Full activation of MEN2B mutant RET by an additional

Oncogene (1998) 16, 2295 ± 2301
 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00
http://www.stockton-press.co.uk/onc
Full activation of MEN2B mutant RET by an additional MEN2A mutation
or by ligand GDNF stimulation
Italia Bongarzone1, Elena Vigano'1, Luisella Alberti1, Maria Grazia Borrello1, Barbara Pasini2,
Angela Greco1, Piera Mondellini1, Darrin P Smith3, Bruce AJ Ponder3, Giovanni Romeo4 and
Marco A Pierotti1
1
Division of Experimental Oncology A and 2Scienti®c Direction, Istituto Nazionale Tumori, 20133 Milan, Italy; 3CRC Human
Cancer Genetics Research Group, University of Cambridge, Cambridge CB2 2QQ England; 4International Agency for Research on
Cancer, 69372 Lyon Cedex 08, France
Germline mutations of RET gene, encoding a receptor
tyrosine kinase, have been associated with the MEN2A
and MEN2B inherited cancer syndromes. In MEN2A
mutations aecting cysteine residues in the extracellular
domain of the receptor cause constitutive activation of
the tyrosine kinase by the formation of disul®de-bonded
homodimers. In MEN2B a single mutation in the
tyrosine kinase domain (Met918Thr) has been identi®ed.
This mutation does not lead to dimer formation, but has
been shown (both biologically and biochemically) to
cause ligand-independent activation of the Ret protein,
but to a lesser extent than MEN2A mutations.
Intramolecular activation by cis-autophosphorylation of
RetMEN2B monomers has been proposed as a model for
activation, although alternative mechanisms can be
envisaged. Here we show that the activity of RetMEN2B
can be increased by stable dimerization of the receptor.
Dimerization was achieved experimentally by constructing a double mutant receptor with a MEN2A mutation
(Cys634Arg) in addition to the MEN2B mutation, and
by chronic exposure of RetMEN2B-expressing cells to
the Ret ligand GDNF. In both cases full activation of
RetMEN2B, measured by ìn vitro' transfection assays
and biochemical parameters, was seen. These results
indicate that the MEN2B phenotype could be in¯uenced
by the tissue distribution or concentration of Ret
ligand(s).
Keywords: RET; MEN2B; GDNF
Introduction
The RET proto-oncogene is a member of the receptor
tyrosine kinase superfamily and consists of an
extracellular ligand binding domain with a cysteinerich region close to the cellular membrane, a single
transmembrane domain, and an intracellular kinase
domain (Takahashi et al., 1988). RET expression in
migrating neural crest cells, in the central and
peripheral neurones and in the developing kidney
(Pachnis et al., 1993), as well as the developmental
defects involving the enteric and peripheral nervous
systems and kidney in mice homozygous for an inactive
RET gene (Schuchardt et al., 1994; Durbec et al., 1996)
Correspondence: I Bongarzone
Received 22 July 1997; revised 1 December 1997; accepted 1
December 1997
indicate that RET is involved in the dierentiation and/
or proliferation of neural lineages and the kidney.
Recently, it has been reported that two neurotrophic
factors, GDNF and NTN, can activate the Ret
receptor tyrosine kinase. Activation of Ret by GDNF
or NTN has been shown to require one of two
accessory proteins, GDNFRa or GDNFRb, otherwise
designated RETL1 and RETL2 or TrnR1 and TrnR2,
respectively (Treanor et al., 1996; Jing et al., 1996;
Suvanto et al., 1997; Baloh et al., 1997; Sanicola et al.,
1997).
Mutations in the RET gene result in human diseases
including familial medullary thyroid carcinoma
(FMTC), multiple endocrine neoplasia type 2A and
2B (MEN2A and MEN2B), and Hirschsprung's disease
(Donis-Keller et al., 1993; Romeo et al., 1994;
Mulligan et al., 1994; Hofstra et al., 1994; Pasini et
al., 1996; Edery et al., 1994). MEN2A and MEN2B are
hereditary neoplastic syndromes characterized by the
presence of medullary thyroid carcinomas and pheochromocytomas. MEN2B is also associated with
skeletal abnormalities, ganglioneuromas of the intestinal tract, and mucosal neuromas. FMTC is a related
cancer disorder characterized by medullary thyroid
carcinoma only (Ponder and Smith, 1996). Mutations
in one of ®ve cysteine codons in the extracellular
domain of the RET gene are associated with most cases
of FMTC and MEN2A (Mulligan et al., 1993; DonisKeller et al., 1993). A single point mutation within the
Ret catalytic domain, which results in a Thr for Met
substitution at codon 918, is responsible for MEN2B
(Carlson et al., 1994; Hofstra et al., 1994).
The mutations tested so far convert RET into a
dominant transforming gene (RETMEN2A and RETMEN2B alleles) by constitutive activation of the
intrinsic tyrosine kinase activity of its product (Asai
et al., 1995; Santoro et al., 1995; Borrello et al., 1995).
The transfection of wild type (WT), MEN2A (Cys634
Arg) and MEN2B (Met918 Thr) RET cDNAs into cell
lines has provided three types of evidence for
constitutive activation. Firstly, under conditions where
RETWT has no eect, RETMEN2A and RETMEN2B
cause transformation of NIH3T3 murine ®broblasts,
and dierentiation of rat pheochromocytoma-derived
PC12 cells (Borrello et al., 1995; Califano et al., 1996).
Secondly, in NIH3T3 transfectants, RetMEN2A and
RetMEN2B proteins but not RetWT are constitutively
phosphorylated on tyrosine.
Thirdly, the ìn vitro' kinase activity of RetMEN2A
and RetMEN2B proteins is signi®cantly higher than
that of the RetWT. However, MEN2A and MEN2B
GDNF stimulation of MEN2B mutant RET
I Bongarzone et al
2296
mutations dier in their mechanism of activation. In
the case of MEN2A mutations activation most likely
results from constitutive receptor dimerization through
the formation of intermolecular disul®de bonds. The
MEN2B mutation does not result in dimerization, and
activation may result from altered conformation of the
kinase domain which also leads to altered substrate
speci®city (Songyang et al., 1995; Santoro et al., 1995).
Insertion of the MEN2B mutation into the constitutively dimerized RET/PTC2 oncogene signi®cantly
increases the enzymatic activity of the tyrosine kinase
(TK) domain, indicating that dimerization may be
necessary for RetMEN2B to be fully active (Borrello et
al., 1995). In addition, transfection experiments with a
chimeric construct in which the ligand-binding domain
of the epidermal growth factor (EGF) receptor was
fused to the catalytic domain of wild type Ret and Ret
carrying the MEN2B mutation showed that EGF
stimulation of the MEN2B mutant resulted in longer
neuritic processes in PC12 cells and a higher
transformation eciency in NIH3T3 cells (Rizzo et
al., 1996). Hence it appears that RetMEN2B may still
be sensitive to ligand stimulation, and therefore that
the phenotype of tissues aected in MEN2B could be
in¯uenced by the tissue distribution or concentration of
ligands. Here we have investigated whether the
biological and biochemical activities of RetMEN2B
can be augmented by the presence of a dimerizing
MEN2A mutation, or by ligand (GDNF) stimulation.
A MEN2A mutation in addition to a MEN2B results
in activity greater than either mutation alone, and
chronic GDNF stimulation of RetMEN2B results in
increased activity.
Results
Biological activity of RETMEN2A/B transfected into
NIH3T3 and PC12 cells
Figure 1 is a schematic representation of the RETWT,
RETMEN2A (Cys634Arg), RETMEN2B (Met918Thr)
(Borrello et al., 1995) and RETMEN2A/B (see
Materials and methods) proteins encoded by cDNAs
cloned into the eukaryotic expression vector pCEP9b
which carries the G418 resistance (G418R) gene. These
constructs were transfected into the mouse ®broblast
cell line NIH3T3 and the rat pheochromocytoma cell
line PC12 in order to compare their biological activity.
In agreement with previous reports (Santoro et al.,
1995; Asai et al., 1995; Borrello et al., 1995) the focus
formation assay in NIH3T3 cells showed that RETWT
was unable to induce transformed foci, and that the
transformation eciency of RETMEN2A was approximately ®vefold greater than RETMEN2B. The
transformation eciency of RETMEN2A/B was also
greater than RETMEN2B, and a little greater than
RETMEN2A (Table 1). In addition, RETMEN2A/B
foci appeared earlier than RETMEN2A foci and,
consequently, were larger 3 weeks after transfection.
Also in agreement with a previous report (Borrello et
al., 1995) RETMEN2A and RETMEN2B were able
induce PC12 cell dierentiation whereas RETWT was
not. RETMEN2A/B also caused PC12 cell differentiation (data not shown).
Biochemical analysis of RetWT, RetMEN2A,
RetMEN2B and RetMEN2A/B in bulk populations of
transfected cells
We have previously noticed that the yield of G418R
colonies expressing RetWT or RetMEN2B proteins is
extremely low, whereas all RETMEN2A G418R
colonies detectably express the protein. RETMEN2A/
B behaves like RETMEN2A (data not shown). To
overcome this problem, we have analysed the
biochemical properties of the various forms of Ret
receptor by using bulk populations of NIH3T3
transfectants. Two dierent doses (0.050 and 2.0 mg)
of RETWT, RETMEN2A, RETMEN2B and RETMEN2A/B constructs were transfected, G418 resistance used to select all stable transfectants, the cells
allowed to reach con¯uence, and then some of the
experimental plates used for the biochemical analysis
and some used to count the number of transformed
foci.
As shown in Table 2, the number of transformed
foci obtained by transfection of 0.050 and 2.0 mg of
both RETMEN2A and RETMEN2A/B was 3 ± 4 times
higher than that obtained with RETMEN2B. In
agreement with the data above, this also indicates
that the constitutive dimerization of the RetMEN2B
receptor as a result of the introduction of the MEN2A
mutation dramatically enhances the transforming
activity of the RetMEN2B kinase. For protein
analysis 36106 G418R cells from a transfection with
0.050 mg of RETMEN2A, RETMEN2B or RETMEN2A/B constructs, and from a transfection with
0.050 mg or 2.0 mg of RETMEN2B construct were
used. The Western blot analysis in Figure 2 shows that
at the lower DNA dose (0.050 mg) similar levels of
RetMEN2A and RetMEN2A/B proteins (of 140 and
160 kDa; partially and fully glycosylated forms,
respectively) could be detected (a), and these showed
a similar level of phosphorylation (b). No RetMEN2B
protein could be detected with this lower DNA dose,
Table 1
Figure 1 Schematic representation of MEN2A and MEN2B
mutations inserted in proto-Ret protein. SP=Signal peptide;
TM=Transmembrane domain; TK=Tyrosine Kinase domain;
KI=Kinase insert. iso51 and iso9 are two possible alternately
spliced isoforms of 9 and 51 amino acids. iso9 was used in these
experiments
Transforming activity of RETWT, RETMEN2A,
RETMEN2B and RETMEN2A/B
Transfected DNAs
RETWT
RETMEN2A
RETMEN2B
RETMEN2A/B
Dose/dish
(mg)
10.0
0.100
0.100
0.100
G418R
No. of
(colonies/mg) transformation foci
0.96104
1.16104
1.46104
0.96104
The values are the average of three experiments
51
6.66103
1.66103
7.66103
GDNF stimulation of MEN2B mutant RET
I Bongarzone et al
Table 2 Transforming activity of dierent RET constructs in bulk populations of G418 resistant NIH3T3
cells
Transfected DNAs
(mg/dish)
RETWT
0.050
2.0
±
±
No. of transformation foci with:
RET2A
RET2B
exp.1
exp.2
exp.1
exp.2
RET2A/B
exp.1
exp.2
17
132
18
104
2
152
3
27
9
35
26
128
Two independent transfection experiments were performed. Values listed represent the mean number of foci
counted on three plates. RET2A, RETMEN2A; RET2B, RETMEN2B; RET2A/B, RETMEN2A/B
b
a
2A 2A/B
1
2
2B
3
2A 2A/B
4
1
2
wt
2B
3
2A
2B
2A/B
4
gp 160
gp 140
≥ 300 KDa
DIMERS —
IP:
BLOT:
αRet
αRet
αRet
αPtyr
Figure 2 Expression and tyrosine phosphorylation of RetMEN2A (2A), RetMEN2B (2B) and RetMEN2A/B (2A/B) in
mass G418-selected NIH3T3 cells transfected with the corresponding constructs. RetMEN2A and RetMEN2A/B protein
extracts were from cells transfected with 0.050 mg of construct
(lanes l and 2); RetMEN2B protein extract was from cells
transfected with 0.050 and 2.0 mg (lanes 3 and 4, respectively). All
protein extracts were immunoprecipitated (IP) with anti-Ret
antiserum (aRet), subjected to 6% SDS ± PAGE, Western
blotted, and the ®lters developed (BLOT) with anti-Ret (aRet,
a) or anti-phosphotyrosine (aPtyr, b) antiserum
and only cells transfected with 2.0 mg of RETMEN2B
vector showed a detectable amount of phosphorylated
receptor (a and b).
Although the number and size of transformed foci is
similar using 0.050 mg of RETMEN2A or RETMEN2A/B and 2.0 mg of RETMEN2B (Table 2) the
expression level and degree of phosphorylation of
RetMEN2B is clearly less. This indicates that the level
of RetMEN2B expression could be less (although
cloned in the same vector), or more likely that
RetMEN2B is intrinsically less stable than RetMEN2A, but the double mutant RetMEN2A/B is as
stable as RetMEN2A (Figure 2).
Cell extracts from NIH3T3 clones stably expressing
RET cDNAs were analysed by SDS ± PAGE under
non-reducing conditions to look for the presence of
disul®de-bonded Ret dimers. Under these conditions,
RetWT and RetMEN2B proteins migrated as monomers of 140 and 160 kDa whereas RetMEN2A and
RetMEN2A/B displayed both monomeric forms and
signi®cant amounts of a dimeric form of about
300 kDa (data not shown). When the blot was
developed with antiphosphotyrosine antibodies, two
distinct patterns of Ret phosphorylation were seen
(Figure 3).
RetMEN2B monomers (both 140 and 160 kDa)
were phosphorylated, whereas only dimers of RetMEN2A and predominantly dimers of RetMEN2A/B
were phosphorylated. It is worth noting that a weak
but de®nite phosphorylation of RetMEN2A/B monomers is detectable on longer exposure (not shown),
suggesting that like RetMEN2B this receptor does not
require dimerization for autophosphorylation activity.
— gp160
MONOMERS
— gp140
IP:
BLOT:
αRet
αPtyr
Figure 3 Dimerization and tyrosine phosphorylation of Ret
proteins. Lysates NIH3T3 cells stably transfected with RETWT,
RETMEN2A, RETMEN2B and RETMEN2A/B were immunoprecipitated with anti-Ret antiserum (aRet) and the resulting
complexes were separated by SDS ± PAGE under non-reducing
conditions. After Western blotting ®lters were developed with
anti-phosphotyrosine (aPtyr) antiserum
Kinase activity of RetMEN2A, RetMEN2B and
RetMEN2A/B
We have previously reported that the RET/PTC2
oncogene (which contains the Ret TK domain fused
to constitutively dimerized RIa) containing the
MEN2B mutation has somewhat decreased autophosphorylation capacity and increased ability to phosphorylate the exogenous substrate myelin basic protein
(MBP) in ìn vitro' immunocomplex kinase assays
(Borrello et al., 1995). Figure 4 shows the result of
one representative experiment in which we have
compared RetMEN2A, RetMEN2B, RetMEN2A/B,
RetPTC2 and RetPTC2-2B proteins in similar assays.
Equal amounts of proteins were immunoprecipitated
from NIH3T3 transfectants with a Ret antibody and
incubated in presence of symbol [g32P]ATP and MBP.
Phosphorylated products were resolved by SDS ±
PAGE, detected by autoradiography and quantitated
by Phosphorimager analysis. We have determined
autokinase activity and kinase activity towards the
2297
GDNF stimulation of MEN2B mutant RET
I Bongarzone et al
2298
2A
2B
Table 3 Transforming activity of RETMEN2B in presence or
absence of GDNF
2A/2B
160 gp
140 gp
Transfected
DNA
RETWT
RETMEN2B
RETH13
MBP
RET
PTC2 PTC2-2B
ratio
3
0.4
2A
2
2B
0.4
2A/B
0.35
wt
1.6
Dose/dish
(mg)
10.0
0.100
10.0
foci/dish
7GDNF
+GDNF
±
13
±
30
135
±
The values are the average of three independent experiments
(auto-phos/MBP-phos)
Figure 4 Ìn vitro' immunocomplex assays of mutant Ret
proteins. RetMEN2A (2A), RetMEN2B (2B) and RetMEN2A/B
(2A/B) proteins were expressed by transient transfection in HeLa
cells, immunoprecipitated with anti-Ret antiserum and subjected
to the immunocomplex assay as described in Materials and
methods. Three replicates following 15% SDS ± PAGE and
autoradiography of the dried gel are shown. Bands representing
autophosphorylated gp 160 and/or gp 140 forms of Ret, and
phosphorylated exogenous substrate myelin basic protein (MBP)
are indicated. These data represent one of two similar
experiments. Phosphorylated bands were quantitated by Phosphorimager analysis, and the ratio counts per minute (c.p.m.) of
autophosphorylated bands/c.p.m. of MBP band is shown.
Included in this analysis are transfection experiments with
RetWT (wt), RetPTC2 (PTC2) and RetPTC2-2B (PTC2-2B)
exogenous substrate MBP, and compared them by
calculating a ratio which is shown at the bottom of
Figure 4. With RetMEN2B, RetMEN2A/B and
RetPTC2-2B kinase activity towards MBP is greater
than autokinase activity. In contrast, with RetMEN2A,
RetWT and RetPTC2 autokinase activity is greater
than activity towards MBP. In such assays even when
there is no dimerization through a mutation (MEN2A)
or a fusion (PTC), Ret proteins (WT, MEN2B) will be
dimerized by immunoprecipitation. Hence these ratios
clearly show that following dimerization by whatever
mean the MEN2B mutation leads to increased activity
towards an exogenous substrate (MBP).
GDNF stimulation of RETWT and RETMEN2B
transfected cells
To address the question of whether RetMEN2B
activity can be increased by the recently identi®ed
Ret ligand (GDNF), we tested the eect of GDNF on
focus formation in NIH3T3 cells transfected with
RETWT or RETMEN2B. GDNF was added the day
after transfection, and maintained in the medium until
foci were counted (at 14 days). The results are shown
in Table 3. In the absence of GDNF RETWT did not
induce foci and RETMEN2B gave a typical number of
foci for the DNA dose used. In the presence of GDNF,
foci were seen with RETWT, and the number of foci
with RETMEN2B was increased tenfold.
Interestingly, the morphology of the RETWT and
RETMEN2B# GDNF-induced foci was dierent
(Figure 5). As is clearly evident at higher magnification of the same dish, RETWT in the presence of
GDNF induced ¯at, transparent foci composed of
rounded cells. In contrast, RETMEN2B produced a
more aggressive phenotype with foci composed of
refractile, spindle-shaped cells and these foci occurred
earlier and in greater number following GDNF
treatment. As a negative control we used a RET
construct (RETH13) carrying a mutation previously
described in a HSRC family, and which has been
shown to substantially inactivate Ret kinase activity
(Pasini et al., 1995). Table 3 and Figure 5 show that
RETH13 is inactive with or without GDNF.
Finally, we assessed the eect of the presence of
GDNF on the phosphorylation status of RetWT and
RetMEN2B. Similar amounts of cell extract from cells
transfected with RetWT or RetMEN2B and with or
without GDNF treatment were immunoprecipitated
with an anti-Ret antibody and after Western blotting
developed with anti-Ret and anti-phosphotyrosine
antibodies (Figure 6). Similar amounts of Ret protein
were present with or without GDNF (anti-Ret lanes).
However, there is a clear and signi®cant increase in
phosphorylation of both RetWT and RetMEN2B with
GDNF stimulation, and this is more evident in the
upper fully glycosylated gpl60 bands (anti-phosphotyrosine lanes). The weak basal level of RetWT
phosphorylation without GDNF is most likely due to
spontaneous dimerization at relatively high levels of
receptor expression.
Discussion
Here we have shown by two dierent approaches that
in order to express its full activity, Ret carrying the
Met918Thr mutation associated with the MEN2B
cancer syndrome, must be stably dimerized. Firstly,
we have demonstrated that RETMEN2B focus forming
eciency in NIH3T3 cells is augmented by creating a
double mutant receptor with both a dimerizing
MEN2A mutation (Cys634Arg) and the MEN2B
mutation. RetMEN2A/B forms covalent dimers
similar to RetMEN2A and has a transformation
eciency much greater than RetMEN2B and a little
greater than RetMEN2A. RetMEN2A/B foci appear
earlier and are larger than those induced by the single
mutants. Secondly, we have demonstrated that RetMEN2B focus forming eciency is increased in the
presence of ligand (GDNF) and that the steady state
level of RetMEN2B tyrosine phosphorylation is
increased in the presence of GDNF. These data
suggest that ligand stimulation of the mutant RetMEN2B receptor will contribute to the development of
MEN2B.
We have previously reported (Borrello et al., 1995)
that the MEN2B mutation in the constitutively
dimerized PTC2 oncogene increases phosphorylation
activity towards the exogenous substrate MBP. Here
we have con®rmed these observations and extended
them to show that the MEN2B mutation also increases
activity towards MBP in RetMEN2A/B.
We and others have shown that the MEN2B
mutation results in constitutive ligand-independent
activation (possibly by stabilising a non-inhibitory
conformation of the kinase domain) and also leads to
altered catalytic substrate speci®city (Santoro et al.,
GDNF stimulation of MEN2B mutant RET
I Bongarzone et al
2299
Focus assay
– GDNF
+ GDNF
ret wt
ret MEN2B
ret H13
Figure 5 Growth of foci following transfection of NIH3T3 cells with RETWT, RETMEN2B and RETH13 constructs. Focus
formation was tested in the absence (7) or presence (+) of GDNF (0.05 mg/ml) as indicated. After 14-days the cells were ®xed and
stained with trypan blue solution. The right +GDNF column is a higher magni®cation view of part of the dish shown in the left
+GDNF column
wt
–
+
2B
–
+
GDNF
–
+
–
+
gp 160
gp 140
IP:
BLOT:
αRet
αRet
αPtyr
αRet
αRet
αPtyr
Figure 6 Phosphorylation of RetWT (wt) and RetMEN2B (2B)
proteins in absence or presence of GDNF. NIH3T3 cells
transfected with the RETWT or RETMEN2B constructs were
treated with (+) or without (7) GDNF for 48 h. Cells were
lysed, precipitated with anti-Ret (aRet) antiserum and the
resulting complexes separated by SDS ± PAGE, Western blotted
and developed with anti-Ret or anti-phosphotyrosine (aPTyr)
antiserum
1995; Songyang et al., 1995; Borrello et al., 1995; Liu
et al., 1996). Our current observations suggest that
dimerization-dependent trans-autophosphorylation increases the activity of RetMEN2B, and hence suggests
that cis-autophosphorylation is unlikely to make a
contribution to RetMEN2B activity. Despite the fact
that MEN2B is a more aggressive neoplastic disease
than MEN2A we consistently ®nd that the focus
formation activity of RetMEN2B is less than Ret-
MEN2A. It has been suggested that this anomaly can
be explained by the altered substrate speci®city of
RetMEN2B, which may contribute to the aggressiveness of MEN2B ìn vivo' and to the developmental
abnormalities seen in the disease. Our data suggest that
in the presence of ligand RetMEN2B may be more
active than RetMEN2A, and this could be an
additional explanation for the earlier onset of tumours
in MEN2B and its peculiar features of MEN2B and
the developmental abnormalities. This would depend
on the availability of Ret ligands and accessory
proteins in the tissues aected in MEN2B. Unfortunately, even though dierent studies have been
reported on the expression of Ret ligands and of the
relative coreceptors (Trupp et al., 1997; Widenfalk et
al., 1997), there are no data concerning MEN2B
aected tissue and the derived tumours.
It has been reported that GDNF stimulation of Ret
autophosphorylation and activity is dependent on one
of two cell-surface-associated accessory proteins,
designated GDNFRa (Jing et al., 1996; Treanor et
al., 1996) and GDNFRb (Suvanto et al., 1997; Baloh et
al., 1997; Sanicola et al., 1997). We have used RT ±
PCR to look for accessory protein expression in our
transfected NIH3T3 cells and in cells from our stock
GDNF stimulation of MEN2B mutant RET
I Bongarzone et al
2300
collection. We have found no evidence for the
expression of GDNFRa, but we have found weak
expression of GDNFb (data not shown). Therefore, if
Ret activation is dependent on accessory proteins,
GDNF in our experiments is most likely signalling in
conjunction with GDNFRb or additional members
(GDNFRg) of the family.
Experiments are currently in progress to assess if
and to what extent GDNF-mediated phosphorylation
of RetWT and RetMEN2B is dependent on accessory
proteins.
A somatic RET Met918Thr is also present in a
substantial proportion (about 40%) of sporadic
medullary thyroid carcinomas (MTCs) (Ponder and
Smith, 1996). When present in the germline this
mutation is clearly associated with initiating events
in MTC tumorigenesis. However, this is not
necessarily the case in sporadic disease where it may
occur as a late event since it was found only in some
microdissected subpopulations of MTCs, and only in
some of multiple metastases (Eng et al., 1996). In
contrast to Met918Thr mutations, MEN2A-like
mutations are rare in sporadic MTC. The altered
substrate speci®city of RetMEN2B has also been
suggested to account for this dierence. Our data
suggest that greater activity of ligand-stimulated
RetMEN2B may be an alternative explanation.
However, our preliminary experiments, would indicate that the extent of the ligand-stimulation of
RetMEN2B is isoform speci®c. In particular, ligandstimulation is very strong for the short isoform of
RetMEN2B, as here reported, but it is scarce for the
long one, characterized by a intrinsic high transforming activity (IB unpublished results; Asai et al., 1996;
Rossel et al., 1997).
Interestingly, a somatic MEN2B-like mutation has
been recently reported in three out of 15 MTCs that
arose in MEN2A/FMTC patients carrying a germline
mutation at codon 634 (Cys634Arg) (Marsh et al.,
1996). The authors suggest that the presence of the
somatic mutation, in addition to the pre-existing
germline mutation in the same gene, may contribute
to tumorigenesis ìn vivo'. They could not determine
whether the two mutations were carried by the same
allele or not. Therefore, two situations may be
envisaged. If they are on dierent alleles, all Ret
receptors in aected cells are mutant. Although this
could be advantageous for oncogenicity, we have
found a lower transformation activity, compared to
control transfection experiments, when we have cotransfected RETMEN2A and RETMEN2B constructs
(IB unpublished results). Perhaps transformation
de®cient heterodimers are formed. The other situation, i.e. both mutations in the same molecule, has
been examined in this report. We have shown that
the combination of the two mutations in the same
receptor augments both the biological and biochemical activities of the RetMEN2B kinase, and may
therefore lead to increased òncogenicity' ìn vivo'.
Analysis of the RET mutations associated with
MEN2 and sporadic tumours will ultimately lead to
the elucidation of the dierent mechanisms of Retmediated neoplastic transformation, and should
provide a conceptual framework for the development
of new tools for the control of RET-related
neoplasias.
Materials and methods
Plasmid construction
The cloning of the RETWT, RETMEN2A, RETMEN2B
has been previously reported (Borrello et al., 1995). The
double mutant RETMEN2A/B cDNA was obtained by
ligating a RET fragment carrying the Cys634Arg mutation
(HindIII ± BglII fragment of 2690 bp) to a RET fragment
carrying the Met918Thr mutation (BglII ± XbaI fragment of
800 bp), followed by cloning into the expression vector
pCEP9b. The mutant RETH13 cDNA (Pasini et al., 1995)
and the RETPTC2 and RETPTC2-2B cDNAs (Borrello et
al., 1995) have been previously described.
Cell culture and transfection
Mouse NIH3T3 cells were grown in Dulbecco's modi®ed
Eagles's medium (DMEM) containing 10% calf serum.
Rat pheochromocytoma PC12 cells were maintained in
RPMI 1640 medium supplemented with 5% fetal calf
serum and 10% horse serum. Transfection experiments
were carried out in NIH3T3 cells by the calcium
phosphate precipitation method as previously described
(Bongarzone et al., 1993). NIH3T3 cells transfected with
the various amounts (0.050 ± 10 mg) of the expression
vectors and 30 mg of mouse genomic DNA as carrier.
G418-resistant (G418R) colonies were selected in DMEM
plus 10% calf serum plus G418 antibiotic (650 mg/ml),
and transformation foci were selected in DMEM plus 5%
calf serum. Where indicated GDNF (0.050 mg/ml) was
added. Foci were ®xed and counted 14 days after
transfection.
Immunoprecipitation and Western blot analysis
Protein samples were prepared as previously described
(Borrello et al., 1994) and immunoprecipitated with
anity puri®ed anti-Ret polyclonal antiserum (Borrello
et al., 1994). The anti-Ret immunoprecipitates were
resolved by electrophoresis on 6% SDS polyacrylamide
gels (PAGE). Proteins were transferred onto nitrocellulose
®lters and immunoblotted with the same anti-Ret anitypuri®ed antibodies or with anti-phosphotyrosine (antiPTyr) antisera essentially as described previously
(Borrello et al., 1994). Immunoreactive bands were
visualized using 125I-labelled protein A (Amersham)
followed by autoradiography or using horseradish
peroxidase conjugated anti-rabbit or anti-mouse antisera
and ECL detection reagents (Amersham). When 125IProtein A was used, the ®lters were exposed to storage
phosphor screen ®lms (Molecular Dynamics) in order to
quantify with a Phosphorimager the counts per minute
(c.p.m.) associated with the Ret-speci®c bands reacting
with anti-Ret or anti-PTyr antisera. For Western blotting
under non reducing conditions 2-mercaptoethanol was
omitted.
In vitro immunocomplex kinase assay
Anti-Ret immunoprecipitates, absorbed on protein ASepharose beads, were washed twice with lysis buer,
once with incubation buer (50 mM HEPES pH 7.2, 20 mM
MnCl2, 5 mM PMSF) and incubated for 15 min at 48C in
20 ml of the same buer containing 0.5 mM DTT, 50 mM
myelin basic protein (MBP) and 4 mCi of [g32P]ATP
(5000 Ci mmol-1 Amersham) diluted with the unlabelled
ATP to a ®nal concentration of 26 pmol ATP per sample.
The reaction was stopped by addition of concentrated
Laemmli buer. Proteins were eluted and subjected to 15%
SDS ± PAGE. 32P-labelled bands were visualized by
autoradiography of the dried gel and quanti®ed by
Phosphorimager analysis.
GDNF stimulation of MEN2B mutant RET
I Bongarzone et al
Acknowledgements
This work was partially supported by Associazione Italiana
per la Ricerca sul Cancro (AIRC), Fondazione Italiana per
la Ricerca sul Cancro (FIRC), CNR Special Project
ÀCRO', Project BIOMED2 No. BMH4-CT97-2157 and
Istituto Superiore di Sanita'. The authors thank Mr Mario
Azzini for professionally preparing illustrations, Mrs Anna
Grassi and Cristina Mazzadi for their assistance in
manuscript preparation.
References
Asai N, Iwashita T, Matsuyama M and Takahashi M.
(1995). Mol. Cell. Biol., 15, 1613 ± 1619.
Asai N, Murakami H, Iwashita T and Takahashi M. (1996).
J. Biol. Chem., 271, 17644 ± 17649.
Baloh RH, Tansey MG, Golden JP, Creedon DJ, Heuckeroth RO, Keck CL, Zimonjic DB, Popescu NC, Johnson
EMJ and Milbrandt J. (1997). Neuron, 18, 793 ± 802.
Bongarzone I, Monzini N, Borrello MG, Carcano C,
Ferraresi G, Arighi E, Mondellini P, Della Porta G and
Pierotti MA. (1993). Mol. Cell. Biol., 13, 358 ± 366.
Borrello MG, Pelicci G, Arighi E, De Filippis L, Greco A,
Bongarzone I, Rizzetti MG, Pelicci PG and Pierotti MA.
(1994). Oncogene, 9, 1661 ± 1668.
Borrello MG, Smith DP, Pasini B, Bongarzone I, Greco A,
Lorenzo MJ, Arighi E, Miranda C, Eng C, Alberti L,
Bocciardi R, Mondellini P, Scopsi L, Romeo G, Ponder
BAJ and Pierotti MA. (1995). Oncogene, 11, 2419 ± 2427.
Califano D, D'Alessio A, Colucci-D'Amato GL, De Vita G,
Monaco C, Santelli G, Di Fiore PP, Vecchio G, Fusco A,
Santoro M, and de Francisci V. (1996). Proc. Natl. Acad.
Sci. USA, 93, 7933 ± 7937.
Carlson KM, Dou S, Chi D, Scavarda NJ, Toshima K,
Jackson CE, Wells SAJ, Goodfellow P and Donis-Keller
H. (1994). Proc. Natl. Acad. Sci. USA, 91, 1579 ± 1583.
Donis-Keller H, Dou S, Chi D, Carlson KM, Toshima K,
Lairmore TC, Howe JR, Moley JF, Goodfellow P and
Wells SAJ. (1993). Human Molec. Genet., 2, 851 ± 856.
Durbec PL, Larsson-Blomberg LB, Schuchardt A, Costantini F and Pachnis V. (1996). Development, 122, 349 ± 358.
Edery P, Lyonnet S, Mulligan LM, Pelet A, Dow E, Abel L,
Holder S, Nihoul-Fekete' C, Ponder BAJ and Munnich A.
(1994). Nature, 367, 378 ± 380.
Eng C, Mulligan LM, Healey CS, Houghton C, Frilling A,
Raue F, Thomas GA and Ponder BA. (1996). Cancer Res.,
56, 2167 ± 2170.
Hofstra RM, Landsvater RM, Ceccherini I, Stulp RP,
Stelwagen T, Luo Y, Pasini B, Hoppener JWM, Ploos
van Hamstel HK, Romeo G, Lips CJM and Buys CHCM.
(1994). Nature, 367, 375 ± 376.
Jing S, Wen D, Yu Y, Holst PL, Luo Y, Fang M, Tamir R,
Anatonio L, Hu Z, Cupples R, Louis J-C, Hu S, Altrock
BW and Fox GM. (1996). Cell, 85, 1113 ± 1124.
Liu X, Vega QC, Decker RA, Pandey A, Worby CA and
Dixon JE. (1996). J. Biol. Chem., 271, 5309 ± 5312.
Marsh DJ, Andrew SD, Eng C, Learoyd DL, Capes AG,
Pojer R, Richardson AL, Houghton C, Mulligan LM,
Ponder BJ and Robinson BG. (1996). Cancer Res., 56,
1241 ± 1243.
Mulligan LM, Kwok JBJ, Healey CS, Elsdon MJ, Eng C,
Gardner E, Love DR, Mole SE, Moore JK, Papl L,
Ponder MA, Telenlus H, Tunnaclie A and Ponder BAJ.
(1993). Nature, 363, 458 ± 460.
Mulligan LM, Eng C, Healey CS, Clayton L, Kwok JBJ,
Gardner E, Ponder MA, Frilling A, Jackson CE, Lehnert
H, Neumann HPH, Thibodeau SN and Ponder BAJ.
(1994). Nature Genetics, 6, 70 ± 74.
Pachnis V, Mankoo B and Costantini F. (1993). Development, 119, 1005 ± 1017.
Pasini B, Borrello MG, Greco A, Bongarzone I, Luo Y,
Mondellini P, Alberti L, Miranda C, Arighi E, Bocciardi
R, Seri M, Barone V, Romeo G and Pierotti MA. (1995).
Nature Genet., 10, 35 ± 40.
Pasini B, Ceccherini I and Romeo G. (1996). Trends Genet.,
12, 138 ± 144.
Ponder BA and Smith D. (1996). Adv. Cancer Res., 70, 179 ±
222.
Rizzo C, Califano D, Colucci-D'Amato GL, De Vita G,
D'Alessio A, Dathan NA, Fusco A, Monaco C, Santelli G,
Vecchio G, Santoro M, and de Franciscis V. (1996). J.
Biol. Chem., 271, 29497 ± 29501.
Romeo G, Ronchetto P, Luo Y, Barone V, Seri M,
Ceccherini I, Pasini B, Bocciardi R, Lerone M,
Kaariainen H and Martucciello G. (1994). Nature, 367,
377 ± 378.
Rossel M, Pasini A, Chappuis S, Geneste O, Fournier L,
Schuenecker I, Takahashi M, van Grunsven LA,
Urdiales JL, Rudkin B, Lenoir GM and Billaud M.
(1997). Oncogene, 14, 265 ± 275.
Sanicola M, Hession C, Worley D, Carmillo P, Ehrenfels C,
Walus L, Robisnon R, Jaworski G, Wei H, Tizard R,
Whitty A, Pepinski RB and Cate RL. (1997). Proc. Natl.
Acad. Sci. USA, 94, 6238 ± 6243.
Santoro M, Carlomagno F, Romano A, Bottaro DP, Dathan
NA, Grieco M, Fusco A, Vecchio G, Matoskova B, Kraus
MH and Di Fiore PP. (1995). Science, 267, 381 ± 383.
Schuchardt A, D'Agati V, Larsson-Blomberg L, Costantini
F and Pachnis V. (1994). Nature, 367, 380 ± 383.
Songyang Z, Carraway III KL, Eck MJ, Harrison SC,
Feldman RA, Mohammadi M, Schlessinger J, Hubbard
SR, Smith DP, Eng C, Lorenzo MJ, Ponder BAJ, Mayer
BJ and Cantley LC. (1995). Nature, 373, 536 ± 539.
Suvanto P, Wartiovaara K, Lindhal M, Arumae U,
Moshnyakov M, Horelli-Kuitunen N, Airaksinen MS,
Palotie A, Sariola H and Saarma M. (1997). Hum. Mol.
Genet., 6, 1267 ± 1273.
Takahashi M, Buma Y, Iwamoto T, Inaguma Y, Ikeda H
and Hiai H. (1988). Oncogene, 3, 571 ± 578.
Treanor JJS, Goodman L, de Sauvage F, Stone DM, Poulsen
KT, Beck CD, Gray C, Armanini MP, Pollock RA, Hefti
F, Phillips HS, Goddard A, Moore MW, Buj-Bello A,
Davies AM, Asai N, Takahashi M, Vandlen R, Henderson
CE and Rosenthal A. (1996). Nature, 382, 80 ± 83.
Trupp M, Belluardo N, Funakoshi H and Ibanez CF. (1997).
J. Neuroscience, 17, 3554 ± 3567.
Widenfalk J, Nosrat C, Tomac A, Westphal H, Hoer B and
Olson L. (1997). J. Neuroscience, 17, 8506 ± 8519.
2301

Download Report