Transformation and Stat activation by derivatives of FGFR1, FGFR3, and FGFR4

Abstract

The fibroblast growth factor receptor (FGFR) family members mediate a number of important cellular processes, and are mutated or overexpressed in several forms of human cancer. Mutation of Lys650→Glu in the activation loop of the FGFR3 kinase domain causes the lethal human skeletal disorder thanatophoric dysplasia type II (TDII) and is also found in patients with multiple myeloma, bladder and cervical carcinomas. This mutation leads to constitutive activation of FGFR3. To compare the signaling activity of FGFR family members, this activating mutation was generated in FGFR1, FGFR3, and FGFR4. We show that the kinase domains of FGFR1, FGFR3, and FGFR4 containing the activation loop mutation, when targeted to the plasma membrane by a myristylation signal, can transform NIH3T3 cells and induce neurite outgrowth in PC12 cells. Phosphorylation of Shp2, PLC-γ, and MAPK was also stimulated by all three ‘TDII-like’ FGFR derivatives. Additionally, activation of Stat1 and Stat3 was observed in cells expressing the activated FGFR derivatives. Finally, we demonstrate that FGFR1, FGFR3, and FGFR4 derivatives can stimulate PI-3 kinase activity. Our comparison of these activated receptor derivatives reveals a significant overlap in the panel of effector proteins used to mediate downstream signals. This also represents the first demonstration that activation of FGFR4, in addition to FGFR1 and FGFR3, can induce cellular transformation. Moreover, our results suggest that Stat activation by FGFRs is important in their ability to act as oncogenes.

Introduction

The fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases contains four members, which share a conserved structure and a high level of homology (56–71% overall identity) (Jaye et al., 1992). Each receptor consists of an extracellular ligand-binding domain containing three immunoglobulin-like repeats, a single membrane-spanning region, and an intracellular split tyrosine kinase domain. This family of RTKs serves as high affinity receptors for the fibroblast growth factors (FGFs), a large family of at least 15 members (Szebenyi and Fallon, 1999) which have been shown to be crucial for the embryonic development of limbs, digits (Martin, 1998), and many other organ systems (Goldfarb, 1996). FGF signaling is important in the regulation of many cellular processes, including proliferation, differentiation, survival, and motility (Basilico and Moscatelli, 1992). Ligand binding to the receptor is potentiated by heparin sulfate proteoglycans and other extracellular matrix factors. Both the FGFs and the FGFRs exhibit distinct expression patterns during development, reflecting their somewhat specialized roles. The ligand-binding specificity of each FGFR family member is complex and only well-defined for certain ligand-receptor pairs. The alternative splicing of FGFR1, FGFR2, and FGFR3 transcripts results in additional ligand-binding specificities, further complicating the study of ligand-activated signaling through the FGFRs (reviewed in Jaye et al., 1992; Johnson and Williams, 1993; Wilkie et al., 1995).

Numerous skeletal and developmental disorders have been shown to result from mutations in the FGFRs (Burke et al., 1998; Webster and Donoghue, 1997b). For example, the lethal human skeletal syndrome thanatophoric dysplasia type II (TDII) results from a mutation of Lys650 to Glu in the activation loop of the FGFR3 kinase domain (Tavormina et al., 1995). A different mutation at this same position, Lys650→Met, leads to a second syndrome, SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans), which involves skeletal malformations, CNS disturbances, and skin dysplasia (Tavormina et al., 1999). Other mutations in FGFR3 lead to different dwarfism syndromes of varying severity, while mutations in the extracellular domains of FGFR1, FGFR2, and FGFR3 lead to craniofacial syndromes such as Crouzon, Pfeiffer, Apert and Jackson-Weiss (Burke et al., 1998; reviewed in Webster and Donoghue, 1997b). Studies in our laboratory and others have demonstrated that many of these mutations lead to ligand-independent activation of the receptors, but through distinct mechanisms (D'Avis et al., 1998; Galvin et al., 1996; Naski et al., 1996; Neilson and Friesel, 1995, 1996; Robertson et al., 1998; Webster and Donoghue, 1996).

Activation of FGFRs has also been implicated in cell proliferation and tumorigenesis. Activating mutations in FGFR3 previously shown to be involved in thanatophoric dysplasia syndromes (TDI, TDII), as well as overexpression of FGFR3 resulting from a t(4;14) translocation, have recently been detected in human multiple myeloma (Chesi et al., 1997; Richelda et al., 1997). The TD mutations are also frequently detected in human bladder and cervix carcinomas (Cappellen et al., 1999). Additionally, at least four FGF genes have been identified as oncogenes in human tumors (van Leeuwen and Nusse, 1995; Wright et al., 1993; Wright and Huang, 1996), and FGFR2 has been implicated in T-lymphocytic malignancies (Hattori et al., 1992). Coexpression of aFGF (FGF-1) and FGFR1 observed in tumor specimens from breast cancer patients suggests a possible autocrine mechanism for the progression of some tumors (Yoshimura et al., 1998). Translocations involving FGFR1 and several novel genes have been detected in myeloproliferative disorders affecting both T- and B-cells (Popovici et al., 1998, 1999; Reiter et al., 1998; Smedley et al., 1998; Xiao et al., 1998).

Activation of FGFRs results in the stimulation of several signaling pathways and, although these pathways have not been completely defined, several proteins involved have been identified. Activation of FGFR1 by aFGF has been shown to result in tyrosine phosphorylation of FRS2, an adaptor protein containing multiple Grb2 binding sites. FRS2 function is dependent on localization to the plasma membrane via lipid modifications at the N-terminus (Kouhara et al., 1997). Shp2, a ubiquitously-expressed tyrosine phosphatase that appears to be the mammalian homolog of Drosophila corkscrew, binds to FRS2 and becomes tyrosine phosphorylated in a FGF-dependent manner (Hadari et al., 1998; Perkins et al., 1996). However, the corresponding substrates of Shp2 in this system have not yet been elucidated. In addition, stimulation of cells with FGFs leads to phosphorylation of MAPK, Shc, c-src, cortactin, Sos, and PLC-γ (Kanai et al., 1997; Klint et al., 1995; Landgren et al., 1998; LaVallee et al., 1998; Spivak-Kroizman et al., 1994; Wang et al., 1994; Zhan et al., 1994). It is not yet known whether all four FGFR family members signal through the same pathways.

Since similar mutations in the different FGFR family members lead to related but distinct developmental syndromes and/or diseases, we set out to compare the signaling pathways utilized by the different receptors to understand how they mediate different effects while being so structurally related. In this study, we have exploited the ability to activate FGFR3 constitutively by use of a point mutation in the kinase domain, and examined whether other FGFRs are also activated in this manner. By generating myristylated derivatives of the various FGFR kinase domains, previously demonstrated to target the kinase domain to the appropriate subcellular location (Webster and Donoghue, 1997a), the signaling activity of the mutated receptors was examined without the additional complication of ligand-dependence. The myristylated and activated derivatives of FGFR1, FGFR3, and FGFR4 all induced transformation of NIH3T3 cells. Experiments described below demonstrate that these three FGFR family members qualitatively utilize the same signaling molecules, including Shp2, PLC-γ, MAPK, Stat1, and Stat3. We also have evidence that all three receptors stimulate PI-3 kinase activity. The myristylated FGFR1 wild-type derivative also stimulated phosphorylation of all the effector proteins examined, suggesting the ability to positively affect signaling activity of this receptor by simply truncating the extracellular and transmembrane domain sequences. These results support the possible involvement of these three FGFRs in proliferative cellular processes.

Results

Generation of activated FGFR constructs

In previous work from this laboratory (Webster and Donoghue, 1997a), derivatives of FGFR3 were described in which a myristylation signal was substituted in place of the extracellular and transmembrane domains, thereby targeting the kinase domain to the plasma membrane; such derivatives were capable of mitogenic signaling. In contrast, kinase-domain derivatives containing a non-functional myristylation signal exhibited a cytoplasmic localization and were biologically inactive. These experiments demonstrated the importance of plasma membrane localization in FGFR3 signaling, and also established the feasibility of this approach for targeting receptor kinase domains to the plasma membrane. A point mutation in the activation loop region of the kinase domain (Lys650→Glu) of FGFR3 has been shown to underlie the lethal development disorder thanatophoric dysplasia type II (TDII) (Tavormina et al., 1995), and has also been detected in several human malignancies (Cappellen et al., 1999; Chesi et al., 1997; Richelda et al., 1997). Incorporation of this mutation into the myristylated FGFR3 derivative generates a protein that is capable of transforming NIH3T3 cells (Webster and Donoghue, 1997a). An alignment of the activation loop sequences of FGFR1, FGFR3, and FGFR4 revealed almost complete amino acid identity within this region (Figure 1a). Therefore, it seemed likely that a similar mutation in the activation loops of FGFR1 and FGFR4 would also result in ligand-independent receptor activation.

Figure 1
figure1

Schematic representation of FGFR derivatives. (a) Diagram of full-length FGFRs and alignment of activation loop sequences. The amino acid sequences are highly conserved between receptor isoforms over the 41-residue region depicted here. All four receptor isoforms contain the `YYKK' motif in the region where the TDII and SADDAN mutations in FGFR3 have been found. SP=signal peptide; Ig=immunoglobulin-like repeat; AB=acid box; TM=transmembrane domain; kinase=each lobe of the intracellular kinase domain, separated by a short insert region. (b) FGFR derivatives generated for this study. A myristylation signal, derived from c-src, was utilized as a targeting sequence to localize the intracellular tyrosine kinase domains of FGFR1, FGFR3, and FGFR4 to the inner surface of the plasma membrane. The targeting signal was appended to the N-terminal end of the entire intracellular domains of FGFR1 (a.a.398–822), FGFR3 (a.a.399–806), and FGFR4 (a.a.391–802), generating the constructs myrR1-WT, myrR3-WT, and myrR4-WT. Additionally, FGFR intracellular domain derivatives containing a Lys→Glu point mutation in the activation loop (residue 656 in FGFR1, 650 in FGFR3, and 645 in FGFR4) were modified to contain the myristylation signal, generating the myrR1-TDII, myrR3-TDII, and myrR4-TDII constructs

In order to facilitate comparison of the signaling properties of FGFR1, FGFR3, and FGFR4, myristylated derivatives were constructed for both the wild-type and point-mutated receptors, as depicted in Figure 1b. These derivatives consist of the 15 a.a. myristylation signal derived from c-src (Aronheim et al., 1994; Buss et al., 1988) and the entire cytoplasmic region of the receptors (a.a.398–822 of FGFR1, a.a.399–806 of FGFR3, and a.a.391–802 of FGFR4). The wild-type derivatives are designated myrR1-WT, myrR3-WT, and myrR4-WT in the text. The ‘TDII-like’ mutation (Lys650→Glu in FGFR3; myrR3-TDII) corresponds to Lys656→Glu in the activation loop of FGFR1 (myrR1-TDII), and Lys645→Glu of FGFR4 (myrR4-TDII).

FGFR2 derivatives are conspicuously absent from this comparative study. Despite intensive efforts to construct a myrR2-TDII derivative, these experiments were unsuccessful due to an apparent toxicity towards host bacterial strains.

Transforming activity of myristylated FGFR derivatives

To confirm proper localization of myristylated FGFR derivatives, immunofluorescence was performed in a number of different cell types transfected with the constructs. In all cases, plasma membrane localization was abundant (Figure 8a, data not shown). Interestingly, in NIH3T3, 293T, and REF-52 cells, the expression of myristylated ‘TDII-like’ derivatives of FGFR1, FGFR3, and FGFR4 resulted in a highly rounded and refractile morphology (data not shown). In fibroblasts and other cell types, a spindle-shaped and refractile morphology is often indicative of morphological transformation. As mentioned previously, myrR3-TDII was shown to have transforming activity when expressed in NIH3T3 cells (Webster and Donoghue, 1997a). To examine if the ‘TDII-like’ mutation in FGFR1 or FGFR4 also resulted in the acquisition of transforming activity, transformation assays were performed using NIH3T3 cells. The ‘TDII-like’ derivatives of FGFR1, FGFR3, and FGFR4 were all transforming in this assay, although the myrR4-TDII derivative yielded only 5% the activity of myrR3-TDII (Figure 2). The wild-type derivatives of myrR1, myrR3 and myrR4 were completely inactive in the focus forming assays (Figure 2). These results demonstrate that all three FGFR family members examined are capable of inducing morphological transformation.

Figure 8
figure2

Induction of neurite outgrowth in PC12 cells by `TDII-like' FGFR1, FGFR3, and FGFR4 derivatives. (a) PC12 cells transfected with indicated constructs were fixed, permeabilized, and subjected to immunofluorescence analysis using FGFR1-, FGFR3-, or FGFR4-specific antisera as indicated. Cells expressing myristylated wild-type receptors displayed very little to no neurite outgrowth (middle column). In contrast, cells expressing the `TDII-like' receptor derivatives exhibited significant outgrowth of long neurite extensions (right column). (b) Quantitation of the percentage of receptor-expressing cells that exhibited neurite extensions greater than or equal to one cell body in length. The myrR1-WT derivative caused a basal level of neurite outgrowth in about 16% of the cells, while the `TDII-like' derivatives of FGFR1, FGFR3, and FGFR4 caused differentiation of between 60–70% of the expressing cells

Figure 2
figure3

Focus-forming activity of myristylated FGFR derivatives. NIH3T3 cells were transfected with the indicated constructs, and subjected to focus-forming assays to analyse the ability of activated FGFR derivatives to confer the transformed phenotype. The results are presented as percentage of myrR3-TDII transforming activity, which was previously reported (Webster and Donoghue, 1997a). These results represent the average of six independent experiments

Kinase activity of myristylated FGFR derivatives

The TDII mutation in FGFR3 has been shown to lead to elevated receptor kinase activity in both the full-length and myristylated forms (Naski et al., 1996; Webster et al., 1996; Webster and Donoghue, 1997a). To examine the effects of this ‘TDII-like’ mutation on the kinase activity of FGFR1 and FGFR4, in vitro kinase assays were performed on FGFRs immunoprecipitated from transiently transfected, serum-starved 293T cells. The TDII mutation in myrR3 leads to an increase in activity approximately sixfold over wild-type (Figure 3a). A similar increase in activity is seen when the TDII mutation is incorporated into full-length FGFR3. The myrR1 and myrR4 derivatives exhibit high levels of tyrosine kinase activity, regardless of whether the activation loop is wild-type or contains the ‘TDII-like’ mutation. The full-length wild-type forms of FGFR1 and FGFR4 also exhibited a high basal level of autophosphorylation activity in these assays (Figure 3a). Equivalent levels of receptor proteins were confirmed to be present in the lysates prior to immunoprecipitation (Figure 3b). Interestingly, myrR1-WT and myrR1-TDII exhibited comparable levels of in vitro kinase activity; nonetheless, myrR1-WT was unable to induce cellular transformation (Figure 2).

Figure 3
figure4

In vitro kinase activity of full-length and myristylated FGFR derivatives. (a) 293T cells transfected with the indicated constructs were lysed and subjected to immunoprecipitation with FGFR1, FGFR3, or FGFR4 antisera as appropriate. Immunoprecipitates were subjected to an in vitro kinase assay using [γ-32P]ATP, then analysed by SDS–PAGE and detected by autoradiography. In vitro kinase activity was high in all samples excluding mock-transfected cells and cells expressing full-length FGFR3-WT or myrR3-WT. (b) Whole cell lysates of the above samples were separated by SDS–PAGE, transferred to nitrocellulose, and were subjected to Western blotting using FGFR1, FGFR3, or FGFR4 antisera, to confirm equivalent levels of receptor expression in each sample. Molecular mass markers in kD are indicated

Phosphorylation of Shp2 and MAPK in response to constitutively-active FGFR constructs

The adapter protein FRS2 is myristylated and membrane-associated. Phosphorylation of FRS2 in response to FGF on tyrosine residues provides numerous binding sites for Grb2, thus linking FGF stimulation to ras activation (Kouhara et al., 1997). In addition to recruiting Grb2/Sos complexes, FRS2 has also recently been shown to recruit Shp2, a protein tyrosine phosphatase, in response to FGF stimulation (Hadari et al., 1998). To determine if FGFR1, FGFR3 and FGFR4 differ in their ability to affect this downstream signaling protein, myristylated FGFR derivatives were expressed in 293T cells, and immunoprecipitated Shp2 was examined for tyrosine phosphorylation by anti-phosphotyrosine immunoblot analysis. Figure 4a demonstrates that expression of all three FGFR derivatives containing the activation loop mutation led to phosphorylation of Shp2. Expression of myrR1-WT also stimulated Shp2 phosphorylation (Figure 4a, lane 2). Shp2 protein levels were similar in all samples (Figure 4b). FGFR expression levels were equivalent in each sample (data not shown). These results suggest that all three FGFRs examined utilize Shp2 as part of their repertoire of downstream effectors.

Figure 4
figure5

The FGF-responsive effector proteins Shp2 and MAPK are phosphorylated in response to `TDII-like' derivatives of FGFR1, FGFR3, and FGFR4, and myrR1-WT. (a) Lysates of transfected 293T cells were subjected to immunoprecipitation with Shp2 antisera, separated by SDS–PAGE, and transferred to nitrocellulose. The membrane was probed with 4G10 antisera to detect tyrosine-phosphorylated Shp2. (b) The same membrane described in (a) was stripped and re-probed with Shp2 antisera to confirm equal recovery of Shp2 protein in each sample. Arrows to the right in (a) and (b) indicate the position of the Shp2 protein. (c) Whole cell lysates from 293T cells transfected with the indicated samples were separated by SDS–PAGE, transferred to nitrocellulose, and probed with Phospho-p44/42 MAP kinase (Thr202/Tyr204) antisera, which detects the phosphorylated, activated forms of p42MAPK and p44MAPK. Arrows to the right indicate the positions of the phosphorylated forms of these two proteins. (d) Samples in (c) were re-probed with ERK1/2 antisera, to confirm equivalent levels of p42MAPK and p44MAPK in each lane. Arrows to the right indicate the positions of the two proteins. In all cases, equivalent levels of FGF receptor expression was confirmed in the lysates by Western blotting (data not shown)

Recruitment of Grb2 by FRS2 leads to activation of ras and the MAPK cascade, a major mitogenic pathway in mammalian cells. To examine whether the different FGFRs stimulate the MAPK cascade, lysates from 293T cells transiently transfected with the myristylated receptor derivatives were analysed for the presence of phosphorylated, activated forms of p42MAPK and p44MAPK. Expression of the ‘TDII-like’ derivatives of myrR1, myrR3, and myrR4, as well as the wild-type myrR1, correlated with phosphorylation, and thus activation, of both forms of MAPK (Figure 4c). The levels of p42MAPK and p44MAPK were similar in all lysates examined (Figure 4d), and receptor expression levels were comparable in each sample (data not shown). These results demonstrate that this important mitogenic signal transduction pathway, involving p42MAPK and p44MAPK, can be stimulated by these three different FGFRs containing a point mutation in their activation loop.

Activation of PLC-γ by myristylated FGFR constructs

PLC-γ is tyrosine-phosphorylated and activated in response to FGF stimulation (Mohammadi et al., 1992), and binds to Tyr766 of FGFR1, which corresponds to Tyr760 of FGFR3 and Tyr754 of FGFR4 (Mohammadi et al., 1991). Phosphorylation of PLC-γ in response to myristylated ‘TDII-like’ derivatives of FGFR1, FGFR3, and FGFR4 was examined using 293T cells. Expression of the activation loop mutants of each receptor, as well as expression of the myrR1-WT derivative, resulted in phosphorylation of PLC-γ, which was detected as a 120 kD band on anti-phosphotyrosine Western blots (Figure 5a). Levels of PLC-γ protein in the samples were shown to be equivalent (Figure 5b), as were the FGFR expression levels (Figure 5c). Thus, these three FGFRs utilize the same signaling molecules (Shp2, PLC-γ, and MAPK) to mediate, at least in part, their signaling activities.

Figure 5
figure6

Phosphorylation of PLC-γ in response to expression of `TDII-like' FGFR derivatives and myrR1-WT. (a) Transfected 293T cells were subjected to immunoprecipitation with PLC-γ antisera. Resulting samples were analysed by Western blotting with 4G10 antisera. PLC-γ was phosphorylated in cells expressing myrR1-WT, myrR1-TDII, myrR3-TDII, and myrR4-TDII derivatives. (b) Samples in (a) were re-probed with PLC-γ antisera, which confirmed equivalent recovery of PLC-γ in each sample. The arrows in (a) and (b) indicate the position of PLC-γ. (c) Whole cell lysates from (a) were separated by SDS–PAGE and subjected to Western blotting with appropriate FGFR1, FGFR3, and FGFR4 antisera. FGFR expression levels were similar in each sample

Activation of Stat proteins by FGFR derivatives

Recently, it was shown that in tissue from patients with TDII, as well as in TDII ‘knock-in’ mice and rat chondrosarcoma (RCS) cells stimulated with FGF, Stat1 becomes activated and translocates to the nucleus (Legeai-Mallet et al., 1998; Li et al., 1999; Sahni et al., 1999; Su et al., 1997). To examine whether FGFR1 and FGFR4 are also able to activate Stat proteins, lysates from cells expressing myristylated FGFR derivatives were analysed for the presence of phosphorylated Stat1 and Stat3. Expression of the ‘TDII-like’ derivatives of FGFR1, FGFR3, and FGFR4, as well as myrR1-WT, led to phosphorylation of both Stat1 and Stat3 (Figure 6a,b, top panels). The cell lysates expressed equivalent levels of Stat1 and Stat3 protein (Figure 6a,b, bottom panels), and equal levels of FGFR derivatives (data not shown). This represents the first evidence that FGFRs can also activate Stat3.

Figure 6
figure7

Activation of Stat1 and Stat3 by FGFR derivatives. (a) Lysates of 293T cells transfected as indicated were analysed by Western blotting using Phospho-Stat1 (Y701) antisera (top) or Stat1 antisera (bottom). (b) The same lysates in (a) were re-examined for phosphorylated Stat3 by Western blotting with Phospho-Stat3 (Y705) (top) or Stat3 antisera (bottom). Arrows in (a) and (b) indicate the positions of phosphorylated Stat1/3 and endogenous Stat1/3 in the lysates. Receptor expression levels were equivalent in each sample. (c) Cells were co-transfected with 4×StRE-luciferase, a Stat-responsive reporter construct, and various FGFR derivatives, and subjected to luciferase assay. The graph shows the average relative luciferase units (RLU) for duplicate readings of samples from four independent experiments

We next examined whether phosphorylation of Stat proteins in FGFR-expressing cells correlated with their ability to translocate to the nucleus and activate transcription. In co-transfection and immunofluorescence experiments, we were able to demonstrate translocation of myc-Stat3 to the nucleus of cells expressing activated FGFR derivatives (data not shown). In addition, we demonstrated that myristylated FGFR derivatives containing the ‘TDII-like’ mutation caused a robust induction of a Stat-responsive luciferase reporter construct (Figure 6c). The TDII derivatives, when compared to their wild-type counterparts, exhibited threefold, 120-fold, and 17-fold induction of luciferase for FGFR1, FGFR3, and FGFR4, respectively. These results demonstrate that all three FGFR family members examined here are able to lead to Stat activation. Interestingly, myrR1-WT was threefold less active than myrR1-TDII in this assay, despite equivalent expression levels (data not shown). Since this is one of the only effector assays where myrR1-WT exhibited a reduced response in comparison to the transforming myrR1-TDII derivative, it suggests that the ability of our myristylated FGFR derivatives to transform cells is at least partially dependent upon Stat activation.

Activation of PI-3 kinase activity

All four FGFR family members contain a consensus PI-3 kinase binding site in the C-terminal portion of their intracellular domains. We examined whether our myristylated, constitutively-active derivatives were capable of stimulating this pathway. As depicted in Figure 7, when compared to mock-transfected cells, we see a significant increase in PI-3 kinase activity in phosphotyrosine immunoprecipitates from cells expressing the ‘TDII-like’ derivatives of FGFR1, FGFR3, and FGFR4. Again, we also see elevated activity in this assay in cells myrR1-WT, while the myrR3-WT and myrR4-WT derivatives do not promote PI-3 kinase activation (Figure 7). These results suggest that FGFRs at least have the potential to signal through the PI-3 kinase pathway.

Figure 7
figure8

PI-3 kinase activity in cells expressing various FGFR derivatives. 293T cells were transfected with the indicated constructs, immunoprecipitated with phosphotyrosine-specific antisera, and subjected to an in vitro kinase assay using phosphatidylinositol (PI) as a substrate. The arrows indicate the positions of the origin and PIP product

Neurite outgrowth in PC12 cells

FGFs are potent inducers of neurite outgrowth in PC12 rat pheochromocytoma cells. PC12 cells were transfected with the myristylated wild-type and ‘TDII-like’ derivatives of FGFR1, FGFR3, and FGFR4, to determine if they differed in their ability to induce neurite outgrowth. Figure 8a shows representative cells expressing the FGFR derivatives. The wild-type derivatives of FGFR1, FGFR3, and FGFR4 induced little or no neurite outgrowth. In contrast, the ‘TDII-like’ derivatives of all three FGFRs were potent inducers of neurite outgrowth. Figure 8b displays the results of a quantitative analysis of the number of cells expressing the various FGFR derivatives that exhibited neurites greater than or equal to one cell body in length, versus the total number of receptor-positive cells. The myrR1-WT derivative induced neurites in approximately 16% of the cells, reflecting its high basal activity in the kinase and other assays. With the ‘TDII-like’ mutation, myrR1 was able to induce neurites in greater than 65% of the cells in which it was expressed. Similar results were seen with the other ‘TDII-like’ derivatives of FGFR3 and FGFR4. Thus, all three of the FGFRs tested are capable of inducing differentiation of PC12 cells.

Discussion

In this work, a unique method is exploited to study the signaling activity of a family of growth factor receptors. Constitutively-activated derivatives of FGFR1, FGFR3, and FGFR4 were constructed that are localized to the plasma membrane via a myristylation signal, eliminating the need for ligand-induced activation. Experiments described here show that all three of the FGFR family members utilize the same effector proteins to facilitate signal transduction, including Shp2, MAPK, and PLC-γ. Interestingly, the activated FGFR derivatives examined here were capable of inducing morphological transformation, as well as differentiation of PC12 cells. We also show that all three family members can stimulate PI-3 kinase activity. In addition, these experiments reveal that all three activated FGFRs are able to stimulate phosphorylation and activation of Stat proteins. This study demonstrates that FGFR1, FGFR3, and FGFR4 proteins, activated by a combination of truncation and point mutation, can all mediate cellular transformation. These results suggest that unregulated signaling through FGFRs can result in diseases that are proliferative in nature.

The finding that FGFR1, FGFR3, and FGFR4 utilize similar effector proteins makes it difficult to determine why FGFR4 is so much less transforming than activated FGFR1 or FGFR3. One possible explanation is that the levels of activation of downstream effectors are much lower when stimulated by FGFR4 (see below). Alternatively, there may be a novel pathway, as yet unidentified, that is activated by FGFR1 and FGFR3, but not FGFR4, and perhaps this pathway makes a significant contribution to transformation.

Involvement of FGFRs in proliferative diseases

FGFRs and their ligands play a major role in autocrine and paracrine signaling loops that have been implicated in a number of malignancies, including cancers of the stomach, breast, thyroid, prostate, pancreas, and leukemias (Basilico and Moscatelli, 1992). A frequent translocation [t(4;14)(p16.3;q32.3)] associated with multiple myeloma involves the FGFR3 gene, and results in increased expression of FGFR3 or selective expression of FGFR3 alleles that contain activating mutations. Interestingly, the same mutations identified in multiple myeloma, Tyr373→Cys and Lys650→Glu, have also been implicated in the skeletal malformation syndromes thanatophoric dysplasia types I and II (Chesi et al., 1997; Richelda et al., 1997). TDI and TDII mutations have also recently been characterized as frequently occurring in bladder and cervical carcinomas in humans (Cappellen et al., 1999). Our results suggest that FGFR1, FGFR3, and to a lesser extent FGFR4, are all capable of causing cellular transformation. This is the first direct demonstration of the transforming potential of these three FGFRs. This result predicts that other mutational events that alter FGFR expression and/or coding sequences will be found to underlie proliferative disorders in humans.

Results reported here and from other laboratories (Vainikka et al., 1994; Wang et al., 1994) suggest that FGFR1 is the most potent mitogenic member of the FGFR family, and has been shown to be involved in several human proliferative disorders. For example, previous studies have identified the translocation t(8;13)(p12;q12) in stem-cell myeloproliferative disorder, which results in a FGFR1/FIM fusion protein that lacks the extracellular domain of FGFR1 (Popovici et al., 1998). An additional translocation [t(6;8)(q27;p11)] has been characterized involving fusion of FGFR1 and a novel gene, FOP (FGFR1 Oncogene Partner) (Popovici et al., 1999). This is predicted to result in the FOP N-terminal leucine-rich region being fused to the catalytic region of FGFR1. The myrR1-WT derivative described here contains a similar deletion of the extracellular domain of FGFR1. Both the FGFR1/FIM fusion and myrR1-WT exhibit constitutive kinase activity (Popovici et al., 1998; this study). In our hands, full-length FGFR1-WT also has a high basal kinase activity (see Figure 3). Additionally, results reported here demonstrate that myrR1-WT leads to activation of downstream effectors such as Shp2, MAPK, PLC-γ, and PI-3 kinase, to levels similar to those stimulated by myrR1-TDII. However, we do see a reduction in the ability of myrR1-WT to induce Stat-responsive transcription (Figure 6c), neurite outgrowth (Figure 8b), and a lack of transforming activity (Figure 2). These results suggest that the loss of the extracellular domain generates a truncated form of FGFR1 that is prone to activation, either through fusion of a heterologous sequence or relief of an inhibitory force normally provided by the extracellular region. This mechanism for activation may involve oligomerization, as suggested by the demonstration that fusion of the catalytic region of FGFR1 to beta-galactosidase results in tetramerization and activation of the chimeric kinase (Kouhara et al., 1995). Additionally, in the crystal structure of FGFR1, the isolated kinase domain preferentially forms a dimeric structure (Mohammadi et al., 1996). Studies are currently in progress to assess the mechanism of constitutive activity of myrR1-WT.

Previous studies have identified a chromosomal rearrangement in rat osteosarcoma cells, which results in a fusion of FGFR2 with a novel gene designated FRAG1. This fusion protein exhibited high constitutive activity due to dimerization induced by the novel C-terminal coding sequence (Lorenzi et al., 1996). Ligand-independent dimerization of FGFR2 also underlies the mechanism of various craniosynostosis syndromes that result from point mutations in FGFR2 (reviewed in Webster and Donoghue, 1997b, 1998). While it is not yet clear whether developmental syndromes involving premature fusion of the cranial structures result from a constitutive activation of mitogenic pathways, or premature activation of differentiation pathways, it seems clear that mis-regulation of FGFR2 dimerization can result in constitutive signaling.

Mutation of FGFR4 activation loop reveals the potential for disease involvement

It is interesting to note that FGFR4 has not yet been found to be mutated in any developmental disease or syndrome, although the results reported here suggest that FGFR4 can be activated in a manner similar to that seen with other FGFRs. FGFR4 up-regulation is detected in late-stage tumors from mouse pancreatic β cells, implicating FGFR4 in the progression of islet cell carcinogenesis; however, this overexpression does not functionally contribute to tumor development (Olson et al., 1998). In this study, we demonstrate that replacement of the extracellular and transmembrane domains of wild-type FGFR4 with a myristylation signal leads to constitutive activation of its in vitro kinase activity. When combined with the point mutation in the FGFR4 activation loop, these modifications generate an FGFR4 derivative capable of morphological transformation. Since FGFR4 is not expressed in developing bone (Partanen et al., 1991; Stark et al., 1991), it is not surprising that diseases such as Apert syndrome or achondroplasia have not been associated with FGFR4 mutations. However, since FGFR4 is highly expressed in skeletal muscle, as well as organs such as the liver, lung, kidney, spleen, intestine, and pancreas (Hughes, 1997; Partanen et al., 1991; Stark et al., 1991), there could be some diseases involving these tissues which result from FGFR4 mutations.

Activation of Stat1 and Stat3 by FGFRs – mediators of transformation?

Recent work has shown that FGFR3-TDII can lead to activation of Stat1, and in some cases Stat5A and 5B (Legeai-Mallet et al., 1998; Li et al., 1999; Sahni et al., 1999; Su et al., 1997). Our results demonstrate that FGFR1 and FGFR4 containing a ‘TDII-like’ mutation can also activate Stat1 and Stat3. Stat activation has been linked to anti-proliferative effects and premature apoptosis in TD patients (Legeai-Mallet et al., 1998), and recently shown to be required for FGF-induced growth inhibition of RCS cells and primary murine chondrocytes (Sahni et al., 1999). Whether FGFR1 and FGFR4 normally signal through Stat proteins, and whether activation of Stat proteins by FGFR3 is relevant in cells other than chondrocytes, are intriguing questions that could be addressed by using the constitutively-active FGFR derivatives described herein.

Stats have also been implicated in proliferative responses in some cell types, such as myeloid cells (Ilaria et al., 1999), T cells (Moriggl et al., 1999), and erythroid cells (Kirito et al., 1998). Indeed, several laboratories have demonstrated that constitutive Stat3 activation contributes to oncogenesis by a variety of activated tyrosine kinases, including src, Eyk, and Ros (Yu et al., 1995; Cao et al., 1996; Besser et al., 1999; Zong et al., 1996, 1998). A number of human cancers exhibit Stat3 activation, including breast carcinoma, head and neck squamous cell carcinoma, and multiple myeloma (Garcia and Jove, 1998; Grandis et al., 1998; Catlett-Falcone et al., 1999). Recent evidence suggests that over-activation of Stat3 protects multiple myeloma cells from apoptosis, thereby contributing to disease progression (Catlett-Falcone et al., 1999).

Interestingly, activated FGFR3 is also associated with multiple myeloma. Our results thus suggest that activated FGFRs can promote tumor progression by two different mechanisms – by stimulation of growth-promoting pathways (such as the MAPK cascade) and/or by preventing or inhibiting apoptosis of damaged or impaired cells.

Potential for naturally-occurring ‘TDII-like’ mutations in other FGFRs

The Lys→Glu mutation in the activation loop has only been detected as a naturally occurring mutation in human FGFR3. Examination of the nucleotide sequence encoding the activation loop Lys in FGFR1, FGFR2, and FGFR4 reveals that, in all cases, a single base change of the first nucleotide of the Lys codon would convert it to Glu. Thus, this mutation could be generated naturally in all FGFRs by spontaneous mutation. These ‘TDII-like’ mutations may actually occur in nature, but may remain undetectable because the resulting receptor protein is so active that development may be compromised at an early embryonic stage. Alternatively, the gene structure of FGFR3 may render this region more susceptible to mutation than the other FGFR genes. The activation loops of several other receptor tyrosine kinases, such as IRK, PDGFR-β, and c-kit, also contain a Lys at this position, suggesting that other RTK families may be susceptible to mutational activation by amino acid substitutions in this region.

Other comparative studies involving FGFRs

In general, prior studies have shown that FGFR1 exhibited higher signaling activity than FGFR4 as determined by different assays, including phosphorylation of Shc, MAPK, induction of immediate-early genes such as fos, and survival of BaF3 cells (Vainikka et al., 1994; Wang et al., 1994). In L6 myoblasts, both FGFR1 and FGFR4 can increase DNA synthesis in response to aFGF (Vainikka et al., 1994). However, FGFR1 leads to much greater phosphorylation of PLC-γ, Shc, and ERK than FGFR4 (Vainikka et al., 1994; Wang et al., 1994). Differential use of signaling proteins between FGFR1, FGFR3, and FGFR4 has also been observed. For example, only FGFR1 stimulated phosphorylation of an 80 kD protein in L6 cells (Wang et al., 1994), while only FGFR4 was shown to be able to co-immunoprecipitate an 85 kD serine/threonine kinase (Vainikka et al., 1994, 1996). Moreover, FGFR1 was shown to be much more potent than FGFR3 in inducing neurite outgrowth of PC12 cells (Lin et al., 1996), and this activity was localized to the juxtamembrane region of FGFR1, which mediated sustained phosphorylation of FRS2 and MAPK (Lin et al., 1998). Results presented here are consistent with the idea that FGFR1 is the most potent member of the FGFR family in mediating mitogenic signals.

We demonstrate that all activated forms of FGFR examined here utilize the same effector proteins, and the differences in mitogenic/transforming potential probably reflect a variation in the level of stimulation of these same effectors. During preparation of this manuscript, another study exploiting PDGFR-FGFR chimeric receptors clearly demonstrated that indeed, FGFR1, FGFR3, and FGFR4 differ in their quantitative ability to activate PLC-γ, Shc, ERK1/2, and FRS2 (Raffoni et al., 1999). These experiments were performed with PC12 cell lines stably-expressing chimeric receptors, and required addition of PDGF ligand to induce responses. In contrast, our studies were performed with constitutively-active, myristylated derivatives of the kinase domains, abrogating the need for addition of ligand. The data obtained using both strategies is consistent, and validates the use of activated kinase domains appropriately localized to the plasma membrane to study FGFR signaling. It is likely that this system would facilitate the study of signaling by other receptor families as well.

Materials and methods

Construction of myristylated FGFR constructs

The myristylated forms of FGFR3 wild-type (myrR3-WT) and FGFR3-TDII (myrR3-TDII) have been previously described (Webster and Donoghue, 1997a). Full-length human FGFR1 (pcDNA3.1-hFGFR1) was received from Pam Maher, and subcloned into pcDNA3. The myristylated FGFR1 wild-type was generated by inserting complementary oligonucleotides (D1844/D1845) into the HindIII-KpnI sites, which encoded a substitution of the 15 a.a. myristylation signal of c-src (Aronheim et al., 1994; Buss et al., 1988) for the extracellular and transmembrane domains of FGFR1 (a.a. 1–397). Construction of the Lys656→Glu mutation first required creation of an intermediate clone by three-part ligation of the EagI-XbaI fragment (containing the 3′ portion of FGFR1) plus the BstXI-XbaI fragment (containing the vector and 5′ portion of FGFR1) with complementary oligonucleotides encoding a ‘stuffer’ region, containing a unique, silent EcoRV site near the sequence to encode the TDII mutation, that generated BstXI-EagI cohesive ends when annealed. This intermediate was utilized to insert complementary oligonucleotides encoding the TDII mutation (D1780/D1781) by a three-part ligation using EcoRV-EagI overhangs for the oligos, and the EagI-XbaI and XbaI-EcoRV fragments of the intermediate.

Full-length FGFR4 (pLTR-FGFR4) was received from Kari Alitalo, and subcloned into pcDNA3(ΔXbaI) as a HindIII-EcoRI insert. Myristylated FGFR4 wild-type (myrR4-WT) was constructed by a three-part ligation using HindIII-EagI oligonucleotides (D1901/D1902) encoding the c-src myristylation signal, plus the EagI-XbaI 5′ fragment of FGFR4 and the XbaI-HindIII fragment containing the 3′ end of FGFR4 and vector. The Lys645→Glu mutation was incorporated by using SstII-EagI complementary oligonucleotides (D1898/D1899) plus the EagI-EcoRI fragment (3′ end of FGFR4) and the EcoRI-SstII fragment (vector plus 5′ portion of FGFR4). All derivatives incorporating synthetic oligonucleotides were confirmed to be correct by dideoxy nucleotide sequencing (Amersham).

Transfections and cell culture

NIH3T3 cells were maintained in DME plus 10% bovine calf serum (BCS) in a 10% CO2 humidified incubator. 293T cells were grown in DME plus 10% fetal bovine serum (FBS), 10% CO2. REF-52 cells were grown in DME plus 10% FBS, 5% CO2. NIH3T3 and 293T cells were transfected using a modified calcium phosphate transfection protocol (Chen and Okayama, 1987). NIH3T3 cells were plated at a density of 2.0×105 per 60 mm plate and transfected the next day using 10 μg total DNA. 293T cells were split approximately 1 : 10 to 10 cm plates and transfected when approximately 60% confluent with 5 μg total DNA. REF-52 cells were plated at a density of 1.0×105 per 60 mm plate and transfected 2 days later with 3 μg total DNA using LipofectAMINE (Gibco-BRL) according to the manufacturer's instructions. PC12 cells were maintained on collagen-coated dishes (Biocoat) in RPMI1640 with 10% horse serum and 5% FBS, 5% CO2.

Transformation assays

Focus forming assays were performed using NIH3T3 cells as previously described (Hart and Donoghue, 1997). Foci were quantitated after 12–14 days. This experiment was repeated six times with similar results.

Immunoprecipitation and Western blotting

Primary antibodies were obtained from the following sources: Flg (C-15; FGFR1), FGFR3 (C-15), FGFR4 (C-16), SH-PTP2 (C-18; Shp2), Stat1 (C-136) – Santa Cruz Biotechnology; Phospho-MAPK (T202/Y204), Phospho-Stat1 (Y701), Phospho-Stat3 (Y705) – New England Biolabs, Inc.; Mouse Anti-MAPK (ERK1+ERK2) – Zymed; 4G10 (anti-phosphotyrosine) – Upstate Biotechnology, Inc.; Stat3 – Transduction Labs; PLC-γ – a kind gift of Jill Meisenhelder (Salk Institute); Secondary antibodies were obtained from the following sources: HRP-conjugated donkey anti-rabbit IgG – and sheep anti-mouse IgG – Amersham; HRP-conjugated anti-goat IgG – Santa Cruz Biotechnology and Sigma; Texas Red-conjugated goat anti-rabbit IgG – Cappel/ICN; fluorescein-conjugated phalloidin – Sigma.

293T cells were transfected as described above. After overnight starvation in 0.2% FBS, cells were harvested, pelleted, washed once with sterile cold PBS, then lysed. For Shp2 and MAPK assays, lysis was performed with RFR Buffer (20 mM Tris pH 8.0, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 25 mM β-glycerophosphate, 1 mM PMSF, 1 μg/ml Leupeptin, 1 μg/ml Pepstatin A, 2.25 μg/ml Aprotinin, 1 mM sodium vanadate). For PLC-γ assays, lysis was performed with NP40 lysis buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1% NP40, 1 mM DTT, 20 μg/ml Leupeptin, 2.5 μg/ml Aprotinin, 0.1 mM sodium vanadate). For FGFR immunoblotting, Stat1, Stat3, and MAPK activation assays, 50 μg total protein from each sample was loaded for SDS–PAGE analysis. Immunoprecipitations were performed o/n at 4°C, and collected by Protein A-Sepharose (Sigma) or Protein G-Plus agarose (Santa Cruz Biotechnology).

After separation on SDS–PAGE, proteins were transferred to nitrocellulose or Immobilon, and membranes blocked in 3% BSA/TBS/0.05% Tween-20 (for anti-phosphotyrosine blots), 3% milk/TBS/0.05% Tween-20/0.02% sodium azide, or 1% ovalbumin/TBS/0.05% Tween-20/0.02% sodium azide. After incubation of membranes with primary antibodies, membranes were washed extensively in TBS/0.05% Tween-20, then incubated with secondary antibodies. Proteins were detected by enhanced chemiluminescence (Amersham) according to manufacturer's instructions.

Kinase assays

Receptor proteins were isolated from transfected 293T cells lysed in RFR buffer by immunoprecipitation as described above. Immunoprecipitates were washed 2–3 times in NP-40 wash buffer (20 mM Tris, pH 7.5, 137 mM NaCl, 1% NP40, 1 mM sodium vanadate, 5 mM EDTA, 10% glycerol, 10 μg/ml Aprotinin), once with kinase assay buffer (20 mM Tris, pH 7.5, 10 mM MnCl2, 5 mM MgCl2) and subjected to in vitro kinase assays using 10 μCi [γ-32P]-ATP, at 37°C for 15 min. Proteins were separated by SDS–PAGE gel electrophoresis, and the resulting gel was dried and exposed to film. Equivalent amounts of receptor protein were detected in the lysates prior to immunoprecipitation.

For PI-3 kinase assays, 293T cells were transfected with FGFR derivatives, starved overnight, washed twice in buffer A (137 mM NaCl, 20 mM Tris, pH 7.5, 1 mM MgCl2, 1 mM CaCl2, 100 μM sodium vanadate), then lysed in lysis buffer (buffer A plus 1% NP-40, 10% glycerol). Equivalent amounts of lysate were immunoprecipitated with anti-phosphotyrosine sera (4G10, Upstate Biotechnology) and collected with Protein A-sepharose. Samples were washed three times with PBS/1% NP-40, twice with 0.1 M Tris pH 7.5/0.5 M LiCl, and twice with TNE (10 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA), all with 100 μM sodium vanadate. Twenty μg per reaction of PI (Avanti Polar Lipids) in CHCl3 was dried, resuspended in lipid sonicating solution (10 mM HEPES, pH 7.5, 1 mM EGTA), and sonicated for 5 min in a water bath sonicator. Twenty μl of immunoprecipitated sample was combined with 10 μl lipid solution, 10 μl ATP solution [20 μCi [32P]ATP (3000 Ci/mM), 40 μM ATP, 10 mM MgCl2, 20 mM HEPES, pH 7.0], and 60 μl TNE, mixed and incubated at room temperature for 10 min. The reaction was stopped by adding 20 μl 8 N HCl and 160 μl 1 : 1 CHCl3/MeOH, then vortexed, centrifuged for 30 s, and the bottom layer saved. Equal amounts of sample were spotted onto TLC plates (silica gel 60, Merck) coated with 10% potassium oxalate, and chromatography performed in the following solvent system: CHCl3/MeOH/H2O/NH4OH 60 : 47 : 11.3 : 2. TLC plates were air-dried and exposed to film.

Reporter assays

NIH3T3 cells were transfected with 2 μg of pKH165 (4×StRE-luciferase) reporter plasmid, containing four copies of the m67 high-affinity binding site for Stat1 and Stat3 (Bromberg et al., 1999), and 8 μg of plasmids encoding FGFR derivatives. Eighteen to 20 h after transfection, plates were starved for approximately 40 h in DME plus 0.2% bovine calf serum. Cells were rinsed twice in cold PBS, and lysed in 400 μl of Reporter Lysis Buffer (Promega), and subjected to luciferase assays according to manufacturer's instructions.

Neurite outgrowth experiments

PC12 cells were plated on polyethylenimine-coated coverslips, and transfected with 0.2 μg DNA using Effectene reagent (Qiagen). Cells were fixed and processed for immunofluorescence 3–4 days after transfection, when neurites were visible by light microscopy. Immunofluorescence was performed as previously described (Hart and Donoghue, 1997).

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Acknowledgements

This work was supported by Public Health Service grant NIH/NIDCR RO1 DE12581. We thank Jill Meisenhelder, Pam Maher, and Kari Alitalo for providing reagents, Pedro Ramos and Melanie Webster for assistance in generating constructs, Michael David and Leon Su for advice and discussions regarding Stat experiments, and Laura Castrejon for editorial assistance.

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Correspondence to Daniel J Donoghue.

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Keywords

  • FGFR
  • transformation
  • Stats
  • signal transduction

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