From INSERM U 440/Université Paris 6, Signalisation et Différenciation Cellulaires dans les Systèmes Nerveux et Musculaire, 17 rue du Fer à Moulin, F-75005 Paris, France
Received for publication, August 11, 2000, and in revised form, December 13, 2000
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ABSTRACT |
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Anaplastic lymphoma kinase (ALK) is a novel
neuronal orphan receptor tyrosine kinase that is essentially and
transiently expressed in specific regions of the central and peripheral
nervous systems, suggesting a role in its normal development and
function. To determine whether ALK could play a role in neuronal
differentiation, we established a model system that allowed us to mimic
the normal activation of this receptor. We expressed, in PC12 cells, a
chimeric protein in which the extracellular domain of the receptor was replaced by the mouse IgG 2b Fc domain. The Fc domain induced the
dimerization and oligomerization of the chimeric protein leading to
receptor phosphorylation and activation, thus mimicking the effect of
ligand binding, whereas the wild type ALK remained as a monomeric
nonphosphorylated protein. Expression of the chimera, but not that of
the wild type ALK or of a kinase inactive form of the chimera, induced
the differentiation of PC12 cells. Analysis of the signaling pathways
involved in this process pointed to an essential role of the
mitogen-activated protein kinase cascade. These results are consistent
with a role for ALK in neuronal differentiation.
The common structural features of a receptor tyrosine kinase
(RTK)1 include an
extracellular ligand binding region, a hydrophobic membrane-spanning
segment, and a cytoplasmic domain that carries the catalytic function.
Following ligand binding, the RTK dimerizes and autophosphorylates (1).
The activated RTK initiates signal transduction cascades through
binding of SH2 domain-containing proteins to specific receptor
phosphotyrosine residues (2). RTKs can regulate a wide variety of
cellular processes involved in cell division, differentiation,
survival, and motility. A number of RTKs play essential roles during
the development of the nervous system by contributing to neuronal
differentiation, survival, and function (reviewed in Ref. 3). Most of
these receptors have specific or shared ligands called neurotrophic
factors that have been identified (reviewed in Ref. 3). However, for
some of them, named orphan receptors, their ligands are still unknown (4-6).
Anaplastic lymphoma kinase (ALK), a novel orphan neuronal receptor, was
originally identified as a member of the insulin receptor subfamily of
receptor tyrosine kinases that acquires transforming capability when
truncated and fused in the t(2;5) chromosomal rearrangement associated
with the non-Hodgkin lymphoma (7). This translocation produces a fusion
gene that encodes a soluble chimeric transforming protein comprised of
the N-terminal portion of the phosphoprotein nucleophosmin (NPM), a
highly conserved RNA-binding nucleolar protein, linked to the
cytoplasmic portion of ALK (7). The NPM-ALK fusion protein was
localized within both the cytoplasm and the nucleoplasm and also within
the nucleoli of t(2;5)-translocation-positive lymphoma cells (8).
However, whereas the NPM sequence is essential for the transforming
activity (9), the nuclear localization, occurring via the shuttling activity of NPM (10), is not required for oncogenesis (11). It has been
demonstrated that the NPM portion was responsible for the dimerization
of the fusion protein leading to the constitutive activation of the
kinase and to the transforming activity (8).
Human and mouse cDNAs encoding full-length ALK have been
characterized (5, 6). The deduced amino acid sequences revealed that
ALK is a novel RTK having an extracellular domain, a single transmembrane domain, and an intracellular domain containing the tyrosine kinase activity. The open reading frame encodes a 1620-amino acid protein that is most closely related to leukocyte tyrosine kinase
(4, 12). Surface labeling studies indicated that the mature form of the
receptor is a 200-kDa glycoprotein exposed at the cell membrane (5),
consistent with the prediction that ALK serves as the receptor for yet
unidentified ligand(s). In situ hybridization analysis
showed that ALK RNA is essentially and transiently expressed in
specific regions of the central and peripheral nervous systems such as
the thalamus, mid-brain, olfactory bulb, and peripheral ganglia and
that it localizes mostly in neuronal cells (5). The neonatal brain
showed the highest expression, suggesting a possible involvement of ALK
in development of the nervous system when axon sprouting and retraction
are occurring. Because ALK expression is maintained, albeit at a lower
level, in the adult brain, it may also play a role in synapse formation and maintenance (5). Thus, ALK is a novel orphan receptor tyrosine kinase that might play an important role in the normal development and
function of the nervous system.
The ligand of ALK is unknown. Therefore, to investigate whether ALK can
play a role in neuronal differentiation, we generated a constitutively
active transmembrane form of ALK by substituting the extracellular
domain of the receptor by the Fc fragment of mouse IgG 2b. We
show here that the ALK.Fc protein expressed in PC12 cells dimerized,
oligomerized, and was tyrosine-phosphorylated. Furthermore, we show
that transient expression of ALK.Fc induced neuronal differentiation
through the mitogen-activated protein (MAP) kinase pathway.
Reagents--
PD98059 was obtained from New England BioLabs
(Beverly, MA). Wortmannin was from Sigma (St. Quentin Fallavier,
France). ET-18-OCH3 was purchased from Calbiochem
(San Diego, CA). Nerve growth factor (NGF) was from Life Technologies
(Cergy Pontoise, France) and basic fibroblast growth factor (bFGF) was
a gift from F. Mascarelli (INSERM U450, Paris). Rabbit anti-ALK
antibody was purchased from Accurate Chemicals Co. (Westbury, NY).
Horseradish peroxidase-conjugated anti-mouse IgG was from Dako
(Copenhagen, Denmark). Goat anti-P-MAPK antibody was obtained from UBI
(Lake Placid, NY). Rabbit anti-phosphotyrosine antibody was from
Transduction Laboratories (Lexington, KY). Rabbit anti-SCG10 antibody
was a gift from S. Ozon (INSERM U440, Paris).
Plasmid Constructions--
The full-length human ALK cDNA in
pBluescript was obtained from the American Tissue Culture Collection
(ATCC). The cDNA, cut with XhoI-NotI and
blunt-ended, was inserted at the EcoRV site of the mammalian
expression vector pcDNA3.1 (Invitrogen, Groningen, The Netherlands)
generating the pcDNA-ALK.wt construct. The cDNA, 6226 bp,
covered the entire coding sequence of the protein. The ATG start codon
is located at nucleotide position 912, the sequence coding for the
transmembrane domain is located between nucleotides 4020 and 4083, and
the stop codon is at position 5774. There is a HincII site
at position 1079 and a PshAI site at position 3892. These
sites were used to delete a major part of the ALK extracellular domain
to generate the pcDNA-ALK.Fc construct (see below).
The 864-bp PstI-EcoRV fragment of the mouse IgG
2b cDNA corresponding to the Fc fragment (GenBankTM accession
number MMIGG7) in pBluescript was a gift from Dr. N. Doyen
(Institut Pasteur, Paris). To construct the cDNA coding for the
chimeric protein (pcDNA-ALK.Fc) containing extracellularly the
mouse IgG 2b Fc domain linked to the membrane-spanning segment and the
whole cytoplasmic domain of ALK, a polymerase chain reaction product
corresponding to the entire sequence of the Fc fragment, flanked at the
5'-region with a HincII site and at the 3'-region with a
PshAI site was produced and inserted at the same sites in
the pcDNA-ALK.wt with its HincII-PshAI
segment deleted.
We prepared a kinase-defective form of the chimera (designated ALK*.Fc,
and the corresponding construct was pcDNA-ALK*.Fc) in which the
invariant lysine residue located in the ATP-binding portion of the
catalytic domain was changed to arginine. This lysine residue,
originally identified as residue 210 of the NPM-ALK fusion protein (7,
8), is located at position 1150 in ALK (5, 6). The mutation was
generated with the QuikChange site-directed mutagenesis kit (Stratagene
Europe, Amsterdam, The Netherlands) using the sense oligonucleotide
primer CTGCAAGTGGCTGTGAGGACGCTGCCTGAAGTG (in which the
underlined G replaced an A in the non-mutated sequence), together with the corresponding antisense oligonucleotide primer and
the pcDNA-ALK.Fc construct as the template. This single base mutagenesis was verified by sequencing (Genset, Paris, France).
Cell Culture and Electroporation--
The PC12 rat
pheochromocytoma cell line (13), purchased from ATCC, was grown, unless
otherwise specified, in RPMI 1640 supplemented with 10% horse serum
and 5% fetal calf serum at 37 °C in an atmosphere containing 5%
CO2.
PC12 cells were electroporated using the EasyJect Plus apparatus
(Equibio, United Kingdom) as recommended by the manufacturer. Briefly,
5 × 106 cells in 0.8 ml of OptiMEM (Life
Technologies) were mixed with 30 µg of DNA and then pulsed (260 V,
950 microfarads). Cells were immediately transferred to fresh culture
media and cultivated on gelatin-coated dishes. In experiments involving
pharmacological inhibitors and Cell Extracts, Immunoprecipitation, and
Immunoblotting--
Following electroporation, cells were grown for
48 h in medium with 10% horse serum and 5% fetal calf serum,
then for 24 h in medium with only 0.5% horse serum. Cell extracts
were prepared by lysing the cells in immunoprecipitation buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 0.5%
deoxycholic acid, 1% Nonidet P-40, 10% glycerol, 1 mM
sodium orthovanadate, 50 mM sodium fluoride, and protease
inhibitor mixture). The lysates were cleared by centrifugation and
subjected to either immunoprecipitation or immunoblotting.
For immunoprecipitation, 0.5 mg of total proteins was incubated at
4 °C in a rotating shaker with protein A-Sepharose beads on which a
rabbit anti-phosphotyrosine antibody had been previously bound. After
4 h of incubation, the beads were washed with lysis buffer and the
bound proteins were eluted by boiling for 5 min in SDS-PAGE sample buffer.
For Western blotting, cell extracts (20 µg) or immunoprecipitated
material were resolved by SDS-PAGE and transferred to nitrocellulose membranes. After blocking the membranes in phosphate-buffered saline
(PBS), 0.1% Tween 20, 5% powdered milk, they were probed with the
antibodies at appropriate dilutions for 1 h at room temperature. The blots were then washed in PBS, 0.1% Tween 20 and incubated with
the appropriate secondary antibody coupled to horseradish peroxidase
(Dako, Glostrup, Denmark) for 1 h. After washing in PBS, 0.1%
Tween 20, the proteins were visualized using the ECL system (Amersham
Pharmacia Biotech, Buckinghamshire, UK).
Immunofluorescence Staining--
PC12 cells transiently
transfected with the different constructs (pcDNA, pcDNA-ALK.wt,
pcDNA-ALK.Fc, or pcDNA-ALK*.Fc) were grown on glass coverslips
for 72 h, washed in PBS, fixed for 10 min at room temperature with
4% formaldehyde in PBS, and then washed 3 × 5 min with PBS, 50 mM NH4Cl. After 1 h of blocking in PBS,
containing 3%BSA, cells were incubated in the same buffer with an
FITC-conjugated goat anti-mouse IgG antibody (1/500 dilution; Jackson
Laboratories, West Grove, PA) to visualize ALK.Fc- or ALK*.Fc-expressing cells. After washing 5 × 5 min with PBS, cells were mounted in Citifluor (UKC Chemical Laboratory, Canterbury, UK)
before viewing on a conventional fluorescence microscope (Provis, Olympus). To analyze and localize SCG10, a rabbit anti-SCG10 antibody was added to the cells after blocking with PBS, 3% bovine serum albumin, 0.05% saponin for 1 h at room temperature. After washing 3 × 5 min with PBS, 0.05% saponin, cells were incubated with an FITC-conjugated anti-rabbit IgG (Jackson Laboratories, West Grove, PA)
for 1 h, washed 5 × 5 min in PBS, mounted, and visualized as
described above.
Because the ALK ligand is unknown, we generated a constitutively
active form of ALK to study the biological function(s) of this
receptor. We thus chose a strategy in which the extracellular domain of
ALK was substituted by the mouse IgG 2b Fc domain that we expected
would dimerize the resulting chimeric protein through disulfide bond
formation between cysteine residues of the Fc domain.
Fig. 1 shows the structures of the
pcDNA-ALK.wt, pcDNA-ALK.Fc, and pcDNA-ALK*.Fc constructs.
The pcDNA-ALK.wt construct encodes the membrane-bound wild type
receptor (see introduction and below). The pcDNA-ALK.Fc construct
codes for a transmembrane protein that contains the 30 N-terminal amino
acids of ALK, the Fc fragment and a juxtamembrane portion of the
extracellular domain (42 amino acids), the transmembrane, and the
entire intracellular domains of ALK. The pcDNA-ALK*.Fc construct
codes for a kinase-defective form of the chimera in which the invariant
lysine residue located in the ATP-binding portion of the catalytic
domain was changed to arginine (see "Materials and Methods"). This
point mutation has been previously shown to completely inhibit the
transforming capability of NPM-ALK (8).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, these supplements
were added daily along with fresh culture media. For experiments with
growth factors, PC12 cells were grown for 48 h in the presence of
either 25 ng/ml NGF or 50 ng of bFGF. Cultures were photographed and neurite-bearing cells counted using an inverted microscope after an
incubation period of 48 or 72 h, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the
pcDNA-ALK.wt, pcDNA-ALK.Fc, and pcDNA-ALK*.Fc
constructs. Diagram shows the positions of the HincII
and PshAI sites used to delete a portion of the
pcDNA-ALK.wt encoding the extracellular domain and to insert the Fc
fragment to generate the pcDNA-ALK.Fc construct. The site of
mutagenesis leading to inactivation of the kinase in the ALK*.Fc
construct through substitution of the lysine residue of the ATP-binding
portion of the catalytic domain for an arginine is indicated by an
asterisk (see "Materials and Methods" and Ref. 8).
EC, extracellular domain; TK, tyrosine kinase
domain; TM, transmembrane domain; SS, signal
sequence.
Expression of ALK.Fc and ALK.wt in PC12 Cells--
The
pcDNA-ALK.wt and the pcDNA-ALK.Fc constructs were transiently
expressed in PC12 cells, and the proteins they encoded were analyzed by
SDS-PAGE and Western blotting with anti-ALK antibody or anti-mouse IgG
(Fig. 2). Under both reducing and
nonreducing conditions, the ALK.wt receptor migrated as a single
200-kDa band, in agreement with previous reports (5). In contrast, the
ALK.Fc protein migrated as a single band of 120 kDa under reducing
conditions and mainly as a doublet of about 240 and 360 kDa under
nonreducing conditions. The 120-kDa band is consistent with that of the
predicted 965-amino acid protein encoded by the pcDNA-ALK.Fc
construct, whereas the 240- and 360-kDa proteins detected in
nonreducing conditions correspond probably to dimers and oligomers (at
least trimers), respectively. It is important to note that the anti-ALK antibody detected both the ALK.wt and the ALK.Fc proteins, whereas the
anti-mouse IgG revealed only the ALK.Fc protein. These results indicate
that the ALK receptor existed as a monomer and that the Fc fragment
allowed the ALK.Fc chimera to dimerize and oligomerize through
disulfide bond formation.
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Expression ALK.Fc Induced Neuronal Differentiation of PC12
Cells--
PC12 cells transiently expressing the pcDNA-ALK.Fc
construct exhibited neurite extensions, whereas cells expressing the
pcDNA-ALK.wt construct, or those that were transfected with the
pcDNA-ALK*.Fc vector or the empty pcDNA3 vector, did not (Fig.
3A). The neurites were visible
as soon as 24 h post-electroporation and reached the size of
severalfold the cell body size at 48 h post-electroporation. To
visualize cells overexpressing the ALK.Fc protein, immunofluorescence staining was performed with an FITC-conjugated goat anti-mouse IgG. As
shown in Fig. 3B, only neurite-bearing cells stained with the antibody and were therefore expressing ALK.Fc. Immunostaining for
the neuronal marker SCG10 (Fig. 3C) revealed an increased expression and a perinuclear localization of the protein (probably in
the Golgi network) of the neurite-bearing cells as previously demonstrated for PC12 cells induced to differentiate with NGF (14, 15).
In contrast, cells expressing the ALK*.Fc kinase-inactive form of the
chimera ALK.Fc failed to extend neurites, although they clearly
expressed the corresponding protein, as demonstrated by immunostaining
with FITC-conjugated goat anti-mouse IgG (Fig. 3B). One can
note that the staining appeared concentrated at the periphery of the
transfected cells, suggesting a plasma membrane localization of the
protein. No specific immunoreactivity was detected in cells
electroporated with both the pcDNA vector or the pcDNA-ALK.wt
construct (not shown).
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When cells transiently transfected with the pcDNA-ALK.Fc construct
were maintained in the presence of 500 µM of the reducing agent -mercaptoethanol, the neurite extension process was almost completely blocked (Fig. 4, A
and B). This indicated that the neurite outgrowth process
was due to ALK.Fc receptor dimerization and oligomerization through
disulfide bond formation, because neurite extension stimulated by
either bFGF or NGF was not affected by the presence of
-mercaptoethanol in the culture medium. These results demonstrate
that overexpression of ALK.Fc induced neuronal differentiation of PC12
cells.
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We were unable to isolate stable transfectants from pcDNA-ALK.Fc-transfected cultures, probably because sustained activation of ALK leads essentially to neuronal differentiation and not to cell proliferation in PC12 cells. However, we easily isolated stable transfectants from pcDNA-ALK.wt cultures (not shown). These cells could be good tools for the isolation of the ALK ligand(s) and for further studies on the neurotrophic activity of ALK.
ALK.Fc-induced PC12 Neuronal Differentiation Was Blocked by the
MEK-1 Inhibitor PD98059--
To analyze the signal transduction
cascade involved in the neuronal differentiation process induced by
ALK.Fc, we used pharmacological inhibitors targeting the major
signaling pathways coupled to RTKs. The MEK1 (a MAP kinase kinase)
inhibitor PD98059, at a concentration of 10 µM,
completely blocked the neurite outgrowth process induced by ALK.Fc
(Fig. 5, A and B).
In contrast, the Phosphoinositide-3 kinase (PI3K) inhibitor wortmannin
and the phospholipase C (PLC
) inhibitor ET-18-OCH3,
used at their active concentrations, 20 nM for each
inhibitor, had no apparent effect on this process (Fig. 5, A
and B). Thus, these data indicate that the PC12 neuronal differentiation induced by ALK.Fc involves mainly the MAP kinase pathway.
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Expression of ALK.Fc Induced Tyrosine Phosphorylation and
Activation of the MAP Kinases ERK1 and ERK2--
To determine whether
ALK.Fc was autophosphorylated and tyrosine phosphorylated downstream
signaling molecules, cell extracts from PC12 cells transiently
electroporated with the empty vector, the pcDNA-ALK.wt, or the
pcDNA-ALK.Fc constructs were subjected to immunoprecipitation with
a rabbit anti-phosphotyrosine antibody, and the immunoprecipitated
proteins were visualized by Western blotting with the same antibody.
Although the ALK.Fc protein was highly tyrosine-phosphorylated, the
ALK.wt protein showed no apparent phosphorylation (Fig.
6A), indicating that
dimerization and/or oligomerization of the receptor via the Fc fragment
induced its autophosphorylation on tyrosines. Several other
tyrosine-phosphorylated proteins, most probably downstream signaling
molecules, with molecular masses in the range 50-110 kDa
coimmunoprecipitated with ALK.Fc (Fig. 6A).
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To confirm the activation of the MAP kinase cascade by ALK.Fc, cell
extracts from PC12 cells transiently electroporated by the empty
vector, the pcDNA-ALK.wt, or the pcDNA-ALK.Fc DNAs were analyzed by immunoblotting with an anti-phospho-MAP kinase antibody (i.e. an antibody reacting with the active forms of MAP
kinases). The results in Fig. 6B show indeed that the
amount of active forms of the MAP kinases (phospho-ERK1 and -ERK2) were
higher in cell extracts from PC12/ALK.Fc cells than in extracts from
PC12/ALK.wt and PC12/pcDNA cells. Thus activation of the ALK
receptor induced by the Fc fragment led to the activation of the MAP
kinase pathway.
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DISCUSSION |
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ALK is a novel orphan receptor tyrosine kinase that is essentially and transiently expressed in the nervous system (mostly in neuronal cells), suggesting an important role for this receptor during normal development and function of the nervous system. However, because the ligand of this receptor has not yet been identified, the normal biological functions of ALK `are still unknown. In particular, it remains to be shown whether it could act as a functional membrane receptor and whether activation of its kinase activity could induce neuronal differentiation.
To answer these questions, and to study the signal transduction pathways involved in the potential neurotrophic activity of ALK, we established a model system that allowed us to mimic the normal activation of this receptor. We generated an ALK.Fc chimera containing extracellularly the mouse IgG 2b Fc domain linked to the membrane-spanning segment and the whole cytoplasmic domain of ALK and expressed it into PC12 cells. PC12 cells are a widely used and well established in vitro model system for the study of neuronal differentiation, because they can differentiate to neuron-like cells upon exposure to neurotrophic factors (16). The ALK.Fc chimera expressed in PC12 cells had, under reducing conditions, an apparent molecular mass of about 120 kDa, which was in good agreement with that of the predicted 965-amino acid protein encoded by the pcDNA-ALK.Fc construct. In agreement with previous reports (5, 6) the wild type ALK appeared as a 200-kDa protein. As expected, this strategy led to a forced dimerization and oligomerization of the chimera induced by inter-chain disulfide bridges of the Fc domains, whereas the ALK.wt did not dimerize or oligomerize. Western blot studies showed that ALK.Fc was hyperphosphorylated on tyrosines, whereas ALK.wt was not, indicating that the dimerized receptor was activated. To our knowledge, this is the first time that this strategy has been used to produce a constitutively activated form of a RTK. Thus it could be applied to other RTKs but also to other types of membrane-bound receptors that need to be dimerized and/or oligomerized as a first step of their activation.
Transient expression of the pcDNA-ALK.Fc construct in PC12 cells, but not of the pcDNA-ALK.wt and pcDNA-ALK*.Fc constructs, induced neurite extensions in a large majority of ALK.Fc-expressing cells, indicating a direct relationship between expression of the chimeric receptor and the biological effect observed. The neurite extension process was visible as soon as 24 h post-electroporation, consistent with the time necessary for high expression of the oligomerized receptor. This process was specifically induced by dimerization and oligomerization of the chimeric receptor, because inhibition of disulfide bond formation strongly prevented neurite extensions induced by ALK.Fc but did not affect those induced by NGF or bFGF. In parallel to the morphological differentiation, overexpression of ALK.Fc induced expression and localization of the neuronal marker SCG10 in a pattern similar to that induced by NGF (14, 15). The fact that the expression of the ALK*.Fc kinase-inactive form of the chimera did not induce neurite outgrowth practically eliminates the possibility that the dimerized constitutively active form (ALK.Fc) simply serves as a docking site for some other kinase that could be responsible for the effect observed with ALK.Fc. The neuronal differentiation induced by the ALK.Fc expression is due to the intrinsic activation of the kinase activity of this chimera.
Additional proof for the specificity of the neurite elongation process driven by ALK.Fc in PC12 cells was given by transient expression of the pcDNA-ALK.Fc construct in epithelial COS cells. COS cells electroporated with pcDNA-ALK.Fc expressed similar levels of phosphorylated dimerized and oligomerized ALK.Fc chimera than PC12 cells but did not exhibit any neurite-like extensions (data not shown).
It has been reported that ectopic expression of the insulin (17), epidermal growth factor (EGF) (18), and platelet-derived growth factor (19) receptors induced neuronal differentiation of PC12 cells only when they are activated by their cognate ligands. In agreement with these results, overexpression of the wild type ALK did not induce neuronal differentiation of PC12 cells, this receptor being monomeric and inactive in the absence of its ligand. In contrast, the ALK.Fc was permanently active. Altogether, these reports and our data stress the importance of the activation of the tyrosine kinase activity of the RTKs for the neuronal differentiation of PC12 cells.
The cytoplasmic molecules that mediate downstream signaling by ALK are
presently unknown. However, analysis of the amino acid sequence of the
intracellular portion of ALK revealed potential sites for binding of
substrates such as Shc, PI3K, rasGAP, IRS1, Grb2, and PLC (5).
Coimmunoprecipitation studies with the NPM-ALK revealed a physical
association of the fusion protein with IRS1, Grb2, Shc, and PLC
(9,
20). Mutants of NPM-ALK that were defective for binding to and
phosphorylation of Shc or IRS-1 could transform NIH3T3 (9) cells,
indicating that these signaling molecules are not essential for cell
transformation. Finally, PLC
appeared to be an important downstream
target of NPM-ALK that contributes to its mitogenic activity and is
likely to be important in the pathogenesis of large-cell anaplastic
lymphomas, since knock-out of this single pathway was sufficient to
significantly impair NPM-ALK-mediated oncogenicity in lymphocytes
(20).
Using pharmacological inhibitors of the classic pathways coupled to
RTKs, we found that the ALK.Fc-induced neurite extension process
requires the MAP kinase but not the PI3K and PLC activities. Studies
at the molecular level confirmed the activation of the MAP kinase
pathway, because the amount of active forms of EKR1 and ERK2 was found
to be higher in PC12 cells expressing the pcDNA-ALK.Fc construct
versus the pcDNA-ALK.wt construct. Thus, these results indicated for the first time that the MAP kinase pathway can be involved in ALK signaling and that it was necessary for the promotion of neurite extension induced by this receptor.
In addition, these data call several remarks:
First of all, sustained activation of the MAP kinase signaling cascade seems to be essential for the differentiation processes induced by various neurotrophic factors in PC12 cells (21). For instance, stimulation of PC12 cells with NGF or bFGF led to sustained activation of MAP kinase and to neuronal differentiation (22). In contrast, stimulation of the same cells with insulin or EGF triggered transient activation of MAP kinase and did not lead to neuronal differentiation but to cell proliferation (22-25). Nevertheless, when insulin or EGF receptors were overexpressed, sustained activation of MAP kinase and neuronal differentiation was obtained with the respective ligands (17, 18), indicating that the level of receptor expression is critical for the induction of a marked and sustained activation of MAP kinase and neuronal differentiation. In our experiments, the constitutive activation of ALK.Fc probably led to sustained activation of MAP kinase. The fact that we were able to detect MAP kinase activation as late as 72 h post-electroporation supports this assertion.
Second, the PI3K pathway has been shown to be nonessential for neuronal differentiation (26) and has been proposed to be required for the prevention from apoptosis (27) but not for neurite extension (28, 29) promoted by NGF. Thus, these results are consistent with our data showing that PI3K is not involved in the neurite extension induced by ALK.Fc.
Third, mutations at either the PLC or Shc binding site on TrkA
(the high affinity NGF receptor) showed no defects in NGF-induced neurite outgrowth and MAP kinase activation, but mutations at both
sites did (30). These results indicate that both PLC
and Shc trigger
the MAP kinase cascade and that they can substitute for each other in
NGF signaling. In agreement with our data, these results point to a
pivotal role of the MAP kinase cascade in neuritogenesis and can
explain why the sole inhibition of PLC
activity with ET-18-OCH3 did not block the neurite extension process
induced by ALK.Fc. Therefore, it seems that PLC
activation is
crucial for the mediation of the oncogenic potential of NPM-ALK (see
above) (20) but nonessential for the neuronal differentiating activity of ALK.
In conclusion, our results showed that activation of the ALK
receptor tyrosine kinase led to neuronal differentiation and this
differentiating effect was mainly achieved through the MAP kinase
signaling pathway. Thus, these results suggest that ALK could be
involved in neuronal differentiation and present the first example for
a biological role assigned to ALK.
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ACKNOWLEDGEMENTS |
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We thank Dr. N. Doyen (Institut Pasteur, Paris) for providing us with the mouse IgG2b cDNA. We are grateful to M. Lambert for helpful discussions and to Drs. A. Sobel and R. Rotundo for comments on the manuscript.
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FOOTNOTES |
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* 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).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received a postdoctoral fellowship from ARC during this work.
§ To whom correspondence should be addressed: INSERM U 440, 17 rue du Fer à Moulin, F-75005 Paris, France. Tel.: 33-1-45-87-61-35; Fax: 33-1-45-87-61-32; E-mail: vigny@ifm.inserm.fr.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M007333200
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ABBREVIATIONS |
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The abbreviations used are:
RTK, receptor
tyrosine kinase;
ALK, anaplastic lymphoma kinase;
MAP kinase, mitogen-activated protein kinase;
PI3K, phosphoinositide-3 kinase;
PLC, phospholipase C
;
NPM, nucleophosmin;
NGF, nerve growth
factor;
bFGF, basic fibroblast growth factor;
bp, base pair(s);
PAGE, polyacrylamide gel electrophoresis;
PBS, phosphate-buffered saline;
FITC, fluorescein isothiocyanate;
ERK1/2, extracellular
signal-regulated kinase 1 and 2;
MEK, mitogen-activated protein
kinase/ERK kinase;
EGF, epidermal growth factor.
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REFERENCES |
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