¹Ludwig Institute for Cancer Research, Box 595, S-751 24, Uppsala, Sweden

²CNRS UMR 146, Institut Curie-Section de Rechèrche, Centre Universitaire, 91405 Orsay, France

Correspondence to: Arne Östman, Ludwig Institute for Cancer Research, Box 595, S-751 24, Uppsala, Sweden

T Sjöblom and A Boureux contributed equally to this paper

The t(5;12) translocation, associated with chronic myelomonocytic leukemia, generates a novel gene encoding a protein, TEL-PDGFR, composed of the 154 amino-terminal amino acids of the transcription factor TEL and the transmembrane and intracellular part of the PDGF -receptor (PDGFR). TEL also occurs as a tumor-associated fusion partner for the tyrosine kinases c-ABL, JAK2 and TRK-C. Previous studies have demonstrated growth promoting activity of TEL-PDGFR and also indicated that the TEL moiety activates the tyrosine kinase of the PDGFR through the formation of TEL-PDGFR oligomers. We demonstrate that tyrosine phosphorylation of the fusion protein can be attenuated through overexpression of the TEL part of TEL-PDGFR, suggesting a strategy for antagonizing the signaling of TEL-PDGFR, and other TEL-fusion proteins containing tyrosine kinase domains. Comparison of BaF/3 cell lines expressing TEL-PDGFR and ligand-stimulated PDGFR revealed that only TEL-PDGFR expression conferred IL-3-independent growth, suggesting differences in signaling capacity of the two proteins. Finally, tyrosine residues 17 and 27 in TEL-PDGFR was identified as autophosphorylation sites in TEL-PDGFR.

chronic myelomonocytic leukemia; chromosomal translocation; platelet-derived growth factor; TEL

Introduction

Chromosomal rearrangements leading to fusion of protein oligomerization domains to tyrosine kinase domains occur in a number of malignancies (Rabbitts, 1994; Sawyers and Denny, 1994). The t(5;12) translocation, associated with chronic myelomonocytic leukemia (CMML), generates a fusion gene encoding a protein composed of the 154 amino-terminal amino acids of TEL and the transmembrane and intracellular tyrosine kinase domain of the PDGF -receptor (PDGFR) (Golub et al., 1994).

The PDGFR is composed of an extracellular part, consisting of five Ig-like domains, a transmembrane segment and an intracellular part containing a split tyrosine kinase domain. PDGF-BB, the ligand of the PDGFR, induces activation of PDGFR through dimerization of receptors. Dimerization of receptors leads to activation of the intrinsic kinase activity and subsequent autophosphorylation of tyrosine residues in the intracellular part, which serve as docking sites for SH-2-domain-containing signaling proteins (Heldin et al., 1998). Dysregulated activation of the PDGFR, through ectopic ligand production, has been suggested to contribute to malignant cell growth in a number of different tumor types, including glioblastomas, mesotheliomas and sarcomas (reviewed in Heldin and Westermark, 1996). Also, in addition to the TEL-PDGFR fusion protein, two other genetic rearrangements giving rise to fusion proteins containing the kinase domain of the PDGFR has recently been identified; the CEV14-PDGFR and HIP-PDGFR genes isolated from an AML and a CMML patient, respectively (Abe et al., 1997; Ross et al., 1998).

TEL, also known as ETV6, is a 452 amino acid residue protein of the ETS family of sequence specific transcription factors (Ghysdael and Boureux, 1997). The ETS family members contain a conserved DNA binding domain, the ETS domain. A subset of ETS proteins, including TEL, also contains a conserved amino-terminal domain termed the pointed domain/B domain. This domain has been shown to be necessary and sufficient for homophilic self-association of TEL-PDGFR (Carroll et al., 1996; Jousset et al., 1997). Malignancy-associated chromosomal rearrangements leading to fusion of the pointed/B domain of TEL to a number of other tyrosine kinases, including c-ABL, JAK2 and TRK-C, have recently been characterized (Golub et al., 1995; Knezevich et al., 1998; Lacronique et al., 1997; Papadopoulos et al., 1995). In vitro and in vivo transforming ability of TEL-PDGFR and TEL-JAK2 has been demonstrated (Carroll et al., 1996; Jousset et al., 1997; Schwaller et al., 1998; Thomasson et al., 1999). TEL has also been described as a fusion partner in human leukemias for other transcriptional regulators like AML1 and CDX2 (Chase et al., 1999; Golub et al., 1995; Romana et al., 1995).

The aim of the present study was to further investigate the importance of TEL-mediated oligomerization for the activation of TEL-PDGFR and to compare the signaling properties of TEL-PDGFR and ligand-activated PDGFR. We show that overexpression of the TEL moiety leads to reduction of tyrosine phosphorylation of the fusion protein. We also demonstrate differences in the signaling capacity between ligand-stimulated PDGFR and TEL-PDGFR and provide evidence for the presence of unique autophosphorylation sites in TEL-PDGFR.

Results

TEL-PDGFR-stimulated IL-3-independent growth of BaF/3 cells is blocked by the PDGF receptor kinase inhibitor AG1296

TEL-PDGF is a constitutively active tyrosine kinase able to relieve the IL-3 requirement of BaF/3 cell survival and proliferation (Carroll et al., 1996; Jousset et al., 1997). To evaluate the role of the tyrosine kinase activity of TEL-PDGFR in its ability to stimulate IL-3 independent growth of BaF/3 cells, the effects of the selective PDGF receptor inhibitor AG1296, which blocks ATP binding of the receptor, was investigated (Kovalenko et al., 1994).

As shown in Figure 1a, culturing in the presence of the inhibitor reduced, in a dose-dependent manner, the growth of TEL-PDGFR-BaF/3 cells. The effect of AG1296 on the growth of TEL-PDGFR-BaF/3 was concluded to be mediated through targeting of the tyrosine kinase activity of TEL-PDGFR since no effect was observed either on BaF/3 cells stably expressing the v-mpl oncoprotein or on TEL-PDGFR-BaF/3 cells cultured in the presence of IL-3 (Figure 1a). The inhibitory effect of AG1296 on TEL-PDGFR tyrosine kinase activity was directly demonstrated by analysing the immunoprecipitated fusion protein with respect to tyrosine phosphorylation (Figure 1b). As shown, inhibition of tyrosine phosphorylation of TEL-PDGFR occurred in the same concentration range as was required to suppress the growth of TEL-PDGFR-BaF/3 cells.

We thus conclude, in agreement with previous studies (Carroll et al., 1996, 1997; Jousset et al., 1997), that the tyrosine kinase activity of TEL-PDGFR is required for its ability to stimulate IL-3-independent growth of BaF/3 cells.

TEL-PDGFR occurs as cross-linkable multimers in vivo

The pointed/B domain of TEL has been shown to be sufficient to mediate homophilic protein - protein interactions (Jousset et al., 1997). To investigate if TEL-PDGFR occurred as dimers and/or multimers in vivo, the fusion protein was transiently expressed in COS cells and analysed by immunoprecipitation and immunoblotting after the addition to the cell lysate of a covalent cross-linker, bis(sulfosuccinimidyl)suberate. Unstimulated and ligandstimulated wildtype PDGFR were analysed in parallel. As shown in Figure 2, dimeric forms of the wild-type PDGFR was detected in a ligand-dependent manner (Figure 2, lanes 3 - 6). In the case of TEL-PDGFR, which as a monomer migrates as a component with an M_r of approximately 90 000 in SDS gel-electrophoresis, addition of cross-linker to cell lysates generated multiple component(s) with M_rs of 250 000 - 500 000. No distinct component of the expected size of a dimer was detected (Figure 2, lanes 7 and 8).

These results thus confirm the ability of the TEL-moiety of TEL-PDGFR to mediate homophilic interactions. However, in contrast to the ligand-stimulated wild-type PDGFR which occurs as a dimer, TEL-PDGFR appears to exist predominantly as oligomeric or multimeric complexes. The observation that TEL-PDGFR eluted very close to the V_o fraction, when analysed by size-exclusion chromatography, lends further support to this conclusion (data not shown).

Overexpression of the TEL-moiety reduces the tyrosine phosphorylation of TEL-PDGFR

The mechanism underlying the constitutive tyrosine phosphorylation of TEL-PDGFR has been suggested to involve pointed/B domain-mediated oligomerization of TEL-PDGFR (Carroll et al., 1996; Jousset et al., 1997). To experimentally test this hypothesis we investigated the effects of co-expression of a truncated form of TEL-PDGFR, lacking the kinase domain (HA TP-0tt) (Jousset et al., 1997), on TEL-PDGFR tyrosine phosphorylation.

COS cells were transiently transfected either with an HA-tagged form of TEL-PDGFR alone, or together with an excess of HA TP-0tt. Expression of the two proteins was detected by immunoprecipitation with an antiserum against the HA-epitope followed by immunoblotting with an HA monoclonal antibody (Figure 3). The expression levels of TEL-PDGFR were similar in both transfections (Figure 3, middle panel). To determine the extent of tyrosine phosphorylation of TEL-PDGFR, the filter was reprobed with phospho-tyrosine antibodies. As shown in the top panel, co-expression of HA TP-0tt significantly reduced the tyrosine phosphorylation of TEL-PDGFR. By comparing the phospho-tyrosine signal from different amounts of immunoprecipitated TEL-PDGFR the specific tyrosine phosphorylation of TEL-PDGFR was estimated to be reduced more than 50% after co-expression of HA TP-0tt (data not shown).

We thus conclude that TEL-mediated homophilic interactions contribute to the constitutive tyrosine phosphorylation of TEL-PDGFR and constitute a candidate target for antagonists.

Ligand-stimulated PDGFR and TEL-PDGFR differ in signaling properties

To compare the signaling properties of ligand-stimulated wild-type PDGFR and TEL-PDGFR, BaF/3 cells were electroporated with a PDGFR expression vector conferring G418 resistance. After selection with G418 in the presence of IL-3, a population of resistant BaF/3 cells were obtained and analysed with regard to PDGFR expression. Cell lysates from unstimulated and PDGF-stimulated cells were subjected to immunoprecipitation with a PDGFR antiserum and subsequently analysed by PDGFR and phospho-tyrosine immunoblotting. As shown in Figure 4, both precursor and mature forms of the PDGFR, with the expected sizes of 160 and 180 kDa, respectively, were detected. The PDGFR was functionally active as evidenced by tyrosine phosphorylation in response to PDGF-BB (Figure 4a). Comparison with TEL-PDGFR expressing BaF/3 cells revealed similar expression levels of the two proteins (Figure 4b).

The PDGFR expressing BaF/3 was used to investigate if ligand-stimulated PDGFR was able to confer IL-3-independent growth of BaF/3 cells. Cells expressing wild-type PDGFR, cells transfected with an empty expression vector and TEL-PDGFR expressing cells were deprived of IL-3, and seeded in 96-well plates in the absence or presence of IL-3 or PDGF-BB. As shown in Table 1, this analysis revealed that ligand-stimulated PDGFR, in contrast to TEL-PDGFR, was unable to stimulate IL-3 independent proliferation of BaF/3 cells.

These findings thus suggest that TEL-PDGFR signaling differs qualitatively from that of ligand stimulated wild-type PDGFR.

Identification of unique tyrosine phosphorylation sites in TEL-PDGFR

To explore the possibility that the differences in signaling between wild-type PDGFR and TEL-PDGFR occurred as a consequence of unique tyrosine phosphorylation sites in TEL-PDGFR, the tyrosine phosphorylation patterns of the two proteins were compared.

TEL-PDGFR and ligand-stimulated wild-type PDGFR were immunoprecipitated from transiently transfected COS cells and autophosphorylation sites were labeled with radioactive phosphate through an in vitro kinase assay. Tryptic peptides derived from the labeled TEL-PDGFR and PDGFR were isolated and separated by two dimensional analysis by high voltage electrophoresis followed by thin layer chromatography (Figure 5a,b). Comparison of the two patterns revealed the presence of two unique ³²P-labeled TEL-PDGFR derived peptides, TP-1 and TP-2 (Figure 5b).

Phosphoamino-acid analysis was performed of TP-1 and TP-2 and demonstrated exclusively phosphorylation of tyrosine residues (Figure 5c). As shown in Figure 5c, both peptides contained radioactively labeled residues at positions 3 and 13. Inspection of the amino-acid sequence of TEL-PDGFR revealed that the only sequence containing tyrosine residues 3 and 13 amino-acid residues downstream of a tryptic cleavage site occurred carboxy-terminal of the arginine residue at position 14. This analysis therefore demonstrates that tyrosine residues at positions 17 and 27 of the TEL-PDGFR serve as autophosphorylation sites in vitro.

To investigate if these sites were also phosphorylated in vivo, TEL-PDGFR and ligand-stimulated PDGFR were ³²P-labeled by incubation of transfected COS cells with [³²P]orthophosphate. After immunoprecipitation, tryptic peptides were generated and analysed by two dimensional analysis (Figure 5d,e). From TEL-PDGFR, but not from PDGFR, two peptides migrating similar to TP-1 and TP-2 were recovered (Figure 5e).

Based on the identification of tyrosine residues 17 and 27 as in vitro autophosphorylation sites and on the migratory pattern of the TP-1 and TP-2 peptides derived from in vivo labelings, we suggest that tyrosine residues 17 and 27 serve as unique TEL-PDGFR in vivo autophosphorylation sites.

Alteration of tyrosine residues 17 and 27 to phenylalanine residues does not affect TEL-PDGFR ability to stimulate IL-3 independent growth or colony formation

To investigate if tyrosine residues 17 and 27 were required for the capacity of TEL-PDGFR to promote IL-3 independent proliferation, BaF/3 cells expressing a mutant form of TEL-PDGFR, in which tyrosine residues 17 and 27 were altered to phenylalanine residues (TEL-PDGFR-Y17,27F) were established. Expression levels of TEL-PDGFR-Y17,27F was demonstrated by PDGFR immunoprecipitation, followed by PDGFR immunoblotting and shown to be similar to that of TEL-PDGFR (Figure 6, upper panel). Comparison of the tyrosine phosphorylation of TEL-PDGFR-Y17,27F and TEL-PDGFR did not reveal any major differences in the extent of tyrosine phosphorylation (Figure 6, lower panel).

BaF/3 cells expressing TEL-PDGFR-Y17,27F were analysed with regard to IL-3 independent growth as well as formation of colonies in methylcellulose. As shown in Table 2, substitution of tyrosine residues 17 and 27 with phenylalanine residues did not affect the functional properties of TEL-PDGFR as determined by these assays.

Thus, phosphorylation of tyrosine residues 17 and 27 of TEL-PDGFR is not required for the ability to confer IL-3-independent proliferation of BaF/3 cells.

Discussion

The findings in the present study extends previous characterization of the chronic myelomonocytic leukemia-associated TEL-PDGFR fusion protein. Interference with pointed/B domain-mediated homophilic interactions were demonstrated to reduce tyrosine phosphorylation of TEL-PDGFR (Figure 3). When ligand-stimulated wild-type PDGFR and TEL-PDGFR were compared with regard to ability to confer IL-3-independent proliferation, differences in signaling potency of the two proteins were revealed (Figure 4, Table 1). Finally, two autophosphorylation sites were identified in the TEL moiety of the fusion protein (Figure 5).

Since the original identification of TEL as a fusion partner for PDGFR, three additional fusion proteins containing the amino-terminal part of TEL fused to tyrosine kinases have been identified; TEL-ABL, TEL-JAK2 and TEL-TRKC (Golub et al., 1995; Knezevich et al., 1998; Lacronique et al., 1997; Papadopoulos et al., 1995). In the cases of TEL-PDGFR, TEL-ABL and TEL-JAK2, biochemicial evidence for TEL-moiety mediated activation of associated tyrosine kinase activity has been demonstrated (Carroll et al., 1996; Golub et al., 1996; Jousset et al., 1997; Lacronique et al., 1997; Papadopoulos et al., 1995). Our finding that overexpression of the TEL-moiety reduces the tyrosine phosphorylation of TEL-PDGFR provides experimental evidence for an antagonistic strategy that has previously only been suggested. Although it remains to be experimentally proven, it is likely that a similar strategy could be used to interfere with signaling of TEL-ABL, TEL-JAK2 and TEL-TRKC. Our findings thus suggest that the pointed/B domain homophilic interaction is a candidate target for drug development. In this context, the recently reported structures of the pointed/B domain of Ets-1, and of the structurally related SAM domains of the Eph4A and EphB2 receptor tyrosine kinases, are of great interest (Slupsky et al., 1998, Stapleton et al., 1999; Thanos et al., 1999).

BaF/3 cells have previously been used as a model system to study PDGFR signaling. IL-3 independent proliferation was observed after co-expression of PDGFR and v-sis, the viral homologue of PDGF B-chain (Drummond-Barbosa et al., 1995). In contrast we observed that ligand-stimulated PDGFR expressing BaF/3 cells fail to proliferate in absence of IL-3. One possible explanation to the difference between our findings and the findings in the study of Drummond-Barbosa et al. (1995) are variations in PDGFR expression levels. Alternatively, the differences occur as a consequence of differences in signaling triggered by receptors stimulated in an autocrine fashion and by receptors stimulated by exogenously added ligands. The notion that signals leading to IL-3 independent proliferation can be transmitted from activated intra-cellular receptors, are supported by the occurrence of IL-3 independent proliferation of 32D cells co-expressing v-sis and PDGF - and -receptor chimeras which did not reach the cell surface (Staebler et al., 1995).

In the case of the comparison between TEL-PDGFR and ligand-stimulated PDGFR, presented in this study, similar expression levels of the two proteins were demonstrated. However, continuous ligand stimulation will most likely lead to ligand-induced downregulation of the PDGFR, and eventually reduced steady-state signaling as compared to the TEL-PDGFR ligand-independent signaling. The differences in subcellular localization of TEL-PDGFR and wild-type PDGFR might provide access to different substrates for the two forms and therefore underlie the phenotypic difference, as discussed above. Finally, the unique autophosphorylation sites in the TEL-PDGFR could lead to recruitment of a different set of SH2-domain-containing signaling proteins. It is noteworthy, that in the well-studied BCR-ABL fusion protein, phosphorylation in the BCR-part creates a GRB2-binding site of importance for the transforming property of the fusion protein (Goga et al., 1995; Pendergast et al., 1993). The observation that the Y17,27F mutant retained the ability to confer IL-3 independent proliferation rules out a role of these tyrosine residues as necessary for this particular phenotype. However, before excluding a functional role of these sites, the mutant and wild-type should be compared in additional models of hematopoetic malignancies, like retrovirally mediated transfer into primary bone marrow cells or in transgenic mice. Whether the same sites are phosphorylated in other TEL-tyrosine kinase fusion proteins also merits further investigation.

Materials and methods

Generation of expression vectors and stable BaF/3 cell lines

cDNA encoding human wild-type PDGFR (Claesson-Welsh et al., 1988) was subcloned into the pcDNA3 neo vector. This construct and pcDNA3 neo control (control) were introduced by electroporation into the murine IL-3-dependent BaF/3 cells, as described previously (Jousset et al., 1997).

To convert tyrosine 17 and 27 of TEL-PDGFR into phenylalanine, M13-TEL-PDGFR recombinant DNA (Jousset et al., 1997) was subjected to oligonucleotide site-directed mutagenesis. The sequence of the mutagenic primer used was 5'-CGAGGAAGCGAAACTCGGGGCACTGGGCTCTCTGGAGGTGTAAATGAAATTCGTTCC-3'. An EcoRI/SstII fragment originatingfrom the site directed mutagenized insert was sequenced and subcloned into EcoRI and SstII restricted pBS-TP-0 (Jousset et al., 1997). The complete TEL-PDGFR Y17,27F insert was subcloned into EcoRI and SalI restricted pBabe puro retroviral expression vector (Morgenstern and Land, 1990). The TEL-PDGFR Y17,27F insert of this construct (pBabe puro TP Y17,27F) was checked by sequencing.

pBabe puro vectors encoding TEL-PDGFR Y17,27F as well as wild-type TEL-PDGFR were introduced by electroporation into the murine IL-3-dependent BaF/3 cells, as described previously (Jousset et al., 1997), and stably transfected cells were selected in the presence of 1 g/ml puromycin and 2% WEHI conditioned medium as source of IL-3. cDNA encoding human wild-type PDGFR was subcloned into the pBabe neo retroviral expression vector. The v-mpl BaF/3 cell line was a kind gift of I Dusanter and S Gisselbrecht (ICGM, Paris) and the PBabe neo TEL-PDLFR BaF/3 cell lines has been described earlier (Jousset et al., 1997).

COS cell transfections

COS cells were maintained in DMEM medium supplemented with 10% fetal calf serum and antibiotics. Plasmids encoding wild-type PDGFR, HA epitope tagged TEL-PDGFR and/or HA TP-0tt construct (Jousset et al., 1997) were transfected into semiconfluent COS cells by the calcium phosphate method. In cotransfection experiments, TEL-PDGFR plasmid was transfected alongside with a tenfold molar excess of HA TP-0tt construct. Cells were harvested for analysis 2 days after transfection.

Immunoprecipitation, immunoblotting and crosslinking procedures

Cells were starved overnight in culture media containing 0.1% FCS, washed once in ice-cold phosphate-buffered saline (PBS) with 0.1% bovine serum albumin (BSA) and stimulated with 100 ng/ml PDGF-BB for 1 h at 4°C followed by rinsing in ice-cold PBS. After extraction with lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 0.5% deoxycholic acid, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 200 M Na₃VO₄) and centrifugation at 10 000 g for 15 min, cell lysates incubated with rabbit antisera raised against the C-terminal part of the PDGFR (Claesson-Welsh et al., 1988) or the HA epitope for 60 min at 4°C. Following incubation with Protein A Sepharose 6MB (Amersham Pharmacia Biotech) for 30 min at 4°C, samples were washed three times in lysis buffer, heated for 5 min at 95°C and subjected to SDS-gel electrophoresis. After semi-dry transfer onto polyvinylidene difluoride (PVDF) membranes and blocking in TBS/0.1% Tween-20, immunoblotting was performed using the PDGFR antibodies P-20 (Santa Cruz Biotechnology) and 06-498 (UBI), phosphotyrosine antibodies PY20 (Transduction Laboratories) and 4G10 (Upstate Biotechnology, Inc.) and HA-epitope antibody (Boehringer Mannheim) in dilutions as recommended by the supplier.

For crosslinking studies, COS cells were transfected with plasmids encoding wild-type PDGFR or TEL-PDGFR, as described above. Cells were starved overnight in DMEM/0.1% FCS, rinsed once in PBS containing 0.1 mg/ml bovine serum albumin and stimulated with 100 ng/ml PDGF-BB for 60 min at 4°C. After lysis in 20 mM HEPES pH 7.5, 1% Triton X-100, 5 mM EDTA, 0.15 M NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1% aprotinin, samples were treated with 0.1 M bis(sulfosuccinimidyl) suberate for 1 h at 4°C. Following incubation in 70 mM methylammonium chloride for 10 min, cell lysates were immunoprecipitated with antisera against the PDGF -receptor, subjected to SDS-gel electrophoresis in 7% gels and immunoblotting using the anti-PDGFR antibody P-20, as described above.

Identification of phosphorylation sites in vitro and in vivo

COS cells were transiently transfected with PDGF -receptor or TEL-PDGFR encoding plasmids. To inhibit the constitutive activity of TEL-PDGFR, cells were incubated with 15 M AG1296 for 24 h before experiments. Thirty minutes prior to lysis, cells were washed with medium to remove the inhibitor. In vitro kinase reactions were performed essentially as described (Hansen et al., 1996). ³²P-orthophosphate labeling, trypsin digestion, two-dimensional phosphopeptide mapping and phosphoamino acid analysis was performed as earlier described (Blume-Jensen et al., 1995).

Culture of BaF/3 cells

BaF/3 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics. Wild-type PDGFR expressing cells were cultured in the presence of 0.5 g/ml G418 and 2% WEHI-conditioned media (CM) as a source of IL-3. AG1296 was added to culture media as a stock solution of 10 mM in DMSO to final concentrations of 1.5, 5 or 15 M alongside with vehicle control. TEL-PDGFR and v-mpl expressing BaF/3 cells were seeded in duplicates at a concentration of 1´10⁵ cells/ml and subsequently grown for 72 h in the presence or absence of AG1296 followed by counting in a Bürker chamber. Inhibition of TEL-PDGFR tyrosine kinase activity by AG1296 was studied by a 2-h incubation of TEL-PDGFR cells with the inhibitor.

To assess the degree of IL-3-independent growth, cells were deprived of IL-3 and seeded in 96-well trays at a concentration of 10⁴ cells per well, as described previously (Lacronique et al., 1997). In the case of PDGFR expressing cells, culturing was performed in the absence or presence of 20 ng/ml PDGF-BB or in the presence of 2% WEHI-CM as a source of IL-3. The number of wells showing proliferating cells were scored after 12 - 20 days in culture.

Colony formation was assessed by seeding TEL-PDGFR and TEL-PDGFR Y17,27F BaF/3 cells in 1% methylcellulose in RPMI supplemented with 10% FCS and antibiotics at a concentration of 5´10⁴ cells per ml. Scoring of colony numbers was done after 12 days in culture.

Acknowledgements

We thank Christer Wernstedt for radio-sequencing of peptides, Dr Alexander Levitzki for providing us with the PDGF receptor kinase inhibitor AG1296, Gudrun Bäckström for valuable contributions to this project and Ingegärd Schiller for expert secretarial assistance.

Abe A, Emi N, Tanimoto M, Terasaki H, Marunouchi T and Saito H. (1997). Blood 90, 4271-4277. MEDLINE

Blume-Jensen P, Wernstedt C, Heldin C-H and Rönnstrand L. (1995). J. Biol. Chem. 270, 14192-14200. MEDLINE

Carroll M, Ohno-Jones S, Tamura S, Buchdunger E, Zimmermann J, Lydon NB, Gilliland DG and Druker BJ. (1997). Blood 90, 4947-4952. MEDLINE

Carroll M, Tomasson MH, Barker GF, Golub TR and Gilliland DG. (1996). Proc. Natl. Acad. Sci. USA 93, 14845-14850. MEDLINE

Chase A, Reiter A, Burci L, Cazzaniga G, Biondi A, Pickard J, Roberts IA, Goldman JM and Cross NC. (1999). Blood 93, 1025-1031. MEDLINE

Claesson-Welsh L, Eriksson A, Morén A, Severinsson L, Ek B, Östman A, Betsholtz C and Heldin C-H. (1988). Mol. Cell. Biol. 8, 3476-3486. MEDLINE

Drummond-Barbosa D, Vaillancourt RR, Kazlauskas A and DiMaio D. (1995). Mol. Cell. Biol. 15, 2570-2581. MEDLINE

Ghysdael J and Boureux A. (1997). In: Oncogenes as Transcriptional Regulators, Ghysdael J. and Yaniv M. (eds). Springer Verlag, pp. 29-88.

Goga A, McLaughlin J, Afar DE, Saffran DC and Witte ON. (1995). Cell 82, 981-988. MEDLINE

Golub TR, Barker GF, Bohlander SK, Hiebert SW, Ward DC, Bray-Ward P, Morgan E, Raimondi SC, Rowley JD and Gilliland DG. (1995). Proc. Natl. Acad. Sci. USA 92, 4917-4921. MEDLINE

Golub TR, Barker GF, Lovett M and Gilliland DG. (1994). Cell 77, 307-316. MEDLINE

Golub TR, Goga A, Barker GF, Afar DE, McLaughlin J, Bohlander SK, Rowley JD, Witte ON and Gilliland DG. (1996). Mol. Cell. Biol. 16, 4107-4116. MEDLINE

Hansen K, Johnell M, Siegbahn A, Rorsman C, Engström U, Wernstedt C, Heldin C-H and Rönnstrand L. (1996). EMBO J. 15, 5299-5313. MEDLINE

Heldin C-H and Westermark B. (1996). The Molecular and Cellular Biology of Wound Repair. Clark RAF. (ed.). Plenum Press: New York, pp. 249-273.

Heldin C-H, Östman A and Rönnstrand L. (1998). Biochim. Biophys. Acta 1378, F79-F113. MEDLINE

Jousset C, Carron C, Boureux A, Quang CT, Oury C, Dusanter-Fourt I, Charon M, Levin J, Bernard O and Ghysdael J. (1997). EMBO J. 16, 69-82. Article MEDLINE

Knezevich SR, McFadden DE, Tao W, Lim JF and Sorensen PH. (1998). Nat. Genet. 18, 184-187. MEDLINE

Kovalenko M, Gazit A, Böhmer A, Rorsman C, Rönnstrand L, Heldin C-H, Waltenberger J, Böhmer F-D and Levitzki A. (1994). Cancer Res. 54, 6106-6114. MEDLINE

Lacronique V, Boureux A, Valle VD, Poirel H, Quang CT, Mauchauffe M, Berthou C, Lessard M, Berger R, Ghysdael J and Bernard OA. (1997). Science 278, 1309-1312. Article MEDLINE

Morgenstern JP and Land H. (1990). Nucl. Acids Res. 18, 3587-3596.

Papadopoulos P, Ridge SA, Boucher CA, Stocking C and Wiedemann LM. (1995). Cancer Res. 55, 34-38. MEDLINE

Pendergast AM, Quilliam LA, Cripe LD, Bassing CH, Dai Z, Li N, Batzer A, Rabun KM, Der CJ, Schlessinger J and Gishizky ML. (1993). Cell 75, 175-185. MEDLINE

Rabbitts TH. (1994). Nature 372, 143-149. MEDLINE

Romana SP, Mauchauffe M, Le Coniat M, Chumakov I, Le Paslier D, Berger R and Bernard OA. (1995). Blood 85, 3662-3670. MEDLINE

Ross TS, Bernard OA, Berger R and Gilliland DG. (1998). Blood 91, 4419-4426. MEDLINE

Sawyers CL and Denny CT. (1994). Cell 77, 171-173. MEDLINE

Schwaller J, Frantsve J, Aster J, Williams IR, Tomasson MH, Ross TS, Peeters P, Van Rompaey L, Van Etten RA, Ilaria Jr R, Marynen P and Gilliland DG. (1998). EMBO J. 17, 5321-5333. Article MEDLINE

Slupsky CM, Gentile LN, Donaldson LW, Mackereth CD, Seidel JJ, Graves B and McIntosh LP. (1998). Proc. Natl. Acad. Sci. USA 95, 12129-12134. MEDLINE

Staebler A, Pierce JH, Brazinski S, Heidaran MA, Li W, Schlegel R and Goldstein DJ. (1995). J. Virol. 69, 6507-6517. MEDLINE

Stapleton D, Balan I, Pawson T and Sicheri F. (1999). Nat. Struct. Biol. 6, 44-49. Article MEDLINE

Thanos CD, Goodwill KE and Bowie JU. (1999). Science 283, 833-836. Article MEDLINE

Thomasson MH, Williams IR, Hasserjian R, Udomsakdi C, McGrath SM, Schwaller J, Druker B and Gilliland DG. (1999). Blood 93, 1707-1714. MEDLINE

Figure 1 AG1296 specifically blocks IL-3-independent growth of TEL-PDGFR BaF/3 cells and decreases tyrosine phosphorylation of TEL-PDGFR. (a) TEL-PDGFR and v-mpl expressing BaF/3 cells were grown for 72 h with or without IL-3 and the indicated concentrations of AG1296. Results are presented as mean cell counts of duplicates. (b) TEL-PDGFR and v-mpl expressing BaF/3 cells were preincubated with the indicated concentrations of AG1296 for 2 h before harvesting. Cell lysates were subjected to immunoprecipitation (IP) with PDGF -receptor antibody followed by SDS-gel electrophoresis in 7% gels. After transfer to PVDF membranes, samples were analysed by immunoblotting (IB) using phosphotyrosine antibody and HA epitope antibody

Figure 2 Evidence for oligomeric complexes of TEL-PDGFR by crosslinking. Analysis was performed using COS cells transfected with TEL-PDGFR (lanes 7 and 8) or wild-type PDGF -receptor in the absence (lanes 3 and 7) or presence (lanes 5 and 6) of PDGF-BB stimulation. After lysis, samples were treated without (-) or with (+) the crosslinker bis(sulfosuccinimidyl) suberate, and the lysates were immunoprecipitated with PDGF -receptor antibody. Following SDS-gel electrophoresis in 7% gels and transfer to PVDF membranes, PDGF -receptor and TEL-PDGFR were detected by PDGF -receptor immunoblotting

Figure 3 Phosphorylation of the TEL-PDGFR protein is reduced by cotransfection with excess HA TP-0tt construct. COS cells were mock transfected, transfected with cDNA encoding HA-epitope tagged TEL-PDGFR alone and/or a tenfold excess of HA TP-0tt. Cell lysates were subjected to immunoprecipitation (IP) with HA epitope antibody followed by SDS-gel electrophoresis in 7% gels and 15% gels. After transfer to PVDF membranes, samples were analysed by immunoblotting (IB) using a phosphotyrosine antibody (upper panel, 7% gel) and HA epitope antibody (middle panel, 7% gel; lower panel, 15% gel)

Figure 4 Expression of PDGF -receptor in BaF/3 cells. (a) Cell lysates of PDGFR BaF/3 cells, incubated without (-) or with (+) 100 ng/ml PDGF-BB for 60 min at 4°C, were subjected to immunoprecipitation (IP) with PDGF -receptor antibody followed by SDS-gel electrophoresis in a 7% gel. After transfer to PVDF membranes, samples were analysed by immunoblotting (IB) with phosphotyrosine antibody and PDGF -receptor antibody. The M_r 180 000 band (upper and lower panel) corresponds to the mature PDGF -receptor and the M_r 160 000 band (lower panel) to the precursor. (b) Cell lysates of BaF/3 cells expressing PDGFR or TEL-PDGFR were subjected to PDGFR immunoprecipitation and immunoblotting

Figure 5 Identification of Tyr17 and Tyr27 as in vitro and in vivo TEL-PDGFR autophosphorylation sites. (a, b) Two-dimensional tryptic phosphopeptide maps of in vitro labeled wild-type PDGFR (a) and TEL-PDGFR (b) from serum-starved transiently transfected COS cells. Following stimulation with 100 ng/ml PDGF-BB (A), lysates were immunoprecipitated with a PDGF -receptor antibody, subjected to an in vitro kinase assay, separated by SDS - PAGE electrophoresis, transferred to Hybond-C membranes and digested in situ with trypsin. Phosphopeptides were separated on cellulose thin-layer chromatography (TLC) glass plates by high voltage electrophoresis followed by ascending chromatography and exposure to film. Phosphopeptides unique to TEL-PDGFR are denoted TP-1 and TP-2. The point of application is indicated by filled arrows and the position of Tyr857 of the PDGF -receptor is indicated with open arrows. (c) Radiosequencing of TP-1 (upper panel) and TP-2 (lower panel). Phosphopeptides TP-1 and TP-2 were eluted from the TLC plate, coupled to a Sequelon-AA membrane through carbodiimide coupling to the carboxyterminus and subjected to Edman degradation. The release of radioactivity in the individual cycles are indicated as arbitrary units. Two-dimensional phosphoamino acid analyses of TP-1 and TP-2 are shown encased in their respective panel. (d, e) Two-dimensional tryptic phosphopeptide maps of wild-type PDGFR (d) and TEL-PDGFR (e) from ³²P-ortophosphate-labeled COS cells. COS cells transiently transfected with PDGFR or TEL-PDGFR were labeled in ³²P-ortophosphate-containing medium. Ligandstimulated PDGFR and TEL-PDGFR were immunoprecipitated using PDGF -receptor antiserum, separated by SDS - PAGE and electrotransferred to a nitrocellulose membrane. Bands corresponding to the phosphorylated PDGF -receptor and TEL-PDGFR protein were cut out and subjected to tryptic cleavage. Phosphopeptides were analysed as in (a) and (b)

Figure 6 Expression and phosphorylation of TEL-PDGFR and TEL-PDGFR Y17,27F in BaF/3 cells. Control BaF/3 cells, TEL-PDGFR BaF/3 cells or TEL-PDGFR Y17,27F BaF/3 cells were lysed and the extracts were subjected to immunoprecipitation with PDGF -receptor antibody. After electrophoresis and transfer, proteins were immunoblotted with PDGF -receptor antibody (upper panel) or anti-phosphotyrosine antibody (lower panel)

Table 1 Analysis of IL-3-independent proliferation of PDGF-BB stimulated PDGFR-expressing BaF/3 cells

Table 2 TEL-PDGFRR Y17,27F can permit IL-3 independent proliferation of BaF/3 cells