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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Summary |
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Key words: ALK, FK506-binding protein, PC12 cells, MAP kinase, Neurite outgrowth, Cell proliferation
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Introduction |
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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., 1997; Loren et al., 2001
; Morris et al., 1997
). 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., 2001
; Stoica et al., 2002
), although several studies do not confirm this hypothesis (Dirks et al., 2002
; Miyake et al., 2002
; Moog-Lutz et al., 2005
; Motegi et al., 2004
). However, a recent report suggests that a truncated form of PTN (and not the full-length PTN) could activate ALK (Lu et al., 2005
). In addition, the protein Jelly Belly (Jeb) has been identified as the ligand of Drosophila ALK (Englund et al., 2003
; Lee et al., 2003
) and Jeb is distinct from the Drosophila homologs of PTN and MK, Miple 1 and 2 (Lee et al., 2003
). 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., 1997; Morris et al., 1997
). In Drosophila, regulated DAlk mRNA and DAlk protein expressions were also described in the developing brain and ventral nerve cord (Loren et al., 2001
). 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., 2001
). 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., 2005
; Motegi et al., 2004
) 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., 2004). 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., 1994
)]. NPM oligomerization leads to the constitutive activation of the ALK kinase domain (Bischof et al., 1997
) and PLC
, 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., 1998
; Bai et al., 2000
; Bischof et al., 1997
; Fujimoto et al., 1996
; Nieborowska-Skorska et al., 2001
; Slupianek et al., 2001
; Zamo et al., 2002
).
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, 2000). 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.
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Materials and Methods |
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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., 2005).
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., 2005). 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.
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Results |
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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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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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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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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., 2001). 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., 1998
) 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., 1992
; Powis et al., 1994
), 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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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, 1976; Rudkin et al., 1989
; van Grunsven et al., 1996a
; van Grunsven et al., 1996b
; Yan and Ziff, 1995
; Yan and Ziff, 1997
) 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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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., 2000; Bischof et al., 1997
; Slupianek et al., 2001
), 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, 1989
). 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., 2000
; Slupianek et al., 2001
). Thus, we tested the effect of two selective and widely used inhibitors of PI 3-kinase activity, wortmannin and LY294002 (Vlahos et al., 1994
), 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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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.
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Discussion |
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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., 2005; Motegi et al., 2004
; Souttou et al., 2001
). 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., 2000
; Ridyard and Robbins, 2003
).
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, 1995). 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., 1994
; Traverse et al., 1994
). 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., 2004
). 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, 2000). 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., 2004
). 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., 2001
) 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., 1989). 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, 1989
). 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., 1994
; Valius and Kazlauskas, 1993
) and that this pathway was essential to enhance proliferation of cells expressing the cytosolic NPM-ALK protein (Bai et al., 2000
; Slupianek et al., 2001
). 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., 1999). 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., 2004
). 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., 2005
) 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.
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Acknowledgments |
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Footnotes |
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References |
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