From Physiological Chemistry I, Biocenter (Theodor Boveri Institute), University of Würzburg, Am Hubland, 97074 Würzburg, Germany
Received for publication, July 24, 2000, and in revised form, October 17, 2000
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ABSTRACT |
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Overexpression of the oncogenic receptor tyrosine
kinase ONC-Xmrk is the first step in the development of
hereditary malignant melanoma in the fish Xiphophorus.
However, overexpression of its proto-oncogene counterpart
(INV-Xmrk) is not sufficient for the oncogenic function of
the receptor. Compared with INV-Xmrk, the ONC-Xmrk receptor displays 14 amino acid changes, suggesting the presence of activating mutations. To
identify such activating mutations, a series of chimeric and mutant
receptors were studied. None of the mutations present in the
intracellular domain was found to be involved in receptor activation.
In the extracellular domain, we found two mutations responsible for
activation of the receptor. One is the substitution of a conserved
cysteine (C578S) involved in intramolecular disulfide bonding. The
other is a glycine to arginine exchange (G359R) in subdomain III.
Either mutation leads to constitutive dimer formation and thereby to
activation of the ONC-Xmrk receptor. Besides, the presence of these
mutations slows down the processing of the Xmrk receptor in the
endoplasmic reticulum, which is apparent as an incomplete glycosylation.
Receptor tyrosine kinases
(RTKs)1 are important
components of the signaling network that controls cell growth and
differentiation. Their enzymatic activity is tightly regulated in
normal cells. After ligand binding and dimerization, they become
activated and a cascade of phosphorylations is initiated inside the
cells (1). Diverse mechanisms have been reported that can lead to the
constitutive activation of these enzymes. These comprise
overexpression, amplification, point mutations, truncations, and
autocrine stimulation. The inappropriate constitutive activation of the
RTKs results in an altered signaling inside the cell and is a widely
documented process implicated in tumor formation (2, 3).
The hereditary melanoma of Xiphophorus fish is a well
established genetic model system for tumor development in which the overexpression of the RTK gene Xmrk
(Xiphophorus melanoma receptor kinase) leads to melanoma formation
(for review, see Ref. 4). Xmrk belongs to the epidermal
growth factor receptor (EGFR) family, but it is an additional member,
clearly distinct from the four receptors (HER1-4) already described in
mammals (5).2 Two copies of
the Xmrk gene have been found. One of them,
INV-Xmrk, is a gene invariably present in all fish
(6).3 It is ubiquitously
expressed at low levels, and, although its physiological role is still
unknown, it appears not to be involved in melanoma formation. It
represents the proto-oncogenic form of Xmrk.
The second oncogenic copy, ONC-Xmrk, is only present in some
species of Xiphophorus. It originated by an ancient gene
duplication event from INV-Xmrk and it is under a different
transcriptional control than the proto-oncogene. Only basal levels of
expression are observed if the regulatory locus R is also
present in the genome. This is the situation found in nonhybrid wild
fish, which are generally tumor-free. In hybrids, due to crossing
conditioned elimination of the R-containing chromosome (7),
the system is deregulated and ONC-Xmrk is overexpressed (8).
This leads to neoplastic transformation of pigment cells. A cell line
(PSM) derived from Xiphophorus melanoma provides an in
vitro system where ONC-Xmrk is also overexpressed.
Here, as in melanoma in situ, the Xmrk receptor is highly
activated, which is apparent as strong tyrosine autophosphorylation (9,
10).
The fact that the highly expressed ONC-Xmrk is constitutively
autophosphorylated in melanoma cells pointed to overexpression, and
thus high concentration of receptors, as one mechanism for activation.
However, the ectopic overexpression of INV- and ONC-Xmrk in
embryos of transgenic fish showed that exclusively those fish expressing ONC-Xmrk were developing tumors with high
incidence, short latency periods, and a specific pattern of affected
tissues, whereas only a basal rate of tumor induction appeared in the
case of INV-Xmrk-expressing fish, comparable to the rate
obtained with the expression of another, nonactivated receptor (6, 11). Besides that, INV-Xmrk was shown to be not phosphorylated when transiently expressed in human cells (HEK293), in contrast to the
strong autophosphorylation of ONC-Xmrk. The different behavior of INV-
and ONC-Xmrk clearly indicates that a mechanism additional to
overexpression is instrumental in Xmrk activation (6).
Comparison of the amino acid sequences of the two versions of Xmrk
revealed that the oncogene differs from the proto-oncogene in 14 residues, including some that are highly conserved in the EGFR family
of RTKs and that are present in INV-Xmrk but substituted in ONC-Xmrk
(6). This fact suggested that mutational alteration could be involved
in the activation of Xmrk. Moreover, the high phosphorylation level
shown by an ONC-INV chimeric receptor containing the extracellular
domain of ONC-Xmrk and the intracellular domain of INV-Xmrk pointed to
one or more of the mutations in the extracellular domain as implicated
in the activation (6). However, this result did neither address a role
of the intracellular mutations in the activation of Xmrk nor could it
identify the extracellular oncogenic amino acid change.
To understand the mechanism of activation of ONC-Xmrk, we have analyzed
the effect of different mutations. We show that two mutations in the
extracellular region of the ONC-Xmrk receptor are responsible for
activation. Both of them independently lead to the constitutive
dimerization of the receptor by aberrant intermolecular disulfide
bonding formation. Additionally, the presence of these mutations slows
down the processing of Xmrk receptor in the endoplasmic reticulum (ER),
which is apparent as an incomplete glycosylation.
Antibodies--
5E.2 (anti-Tyr(P)) is a mouse monoclonal
antibody directed against phosphotyrosines (12). Anti-mrk is a
polyclonal antibody raised against ONC-Xmrk (9).
Construction of Expression Plasmids--
The INV-ONC chimera was
generated by replacing an EcoRI fragment from pRK5 ONC (pRK5
Xmrk; Ref. 13) containing the extracellular, transmembrane, and
juxtamembrane domains from the ONC-Xmrk receptor with an INV-Xmrk
fragment corresponding to the same domains (6). An
EcoRI-Eco47III and an
Eco47III-SalI fragment from ONC-Xmrk were used to replace the corresponding fragments in pRK5 INV and thus
generate the Xmrk(G359R,P388T) and Xmrk(P470L,S476N,C578S,M595I) chimeras, respectively. The different point mutations were created using a Muta-Gene phagemid in vitro mutagenesis kit (version
2, Bio-Rad). Different fragments of INV- or ONC-Xmrk cloned
in pBlueScript were used as templates. The following primers
were used to generate the mutations: 5'-GAAGGATCCGATGTTGGTTG-3' for the
Xmrk(P388T) mutant, 5'-GGACCTGATGTTGGTCGAGTTG-3' for the INV(G359R)
mutant, and 5'-TGCACACTTCGAGCAGTTGG-3' for the Xmrk
(P470L,S476N,M595I) mutant. The INV(C578S) mutant was generated by
replacing a CpoI-NsiI fragment from
INV-Xmrk by the corresponding one from ONC-Xmrk containing the C578S mutation. All constructs containing point mutations were sequenced to ensure that the desired mutation was present.
Cosmid Clones--
Three clones from a X. maculatus
cosmid library were used as templates for sequencing the mutations.
Cosmid L11 091 contains the X-ONC-Xmrk allele, M08 036 contains the Y-ONC-Xmrk allele, and G01 008 contains
INV-Xmrk (14). Oligonucleotides designed from introns 4, 8, 9, 11, and 14 were used as sequencing primers. Cycle sequencing was
performed using a Cycle Sequencing Kit (Amersham Pharmacia Biotech).
The sequence of these cosmid clones revealed the existence in INV- and
ONC-Xmrk of an additional codon in positions 1744 and 1573, respectively. This leads to the insertion of a glycine between Trp-512
and Pro-513. Therefore, the amino acid positions higher than 512 are
increased by 1 as compared with the numbers published by Dimitrijevic
et al. (6). The INV- and ONC-Xmrk sequences in
the GenBankTM data base with accession numbers U53471 for
INV-Xmrk and X16891 for ONC-Xmrk have been
updated accordingly.
Transfections and Cell Culture--
HEK293 cells (human
embryonic kidney fibroblasts) were grown in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) containing 10% fetal calf
serum and 1% penicillin-streptomycin. Cells were transiently
transfected using a modified calcium phosphate transfection method
(15). For the transfections, 1 µg of plasmid DNA encoding each
expression construct was used per 2.3-cm dish and 3-10 µg per 9-cm
dish. Following overnight transfection the medium was changed. Cells
were harvested 48 h after transfection.
Ba/F3 (mouse pro-B-cell line; Ref. 16) cell culture, transfection, and
colony selection were done as described previously (10). BaF ONC and
BaF INV-ONC stable clones express pRK5 ONC (pRK5 Xmrk; Ref. 13) and the
INV-ONC chimera, respectively.
Immunoprecipitations and Western Blotting--
After harvesting,
the cells were washed twice with cold PBS and lysed in Triton lysis
buffer as described (17). Immunoprecipitations were done by using
protein A-Sepharose and anti-Xmrk serum. Immunoprecipitates and cell
lysates were electophoresed through a 6.5% or 7.5% SDS-PAGE gel or a
3-8% gradient SDS-PAGE gel. Laemmli buffer either containing or
lacking 2-mercaptoethanol was added to the samples to be run under
reducing or nonreducing conditions, respectively. The separated proteins were blotted onto a nitrocellulose membrane (Schleicher & Schuell) using standard protocols. Filters were blocked for 5 min in 10 mM Tris-Cl, pH 7.9, 0.5% Tween, 1.5% BSA and incubated 1 h with the first antibody. Horseradish peroxidase-coupled second antibodies were used and developed by enhanced chemiluminescence (ECL;
Amersham Pharmacia Biotech) according to manufacturer's instructions.
When necessary, the filters were stripped in 62.5 mM
Tris-Cl, pH 6.7, 2% SDS, and 100 mM 2-mercaptoethanol for
20 min at 50 °C, following three washes with PBS before blocking with 1.5% BSA previous to the second probing.
Endoglycosidase H Digestion--
Equal amounts of protein
extracts were denatured at 95 °C for 5 min in a digestion buffer
containing 50 mM tribasic sodium citrate (pH 5.5), 0.5%
SDS, 0.1 M 2-mercaptoethanol, and 0.5 mM phenylmethylsulfonyl fluoride. 5 milliunits of endoglycosidase H (Roche
Molecular Biochemicals) were added to half of the extracts, and all
were incubated at 37 °C for 20 h. Digestion products were analyzed on Western blots detected with an anti-Xmrk antibody.
Immunohistochemistry--
105 BaF INV-ONC or BaF ONC
cells were suspended in 500 µl of ice-cold PBS and spun onto slides
at 220 × g for 5 min using a cytospin device
(Hettich). After removal of the liquid, cells were consecutively fixed
for 10 min at The Mutations Responsible for Xmrk Activation Are Exclusively
Located in the Extracellular Domain--
From an earlier study (6), it
was known that the activation of ONC-Xmrk is due not only to
overexpression but also to one or several mutations located in the
extracellular domain of ONC-Xmrk that might contribute to the oncogenic
potential of the receptor. It was shown that an ONC-INV chimera
containing the extracellular part of ONC-Xmrk fused to the
intracellular domains of INV-Xmrk is strongly autophosphorylated when
transiently expressed in human 293 cells.
However, there are five mutations in the carboxyl terminus of ONC-Xmrk
whose role was still unknown. Although the carboxyl terminus is the
most divergent region in this family of receptors, two of the
substitutions found there correspond to highly conserved residues in
all subclass I RTKs. One is the exchange of a proline for leucine
(P984L), and the other is a tyrosine for asparagine (Y1038N) (6). To
exclude a possible contribution of the intracellular domain of ONC-Xmrk
in its oncogenic activation, an INV-ONC chimera containing the
extracellular sequences of INV-Xmrk and the intracellular region of
ONC-Xmrk was generated. After transfection of 293 cells and
immunoprecipitation of the chimeric receptor, the INV-ONC chimera
showed a level of phosphorylation clearly lower than the ONC-INV
construct and similar to that of INV-Xmrk (Fig.
1). This indicates that all activating
mutation(s) should be located in the extracellular part and none of the
intracellular mutations of ONC-Xmrk is involved in activation. This was
confirmed by introducing the five COOH-terminal amino acid changes
(P984L, N1025T, A1035T, Y1038N, and L1156Q) into INV-Xmrk. All mutant
receptors did not show enhanced autophosphorylation when compared with
INV-Xmrk (data not shown).
Evaluation of Species-specific Changes--
For technical reasons,
the cDNAs from INV-Xmrk and ONC-Xmrk genes
were originally isolated from two different species of
Xiphophorus. The cDNA from INV-Xmrk was
isolated from the Xiphophorus xiphidium-derived A2 cell line
and ONC-Xmrk gene from the PSM cell line derived from
Xiphophorus maculatus melanoma (18). The different species origin of these two genes could account for some of the effective nucleotide differences between them. To distinguish which of the changes in the extracellular domains were due to a species-specific variation and which were potentially functional mutations of the oncogenic Xmrk, we sequenced a series of cosmid clones that
contain genomic DNA from different alleles of X. maculatus
INV- and ONC-Xmrk. The alignment on Fig.
2 shows that none of the nucleotide
polymorphisms results in an amino acid difference between X. maculatus and X. xiphidium INV-Xmrk. However, two of
the amino acid changes noted earlier (P195H and S446R) probably are
irrelevant as they do not appear in the ONC-Xmrk sequence
obtained from X. maculatus DNA. They may be a cell
line-specific characteristic as they appear exclusively in the PSM
cells. They were not considered further; thus, the number of supposed
effective mutations in the extracellular region of ONC-Xmrk is reduced
to six. Four of these six mutations (G359R, P470L, S476N, and M595I)
involve amino acids that are not conserved in other members of the EGFR
family, the fifth includes the loss of a semiconserved proline (P388T),
and the sixth eliminates a highly conserved cysteine (C578S) from the
second cysteine-rich domain of the receptor.
Mutations in the Extracellular Domain Promote Covalent
Dimerization--
One mechanism described for RTKs resulting in
constitutive activation is ligand-independent dimerization. Using a
heterologous system (293 cells) for transient expression of the
different Xmrk constructs, we had observed traces of a high molecular
weight phosphorylated form of the molecule that, however, could not be resolved in the routine SDS-polyacrylamide gels. This form appeared exclusively in the constructs where the extracellular domain of ONC-Xmrk was present (see Fig. 1). To test whether this could correspond to a dimeric form of the ONC-Xmrk receptor, electrophoreses in gradient denaturing gels under reducing and nonreducing conditions were performed. After blotting and detection of the proteins with an
Xmrk antibody for both INV- and ONC-Xmrk, the appearance of a 160-kDa
form that corresponds to the Xmrk monomer was recorded (Fig.
3A, lanes
1 and 2). In addition, for ONC-Xmrk under
nonreducing conditions, another signal with a higher molecular weight
was appearing that was consistent with the size of a dimer. This signal was not present under reducing conditions. When the ONC-INV and INV-ONC
chimeras were subjected to the same analysis, the signal corresponding
to the dimer was only present in the case of the ONC-INV chimera under
nonreducing conditions (Fig. 3A, lanes
3 and 4). These data point to the presence of one
or several mutations in the extracellular part of ONC-Xmrk allowing
ligand-independent covalent dimerization.
To investigate whether disulfide-linked dimers were also present in
ONC-Xmrk from fish melanoma cells, we analyzed PSM cells and melanoma
tissue extracts on a gradient gel. The detection with an anti-mrk serum
showed, in both cases, the presence of dimers under nonreducing
conditions, whereas these were not present under reducing conditions
(Fig. 3B). This suggests that in vivo the same
mechanism is present as studied after overexpression of ONC-Xmrk in 293 cells.
More than One Activating Mutation Is Present in the Extracellular
Domain--
To identify the extracellular mutation(s) present in
ONC-Xmrk responsible for dimerization, additional chimeric constructs were made. In these constructs portions of the ONC-Xmrk extracellular domain were used to replace the corresponding regions in INV-Xmrk, thus
introducing groups of mutations in the backbone of the proto-oncogene. Two new chimeras were constructed, Xmrk(G359R,P388T), which contains the two most amino-terminal mutations in the extracellular domain, and
Xmrk(P470L,S476N,C578S,M595I), which contains the four last extracellular mutations of ONC-Xmrk. When these chimeras were subjected
to Western blot analysis, dimer formation under nonreducing conditions
was observed in both cases (Fig. 4),
suggesting that more than one activating mutation exists in ONC-Xmrk
that leads to ligand-independent receptor dimerization.
The C578S Mutation Is Involved in Dimer Formation and Constitutive
Activation of Xmrk--
It has been already described for RTKs (19,
20) that when one conserved cysteine involved in intramolecular
disulfide bonding is lost the remaining cysteine of the pair is able to form an intermolecular disulphide bridge. This aberrant bonding leads
to the formation of receptor dimers that can be observed under
nonreducing conditions. In the case of ONC-Xmrk, a cysteine in position
578 is lost and substituted by a serine. To verify whether this change
was one of the mutations involved in activation, an INV-Xmrk receptor
containing the Cys-to-Ser mutation was constructed. After transient
expression in 293 cells, the INV(C578S) mutant was immunoprecipitated
with an anti-mrk serum and detected with an anti-phosphotyrosine
antibody. This analysis showed increased tyrosine phosphorylation of
the mutant receptor compared with INV-Xmrk, demonstrating that the
introduction of this mutation was sufficient to activate Xmrk receptor
(Fig. 5A). When the INV(C578S) receptor was analyzed on a denaturing gradient gel under nonreducing conditions, the appearance of a band was observed consistent with the
size of the dimer as it appeared also for ONC-Xmrk (Fig.
5B). However, the extent of dimerization was lower than that
observed for ONC-Xmrk, suggesting additional differences between the
two receptors.
To further limit the mutations being involved in dimer
formation, a new construct was made wherein the
Xmrk(P470L,S476N,C578S,M595I) chimera S578 was reverted to the wild
type cysteine. The expression of the Xmrk(P470L,S476N,M595I) variant in
293 cells and subsequent analysis of the receptor protein showed that
this receptor is not able to produce dimers (Fig. 5C). This
finding confirms that P470L, S476N, and M595I do not contribute to
ONC-Xmrk activation.
An Additional Mutation in the Extracellular Part of Xmrk Is
Involved in Dimer Formation and Activation--
As shown by the
Xmrk(G359R,P388T) chimera, there exists a second activating mutation in
ONC-Xmrk that also leads to aberrant disulfide bridging and
dimerization. To find out which of these two mutations was responsible
for dimer formation, the Arg in position 359 was reverted to the wild
type Gly. The analysis in gradient denaturing gels of 293 extracts
expressing the Xmrk(P388T) mutant showed an almost complete
disappearance of the dimer band, suggesting that the Arg in position
359 was the second activating mutation (Fig.
6A). To further support this,
we introduced the Arg mutation in the backbone of INV-Xmrk and studied
its level of phosphorylation. Cellular extracts from 293 cells
transiently transfected with the INV(G359R) mutant were
immunoprecipitated with an anti-mrk serum and subsequently detected
with an anti-Tyr(P) antibody. The INV(G359R) mutant appeared strongly
phosphorylated (Fig. 6B), showing as in the case of the
C578S mutation that covalent dimer formation correlates with increased
receptor phosphorylation.
Different Glycosylation Patterns due to ONC-Xmrk
Mutations--
Analysis of INV- and ONC-Xmrk with the same antibody on
Western blots revealed that both receptors had slightly different electrophoretic mobilities in SDS denaturing gels. ONC-Xmrk always migrated as a smaller molecular weight form than INV-Xmrk (Fig. 7A). This different extent of
migration was obviously associated with the activating mutations. The
ONC-INV chimera showed the same mobility as ONC-Xmrk (data not shown).
However, when the chimeras Xmrk(P470L,S476N,C578S,M595I) and
Xmrk(G359R,P388T) containing only one of the activating mutations each
were analyzed in Western blots, a mixture of the two forms
characteristic of ONC- and INV-Xmrk appeared (Fig. 7B). When
both activating mutations were reverted to the proto-oncogenic
residues, the resulting Xmrk(P388T) and Xmrk(P470L,
S476N,M595I) proteins migrated in the SDS denaturing gels like the
proto-oncogenic form of the receptor. As all these proteins contain the
same number of amino acids, this could reflect a different
post-translational modification. The main post-translational modification in the extracellular domain of a receptor tyrosine kinase
from the family of the EGFR is the glycosylation of certain asparagine
residues. However, none of the mutations affects a canonical
glycosylation sequence. It has been reported that proteins that expose
reactive thiol groups can be retained in the ER (21). Because of
incorrect folding, they are kept there by some kind of quality control
mechanism and they are not processed further to the Golgi (22, 23).
Glycoproteins stopped in this cellular compartment contain high mannose
type oligosaccharides and are therefore sensitive to endoglycosidase H
(endo H) digestion. In contrast, fully N-glycosylated
receptors anchored in the membrane are endo H-resistant. In the case of
ONC-Xmrk, we have demonstrated that the activating mutations lead to
the formation of receptor dimers by intermolecular disulfide bridging
between unpaired cysteines. To test whether these proteins containing
activating mutations and thereby leaving active thiol groups were endo
H-sensitive, we performed digestions on extracts corresponding to
transient transfections of INV- and ONC-Xmrk in 293 cells. The ONC-Xmrk form of approximately 155 kDa was completely digested, and a band of
about 135 kDa appeared (Fig. 7A), which is consistent with the size of the unglycosylated form of ONC-Xmrk as already described (9). However, the INV-Xmrk form of 160 kDa is barely affected by the
action of endo H (Fig. 7A). Endo H digestion of the
Xmrk(G359R,P388T) and Xmrk(P470L,S476N,C578S,M595I) chimeras also
showed the existence of the ONC-Xmrk endo H-sensitive form that was
not present when the mutations were reverted to the wild type
amino acids as shown in the Xmrk(P388T) and Xmrk(P470L,S476N,M595I)
chimeras (Fig. 7B). These results suggest that ONC-Xmrk
containing both mutations is retained in the ER and is not properly
transported and located in the outer cellular membrane.
To elucidate the cellular localization of the different Xmrk receptors,
we performed immunohistochemical analysis of stable Ba/F3 clones
expressing the ONC or INV-ONC receptors. The BaF INV-ONC cells showed
stronger membrane and weaker cytoplasmic anti-mrk staining, indicating
a correct transport of the receptor to the plasma membrane (Fig.
8). However, in BaF ONC cells, the anti-mrk signals were preferentially detected directly around the
nuclear membrane, suggesting a localization in the ER and confirming
that the processing of the Xmrk receptor is affected when the
extracellular mutations are present.
ONC-Xmrk activation results from at least two mechanisms:
overexpression and mutational alteration. In the present study, we have
identified the activating mutations and characterized their mode of action.
The role of the mutations in the carboxyl terminus of Xmrk was unknown.
Earlier experiments with a Xmrk chimera (HER-mrk) that contained the
intracellular compartment of ONC-Xmrk fused to the extracellular region
of the human EGFR showed that this was inactive when expressed in 293 cells (13). Nevertheless, the existence of a determinant in the
extracellular domain of HER that was inhibitory for receptor activation
could not be excluded. It has been reported for the Ret receptor that
the MEN2B mutation located in the catalytic domain when cloned in a
EGFR/ret construct was less effective in activating the Ret function as
compared with full length Ret receptor (24). The fact that the level of
autophosphorylation of the INV-ONC chimera was comparable to that of
INV-Xmrk when expressed in 293 cells showed that none of these
mutations is responsible for the high phosphorylation level observed in
ONC-Xmrk and proved the assumption that the activating mutations are
exclusively located in the extracellular domain. Furthermore, none of
the mutations is located in any of the described substrate binding
sites of Xmrk (25). This excludes the possibility of interaction of the
oncogene receptor with intracellular substrates different to those of
the physiological receptor, a phenomenon already described for other
RTKs (26-29).
The extracellular domain of the EGFR family members can be subdivided
into four regions (1). Subdomains I and III are involved in ligand
binding in the EGFR (30), subdomain III being the one containing the
major ligand binding site (31). Subdomains II and IV are cysteine-rich
domains characterized by the existence of highly conserved cysteines
involved in intramolecular disulfide bonding (32). In the extracellular
domain of ONC-Xmrk, six effective substitutions were found. These six
mutations are not scattered throughout the whole extracellular region
but are clustered in subdomains III and IV. Activating mutations in the
extracellular domain of different RTKs have been described. Many
stabilize a dimeric conformation and lead to ligand-independent
stimulation of the tyrosine kinase activity. Others abolish ligand
binding, and a failure in receptor down-regulation is the major
mechanism that enhances tumorigenicity (33). The permanently active
chimera ONC-INV showed not only a high level of autophosphorylation but was also able to form covalent dimers in the absence of a ligand due to
the presence of two independent activating mutations. One of the
mutations (C578S) substitutes a serine for a cysteine. By introducing
this change in the backbone of INV-Xmrk, we could demonstrate that it
was enough to activate the receptor through the formation of
disulfide-linked dimers. Such a mechanism for receptor activation is
consistent with reports on mutant Ret receptors (19, 34-36), different
members of the fibroblast growth factor receptor family (reviewed in
Ref. 37), Ron receptor (38), and Epo receptor (39), which all show
ligand-independent dimerization and activation by mutations that create
an unpaired cysteine. In most of these cases, as in ONC-Xmrk, the
mutation destroys an intramolecular disulfide bond-forming cysteine and
the remaining cysteine of the pair is then free to form intermolecular
links. Taking the disulfide bond structure of the human EGFR as
reference (32), we can predict that Cys-586 in ONC-Xmrk is the
intramolecular partner of the mutated cysteine and thus the unpaired
residue responsible for the formation of the aberrant links.
The effect of this kind of mutation has also been described in other
members of the EGFR family. Deletions affecting different cysteines
within the extracellular domain of Neu were found in mammary tumors of
transgenic mice carrying a mouse mammary tumor virus (MMTV)/wild type
neu fusion (40). In Let-23, the Caenorhabditis elegans EGFR homolog, the sa62 mutation found in
mutagenized animals involves the loss of a cysteine and leads to excess
vulval differentiation (41). In the human EGFR, introduction of an
extra cysteine near the transmembrane domain was enough to promote
dimer formation in transient expression assays (42). The case reported
here of ONC-Xmrk activation and ligand-independent dimerization by loss
of a cysteine residue is the first naturally occurring and disease-related mutation of a cysteine described in the EGFR family (43).
It has been shown for several RTKs that receptor dimerization does not
always lead to activation (35, 44, 45). It can happen that, although
dimers are formed, the two receptor molecules are brought together in
an incorrect spatial configuration preventing cross phosphorylation of
the receptors. In the case of ONC-Xmrk, the one link that the C578S
mutation provides for dimer formation is enough to activate the
receptor, leading to the suggestion that this mutation can bring the
receptors together in a tight and correct way. Moreover, if the
physiological ligand-mediated dimer interface is maintained, additional
residues that interact in natural conditions may also play a role to
stabilize these aberrant dimers.
The second activating mutation found in ONC-Xmrk is the substitution of
a glycine by an arginine in position 359. This mutation is located at
the beginning of subdomain III of the receptor, which in the EGFR
contains the major ligand binding site (31). The mechanism of action of
this mutation seems to be similar to the one found for the C578S
substitution, namely the formation of disulfide-linked dimers. The
ability to dimerize under nonreducing conditions implicates a role for
a cysteine responsible for building intermolecular bonds. This normally
happens when a mutation leads to the appearance of an unpaired
cysteine, but in the case of the G359R mutation, no cysteine is being
lost or gained. Therefore, the more feasible explanation for the
appearance of the dimers would be that this mutation provokes a
structural change that could disrupt a disulfide bond enabling the
cysteines involved to establish intermolecular interactions. A similar
mechanism has been demonstrated for two activating mutations of FGFR2.
In this case two different mutations not involving cysteines but being
structurally close to a disulfide bond alter the local conformation and
prevent the formation of the intramolecular bond (46). Additionally, in
the case of the Ret receptor, a deletion involving Glu-632-Leu-633 promotes disulfide-linked dimer formation. This deletion is placed just
upstream of Cys-634, whose substitution is responsible for the MEN2A
syndrome (36).
In addition to inducing activation of the receptor, the point mutations
at positions 359 and 578 were found to bring about partial inhibition
of glycosylation and processing of ONC-Xmrk. The presence of only one
of the activating mutations was sufficient for the appearance of an
endo H-sensitive form of the molecule. In the case of ONC-Xmrk and
ONC-INV, containing both activating mutations, the rate of maturation
appears to be very slow due to the strong retention observed in the ER,
and thus only the endo H-sensitive form is present. This leads to the
conclusion that ONC-Xmrk is able to form homodimers in the ER;
furthermore, it can signal from there. This is in agreement with data
reported for other mutated receptors. Two different Ret receptor
mutants causing MEN2A syndrome were shown to be retained in the ER and were able to signal from there (36). A similar situation was described
for a splicing variant of the RON receptor (38) and for a mutant of the
cytokine receptor EPO-R (39).
In conclusion, we have found that ONC-Xmrk contains two mutations that
are able to activate the receptor independently. Although different
activating mutations have been described in the extracellular domain of
several RTKs, thus far only one activating mutation was present in each
reported case. A MEN2A case caused by two de novo mutations
of the RET gene was published recently, but the role of one
of the mutations in activation is still not clear (47). Thus, the case
of ONC-Xmrk is the first RTK described where two independently
activating mutations are present and inherited.
The presence of two independent activating mutations in
ONC-Xmrk is also intriguing from an evolutionary point of
view. The wild-type nonhybrid fish found in nature are polymorphic for
the presence of ONC-Xmrk; in fact, in some populations or
species, it is totally absent. Thus far, no physiological function can be ascribed to ONC-Xmrk. The gene duplication that generated
ONC-Xmrk was dated several million years ago (48). It could
have been expected that a dispensable second copy of Xmrk
would accumulate missense mutations or mutations that impair its
biochemical function as an RTK. However, ONC-Xmrk does not contain
missense mutations and most amino acid changes found are obviously
neutral in effect. The two activating mutations described here make it
even a "better, more active receptor," at least as an oncoprotein.
This is consistent with data on the rate of synonymous
versus nonsynonymous nucleotide changes in ONC- and
INV-Xmrk, which indicated that ONC-Xmrk is not
evolving as a pseudogene (6). However, the selective forces that could
maintain the gene product of ONC-Xmrk functional and oncogenic over long evolutionary times and perpetuate an intact gene in
the germline of these fishes are still to be identified.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C in methanol and for 2 min at
20 °C in
acetone. The fixed cells were then blocked with 1% BSA in PBS for 20 min at room temperature, washed three times with PBS, and incubated
with anti-mrk serum (40 µg/ml) for 60 min. After washing, another
incubation with dichlorotriazinaminofluorescein-conjugated anti-rabbit
IgG (Dianova; 30 µg/ml) for 60 min followed. Cells were embedded in
mounting medium containing DAPI (Vectashield, Vector Laboratories).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (43K):
[in a new window]
Fig. 1.
Tyrosine phosphorylation state of different
Xmrk receptor variants. Whole cell lysates from 293 cells
transfected with the indicated constructs (ONC, ONC-Xmrk;
INV, INV-Xmrk; ONC-INV, chimera bearing the
extracellular domain of ONC and the intracellular domain of INV;
INV-ONC, chimera bearing the extracellular domain of INV and
the intracellular domain of ONC) were used for immunoprecipitation
(Ip) with anti-mrk. The immunoprecipitates were subsequently
analyzed with anti-phosphotyrosine (anti-ptyr) on a Western
blot. Before immunoprecipitation lysates were checked for the same
receptor expression level by Western blot analysis using anti-mrk (data
not shown).
View larger version (18K):
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Fig. 2.
Amino acid differences between INV- and
ONC-Xmrk of different origins: comparison with other members of the
EGFR family. The number on the top of each
residue column indicates its position in INV-Xmrk. ONC-Xmrk sequences
are from PSM cells and X. maculatus. INV-Xmrk sequences are
from X. maculatus or A2 cell line derived from X. xiphidium. Xegfr corresponds to Xiphophorus
EGFR (see Footnote 2). Her (49), Her2 (50), Her3 (51), and Her4 (52)
are the human members of the EGFR family. Mouse (53), chicken (54),
Drosophila (55), and C. elegans (56) correspond
to the residues of the EGFR in these organisms.
View larger version (42K):
[in a new window]
Fig. 3.
The extracellular domain of ONC-Xmrk promotes
receptor dimerization. A, cell extracts from 293 cells
expressing INV-Xmrk, ONC-Xmrk, and the ONC-INV and INV-ONC chimeras
were separated on 3-8% gradient SDS-polyacrylamide gels under
reducing (left panel) or nonreducing
(right panel) conditions. Proteins were analyzed
with an anti-mrk serum on a Western blot. Monomers and dimers of the
Xmrk receptor are indicated. B, protein lysates from PSM
cells and malignant Xiphophorus melanoma (mel)
were electrophoresed on a 3-8% gradient SDS-PAGE gel, blotted to
nitrocellulose, and detected with anti-mrk. Dimeric and monomeric forms
of Xmrk are indicated.
View larger version (48K):
[in a new window]
Fig. 4.
Different mutations promote
independently covalent dimerization of the Xmrk receptor. 293 cells transiently expressing two different constructs were lysed, and
cellular proteins were resolved in 3-8% gradient SDS-polyacrylamide
gel under reducing (left panel) and nonreducing
(right panel) conditions. Proteins were
transferred to nitrocellulose and subjected to Western blotting using
an anti-mrk serum. Monomeric and dimeric forms are indicated.
Lanes 1 and 3, Xmrk(G359R,P388T);
lanes 2 and 4,
Xmrk(P470L,S476N,C578S,M595I).
View larger version (24K):
[in a new window]
Fig. 5.
Analysis of Xmrk receptor containing
the C578S mutation. A, cell extracts from transiently
transfected 293 cells were immunoprecipitated with anti-mrk serum and
subjected to immunoblot analysis with anti-phosphotyrosine
(anti-ptyr) (upper panel),
and the blot was reprobed with an anti-mrk (lower
panel). B and C, protein lysates of
293 cells expressing different constructs were separated on a 3-8%
gradient denaturing gel under reducing (left
panels) and nonreducing (right panels)
conditions and analyzed on a Western blot using anti-mrk. Xmrk monomers
and dimers are indicated. C, lanes 1 and 3, Xmrk(P470L,S476N,C578S,M595I); lanes
2 and 4, Xmrk(P470L,S476N,M595I).
View larger version (25K):
[in a new window]
Fig. 6.
Analysis of the Xmrk receptor with the G359R
mutation. A, cellular lysates of transiently
transfected 293 cells were resolved in a 3-8% gradient denaturing gel
under reducing (left panel) and nonreducing
(right panel) conditions and subsequently blotted
and detected with an anti-mrk serum. Lanes 1 and
3, Xmrk(G359R,P388T); lanes 2 and
4, Xmrk(P388T). B, anti-mrk immunoprecipitates
(Ip) of transfected 293 lysates were separated on a 7.5%
SDS-polyacrylamide gel, blotted, and detected with anti-phosphotyrosine
(anti-ptyr, right panel); the blot was
reprobed with an anti-mrk antibody (left
panel).
View larger version (54K):
[in a new window]
Fig. 7.
Presence of different monomeric glycosylated
receptor forms. A, equal amounts of cell lysates from
293 cells transfected with INV- or ONC-Xmrk were incubated with endo H
(+), without endo H ( ), or left untreated. Proteins separated in a
6.5% SDS-polyacrylamide gel were subjected to Western blot analysis
using anti-mrk. B, 293 cells transfected with different Xmrk
constructs were lysed and cellular extracts were incubated with endo H
(lanes 2, 4, 6, and
8) or without endo H (lanes 1,
3, 5, and 7). Lanes
1 and 2, Xmrk(G359R,P388T); lanes
3 and 4, Xmrk(P470L,S476N,C578S,M595I);
lanes 5 and 6, Xmrk (P388T);
lanes 7 and 8,
Xmrk(P470L,S476N,M595I). Protein extracts were analyzed with anti-mrk
by Western blot.
View larger version (33K):
[in a new window]
Fig. 8.
Localization of ONC and INV-ONC in stably
expressing Ba/F3 cell lines. BaF INV-ONC and BaF ONC cells were
spun onto slides and fixed in methanol/acetone. Fixed cells were
incubated with anti-mrk and with
dichlorotriazinaminofluorescein-conjugated anti-rabbit IgG and embedded
in a DAPI-containing mounting medium to visualize the nuclei.
Upper panels show the anti-mrk stain,
lower panels the corresponding DAPI nuclear
stain. Note that Ba/F3 cells have a very high nucleus/cytoplasm
ratio.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank M. Buehner for fruitful discussions and P. Fischer for technical assistance.
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FOOTNOTES |
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* This work was supported by grants from Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 487: Regulatorische Membranproteine, Graduiertenkolleg: Zellwachstum) and Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Inst. de Acuicultura Torre la Sal, 12595 Castellón, Spain.
§ Present address: Augsburg Hospital, Inst. for Pathology, D-86156 Augsburg, Germany.
¶ To whom correspondence should be addressed. Tel.: 49-931-8884148; Fax: 49-931-8884150; E-mail: phch1@biozentrum.uni- wuerzburg.de.
Published, JBC Papers in Press, October 18, 2000, DOI 10.1074/jbc.M006574200
2 A. Gómez, unpublished data.
3 A. Gómez, M. Schartl, C. Winkler, and Y. Hong, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: RTK, receptor tyrosine kinase; EGFR, epidermal growth factor receptor; PAGE, polyacrylamide gel electrophoresis; endo H, endoglycosidase H; ER, endoplasmic reticulum; BSA, bovine serum albumin; PBS, phosphate-buffered saline; DAPI, 4',6-diamidino-2-phenylindole; ONC, oncogenic version of Xmrk; INV, Xmrk proto-oncogene product.
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