 12, 855-862.[Medline]

Vlahos, C. J., Matter, W. F., Hui, K. Y. and Brown, R. F. (1994). A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269, 5241-5248.[Abstract/Free Full Text]

Yan, G. Z. and Ziff, E. B. (1995). NGF regulates the PC12 cell cycle machinery through specific inhibition of the Cdk kinases and induction of cyclin D1. J. Neurosci. 15, 6200-6212.[Abstract]

Yan, G. Z. and Ziff, E. B. (1997). Nerve growth factor induces transcription of the p21 WAF1/CIP1 and cyclin D1 genes in PC12 cells by activating the Sp1 transcription factor. J. Neurosci. 17, 6122-6132.[Abstract/Free Full Text]

Yang, J., Symes, K., Mercola, M. and Schreiber, S. L. (1998). Small-molecule control of insulin and PDGF receptor signaling and the role of membrane attachment. Curr. Biol. 8, 11-18.[CrossRef][Medline]

Zamo, A., Chiarle, R., Piva, R., Howes, J., Fan, Y., Chilosi, M., Levy, D. E. and Inghirami, G. (2002). Anaplastic lymphoma kinase (ALK) activates Stat3 and protects hematopoietic cells from cell death. Oncogene 21, 1038-1047.[CrossRef][Medline]





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