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Molecular and Cellular Biology, February 1999, p. 1401-1409, Vol. 19, No. 2
Laboratory of Molecular
Oncology1 and
Laboratory of Molecular
Cell Biology,2 The Rockefeller University,
New York, New York 10021
Received 10 August 1998/Returned for modification 9 October
1998/Accepted 27 October 1998
The receptor tyrosine kinase Eyk, a member of the Axl/Tyro3
subfamily, activates the STAT pathway and transforms cells when constitutively activated. Here, we compared the potentials of the
intracellular domains of Eyk molecules derived from c-Eyk and v-Eyk to
transform rat 3Y1 fibroblasts. The v-Eyk molecule induced higher
numbers of transformants in soft agar and stronger activation of Stat3;
levels of Stat1 activation by the two Eyk molecules were similar. A
mutation in the sequence Y933VPL, present in c-Eyk, to the
v-Eyk sequence Y933VPQ led to increased activation of Stat3
and increased transformation efficiency. However, altering another
sequence, Y862VNT, present in both Eyk molecules to
F862VNT markedly decreased transformation without impairing
Stat3 activation. These results indicate that activation of Stat3
enhances transformation efficiency and cooperates with another pathway to induce transformation.
The oncogene v-eyk was
first isolated from an acute avian retrovirus, RPL30 (38).
It encodes a transmembrane receptor-type tyrosine kinase,
p69gp37v-Eyk, in which the intracellular region of the
putative receptor tyrosine kinase (RTK) is fused to the viral gp37
glycoprotein, leading to dimerization and activation of v-Eyk
(38). The chicken proto-oncogene c-eyk, from
which v-eyk is derived, codes for an RTK with a distinct extracellular region, containing two immunoglobulin-like domains and
two fibronectin type III-like repeats (37). Based on its extracellular structure and the kinase domain sequence, c-Eyk is
classified in the same subfamily of tyrosine kinases as Axl/Ark/UFO (36, 49; reviewed in reference
67). In addition to Axl/Ark/UFO, members of this
subfamily include Sky/Rse/Tif/Brt/Tyro3 (24, 40, 43, 51),
Rek (4), and Mer (31), the last of which is the
closest mammalian homologue of c-Eyk. Members of this subfamily of RTKs
are expressed in a variety of tissues and cell types: Tyro3 and Rek in
different regions of the brain (24, 43, 51), Tyro3 in
ovaries and testes (14, 45), Mer in monocytes and bone
marrow cells (31), Axl in cell types of mesodermal origin such as thymic stromal cells (20, 53), and c-Eyk in adult spleen (37). Several of these RTKs are expressed during
embryogenesis in different tissues (30, 37, 50). The ligands
for these RTKs were identified as the anticoagulation factor protein S
and the growth arrest-specific gene product Gas6 (47, 63,
68). The results of several studies addressing the physiological
functions of these RTK-ligand systems have indicated roles in the
growth and survival of cells (2, 28, 41) and the adhesion
and migration (1, 23) of cells. In several cases, members of this subfamily have been implicated in transformation and tumor formation of a variety of cells by overexpression or constitutive activation of the RTK (36, 38, 54). v-Eyk transformation by
infection with the RPL30 virus induces chicken erythroblastosis, fibrosarcomas, endotheliomas, visceral lymphomatosis, and hemorrhage (21).
We reported that chimeric forms of Eyk that are constitutively
dimerized and activated can transform fibroblasts in vitro (74) without significant stimulation of the Ras/ERK
(extracellular signal-regulated kinase) pathway which is characteristic
for transformation by many oncogenes (60). In contrast, Eyk
strongly stimulated molecules of the Jak (Janus kinase)/STAT (signal
transducers and activators of transcription) pathway, namely, Stat1,
Stat3, and Jak1 (74). However, most of the activation of
STATs was assessed in COS cells, and transformation frequencies of
Stat1 and Stat3 activation were not compared quantitatively.
Activation of the Jak/STAT pathway by cytokine receptor activation is
well established (reviewed in references 16, 34, and
59). Activation of Stat3 by treatment of cells with
interleukin 6 (IL-6), a ligand for the gp130 receptor, and activation
of Stat1 by treatment with interferons have been well characterized.
Stat3 has been implicated in proliferation of cells (13,
25), whereas Stat1 activation correlates more with immune
responses and growth arrest of cells (8, 12). These notions
are supported by the phenotypes of mice harboring targeted disruptions
of Stat1 or Stat3. Stat1 In our initial study (74), transient-transfection data for
activated Eyk molecules in COS cells and the fact that a dominant negative Stat1, Stat1 Reagents.
Sources of reagents were as follows:
[
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Single Amino Acid Substitution in the v-Eyk
Intracellular Domain Results in Activation of Stat3 and Enhances
Cellular Transformation
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
/ mice are viable and show
severe deficiencies in acute response to infections by a variety of
pathogens (19, 46), whereas Stat3/ mice die
very early in embryonal development, suggesting a possible function for
Stat3 activity in the proliferation of cells (64). The
activation of various STAT factors by oncogenes and RTKs has been
demonstrated in a growing number of cases, e.g., v-Src (73), v-Abl (15), Bcr-Abl (35), v-Fps and c-Fes
(26, 48), the epidermal growth factor (EGF) receptor
(55, 56), v-Eyk and c-Eyk (74), the hepatocyte
growth factor/scatter factor receptor c-Met (5), v-Sis, a
ligand activating the platelet-derived growth factor-receptor, and
polyomavirus middle-T antigen (26). Recently, we and others
have shown that in the case of v-Src, the activation of Stat3 is
required for transformation (7, 66).
, had a negative effect on transformation efficiency suggested that the activation of Stat1 may have been primarily responsible for transformation. In this study, we examined more extensively and quantitatively the importance of Stat1 and Stat3
activation in the transformation of rat 3Y1 fibroblasts by
constitutively active c-Eyk and v-Eyk. The constitutively active receptor containing the intracellular portion of the v-Eyk molecule induced strong activation of Stat3, but not of Stat1, and resulted in a
higher transformation efficiency. A single amino acid residue (glutamine 936) in the COOH-terminal tail of v-Eyk was found to be
responsible for constitutive Stat3 activation and to be required for
transformation. The transforming ability of c-Eyk could be converted to
that of v-Eyk by changing this single residue from leucine to
glutamine. Finally, we conclude from modification in another tyrosine
residue in the intracellular domain of the Eyk molecules that Stat3
activation alone is not sufficient for transformation.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
-32P]ATP (3,000 Ci/mmol),
[
-32P]deoxynucleoside triphosphates (3,000 Ci/mmol),
and the Renaissance immunodetection system, New England Nuclear;
luciferin, Boehringer Mannheim; GammaBind G-Sepharose and
glutathione-Sepharose 4B (glutathione-beads), Pharmacia; and
Lipofectamine and Opti-MEM, Gibco BRL; all other reagents, Sigma.
Cell lines. Nontransformed 3Y1 rat fibroblasts (39) and 3Y1 cells transformed with CD8-c-Eyk, CD8-v-Eyk, or v-Src were cultured in Dulbecco's modified Eagle medium (DMEM; Gibco BRL) supplemented with 5% calf serum (Gibco BRL). CD8-c-Eyk (74)- and CD8-v-Eyk- and v-Src-transformed cell lines (57) were described elsewhere. AD5-transformed simian virus 40 (SV40) large-T-antigen-containing 293T human embryonic kidney cells (52) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; HyClone).
Plasmid and probes. Luciferase reporter constructs used were pTATA-tk-Luc containing a minimal thymidine kinase promoter (44), pSV40-Luc containing the SV40 promoter and enhancer (pGL2 control; Promega), and p4xM67-tk-Luc. To obtain p4xM67-tk-Luc, an oligonucleotide containing four copies of the sequence GGTTCCCGTAAATGCATCA (underline denotes the STAT-binding site [33]) was introduced in the AccI-BamHI sites of pTATA-tk-Luc upstream of the minimal promoter.
Expression plasmids for constitutively active v-Src and Ras61L (RasLeu61) were pBabe/v-src (57) and pRSV/H-ras61L (18), respectively. Expression constructs used were pEBG/erk1 (65), pRcCMV/stat3-FLAG (7), and pRcCMV/stat1-FLAG (constructed as described for pRSVstat3-FLAG except using the coding sequence of Stat1). pMEXneo-based expression constructs for c-Eyk, CD8-c-Eyk, CD8-v-Eyk, and CD8-c-EykM have been described elsewhere (74). Point mutations in CD8-Eyk molecules were made via a site-directed mutagenesis protocol (QuickChange; Stratagene) as suggested by the manufacturer, giving rise to the constructs pMEX/CD8-c-EykL936Q (CTG to CAG, bp 2832 in c-Eyk [GenBank accession no. L21719 {37}]), pMEX/CD8-c-EykY933F (TAG to TTG, bp 2823), pMEX/CD8-c-EykY862F (TAG to TTG, bp 2610), pMEX/CD8-c-EykK609M (AAG to ATG, bp 1851), pMEX/CD8-v-EykY933F (TAG to TTG, bp 1973 in v-eyk/v-ryk [accession no. M92847 {38}]), pMEX/CD8-v-EykY862F (TAG to TTG, bp 1760), or pMEX/CD8-v-EykK609M (AAG to ATG, bp 1001). The amino acid numbers refer to the c-Eyk sequence (accession no. 438523 [37]). The construct pMEX/CD8-v-Eyk
890 was obtained by chance in an attempt to construct
pMEX/CD8-v-EykY862F but with an additional thymidine introduced in the
tyrosine codon (TAC-TTTC). This construct gave rise to a shortened Eyk
protein which carried a Y862F frameshift followed by 28 random
non-Eyk-related amino acids and a stop codon after amino acid 890. All
mutations mentioned above were confirmed by sequencing of the
constructs (Rockefeller University DNA Technology Center).
Oligonucleotides used for cloning or electrophoretic mobility shift
assay (EMSA) were custom made by The Rockefeller University DNA
Technology Center or by Genelink. The oligonucleotide M67 (upper
sequence, GATCGGTTCCCGTAAAT; underline denotes
STAT-binding site [37]) used in EMSAs was
32P labeled as described elsewhere (72).
Cell transfection and in vitro assays. 3Y1 fibroblasts, 293T cells, and transformed derivatives were transfected by using Lipofectamine as described by the manufacturer. The cells were either harvested for preparation of whole-cell extracts (extraction buffer consisted of 50 mM HEPES · NaOH [pH 7.4], 150 mM NaCl, 1% Triton X-100, 10% glycerol, 10 mM EDTA, 1 mM Na3VO4, 0.1 mM Na2MoO4, 1 mM phenylmethylsulfonyl fluoride, and 0.1 U of aprotinin per ml; ERK assay, Western blotting, and immunoprecipitation), cytoplasmic and nuclear extracts (74) (EMSA and related Western blot analyses), or luciferase extracts (3) or subjected to transformation assays 24 h later.
EMSA was carried out with 2 µg of nuclear extract from nontransfected (Fig. 1) or transfected (Fig. 3 to 5) cells as described previously (74), using 32P-labeled oligonucleotide M67. Western blotting of the resultant cytoplasmic extracts (20 µg) as a measure of expression of certain proteins was carried out in parallel according to standard methods (58) and instructions provided by the manufacturer of the immunodetection system. Luciferase reporter assays with 10 µg of extract were performed as described elsewhere (3). Results were plotted as means ± standard deviations (SD) of experiments performed in duplicate. Immunoprecipitations were done with various amounts of whole-cell extracts with the different antibodies. Briefly, for each precipitation, 1 µg of the antibody was incubated with 20 µl of GammaBind G-Sepharose on a rocker table at 4°C for 2 h. The antibody fraction bound to Sepharose was transferred to 200 µg of whole-cell extracts (adjusted to 1 mg of total protein/ml) and incubated overnight as described above. The next day, the Sepharose beads were washed four times with extraction buffer and the bound proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting as described above. Whole-cell extracts (100 µg/100 µl) from cells transfected with pEBG or pEBG/erk1 (expressing GST-ERK1) together with indicated constructs were precipitated with 20 µl of glutathione-beads for 1 h. The beads were washed once with extraction buffer and three times with kinase buffer (30 mM HEPES · NaOH [pH 7.4], 20 mM MgCl2, 2 mM MnCl2, 1 mM dithiothreitol). The kinase reaction was carried out for 30 min at 30°C in 20 µl of kinase buffer supplemented with 5 µg of myelin basic protein (MBP), 2 µCi of [-32P]ATP (3,000 Ci/mmol), and 10 µM ATP.
The kinase reaction was subjected to SDS-PAGE (8% gel) and
electroblotted to polyvinylidene difluoride membranes (Millipore), and
the 32P-labeled MBP was detected by autoradiography.
Transformation assay. Cells transfected with the various constructs were trypsinized, diluted in 7.5 ml of top agar (0.4% Bacto Agar in DMEM-10% FBS), and spread onto 60-mm-diameter plates containing 5 ml of 0.75% bottom agar (0.75% Bacto Agar in DMEM-10% FBS). The number of colonies of cells which were able to grow in the top agar was determined 2 to 3 weeks after plating.
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RESULTS |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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Stable v-Eyk-transformed cells show increased Stat3 DNA binding and
STAT-specific transcriptional activation.
Constitutively active
forms of c-Eyk and v-Eyk activated by dimerization through linking the
CD8 extracellular domain to the c-Eyk or v-Eyk internal domain are
able to transform immortalized 3Y1 fibroblasts in culture. Furthermore,
these dimeric receptor molecules when expressed transiently in COS
cells are tyrosine phosphorylated and able to activate Stat1, Stat3,
and Jak1 (74).
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Expression of different Eyk constructs induces transformation and increased tyrosine phosphorylation but no activation of ERK1. To further analyze the ability of CD8-Eyk molecules to induce transformation and to score their relative transformation efficiencies, we transfected a series of constructs into 3Y1 cells and after 3 weeks determined the number of transformed cells leading to colonies in soft agar were (Fig. 2A). The transformation assay clearly showed greater transforming ability by CD8-v-Eyk than by CD8-c-Eyk (99 ± 33 and 9 ± 4, respectively. A kinase-dead CD8-c-Eyk construct (CD8-c-EykM) included as a negative control did not lead to the formation of soft agar colonies.
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) or pEBG/erk1 (+) together with 500 ng of plasmid expressing
the indicated protein. Two days later, the cells were either treated
with 50 ng of EGF per ml (+EGF) for 10 min or left untreated, and
whole-cell extracts were prepared. GST-ERK1 was precipitated by using
glutathione-beads, and kinase activity toward MBP was determined (upper
panel). GST-ERK1 was detected with a GST-specific antibody (Santa Cruz)
(lower panel). In the rightmost lane, one-fifth of the kinase reaction
for Ras61L was loaded to avoid overexposure.
Transient expression of Eyk proteins leads to differential DNA binding of Stat3 and Stat1, resulting in activation of a Stat1/3-responsive promoter construct. Since stably transformed CD8-v-Eyk cells exhibited constitutive activation of Stat3, we determined the effects of transient transfection of CD8-c-Eyk and CD8-v-Eyk on Stat1/3-specific DNA binding and Stat1/3-specific transcriptional activation (Fig. 3). As previously shown in studies using COS cells (74), CD8-c-Eyk activates Stat1 and some Stat1/3 heterodimer, indicating that Stat3 is activated but to a lesser extent than Stat1. CD8-v-Eyk led to similar activation of Stat1 but much stronger activation of Stat3 compared to CD8-c-Eyk. A mutation in the kinase domain of Eyk in the construct CD8-c-EykM obliterated the activation of Stat1 and Stat3. As shown for v-Src-transformed 3Y1 cells (Fig. 1A, upper panel), v-Src activates Stat3 preferentially in 3Y1 cells when transiently expressed (Fig. 3A). The nature of the complexes observed in the EMSA has been confirmed by supershift analyses using Stat1- and Stat3-specific antibodies (data not shown).
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A YVPQ sequence in the COOH terminus of CD8-v-Eyk is responsible
for increased Stat3 activation and increased transformation
efficiency.
The coding sequences of c-Eyk and v-Eyk differ in
several regions (37). CD8-c-Eyk contains the transmembrane
domain of c-Eyk including 21 intracellular amino acids, whereas
CD8-v-Eyk contains the transmembrane domain of CD8 but not the
first 21 intracellular amino acids of c-Eyk. This difference accounts
for the slightly smaller molecular weight of CD8-v-Eyk than of
CD8-c-Eyk observed in the Western blot assays using the Eyk antibody.
The sequence of the remaining intracellular domains are identical
between CD8-c-Eyk and CD8-v-Eyk with the exception of two point
mutations. Methionine 631 in the kinase domain of c-Eyk corresponds to
an isoleucine in v-Eyk, and leucine 936 in the COOH terminus of c-Eyk
corresponds to glutamine in v-Eyk.
and the 21 intracellular amino acids of c-Eyk are not responsible for the
differences in Stat3 activation and transformation (data not shown). We
then tested whether the exchange of the COOH termini between CD8-c-Eyk
and CD8-v-Eyk influenced the activation of Stat3 and the
transformation efficiency. Interestingly, the COOH terminus of
CD8-v-Eyk (amino acids 918 to 974) when linked to the NH2
terminus CD8-c-Eyk could confer Stat3 activation and high
transformation capability (data not shown), suggesting that the
mutation L936Q is responsible for these abilities. Amino acid 936 is
juxtaposed to a potentially phosphorylated tyrosine residue at position
933, corresponding to the sequences Y933VPL in CD8-c-Eyk
and Y933VPQ in CD8-v-Eyk.
To test the hypothesis that Y933VPQ in CD8-v-Eyk is
responsible for Stat3 activation and higher transformation efficiency,
we performed site-directed mutagenesis to obtain mutated CD8-Eyk molecules (Fig. 4A). CD8-c-EykL936Q
showed strong activation of Stat3 as tested by DNA binding (Fig. 4B),
STAT-responsive transcription (Fig. 4C), and increased number of soft
agar colonies (Fig. 4D). CD8-v-EykY933F lost the ability to activate
Stat3 and showed transformation efficiency as low as that observed for
CD8-c-Eyk. As mentioned above, CD8-c-Eyk can activate Stat1 and
minimally Stat3 (Fig. 3A), leading to Stat1/3-specific transcriptional
activation (Fig. 3B). To further test the importance of tyrosine 933, we also introduced a Y933F mutation in CD8-c-Eyk. The construct
CD8-c-EykY933F did not show any difference from the wild-type
CD8-c-Eyk, suggesting that Y933 is important for Stat3 activation only
in conjunction with a glutamine in the +3 position. As expected from
the results with CD8-c-EykM described above, two kinase-dead
constructs, CD8-c-EykK609M and CD8-v-EykK609M, neither activated the
STAT pathway nor led to transformation of 3Y1 fibroblasts.
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890 contains a Y862F
mutation, followed by 28 nonrelated amino acids and a stop codon at
amino acid 890. Interestingly, this construct could still activate
Stat1 and Stat1/3-responsive transcription to the same level as
CD8-c-Eyk level, although the COOH terminus of CD8-v-Eyk including
Y933 responsible for high Stat3 activation is missing (Fig. 4B and C).
The antibody against the COOH terminus of Eyk used in Western blot
analyses (Fig. 4B, lower panel) did not recognize this construct. These
results indicated that the site(s) responsible for the activation of
Stat1 and the residual Stat3 activation is not located in the COOH
terminus and is either a tyrosine in the kinase domain or a
non-tyrosine-binding site.
A YVNT sequence is required for transformation. Two of the ten Y-to-F mutants that were generated in the CD8-c-Eyk and the CD8-v-Eyk backgrounds to identify the site responsible for Stat1 activation (see above), namely, CD8-c-EykY862F and CD8-v-EykY862F, showed interesting phenotypes. Both constructs were able to activate the STAT pathway similarly to their wild-type counterparts (Fig. 4B and C) but showed a greatly reduced ability to transform 3Y1 fibroblasts (Fig. 4D). These results indicated that activation of Stat3 by CD8-v-Eyk cooperates with another independent signaling pathway to transform 3Y1 cells in culture. Note that all transfectants analyzed in soft agar assays were also grown on plates containing selective medium. G418-resistant clone numbers were comparable between wild-type and mutant receptor constructs, indicating that there is no difference in the apoptosis in cells bearing the various receptors (data not shown).
The YVPQ sequence is required for binding of Stat3 but not of Stat1. The sequence Y933VPQ in CD8-v-Eyk fits the determined consensus binding sequence YXPQ in the gp130 receptor for the Stat3 SH2 domain (32). Therefore, we determined whether Stat3 interacts with CD8-Eyk molecules harboring the Y933VPQ sequence.
Different mutants of CD8-c-Eyk and CD8-v-Eyk in conjunction with FLAG-tagged Stat1 (Fig. 5A) or Stat3 (Fig. 5B) were transiently expressed in 293T cells. To detect an interaction between CD8-Eyk molecules and the various STAT molecules, we immunoprecipitated the STAT molecules with the FLAG epitope-specific antibody M2 and then determined the levels of CD8-Eyk and Stat1/3 molecules in the immunoprecipitates (Fig. 5). Expression of CD8-Eyk molecules was determined in whole-cell extracts from the transfected cells (Fig. 5A and B, upper panels). All CD8-Eyk molecules including the kinase-dead mutants CD8-c-EykK609M and CD8-v-EykK609M were able to interact with Stat1 (Fig. 5A). It is noteworthy that the expression level of the Stat1 molecules is influenced by the coexpression of kinase-active CD8-Eyk molecules.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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We have analyzed the signaling pathways downstream of activated Eyk molecules leading to transformation of 3Y1 fibroblasts and demonstrated the importance of Stat3 activation. Although Stat1 is activated by both Eyk molecules, this activation did not correlate with cellular transformation or with transcriptional activation.
The activation of Stat3 by the RTK CD8-v-Eyk presumably occurs directly after binding of Stat3 via its SH2 domain to a Y933VPQ sequence present in v-Eyk and by the subsequent tyrosine phosphorylation of Stat3 (Fig. 4 and 6). Similar sequences (YXXQ) have been shown to be important for Stat3 activation by Jak kinases in several cytokine receptors, e.g., the IL-10 receptor (71), the granulocyte colony-stimulating factor receptor (17), and gp130, the receptor activated after IL-6 treatment (32, 62). Unlike the cytokine receptors, the Stat3 docking sites for growth factor (e.g., EGF and platelet-derived growth factor) receptors which activate Stat3 had not been clearly defined. Stat3 activation by the hepatocyte growth factor/scatter factor receptor c-Met has been shown to be involved in epithelial tubule formation. A putative sequence for Stat3 binding in c-Met was suggested to be Y1356VNV, which also binds several other proteins (5). Interestingly, a similar sequence, Y862VNT, is present in Eyk, but appears not to be responsible for Stat3 activation although it is required for transformation (see below). c-Met also contains a YXXQ sequence, which raises the question of whether a synergistic effect similar to that described here for the transformation by CD8-v-Eyk is responsible for epithelial tubule formation by c-Met. It is noteworthy that v-Src does not contain a YXXQ sequence, suggesting that the activation of Stat3 by v-Src could involve another binding site for Stat3 (11).
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The observed activation of Stat1 by the CD8-Eyk molecules (Fig. 3) does not require the Y933VPQ sequence; therefore, Stat1 must bind to some other sites in both c-Eyk and v-Eyk (Fig. 5). We do not know whether Stat1 activation can have a negative effect on transformation efficiency, since Stat1 activation has been previously implicated in growth arrest of cells (8, 12). In fact, comparing the transformation efficiencies of CD8-v-Eyk and v-Src (data not shown and Fig. 1), we found a significantly higher number of colonies with v-Src-expressing cells. While v-Src and CD8-v-Eyk are comparable in the ability to activate Stat3, CD8-v-Eyk also activates Stat1. Thus, it is conceivable that the activation of Stat1 by CD8-v-Eyk can decrease transformation by v-Eyk or that additional signaling pathways involved in transformation are activated by v-Src but not by CD8-v-Eyk.
Wild-type and dominant negative Stat3 molecules have been shown to be efficiently block transformation by v-Src in NIH 3T3 cells (7, 66). We tried to examine the effect of these molecules and the associated Stat1 molecules on the transformation of 3Y1 cells. Coexpression of CD8-Eyk molecules with wild-type Stat1 or Stat3 greatly increased DNA-binding activity, and coexpression of CD8-Eyk with Stat3 led to increased Stat1/3-specific transcriptional activation (data not shown). Nevertheless, neither Stat1Y-F nor Stat3Y-F molecules (7) had a dominant negative influence on the DNA binding of the endogenous STAT molecules, or transcriptional activation, or on transformation by the CD8-Eyk and the v-Src molecules. All STAT molecules in these assays were expressed to detectable levels. We do not know why these dominant negative molecules are not functional under the conditions used here.
We previously reported that Stat1 acted as a suppressor of Eyk
transformation in 3Y1 cells. Stat1 and Stat1 can form heterodimers with Stat3, and eventually the dominant negative construct Stat1 blocks the activation of Stat1 and Stat3 to the same extent and leads
to a reduced number of soft agar colonies because of the blockage of Stat3.
The activation of Jak1 has been shown to be important for the activation of STAT molecules. We reported previously that CD8-Eyk molecules can activate Jak1 (74). The function of this activation for the activation of Stat3 and the transformation remains to be elucidated. Interestingly, Jak1 is also activated in v-Src-transformed fibroblasts, although the importance of this activation for transformation has not been investigated (10).
We present evidence that maximal transformation frequency by CD8-v-Eyk not only is dependent on Stat3 activation but also requires the activation of another pathway activated through the Y862VNT sequence that is present in both CD8-Eyk proteins.
The amino acid sequence following the tyrosine at position 862 is VNT.
The asparagine at +2 is highly conserved in the homologous sequence
among all members of the Axl/Tyro3 subfamily. A YXNX motif has been
reported to be a docking site for the SH2 domain of Grb2
(61). Grb2 as well as other molecules can bind to this motif
in Axl (6). Since docking of Grb2 to receptors leads to the
activation of the Ras/ERK pathway (42), one might expect activation of this pathway downstream of CD8-Eyk via the
Y862VNT sequence. Several reports demonstrated the
activation of ERK downstream of other members of the Axl subfamily in
32D cells and in NIH 3T3 fibroblasts (22, 41). The
involvement of the activation of the Ras/ERK pathway for the
physiological function of these receptors has not been addressed.
Although we did not observe activation of ERK1 in 3Y1 cells (Fig. 2C),
we found that in NIH 3T3 cells, CD8-c-Eyk and CD8-v-Eyk are able to
modestly activate GST-ERK1, suggesting cell line specific regulation of this pathway (data not shown). However, increased ERK activity does not
appear to occur in the transformation of 3Y1 cells by CD8-Eyk. A
homologous tyrosine in c-Axl (Y821VNM) seems to be involved
in the activation of phosphatidylinositol (PI) 3-kinase, S6 kinase and
protein kinase B (PKB)/Akt (6, 29), and we reported that the
homologous tyrosine in the c-Mer receptor, the closest mammalian
homologue of c-Eyk, leads to activation of NF-
B (27). A
very recent study shows that this sequence in c-Axl is required for
transformation (9). We are investigating the potential role
of these molecules in the signaling events downstream of Y862 in
CD8-Eyk.
In conclusion, we propose the following model for the signaling events downstream of CD8-c-Eyk and CD8-v-Eyk leading to the transformation of immortalized 3Y1 fibroblasts (Fig. 6). Both molecules are able to bind and activate Jak1 (74), Stat1, and minimally Stat3 via a region residing in the kinase domain or the intracellular region N terminal to the kinase domain. The major difference between the cellular form CD8-c-Eyk and the viral form CD-v-Eyk is the strong binding of Stat3 to CD8-v-Eyk via a YVPQ sequence (Fig. 5), DNA binding, and transcriptional activation (Fig. 3). A second pathway which appeared to be important for the transformation events by both CD8-Eyk molecules involves Y862VNT. This activation seems to be responsible for the transformation events downstream of CD8-c-Eyk since a construct missing this tyrosine is virtually unable to induce transformation. In CD8-v-Eyk, this pathway synergizes with the activation of Stat3, resulting in transformation efficiency 10-fold higher than that of CD8-c-Eyk.
Our model illustrates how two independent signaling pathways cooperate in inducing transformation in 3Y1 fibroblasts. We speculate that the mutation of Y933VPL to Y933VPQ upon retroviral transduction of the RPL30 virus, harboring v-Eyk, gained a biological advantage by leading to strong activation of Stat3.
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ACKNOWLEDGMENTS |
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We thank Inés Ibañez Tallon and Raymond Birge for discussion and critical reading of the manuscript, Sheila Harroch and all members of the Hanafusa laboratory for helpful advice, and those mentioned in the text for expression plasmids.
This work was supported by Counsel for Tobacco Research grant 4438R1 (to H.H.) and NIH grants CA44356 (to H.H.) and AI32489 and AI34420 (to J.E.D.). D.B. was supported by a fellowship from the Swiss National Foundation, and J.F.B. was supported by a Howard Hughes postdoctoral fellowship for physicians and NIH K08 grant CA67950.
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FOOTNOTES |
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* Corresponding author. Mailing address: The Rockefeller University, Laboratory of Molecular Oncology, 1230 York Ave., New York, NY 10021. Phone: (212) 327-8805. Fax: (212) 327-7943. E-mail: besserd{at}rockvax.rockefeller.edu.
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REFERENCES |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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