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Originally published In Press as doi:10.1074/jbc.M501972200 on May 10, 2005

J. Biol. Chem., Vol. 280, Issue 28, 26039-26048, July 15, 2005
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Activation and Inhibition of Anaplastic Lymphoma Kinase Receptor Tyrosine Kinase by Monoclonal Antibodies and Absence of Agonist Activity of Pleiotrophin*{boxs}

Christel Moog-Lutz{ddagger}§, Joffrey Degoutin{ddagger}§, Jean Y. Gouzi{ddagger}, Yvelyne Frobert¶, Nicole Brunet-de Carvalho{ddagger}, Jocelyne Bureau{ddagger}, Christophe Créminon¶, and Marc Vigny{ddagger}||

From the {ddagger}INSERM, Unité 706/Université Pierre et Marie Curie, Paris F-75005 and the Service de Pharmacologie et d'Immunologie, Direction de la Recherche Médicale, Commissariat à l'Energie Atomique-Saclay, Gif/Yvette 91191, France

Received for publication, February 22, 2005 , and in revised form, May 6, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is transiently expressed in specific regions of the central and peripheral nervous systems, suggesting a role in its normal development and function. The nature of the cognate ligands of ALK in vertebrate is still a matter of debate. We produced a panel of monoclonal antibodies (mAbs) directed against the extracellular domain of the human receptor. Two major species of ALK (220 and 140 kDa) were identified in transfected cells, and the use of our mAbs established that the 140-kDa species results from a cleavage of the 220-kDa form. Two mAbs, in the nM range, induced the differentiation of PC12 cells transiently transfected with ALK. In human embryonic kidney 293 cells stably expressing ALK, these two mAbs strongly activated the receptor and subsequently the mitogen-activated protein kinase pathway. We further showed for the first time that activation of ALK also resulted in a specific activation of STAT3. In contrast, other mAbs presented the characteristics of blocking antibodies. Finally, in these cell systems, a mitogenic form of pleiotrophin, a proposed ligand of ALK, failed to activate this receptor. Thus, in the absence of clearly established ligand(s) in vertebrates, the availability of mAbs allowing the activation or the inhibition of the receptor will be essential for a better understanding of the biological roles of ALK.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Receptors tyrosine kinase (RTKs)1 play essential roles during the development of the nervous system, regulating a wide range of cellular processes such as proliferation, survival, differentiation, and synaptogenesis. Generally, after ligand binding, RTK dimerizes, autophosphorylates, and initiates signal transduction cascades that subsequently lead to cellular responses (for review, see Ref. 1).

Anaplastic lymphoma kinase (ALK) was originally identified as a RTK that acquires transforming capability when truncated and fused in the t(2;5) chromosomal rearrangement associated with the non-Hodgkin lymphoma (2). This translocation produces a fusion gene that encodes a soluble chimeric transforming protein comprising the N-terminal portion of the phosphoprotein nucleophosmin (NPM) linked to the cytoplasmic portion of ALK. It has been demonstrated that the NPM portion is responsible for the dimerization of the fusion protein leading to the constitutive activation of the kinase and to the transforming activity. Phospholipase C{gamma}, phosphatidylinositol 3-kinase, STATs, and Src appear to be important downstream targets of NPM-ALK which contribute to its mitogenic and antiapoptotic activities (3–7). ALK is also involved in different variant chromosomal translocations (for review, see Ref. 8), all leading to the expression of fusion proteins exhibiting a constitutive activation of the kinase.

Human, mouse, and Drosophila cDNAs encoding full-length ALK have been characterized (9–11). 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. ALK belongs to the insulin receptor subfamily of RTKs and is most closely related to leukocyte tyrosine kinase receptor. In situ hybridization analysis performed in rodents showed that alk mRNA 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 (9, 10). The neonatal brain showed the highest expression, suggesting a possible involvement of ALK in the development of the nervous system, and a recent report indicates that Caenorhabditis elegans ALK may play a role in presynaptic differentiation of the worm neuromuscular junctions (12). Because ALK expression is maintained, albeit at a lower level, in the adult brain, it might play an important role in both the normal development and function of the nervous system. In this context, we previously established a construct coding for a chimeric protein, named Fc.ALK, in which the extracellular domain of ALK was replaced by the mouse IgG Fc domain. The Fc domain induced the dimerization of the chimera leading to the constitutive activation of the kinase activity. Expression of Fc.ALK chimera in PC12 cells induced their differentiation (13). Analysis of the transduction pathways involved in this differentiation pointed out the essential role of the MAP kinase cascade.

Pleiotrophin (PTN) and midkine are developmentally regulated proteins forming a family of heparin-binding molecules with putative functions during cell growth and differentiation. PTN and midkine have been proposed as potential ligands of ALK (14–16), but recent studies performed by different groups do not confirm this hypothesis (17–19). In addition, the protein jelly belly (Jeb) has been identified as the ligand of Drosophila ALK (20, 21), and Jeb is distinct from the Drosophila homologs of PTN and midkine, Miple 1 and 2 (21). Finally, no obvious vertebrate homolog has been identified for Jeb in the sequence databases.

In the absence of clearly established cognate ligand(s) for the ALK receptor, the availability of ligand substitutes allowing the activation of the receptor can be essential for a better understanding of its biological roles. In the case of the RTKs that are activated after dimerization upon ligand binding, monoclonal antibodies directed against the extracellular domain of membrane-bound receptors can mimic the effect of the ligand. Thus, they have been largely used to study the mechanisms of receptor activation (22–28), and recently a report describing the agonist properties of a rat mAb reacting with the mouse ALK receptor has been published (19).

In this paper, we describe the characterization of a panel of mouse monoclonal antibodies directed against the human receptor. Two major species of ALK (220 and 140 kDa) were identified in transfected cells, and the use of our mAbs established that the 140-kDa species results from a cleavage of the 220-kDa form, leading to the release of an 80-kDa fragment into the culture medium. Two mAbs, in the nM range, strongly activate the receptor and downstream signaling pathways. In contrast, other mAbs present the characteristics of antagonist antibodies. Finally, in our cell systems, a mitogenic form of PTN failed to activate ALK.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies—Rabbit anti-insulin receptor phosphospecific (pY/pY1162/1163) was purchased from Biomol (Plymouth Meeting, PA). Rabbit anti-STAT3, rabbit anti-ERK1/2, and mouse anti-phospho-STAT5 (clone 8-5-2) were from Upstate%20Biotechnology">Upstate Biotechnology, Inc. (Lake Placid, NY). Rabbit anti-TrkA phosphospecific (pY674/pY675), rabbit anti-phospho-STAT1 (Y701), rabbit anti-STAT1, and rabbit anti-phospho-STAT3 (Y705) were from Cell Signaling Technology (Beverly, MA). Mouse anti-STAT5 (clone 89) and mouse anti-ALK (clone ALK1) were from BD Biosciences. Mouse anti-phospho-ERK1/2 (clone from MAP kinase-YT) was from Sigma. Rat anti-HA (clone 3F10) was from Roche Applied Science.

Human PTN synthesized by stably transfected SW13 cells was produced as described previously (29). Commercial human PTN expressed in Sf1 insect cells was obtained from Sigma. SDS-PAGE and Western blot analysis indicated that both PTNs essentially corresponded to the full-length molecule of 18-kDa apparent molecular mass.

Production of Anti-ALK Antibodies—The entire extracellular domain of human ALK linked to a His tag (RECA.His) was used previously to produce rabbit polyclonal antibodies (30), which were purified further and named REAB. Mice were immunized with the purified recombinant protein RECA.His. Immunization of mice, cell fusion, and screening of the hybridoma supernatants by a specific enzyme immunoassay using RECA.His either as immunogen or biotin-labeled as tracer were performed as described previously (31). The positive supernatants were analyzed further by immunocytochemistry on HEK 293 cells expressing human ALK protein. mAbs were purified from ascites fluids after precipitation with caprylic acid (32).

F(ab')2 fragments were obtained from the purified mAb 48 by treatment with pepsin in acidic medium. We then obtained Fab' fragments by reduction of F(ab')2 in the presence of 10 mmol/liter 2-mercaptoethylamine. Biotinylation of the Fab' fragment was performed as described previously (33). The purity of the different prepared fragments was analyzed by SDS-PAGE.

Plasmid Constructions—The full-length human ALK cDNA as well as a construct coding for a kinase-defective form in which the invariant lysine residue located in the ATP-binding portion of the catalytic domain was changed to arginine were inserted previously in the mammalian expression vector pcDNA3.1 (13). To generate stably transfected HEK cells, we used the dicistronic pCBC vector engineered to allow the coordinated expression of a protein and the pac gene which confer puromycin resistance to mammalian cells (a kind gift of Dr. W. Dirks, Germany). We generated constructs allowing the expression of both the wild type and the kinase-defective form of the receptor linked to an HA tag at its C-terminal end by directed mutagenesis. The proteins coded by these constructs were named, respectively, ALK.H for the active receptor and dALK.H for the dead mutant.

Cell Culture—HEK 293 cells obtained from ATCC (Manassas, VA) were maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen) at 37 °C in atmosphere containing 5% CO2. Stably transfected cells were cultured continuously in the same medium complemented by 2 µg/ml puromycin (Sigma). Cells plated at a density of 3 x 104 cells/cm2 were cultured for 2 days, serum-deprived for 12–16 h, and then treated or not with mAbs or PTN directly added in the medium.

Rat pheochromocytoma (PC12) cells (34) (kindly provided by T. Galli, INSERM U536, Paris, France) were grown in complete medium, which is RPMI 1640 supplemented with 10% horse serum and 5% fetal calf serum (Invitrogen) at 37 °C in atmosphere containing 7.5% CO2. Cells were cultured in flasks or plastic dishes coated with rat tail collagen (1 µg/cm2) (Roche Applied Science).

Cell Transfections—PC12 cells were transfected using the EasyJect Plus electroporation system as recommended by the manufacturer (Equibio, Ashford, UK). Cells were trypsinized, washed, and resuspended at 6.25 x 106 cells/ml of complete medium supplemented with 15 mM Hepes, pH 7.2. Electroporation was performed in a final volume of 0.8 ml of cell suspension using 30 µg of plasmids with one shock at 1,350 microfarads and 850 volts. Cells were transferred immediately to fresh culture medium and cultivated for 1 day before serum starvation. Before treatment with mAbs, cells were cultured in a low serum medium (1% horse serum) for 16 h and then treated or not with the different mAbs for 2 days.

HEK 293 cells were transfected using calcium phosphate coprecipitation (35) of 3 µg DNA vectors adjusted to 14 µg/10-cm Petri dish with pBluescript carrier DNA. Two days after transfection, cells were selected for their puromycin resistance, allowing the selection of stable cells expressing either the ALK.H or the dALK.H protein.

Neurite Outgrowth Assay—Transfected PC12 cells treated or not with mAbs for 2 days were fixed for 15 min with 2% paraformaldhehyde and 30 mM sucrose and washed three times with PBS before being permeabilized in 0.5% PBS and Triton X-100 for 5 min and washed with 0.1 M PBS and glycine for 15 min. After 1 h of blocking in PBS containing 1.5% bovine serum albumin, cells were incubated in the same buffer with rat anti-HA (3F10, 0.5 µg/ml) to visualize ALK.H-expressing cells. Then cells were washed five times with PBS before and after incubation with anti-rat IgG TRITC-conjugated secondary antibody selected for their minimal cross-reaction with mouse immunoglobulins (Jackson ImmunoResearch Laboratories, West Grove, PA). Then cells were mounted in Mowiol 4-88 supplemented with Hoechst 33258 nucleic acid stain (0.5 µg/ml, Molecular Probes) to visualize nuclei. Conventional fluorescence microscopy was performed on a Leica microscope (Roche Applied Science) equipped with a MicroMax CCD camera (Princeton Instruments, Roper Scientific, Trenton, NJ). Images were assembled using Adobe Photoshop software. The Metamorph software was used (Roper Scientific) for quantification; 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.

Cell Extracts and Immunoblotting—Cells washed rapidly with cold PBS buffer (containing 5 mM sodium fluoride and 100 mM sodium orthovanadate) were lysed in a radioimmune precipitation assay 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 {beta}-glycerol phosphate, 50 mM sodium fluoride, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and protease inhibitor Complete, Roche Applied Science). Lysates were clarified by centrifugation at 21,000 x g for 10 min at 4 °C. Protein concentration was determined by the method of Bradford using the Micro BCA Protein Assay Reagent Kit (Pierce). Cell extracts were denatured in NuPAGE LDS sample buffer (Invitrogen), resolved on 4–12% NuPAGE BisTris gels (Invitrogen), and then transferred to nitrocellulose membranes (Schleicher & Schuell). Analysis were performed either under nonreducing conditions or after reduction of the disulfide bridges by the reducing agent provided by Invitrogen. Membranes were blocked and incubated with antibodies as recommended by the manufacturers. Primary antibodies were detected using peroxidase-conjugated anti-mouse or anti-rabbit antibodies (Dako, Glostrup, Denmark) or anti-rat selected for its minimal cross-reaction with mouse immunoglobulins (Jackson ImmunoResearch Laboratories). Bound proteins were visualized using the ECL system (Amersham Bioscience). Alternatively, primary antibodies were detected using IRDye 800- or Alexa Fluor 688-conjugated second antibodies (Rockland). Binding of the fluorescent antibodies was visualized and quantified using the Odyssey Imaging System (LI-COR biosciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
To understand further the biological activities of endogenous ALK we developed a panel of mAbs directed against the entire extracellular domain of human ALK. The nature of the IgG types of the different mAbs is listed Table I.


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  		TABLE I
Properties of anti-ALK mAbs

The corresponding isotypes of the different anti-ALK mAbs are indicated. These mAbs were analyzed for their capacity, in immunoblot experiments, to detect (+) or not (–) the 220- and 140-kDa proteins in cell lysates and the 80-kDa fragment in the conditioned medium of 293/ALK.H cells. Electrophoresis of denatured cell lysates was performed under nonreducing conditions as described under "Experimental Procedures." The working dilution indicates those used to detect ALK protein(s) by Western blot analysis performed after NuPAGE electrophoresis of 10 µg of 293/ALK.H cell lysate. a, 10–50 ng/ml; b, 50–250 ng/ml; c, 250–750 ng/ml; and d, >750 ng/ml. mAb effects on ALK phosphorylation were analyzed by immunoblotting experiments of 10 µg of lysates of 293/ALK.H cells treated with 6 nM different mAbs for 30 min. ALK phosphorylation was revealed using an anti-phosphoinsulin receptor antibody and quantified using the Odyssey Imaging System (see "Experimental Procedures"). The experiments were performed in triplicate, and values are expressed as the mean ± S.E." The basal level of ALK phosphorylation in nontreated cells was scored as 1. The asterisk indicate that mAbs 14 and 58 only induced the phosphorylation of the 220-kDa protein.

 
Molecular Characterization of the ALK Receptor—To analyze the properties of the mAbs we established (see "Experimental Procedures") HEK 293 cells stably expressing human ALK linked to an HA tag at the C terminus. The stably transfected cells, named 293/ALK.H, were lysed, and cell extracts were first separated, after denaturation, in NuPAGE electrophoresis under nonreducing conditions and analyzed by Western blotting. We first used rabbit polyclonal antibodies (named REAB) directed against the entire extracellular domain of human ALK (30). In 293/ALK.H cell extract these antibodies showed two major species of ALK, 220 and 140 kDa (Fig. 1A). The same species had been reported previously in rat and mouse brain (9, 10) and in NIH-3T3 stably transfected with the mouse receptor (19). Two monoclonal antibodies directed against the intracellular domain of ALK.H (an anti-HA tag and ALK1 anti-human NPM-ALK (36)) revealed the two same forms (Fig. 1A). The presence of the HA tag in these two forms indicates that they both contain the C terminus and that they differ in their extracellular domains. The existence of these two forms in cells transfected with the ALK construct also indicated that they did not correspond to splicing products but that the lower molecular mass form could arise from a proteolytic cleavage of the full-length receptor. Interestingly, some mAbs reacted with the two bands of 220 and 140 kDa, whereas others recognized only the higher molecular mass (Fig. 1A and Table I). This result showed that the corresponding epitopes were located differently on the ALK molecule. Because the 140-kDa form likely resulted from a cleavage of the 220-kDa form, we hypothesized that resulting proteolytic product(s) could be released in the conditioned medium of the 293/ALK.H cells and should bear the epitopes of the mAbs reacting only with the 220-kDa form. To address this question, conditioned medium of 293/ALK.H cells cultured overnight in serum-free medium was collected and subjected to Western blot analysis with the different mAbs (Fig. 1B and Table I). A major band of about 80 kDa was clearly recognized by the mAbs which only reacted with the 220-kDa form of ALK. Thus, the 140-kDa species resulted from a proteolytic cleavage at a specific site of the 220-kDa form (Fig. 1C). The parental HEK 293 cells do not express a detectable amount of endogenous ALK receptor as revealed by the different antibodies (Fig. 1A).

The immunoreactivity of the different mAbs, analyzed by Western blotting, varied greatly among the different mAbs (Table I). However, these differences probably resulted from modifications of their corresponding epitopes caused by the SDS-PAGE and the Western blotting procedures. Immunocytochemical experiments or binding on living cells analyzed by a fluorescence-activated cell sorter did not reveal any major differences in their affinities for the native receptor expressed on the cell surface (not shown).

The extracellular domain of ALK contains 26 cysteine residues that could form intrachain disulfide bridges. Thus, reduction of such disulfide bridges could modify the binding of the different mAbs. Therefore, 293/ALK.H cell extracts were also analyzed, after denaturation, by Western blotting under reducing conditions as described under "Experimental Procedures." In these conditions, with the noticeable exception of mAbs 7 and 10, the immunoreactivity of all the other mAbs was either reduced greatly or totally inhibited (not shown). This finding indicates that the corresponding epitopes involved intrachain disulfide bridges that are likely essential for the conformational structure of the ALK extracellular domain. In good agreement with a previous report (10), treatment with tunicamycin of the 293/ALK.H cells reduced the apparent molecular mass to about 170 kDa, thus confirming that the 140-kDa species does not correspond to a precursor of the full-length 220-kDa form. In addition, deglycosylation did not impair the binding of the different mAbs, indicating that the corresponding epitopes did not involve N-linked glycans (not shown).

mAbs 46 and 48 Induced Neurite Extension of ALK-expressing PC12 Cells—We demonstrated previously that a constitutive dimerized and active form of ALK tyrosine kinase domain induced the neuron-like differentiation of PC12 cells (13). We reasoned that activation of the wild type ALK receptor expressed in PC12 cells should also lead to their differentiation. We therefore investigated whether some of our mAbs could induce the differentiation of the PC12 cells transiently transfected with the wild type receptor ALK.H construct.



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  		FIG. 1.
Characterization of anti-ALK monoclonal antibodies. A, immunodetection of ALK protein in 293/ALK.H cell extracts. 5 µg of protein lysates from HEK 293 cells stably transfected with HA-tagged human ALK expression vector (+) or with the corresponding empty vector (–) was denatured and separated by NuPAGE electrophoresis under nonreducing conditions and immunoblotted with anti-ALK (REAB, ALK1, and mAbs 46, 48, 7, 10) or anti-HA antibodies. 220- and 140-kDa ALK proteins are indicated by arrowheads. B, immunodetection of an 80-kDa protein in conditioned medium of HEK 293 cells stably expressing human ALK protein. Serum-free conditioned medium from 293/ALK.H cells cultured for 24 h was collected, separated by NuPAGE electrophoresis under nonreducing conditions, and immunoblotted with the indicated mAbs. C, schematic representation of ALK.H regions recognized by the different antibodies. The potential cleavage site of the ALK extracellular domain is indicated. TK is the tyrosine kinase domain of ALK, and the diamond represents the HA tag.

 
There was no discernible difference between cells transfected with the kinase-defective form of the receptor, dALK.H, and nontransfected cells. Only few of them, less than 5%, extended short and thin neurites, and whatever the mAbs used no differentiation process was observed (Fig. 2, A and B). In contrast, cells transfected with the construct coding for the wild type ALK.H receptor exhibited a low but significant level of basal differentiation of about 15%. This spontaneous differentiation likely resulted from the spontaneous dimerization of the receptor and the subsequent basal activation of its tyrosine kinase activity caused by the overexpression of the construct in some transfected cells (see below). In a first approach, PC12 cells expressing the wild type receptor were treated with increasing concentrations of the different mAbs ranging from 1 to 60 nM (not shown). This preliminary experiment led us to analyze quantitatively the effect of increasing concentrations of five selected mAbs, all reacting with the two forms of the receptor, on the differentiation of the transfected PC12 cells (Fig. 2B). When treated with mAbs 46 and 48 the percentage of transfected cells extending neurites was greatly increased, reaching a maximum of about 50% at a concentration of 6 nM (~1 µg/ml). mAb 15 also enhanced the neurite extension of the transfected cells but to a much smaller extend. At the highest concentration of this mAb (60 nM) only 30% of the transfected cells extended neurites. mAb 13 displayed no discernible effect whatever the concentration used and was chosen as a control mAb for the subsequent experiments. Finally and interestingly, mAb 30 clearly decreased the basal level of differentiation of the ALK.H-expressing cells reaching a level similar to that obtained with cells expressing the kinase-defective dALK.H protein. This strongly suggests that mAb 30 inhibited the basal ALK tyrosine kinase activity in transfected cells.

Effects of Antibodies on ALK Tyrosine Phosphorylation and MAP Kinase Activation—To analyze further the properties of the different mAbs we use the HEK 293 cell line. It is one of the most widely used cell lines to study signaling pathways, and in addition the HEK 293 cells have unexpected properties of neuronal lineage (37). HEK cells stably expressing the wild type human ALK receptor (293/ALK.H) and the kinase-defective form (293/dALK.H) were used.

To analyze the specific activation of ALK upon treatment with the different mAbs, we sought specific anti-phosphotyrosine antibodies revealing only or essentially the tyrosine phosphorylation of ALK. Classical anti-phosphotyrosine antibodies, such as the mAb 4G10, revealed the basal or induced phosphorylation of several proteins unrelated to ALK (not shown). ALK, like the Trk neurotrophin receptors, is a member of the insulin receptor subfamily. These receptors are all characterized by the presence in their catalytic sites of two adjacent tyrosine residues surrounded by conserved amino acids. We hypothesized that antibodies reacting either against the insulin receptor or against TrkA (the nerve growth factor receptor) when they are phosphorylated on these tyrosine residues could also cross-react with the phosphorylated ALK receptor. Similar results were indeed obtained with the two antibodies.



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  		FIG. 2.
mAb effects on neurite extension of PC12 cells expressing ALK.H. A, PC12 cells transfected with plasmids encoding wild type (ALK.H) or the kinase-defective (dALK.H) ALK protein were cultured for 2 days in the presence of 6 nM mAb 48. Transfected cells were fixed and labeled with the anti-HA antibody, and nuclei were counterstained with Hoechst. Bar = 20 mm. B, quantification of mAb effects on neurite outgrowth of PC12 cells expressing ALK.H or dALK.H was performed as indicated under "Experimental Procedures." PC12 cells transfected with plasmid encoding ALK.H protein were cultured for 2 days in the absence or presence of increasing concentrations of the indicated antibodies. Transfected PC12 cells expressing kinase-defective dALK.H protein were cultured for 2 days in the absence or presence of increasing concentrations of mAb 48. The experiment was performed in triplicate, and values are expressed as the mean ± S.E. (%).

 
We first incubated for 30 min 293/ALK.H cells with 6 nM concentrations (~1 µg/ml) of the five mAbs tested previously in the PC12 cell neurite extension assay. We showed previously that a constitutive active form of ALK, the Fc.ALK chimera, induced the activation of the MAP kinase pathway (13). We thus analyzed the level of ALK tyrosine phosphorylation and that of ERK activation triggered by the different mAbs (Fig. 3A). There was a weak but significant level of basal phosphorylation of ALK in untreated 293/ALK.H cells. This basal phosphorylation of ALK was correlated with a weak basal activation of ERK1/2. This basal activation of the tyrosine kinase activity likely resulted from the spontaneous dimerization of ALK in untreated cells. In fact, mAb 30 reduced both the degree of basal phosphorylation of ALK and the basal activation of ERK1/2 (see also the section "Blocking Effects of mAb 30"). mAb 13 had no discernible effect. In contrast, mAbs 46 and 48 strongly enhanced the phosphorylation of both the 220-kDa and the 140-kDa forms in the cells. No other phosphorylated proteins were detected (using the specific antibodies described above), and immunoprecipitation experiments indicated that these two bands indeed corresponded to ALK (not shown). Treatment with the two agonist mAbs 46 and 48 also resulted in a strong activation of ERK1/2. Finally, mAb 15 induced a much weaker phosphorylation of ALK (compared with mAb 46 or 48) correlated with a weaker activation of ERK1/2 activation.

Table I summarizes the effects of the different mAbs on the phosphorylation of ALK. Note that mAbs 14 and 58, which reacted only with the 220-kDa species in Western blot experiments, only increased the tyrosine phosphorylation of this form.

No basal or induced ALK phosphorylation and ERK activation was detected in HEK cells stably expressing the kinase-defective form (293/dALK.H) (Fig. 3B). These results indicated that the specific antibodies described above only revealed ALK phosphorylation and that ERK activation indeed resulted from the kinase activity of ALK.

Characterization of the Agonist Properties of mAb 48 —Because the preceding data indicated that mAb 48 exhibited a high agonist activity, we essentially analyzed the properties of this mAb.

We first investigated the mechanism of activation by this agonist mAb. Two possible mechanisms of ALK activation could be proposed. Either the antibody induced an adequate conformational change of the receptor, or more likely, the bivalent antibody led to the formation of receptor homodimers and subsequent activation of the kinase similar to that obtained with the Fc.ALK chimera. To answer this question we produced the monovalent fragment Fab' of mAb 48. 293/ALK.H cells were treated for 30 min with 6 nM Fab' fragment. Compared with mAb 48 used at the same concentration, no induced phosphorylation of ALK or activation of the MAP kinases ERK1/2 was noticed. Furthermore, preincubation of the cells with the Fab' fragment before the addition of the agonist mAb led to a strong inhibition of both the phosphorylation of ALK and the activation of the MAP kinases (Fig. 3C). Thus, the Fab' fragment competed with the mAb for the binding to ALK. These data indicate that the agonist mAb acted as a dimerizing agent.



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  		FIG. 3.
mAb effects on ALK and ERK1/2 activation. A, analysis of ALK and ERK1/2 phosphorylation induced by the different mAbs. 293/ALK.H cells were untreated (–) or treated for 30 min with a 6 nM concentration of the indicated mAbs. B, ALK kinase activity is required for basal and induced ALK phosphorylation and ERK1/2 activation. ALK and ERK1/2 phosphorylation was analyzed in 293/ALK.H or 293/dALK.H cells untreated or treated with 6 nM mAb 48 for 30 min. C, bivalent mAb 48 is required for ALK activation. 293/ALK.H cells were pretreated (+)or not (–) for 1 h with 20 nM Fab' 48 and cultivated further for 30 min in the absence (–) or presence (+) of 6 nM mAb 48. In A–C,15 µg of cell lysates separated by NuPAGE electrophoresis was blotted on two membranes. The upper part of one membrane was probed with anti-phosphoinsulin receptor, and the lower part with anti-phospho-ERK1/2 antibodies. The other membrane was probed with anti-HA for the upper part and with anti-ERK1/2 for the lower part.

 
To perform a dose-response analysis, we incubated 293/ALK.H cells with increasing concentrations of mAb 48, ranging from 0.05 to 150 nM, for 30 min (see supplemental Fig. S1). The degree of induced tyrosine phosphorylation of ALK by the agonist mAb was a function of the concentration of mAb used, and a maximum effect was achieved with a concentration of 6 nM. We then analyzed the time course of ALK activation in 293/ALK.H cells triggered by 6 nM mAb 48 (Fig. 4A). A significant increase of phosphorylation was detected after 5 min, and the maximum was reached after 15–30 min. Interestingly, only a slight decrease in the degree of phosphorylation was noticed after a prolonged exposure (24 h) to the mAb.

We next analyzed the phosphorylation of the MAP kinases ERK1/2 resulting from ALK activation. We first analyzed the degree of phosphorylation of ERK1/2 in 293/ALK.H cells treated for 30 min with increasing concentrations of mAb 48. The maximum of ERK activation was achieved with 1.2 nM mAb 48, whereas that of ALK required 6 nM (see supplemental Fig. S1). We next analyzed the kinetic of activation of the MAP kinases ERK1/2 in 293/ALK.H cells treated with 6 nM mAb 48 for varying periods of time (Fig. 4A). Similarly, there was a shift between the kinetic of activation of the receptor and that of the kinases ERK1/2. The activation of ERK1/2 reached a maximum after 10 min, whereas that of the receptor was only achieved after 15–30 min (Fig. 4A). The simplest explanation is that the maximal activation of ERK1/2 can be reached as soon as a small fraction of ALK receptor molecules is activated. The activation of ERK1/2 persisted after continuous exposure to the mAb. A slight decrease, however, was observed after 6 h, which appeared more pronounced than that of the receptor. Similar results were obtained with mAb 46 (not shown).

STAT3 Is Specifically Activated by the Agonist mAbs—The STAT pathway has been related to the activation of some RTKs (38, 39), and the oncogenic NPM-ALK protein has been shown to trigger the activation of both STAT3 and STAT5 (5, 40–42). However, ALK is involved in various chromosomal translocations, and a recent report showed that some but not all resulting fusion proteins differentially induced STAT3 phosphorylation (43). Because STAT1, 3, and 5 are expressed by HEK cells, we investigated whether the full-length form of ALK could activate these factors. 293/ALK.H cells were incubated with 6 nM mAb 48 for varying periods of time. Activation through phosphorylation of STAT1, 3, and 5 was revealed by immunoblotting with specific phosphoantibodies. There was no activation of STAT1 and STAT5 in response to ALK activation by mAb 48 (Fig. 4B). To demonstrate that STAT1 was functional, we treated 293/ALK.H cells with interferon-{gamma} (44) (Fig. 4B). As a positive control for STAT5 phosphorylation, we treated with prolactin HEK 293 cells expressing the prolactin receptor after transfection (45) (Fig. 4B). Thus, both STAT1 and 5 can indeed be activated in HEK 293 cells, but no activation of these factors was detected when 293/ALK.H cells were treated with mAb 48 (Fig. 4B), and similar results were obtained with mAb 46 (not shown). In contrast, in 293/ALK.H cells, we detected a basal level of STAT3 phosphorylation that was increased strongly upon ALK activation. This induced phosphorylation was detected after 5 min, reached a maximum after 30 min (Fig. 4B), and was sustained for 24 h. There was a strong correlation between ALK and STAT3 phosphorylation in both kinetic (Fig. 4, A compared with B) and dose-response (not shown) experiments of 293/ALK.H cells treated with mAb 48. Similar results were obtained with 293/ALK.H cells treated with mAb 46 (not shown).



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  		FIG. 4.
Time course analysis of ERK1/2 and STAT phosphorylation induced by mAb 48 activation of ALK. A, ALK and ERK1/2 phosphorylation was analyzed in 293/ALK.H cells untreated or treated with 6 nM mAb 48 for the periods indicated (2 min–24 h). B, specific activation of STAT3 induced by mAb 48. 293/ALK.H cells were untreated or treated with 6 nM mAb 48 for the indicated times. C shows the activation of STAT1 in 293/ALK.H cells treated for 15 min with 10 ng/ml interferon-{gamma}. C' shows the activation of STAT5 in HEK 293 cells expressing the prolactin receptor after transfection and treated for 10 min with 1 µg/ml prolactin. C'' shows the activation of STAT3 in HeLa cells treated for 5 min with 100 ng/ml interferon-{alpha}.

 
In HEK cells stably expressing the kinase-defective form no basal or induced phosphorylation of STAT3 was detected (not shown). Thus, both basal and induced ERK and STAT3 phosphorylation in 293/ALK.H cells indeed resulted from the kinase activity of ALK.

Blocking Effects of mAb 30 —mAb 30 reduced the basal differentiation of the PC12 cells transfected with ALK (Fig. 2B) and both the degree of basal phosphorylation of ALK and the basal activation of ERK1/2 in 293/ALK.H cells (Figs. 3A and 5B). Together these results strongly suggested that the basal phosphorylation of ALK resulted from a weak spontaneous dimerization of the receptor occurring in the absence of mAbs, which is correlated with a basal activation of ERK1/2. Thus, mAb 30 likely dimerized and blocked two receptor molecules in a conformational state in which no trans-activation of the tyrosine kinase domain can occur.

Thus, this mAb could potentially act as a blocking antibody. In the absence of available cognate ligand, we first investigated whether mAb 30 could antagonize the agonist activity of mAb 48 in the model of PC12 cells expressing ALK.H. Transfected PC12 cells were preincubated for 1 h with increasing concentrations (0.6–60 nM) of mAb 30 before the addition of 6 nM agonist antibody 48. There was a clear inhibition effect of mAb 30 upon the induced neurite outgrowth triggered by mAb 48 (Fig. 5A). However, even at the highest concentration of mAb 30 the inhibition was not total, and the percentage of transfected cells extending neurites was higher than in the control or than in cells incubated with mAb 30 only. During the time course of the experiment (48 h) there was a continual synthesis and incorporation of new ALK receptors into the plasma membrane, and there was likely a competition between the agonist mAb 48 and the antagonist mAb 30 for the new synthesized receptors. We also analyzed the blocking effects of mAb 30 in 293/ALK.H stably expressing this receptor. 293/ALK.H cells were incubated for 1 h with increasing concentrations of mAb 30 ranging from 6 to 60 nM. mAb 48 was then added at a concentration of 6 nM for an additional hour. Fig. 5B shows the tyrosine phosphorylation of the receptor and ERK activation in the presence or absence of mAb 30. mAb 30 clearly inhibited both the phosphorylation of the receptor and the activation of ERK1/2 induced by mAb 48. Control mAb 13 did not exhibit any inhibitory effect. Thus, mAb 30 has the ability to block the agonist activity of the mAb 48.

We next investigated whether mAb 30 and 48 reacted with different epitopes. To address this question we used, in immunocytochemical experiments, a biotinylated form of the monovalent fragment Fab' of mAb 48. 293/ALK.H cells were first incubated or not with saturating concentrations of mAb 30 or 48 and then incubated with the biotinylated Fab' fragment. Preincubation with mAb 48 completely inhibited the binding of the Fab' fragment, whereas mAb 30 had no discernible effect (data not shown). Thus, this result demonstrates that mAbs 30 and 48 reacted with different epitopes and that mAb 30 inhibited the agonist activity of mAb 48 in a noncompetitive manner.

The same inhibitory effect of mAb 30 was obtained using the mAb 46 as an agonist mAb (not shown), and mAb 11 had the same blocking properties (not shown).



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  		FIG. 5.
mAb 30 acts as a blocking antibody. A, mAb 30 blocks the differentiation of transfected PC12 cells induced by mAb 48. PC12 cells transfected with plasmids encoding wild type (ALK.H) receptor were preincubated or not for 1 h with increasing concentrations (0.6–60 nM) of mAb 30 before the addition of 6 nM agonist antibody 48. Quantification of mAb effects on neurite outgrowth of PC12 cells was performed 2 days later as indicated under "Experimental Procedures" and in Fig. 2. Percentages of PC12 cells extending neurites cultured for 2 days in the absence of any mAb or only with mAb 30 (6 nM) are shown as a control. B, mAb 30 antagonizes the agonist activity of mAb 48. ALK, ERK1/2, and STAT3 activation was analyzed in 293/ALK.H cells untreated or treated for 1 h with either a single mAb: 6 or 60 nM mAb 30, 6 nM mAb 13, and 6 nM mAb 48 (five left lanes) or preincubated for 1 h with 6 and 60 nM mAb 30 or 6 and 60 nM mAb 13 and further cultivated for 1 h in the presence of 6 nM mAb 48 (right four lanes).

 
PTN, Ligand of ALK?—Recently, PTN has been proposed as a potential ligand of ALK (14, 16, 46), but three recent reports did not confirm this hypothesis (17–19). We tested the potential agonist activity of two batches of PTN: a recombinant eukaryotic PTN purified from the conditioned medium of stably transfected SW13 (named PTN-SW13) and a commercial PTN expressed in Sf1 insect cells (named PTN-SF1). We showed previously that PTN-SW13 was mitogenic for a variety of endothelial cells (47), and PTN-SF1 has been recently reported to activate ALK (48). These two batches of PTNs were first added to ALK.H-transfected PC12 cells. We reasoned that if PTN was a ligand of ALK, the addition of PTN should activate this receptor and induce the differentiation of PC12 cells similar to that obtained with the agonist mAbs. No differentiating effect was visualized (supplemental Fig. S2A).



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  		FIG. 6.
Analysis of PTN effects. Analysis of ALK phosphorylation, ALK expression, and ERK activation in 293/ALK.H cells and nontransfected HEK 293 cells. Left four lanes, 293/ALK.H cells were untreated or treated for 10 min with 100 ng/ml PTN-SW13 with or without preincubation (30 min) with 60 nM blocking antibody mAb 30. Right five lanes, HEK 293 cells were untreated or treated for 30 min with 6 nM agonist mAbs 46 and 48. HEK cells were cells preincubated or not for 30 min with 60 nM mAb 30 and then treated for 10 min with 100 ng/ml PTN. Note that PTN-SW13, in contrast to mAbs 46 and 48, induced the weak phosphorylation of a 150-kDa protein both in 293/ALK.H cells and in the parental cell line (asterisk, *).

 
The ERK activation resulting from the activation of ALK in the 293/ALK.H cells by the agonist mAbs appeared as a highly sensitive assay (see Figs. 3A and 4A). The same two batches of PTN were first added at various concentrations to the 293/ALK.H cells for 10 min, and activation of ERKs kinases was analyzed by Western blotting. The commercial PTN was found inactive. The PTN purified from the conditioned medium of stably transfected SW13 induced a strong activation of ERK1/2 but no modification of phosphorylation of ALK and STAT3 (supplemental Fig. S2B). In addition, preincubation with the antagonist antibody mAb 30 did not inhibit the ERK activation triggered by the PTN-SW13 batch (Fig. 6). The parental nontransfected HEK 293 cells did not express detectable amounts of ALK, at least analyzed by Western blotting (see Figs. 1 and 6), and subsequent addition of the agonist mAbs 46 and 48 to these cells did not result in a measurable ERK activation (Fig. 6). If PTN corresponds to the ALK ligand it should activate ERKs in the stably 293/ALK.H-transfected cells and not in the parental cell line. Activation of ERK1/2 by PTN-SW13 was found similar in the 293/ALK.H cells, and the parental cell line and the antagonist antibody mAb 30 did not exhibit any blocking effect (Fig. 6). Finally PTN-SW13, in contrast to mAbs 46 and 48, induced the weak phosphorylation of a 150-kDa protein both in 293/ALK.H cells and in the parental cell line (Fig. 6). Thus, in this cell system ALK did not transduce the biological activity of PTN-SW13.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Monoclonal antibodies directed against the extracellular domain of membrane-bound receptors always constitute useful tools to analyze their biochemical properties further. In addition, in the case of RTKs, these mAbs could mimic the agonist activity of the natural ligand. The classic working hypothesis is that the bivalent antibodies led to the formation of receptor homodimer and subsequent activation of the kinase similar to that usually triggered by the natural ligand. To date several agonist mAbs directed against various RTKs have been produced (22–28). In the case of the ALK receptor, in the absence of clearly established cognate ligand(s), the availability of ligand substitutes allowing the activation of the receptor can be essential to a better understanding of its biological roles. For all of these reasons, we decided to produce mAbs directed against the extracellular domain of human ALK. Several mAbs enhanced at different degrees the tyrosine phosphorylation of ALK, but two of them exhibited a much higher activity. In addition, we showed further that the agonist activity of mAb 48 indeed required the formation of receptor dimer. However, the dimerization by itself is not sufficient to explain the agonist properties of some of our mAbs. They also should induce an adequate conformational change allowing the activation of the tyrosine kinase domain. This can explain the poor agonist activity of mAb 15 compared with mAb 46 or 48.

Besides the agonist or antagonist properties of our mAbs, the characterization of the ALK receptor by Western blotting deserves several comments. Two major species of ALK of 220 and 140 kDa were identified in the stably transfected HEK cells. These two forms do not correspond to splicing products, and our analysis indicates that the 140-kDa species results from a cleavage at a specific site of the full-length receptor and that the product of this cleavage was released into the medium. Because the two species were found in brain extracts (9, 10), this cleavage could correspond to a physiological process, and the biological significance of the existence of these two forms of ALK remains to be determined.

While this work was in progress, a report by Motegi and co-workers describing the agonist properties of a rat mAb reacting with the mouse ALK receptor was published (19). These authors also used a model of cells (NIH-3T3) stably expressing the mouse receptor. The level of expression of ALK in their clone compared with our 293/ALK.H cells is of course unknown, which does not allow a direct comparison between the two studies. However, the agonist properties of this mAb in term of concentration range inducing the activation of both ALK and downstream signaling pathways appeared similar to those of mAb 48. As pointed out by these authors (19), mAbs reacting with different RTKs, so far described in the literature, usually promote phosphorylation of receptors at much higher concentrations, and few evoke downstream signaling. Thus, the structure of the extracellular domain ALK may be more appropriate than those of other RTKs to obtain powerful agonist mAbs. In cell extracts of NIH-3T3 transfected with the mouse receptor, Motegi and co-workers identified an additional form of ALK of about 85 kDa, which exhibited kinetics of induced phosphorylation upon ALK activation similar to that of the 140- and 220-kDa species (19). In our 293/ALK.H cell extracts no band around 85 kDa was identified with antibodies reacting against either the extra- or the intracellular domain of ALK, and of course no corresponding phosphorylated band was visualized after activation of ALK. Two logical hypotheses can be proposed to explain this apparent discrepancy. Either the mouse and human receptors differ in their potential sites of proteolytic cleavages, or the proteases expressed in HEK and NIH-3T3 cells are different.

ALK, being essentially expressed during the development of the nervous system (9, 10), could control both neuronal differentiation and survival. We essentially investigated two downstream signaling pathways usually involved in theses processes: the MAP kinases and the STAT pathways. Activation of ALK resulted in a fast and sustained activation of MAP kinases. This pathway is required for neuronal differentiation of the PC12 cells (49, 50). In this context the present data concerning the controlled activation of ALK in transfected PC12 cells corroborate those obtained previously in PC12 cells expressing the Fc.ALK chimera (13). NPM-ALK has been shown to activate the transcriptional factors STAT3 and STAT5 (5, 40, 41). We demonstrated that the full-length membrane-bound receptor is actually able to activate STAT3 but not STAT1 and STAT5. Thus, it remains to be determined whether the activation of STAT5 is an intrinsic property of the cytosolic NPM-ALK which is not shared by the membrane-bound ALK receptor or whether it depends on the cell type in which these proteins are expressed. Upon activation of ALK, STAT3 was indeed phosphorylated, and the kinetics of STAT3 phosphorylation roughly paralleled that of the receptor. The mechanism of activation of STAT3 remains to be determined. It could be phosphorylated on tyrosine residues either directly by the receptor or by cytoplasmic kinases such JAKs or Src. In this context it has been reported previously that STAT3 activation triggered by NPM-ALK did not require JAK3 or Src family kinase (41). The STAT pathways have been involved in neuronal survival (51–53). Thus, ALK, through this signaling pathway, could contribute to neuronal survival during development. Two reports demonstrated that NPM-ALK can up-regulate phosphorylated AKT (4, 54). However, we did not detect any significant change of the phosphorylated level of AKT in 293/ALK.H cells. The level of phosphorylated AKT was identical in the parental HEK cell line, in the HEK cells expressing the dALK.H kinase-defective form, or in the 293/ALK.H cells treated or not with our agonist mAbs (not shown). Thus, this signaling pathway may only be operational in specific cell types. Indeed, the absence of regulation of AKT phosphorylation by NPM-ALK in different cell lines has been documented previously (41).

Another important finding of our study is the potential antagonist activity of several mAbs. mAb 30, for example, reduced the degree of basal phosphorylation of ALK in the absence of any treatment. This latter result strongly suggests that the basal phosphorylation of ALK resulted from a weak spontaneous dimerization of the receptor occurring in the absence of mAbs. More interestingly, mAb 30 clearly inhibited both the phosphorylation of the receptor and the activation of ERK1/2 induced by the agonist mAbs. This mAb could constitute a blocking antibody of the cognate ligand when its identity will be fully confirmed. In addition, this mAb could interfere with the basal activation of ALK and downstream signaling pathways in particular when this receptor is overexpressed like in some neuroblastoma cell lines as described by Miyake and co-workers (18). In this case, the inhibition of the basal activation of ALK could lead to the inhibition of both downstream signaling pathways and cell proliferation.

Recently, PTN has been proposed as a potential ligand of ALK (14, 16, 46), but three recent reports do not confirm this hypothesis (17–19). However, the commercial PTNs used by these different authors likely did not display any mitogenic activity. The commercial PTN used in this work, expressed in Sf1 insect cells, was found inactive, in good agreement with several reports indicating that only PTNs processed by high eukaryotic cells exhibit mitogenic or angiogenic activities (55–57). More puzzling, PTN purified from the conditioned medium of stably transfected SW13 cells clearly induced a strong and rapid activation of ERK1/2, but this activation did not depend on ALK. This activation was effective at the same level and with the same kinetics (not shown) both in the parental HEK 293 cell line, which did not express ALK, and in the ALK stably transfected 293/ALK.H cells. Furthermore in the nontransfected HEK 293 cells, the agonist mAbs failed completely to activate the ERK pathway. Finally our potential antagonist mAbs did not interfere with this activation in the two cell lines. Thus, in our cell systems ALK did not correspond to the PTN receptor. We have no simple hypothesis to explain the discrepancies between our data and the strong evidence reported by the Wellstein group (14, 16, 46) because the PTN used in the two studies had the same origin. One can propose that PTN is acting in the HEK 293 cells through a different receptor expressed endogenously. This hypothesis supposes that PTN binds with a much stronger affinity to that unknown receptor than to ALK because expression of ALK in the 293/ALK.H cells did not modify the PTN activity. A second possible and complementary explanation, as proposed by Motegi and co-workers (19), is the requirement of a specific coreceptor not expressed in PC12, HEK (our study), and NIH-3T3 cells (19). This point also seems unlikely because these cell lines were actually used in the different reports describing the activation of ALK by PTN (15, 16).

Preliminary experiments indicate that some of our mAbs (including mAbs 30, 46, and 48) cross-react with the rat and mouse receptor. We are currently studying the localization of the ALK protein in rodents. It will be essential to have present indications, at the protein level, of the tissue and cellular localization of ALK during mouse development. Taking account of this localization and in the absence of clearly established ligand(s) in vertebrates, our mAbs will allow the activation or the inhibition of the ALK receptor in neural cells where it is endogenously expressed and therefore will be essential to a further understanding of the biological roles of this receptor during the development of the nervous system.


   FOOTNOTES
 
* 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 and the Association Française contre les Myopathies. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. Back

§ These authors contributed equally to this work. Back

|| To whom correspondence should be addressed: INSERM Unité 706/UPMC, Institut du Fer à Moulin, 17 rue du Fer à Moulin, Paris F-75005, France. E-mail: vigny{at}fer-a-moulin.inserm.fr.

1 The abbreviations used are: RTK, receptor tyrosine kinase; ALK, anaplastic lymphoma kinase; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ERK, extracellular signal-regulated kinase; HA, hemagglutinin; HEK, human embryonic kidney; mAb, monoclonal antibody; MAP, mitogen-activated protein; NPM, nucleophosmin; PBS, phosphate-buffered saline; PTN, pleiotrophin; STAT, signal transducer and activator of transcription; TRITC, tetramethylrhodamine isothiocyanate. Back


   ACKNOWLEDGMENTS
 
We thank Dr. I. Dusanter-Fourt for the positive control of STAT5 activation. We are grateful to Dr. W. Dirks for the suggestion concerning the use of antibodies directed against P.TrkA. We thank P. Lamourette, K. Moreau, and M. Plaisance for expert technical assistance. We are particularly indebted to Dr. A. Sobel for helpful comments on the manuscript.



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