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Original Studies |
Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare (D.S., M.V.B., G.S., R.M., G.V., M.S.); Istituto Nazionale dei Tumori di Napoli, Fondazione Senatore Pascale (G.C., A.M.); and Dipartimento di Endocrinologia ed Oncologia Molecolare e Clinica, Facoltà di Medicina e Chirurgia, Università Federico II (D.S., G.F.), 80131 Naples, Italy; and Dipartimento di Medicina Sperimentale e Clinica, Facoltà di Medicina e Chirurgia, Università Magna Graecia (A.F.), 88100 Catanzaro, Italy
Address all correspondence and requests for reprints to: Dr. Massimo Santoro, Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Università di Napoli Federico, Via S. Pansini 5, 80131 Naples, Italy. E-mail: masantor{at}unina.it
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit (GFR) and Ret. The GFR coreceptors are glycosyl
phosphatidylinositol-linked polypeptides, and to date, four GFR
proteins, i.e. GFR1–4 have been identified. GFR1, -2,
-3, and -4 bind predominantly GDNF, neurturin, artemin, and persephin,
respectively. Ret functions as a common intracellular
signal-transducing component in conjunction with each of the GFR
subunits (2). RET is also a protooncogene; activation of its transforming potential is responsible for the inheritance of multiple endocrine neoplasia type 2A and type 2B (MEN2A and MEN2B) and familial medullary thyroid carcinoma (FMTC). Although there is a certain degree of overlap, each disease has a distinct phenotype. MEN2A is characterized by medullary thyroid carcinoma (MTC), pheochromocytoma, and parathyroid hyperplasia. The MEN2B phenotype is more severe, being characterized by an earlier occurrence of more aggressive MTC. Finally, FMTC consists of an inherited predisposition for MTC (for a review, see Ref. 3). Involvement of different tissues corresponds to differences in the nature and position of the underlying RET mutation. In most MEN2A cases, the RET mutation consists of the loss of one of the six cysteines localized in the extracellular domain, and this causes constitutive dimerization and activation (4, 5). Conversely, in more than 95% of patients, MEN2B is caused by the substitution of methionine 918 with a threonine (M918T) (6). Interestingly, the same mutation occurs, at the somatic level, in sporadic MTC (7). FMTC can be caused by mutations affecting extracellular cysteines or different residues of the Ret intracellular domain (3). On the other hand, somatic rearrangements of the RET gene are found in a large fraction of human papillary thyroid carcinomas (PTC). These rearrangements are caused by chromosomal inversions or translocations and consist of the deletion of the amino-terminal ligand-binding domain of RET, and fusion of the remaining tyrosine kinase region with the 5'-end of unrelated genes, thus determining the generation of RET/PTC oncogenes (8). At least seven RET/PTC oncogenes, differing in RET fusion partners, have been identified (9, 10, 11, 12, 13, 14, 15). RET/PTC1 and RET/PTC3 are the most prevalent RET/PTC variants (8). RET/PTC oncogenes are consistently found in radiation-associated PTC (16, 17, 18, 19, 20, 21, 22, 23) and are found with a high frequency in clinically silent PTCs (24), which suggests that they may arise in early tumorigenesis.
Despite the large body of evidence supporting the role played by RET in
several endocrine malignancies, the mechanisms of RET-mediated
tumorigenesis are still largely unknown. Potentiation of the kinase
activity is the end result of the different oncogenic mutations of RET,
and this event is predicted to be critical for the transforming ability
of RET-derived oncogenes (4, 5, 8). Indeed, receptor tyrosine kinases
(RTK), of which Ret is one, exert their biological effects mainly
through the autophosphorylation of tyrosine residues, frequently
localized in the carboxyl-terminal tail of the receptor. These
phosphoresidues, in turn, act as docking sites to recruit intracellular
signaling molecules that mediate their biological effects (for a
review, see Refs. 25, 26). The cytoplasmic domain of RET contains 14
tyrosine residues; the longer form (1114 residues long), which arises
due to alternative splicing, contains 2 additional tyrosines. Among
these phosphotyrosines, tyrosines 1015 and 1062 seem to play an
important role in RET signaling. Phosphorylated tyrosines 1015 and 1062
are docking sites for phospholipase C
(27) and Shc (28, 29, 30),
respectively. Phospholipase C is a common substrate of several RTK;
Shc is a docking protein involved in the coupling of several receptors
to the Ras/mitogen-activated protein kinase (MAPK) pathway. Finally,
even in the unphosphorylated state, Y1062 was found to associate to
Enigma, a protein containing the PDZ (named for three of the
proteins containing it, i.e PSD-95, Disk-Large
and ZO1) (26) and LIM (named for three of the
proteins containing it, i.e. lin-11, is1-1 and
mec-3 (26) domains that has been implicated in recruitment
and clustering of RET protein products at the membrane level (31, 32).
The evidence that Y1015 and 1062 are RET autophosphorylation sites is
based solely on in vitro kinase assays (33), and nothing is
known about the in vivo phosphorylation of these sites in
RET oncoproteins.
The advent of phosphorylation site-specific antibodies represented a breakthrough in studies of the phosphorylation states of various classes of signaling proteins. These reagents have enabled in vivo detection of constitutive and dynamic phosphorylation events with a sensitivity and rapidity previously impossible. In the present study we have produced phosphorylation-specific antibodies able to reveal the phosphorylation of Ret Y1015 and Y1062. We show that both tyrosines are rapidly phosphorylated in vivo upon ligand triggering. We also demonstrate that phosphorylation on both residues is common to all Ret oncoproteins expressed in human cancerous cells and tumoral samples. Thus, these antibodies represent a useful tool with which detect oncogenic Ret in human cells. Furthermore, we show that the microinjection of anti-pY1062 antibody blocks Ret-induced DNA synthesis in fibroblasts and in a thyroid carcinoma cell line spontaneously harboring the RET/PTC1 oncogene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-pY mouse monoclonal antibody was obtained from
Upstate Biotechnology, Inc. (Lake Placid, NY).
Antiphospho-extracellular regulated kinase (ERK) antibodies were
purchased from New England Biolabs, Inc. (Beverley, MA).
Rabbit polyclonal anti-Ret antibodies have been described previously
(12). Epidermal growth factor receptor (EGFr) and p185
neu/ErbB-2 were gifts from P. P. Di Fiore, TRK was provided by M.
Barbacid, and platelet-derived growth factor receptor was
obtained from C. H. Heldin. GFR1-expressing vector was provided
by S. Jing. The expression vectors pBabeRET//PTC1, long terminal repeat
(LTR)-RET, LTR-RET C634Y (RET-MEN2A), and LTR-RET M918T (RET-MEN2B)
were described previously (4). The TPC-1 cell line derives from a human
thyroid papillary carcinoma harboring a RET/PTC1 rearrangement (34).
The NPA and ARO (35) cell lines are from human papillary and anaplastic
carcinomas, respectively, and are negative for RET activation. The
three cell lines were grown in DMEM supplemented with 10% FCS
(Life Technologies, Inc., Gaithersburg, MD). The TT cell
line was established from an aggressive sporadic medullary thyroid
carcinoma (36) and was previously characterized for the presence of an
activating MEN2A-type mutation (C634W) (37). TT cells were grown in
RPMI medium supplemented with 16% FCS (Life Technologies, Inc.). NIH-3T3 cells expressing the EGFr-Ret chimera were
described previously (38). To obtain mutants LTR-RET-MEN2A Y1015F, the
LTR-RET-MEN2A Y1062F, and LTR-RET-MEN2A Y905F, PCR fragments containing
the required mutation were generated by recombinant PCR. The resulting
plasmids were sequenced on both strands in the region that underwent
genetic manipulations.
Cells and transfection experiment
COS-7 cells were grown in DMEM (Life Technologies, Inc.) supplemented with 10% FCS and were transfected using the calcium phosphate precipitation method as previously described (39).
Peptide synthesis and antiphosphopeptide antibodies production and purification
Synthetic phosphotyrosine-containing peptide 1 (VKRRDpYLDLAASTPSDC) and peptide 2 (WIENKLpYGRISHAFTR) together with the identical nonphosphate-containing peptides were commissioned from Neosystem S. A. (Strasbourg, France). Those peptides contain Ret Y1015 and Y1062, respectively. Phosphopeptides were coupled to BSA via m-maleimidobenzoyl-N-hydroxysuccinimide ester and injected into New Zealand rabbits. Nonphosphorylated peptides were coupled to ovalbumin via succinimidyl 4-N-maleimidomethylcyclohexane-1- carboxylate for the purification process. Serum samples were first screened for the ability to detect the EGFr-Ret chimera expressed in NIH-3T3 cells, compared to standard detection by anti-Ret antibodies. Next, antiserum was affinity purified following previously described methods (40), including a first passage through a column (Affi-Gel 10, Pierce Chemical Co., Rockford, IL) containing the nonphosphorylated peptide and then affinity purification on an Affi-Gel 10 column coupled to the phosphopeptide. The bound antibodies were eluted, dialyzed, and used in the present study.
Protein studies and calf intestine phosphatase treatment
Cells were lysed in a lysis buffer containing 50 mmol/L HEPES (pH 7.5), 1% (vol/vol) Triton X-100, 50 mmol/L NaCl, 5 mmol/L ethylene glycol-bis-(ß-amino-ethyl ether)-N,N,N',N'-tetraacetic acid, 50 mmol/L NaF, 20 mmol/L sodium pyrophosphate, 1 mmol/L sodium vanadate, 2 mmol/L phenylmethylsulfonylfluoride, and 0.2 mg/mL each of aprotinin and leupeptin. Lysates were clarified by centrifugation at 10,000 x g for 15 min, and the supernatant was processed for immunoblotting or immunoprecipitation. Comparable amounts of total cellular proteins, estimated by a modified Bradford assay (Bio-Rad Laboratories, Inc., Munich, Germany), were subjected to immunoprecipitation or direct Western blot. Proteins were revealed using an enhanced chemiluminescence detection kit (ECL, Amersham Pharmacia Biotech, Aylesbury, UK).
For the phosphatase treatment, lysates were dialyzed twice with phosphatase reaction buffer [50 mmol/L Tris-Cl (pH 8.0), 10 mmol/L MgCl2, and 150 mmol/L NaCl] supplemented with 0.1% Triton X-100/0.05% SDS, 2 mmol/L phenylmethylsulfonylfluoride; suspended in 20 µL phosphatase reaction buffer containing 1% SDS, 1% 2-mercaptoethanol, and 2 mmol/L phenylmethylsulfonylfluoride; and heated (60 C, 3 min). Samples were then divided in half (10 µL), diluted with 40 µL phosphatase reaction buffer, and incubated for 3 h at 37 C with or without 3 U molecular biology grade calf intestinal alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany). The reaction was terminated by the addition of sample buffer; samples were electrophoresed on 7.5% acrylamide SDS-PAGE and analyzed by immunoblotting.
Immunohistochemical analyses
Cells were grown on slide chambers, air-dried for 2 h at room temperature, then placed in a buffer bath [phosphate-buffered saline (PBS)] for 5 min before immunoperoxidase staining. The slides were incubated overnight at 4 C in a humidified chamber with the antibodies diluted 1:500 in PBS and subsequently with biotinylated goat antirabbit IgG for 20 min (Vectostain ABC kits, Vector Laboratories, Inc., Burlingame, CA) and for an additional 20 min with premixed reagent ABC (Vector Laboratories, Inc.). The immunostaining procedure was performed with diaminobenzidine (DAKO Corp., Carpenteria, CA), and micrographs were taken on Kodak Ektachrome film (Eastman Kodak Co., Rochester, NY) with a photo Carl Zeiss system (New York, NY).
Microinjection experiments
NIH-3T3 cells expressing EGFr-Ret were seeded on glass coverslips and grown to 60% confluence. Ab-pY1062 (2 µg/µL) or preimmune IgG were injected into cells using an automated microinjection system (Ais Carl Zeiss, Oberkochen, Germany) with and without EGF (50 ng/mL) and serum (0.5% calf serum). Typically, 100–150 cells were injected per coverslip. Bromodeoxyuridine (BrdU; Sigma, St. Louis, MO) was added to the culture medium (100 µmol/L) and left for 18–20 h. Cells were fixed in paraformaldehyde and incubated with secondary fluorescein-conjugated antirabbit IgG to detect microinjected cells. Anti-BrdU mouse monoclonal antibody followed by a Texas Red-conjugated antibody (Roche Molecular Biochemicals) were used to detect the fraction of cells in S phase. The fluorescent signal was visualized with an epifluorescent microscope (Axiovert 2, Carl Zeiss). All coverslips were washed in PBS containing Hoechst 33258 (final concentration, 1 µg/mL; Sigma), rinsed in water, and mounted in Moviol on glass slides. BrdU incorporation was calculated in injected vs. noninjected cells. Human thyroid carcinoma TPC-1 and NPA cell lines were microinjected with Ab-pY1062 (2 µg/µL) or preimmune IgG, as described above. Cells were maintained in serum-free medium starting from 12 h before microinjection. DNA synthesis was monitored by BrdU incorporation. In each experiment, at least 60 microinjected cells were counted and compared to 400 nonmicroinjected cells from the same coverslips.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We chose Ret tyrosines Y1015 and Y1062, two putative Ret
autophosphorylation sites, as specific target epitopes to generate
antiphosphorylated Ret-specific antibodies (Fig. 1A
). Two phosphopeptides: 1)
VKRRD(pY)LDLAASTPSDC, identical to amino acids 1010–1025; and 2)
IENKL(pY)GRISHAFTRC, corresponding to amino acids 1057–1072 of
the Ret sequence, were individually coupled to albumin and injected
into rabbits. The resulting antisera were purified by two-step affinity
chromatography (see Materials and Methods) to recover
antibodies specifically recognizing the phosphorylated Ret
peptides.
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coreceptors, which, in turn, mediate Ret activation. GFR
molecules mediate Ret-independent signaling through the activation of
as yet uncharacterized tyrosine kinases (41). We previously showed that
an EGFr-Ret chimera, generated by fusing the ligand-binding
extracellular domain of EGFr to the intracellular domain of Ret, is a
useful model system with which to trigger Ret kinase activity; EGF is
simply added to the culture medium, thus avoiding possible interference
from parallel activated kinases (38). We initially used the EGFr-Ret
system to study Ret phosphorylation. The purified antibodies
(i.e. Ab-pY1062 and Ab-pY1015) were assayed by immunoblot
analyses against lysates from NIH-3T3 cells expressing the EGFr-Ret
chimera. As shown in Fig. 1B
, both Ab-pY1062 and Ab-pY1015 detected the
160-kDa EGFr-Ret specifically in the stimulated cells. The very weak
band visible in lysates from unstimulated cells (predominantly with
Ab-pY1062) reflects the low tyrosine phosphorylation levels detected
under basal conditions, as indicated by parallel analysis with
antiphosphotyrosine monoclonal antibody (Ab-pY; Fig. 1B). In fact,
dephosphorylation of cell lysates with calf intestinal alkaline
phosphatase completely abolished the signal (Fig. 1C). To determine the
specificity of our antibodies, we prepared mutant versions of EGFr-Ret
in which the phosphorylated tyrosines were mutated to phenylalanine
(EGFr-Ret-Y1015F and EGFr-Ret-Y1062F). Ab-pY1015 and Ab-pY1062 were
unable to recognize the corresponding mutant protein (Fig. 2, A and B), indicating that the tyrosine
residues are required for antibody recognition.
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), although it
was efficiently expressed, as demonstrated by immunoblotting with an
antibody directed against the entire intracellular domain of the Ret
protein (Ab-Ret; Fig. 2C). Subsequently, to determine whether the
phosphorylation events observed with the EGFr-Ret chimera model also
occurred when wild-type Ret was stimulated by its physiological ligand,
we cotransfected COS-7 cells with Ret and the coreceptor (GFR1) for
GDNF. After transfection, we measured Ret phosphorylation on both Y1015
and Y1062 stimulated with GDNF (50 ng/mL, 10 min). As shown in Fig. 2D, Y1015 and Y1062 were promptly phosphorylated (
10-fold over the basal
condition) when wild-type Ret was stimulated by GDNF. As previously
reported (2), Ret products appeared as a 170- to 150-kDa doublet. The
phosphorylation (detected with both antibodies) of the 170-kDa form was
relatively more intense than that of the immature 150-kDa form,
consistent with the idea that the former represents the mature Ret
protein expressed on the cell surface, interacting with the ligand.
These findings strongly suggest that both Y1015 and Y1062 are in
vivo autophosphorylation sites in the ligand-stimulated Ret
receptor. Given the known homologies of the intracellular domains of various RTK, we tested the specificity of Ab-pY1062 and Ab-pY1015 with lysates from COS-7 cells transiently transfected with the related receptors EGFr, p185 neu/ErbB-2, platelet-derived growth factor receptor, and TRK. While the antiphosphotyrosine blot showed an appropriate band for each receptor, no reactivity was seen with either Ab-pY1062 or Ab-pY1015 (data not shown).
The autophosphorylation process of RTK is generally a transient event
that occurs upon ligand activation. Ret triggering induces prompt
intracellular events, including activation of ERKs or MAPK (43). We
next addressed the question of whether the observed phosphorylation
events are relevant in early intracellular signaling or are merely an
epiphenomenon of Ret activation. Accordingly we analyzed, in a
time-course experiment, the phosphorylation state of the two tyrosines
in the EGFr-Ret system. NIH-EGFr-Ret cells were serum starved for
16 h and then pulsed for 2 min with EGF, after which the
dephosphorylation rate was determined at various time points. As shown
in Fig. 3, the phosphorylation of both
tyrosines was readily detected after 2 min of EGF treatment.
Thereafter, the phosphorylation of Y1015 and Y1062 declined, albeit
with slightly different kinetics. Phosphorylation of Y1015 was high
during the first 15 min of chase and then rapidly declined to basal
levels in 120 min. Phosphorylation of Y1062 started to decline as early
as after 5 min of chase, but it reached basal levels in 180 min. Next,
we examined ERK activation by blotting the same lysates with antibodies
specific for the phosphorylated active form of ERK. The very rapid
timing of ERK activation induced by EGFr-Ret (Fig. 3) paralleled the
pattern observed with both antiphospho-Ret antibodies. This suggests
that Y1015 and Y1062 play a significant role in the early events of the
Ret signaling cascade.
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Uncontrolled activation of kinase function is the hallmark of
oncogenic variants of RTK. Ret-derived oncogenes generated by different
kinds of mutations are found in human tumors. To analyze whether the
constitutive phosphorylation of Y1015 and Y1062 is involved in the
transforming activity of Ret-derived oncoproteins, we transfected COS-7
cells with constructs expressing wild-type RET or with different
RET-derived oncogenes: RET-MEN2A, RET-MEN2B, and RET/PTC1; the latter
is the chimeric oncogene resulting from the fusion between RET and the
H4 gene (9). Equal amounts of protein lysates were immunoblotted with
Ab-pY1015 and Ab-pY1062. Both tyrosines are phosphorylated in all
RET-derived oncogenes, but not in the wild-type protein (Fig. 4, A and B). These results show that
phosphorylation of Y1015 and Y1062 is a common event in Ret-derived
oncogenes and may be considered a tracer of Ret activation itself.
Furthermore, phosphorylation of Ret-MEN2B was relatively higher
vs. that of Ret-MEN2A, especially on Y1062 (Fig. 4A).
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),
demonstrating that both residues are phosphorylated in Ret-derived
oncoproteins activated by different mechanisms even when expressed at
endogenous levels. In the same blots we also analyzed ARO cells
(derived from a RET-negative thyroid anaplastic carcinoma) (35), which
did not react with both antibodies, consistent with the
absence of an activated Ret (Fig. 4, A and B).
To analyze the phosphorylation of Ret oncoproteins in human
malignancies, we analyzed lysates from surgical samples of thyroid
carcinomas (Fig. 4B, lanes 1–10) and MTC (Fig. 4, lanes M1-M3) by
immunoblotting; the surgical samples had previously been characterized
by Southern blot for Ret rearrangements (45) or direct sequencing (46).
Protein extracts from human thyroid carcinoma cell lines contained a
70-kDa protein recognized nonspecifically by Ab-pY1015. Although the
nature of this protein is still unclear, it does not seem to be
ubiquitous, as it does not appear in simian COS-7 cells (Fig. 4A, left panel). For the presence of this nonspecific band, we
selected the Ab-pY1062 to analyze those lysates; Fig. 4B
(bottom) shows the results obtained by immunoblotting
protein lysates (50 µg) with Ab-pY1062. Samples 8 (positive for the
RFG-RET rearrangement, RET/PTC3), 6 (positive for the H4-RET
rearrangement, RET/PTC1), and M2 (containing the Ret C634W MEN2A
allele) (46) scored positively at this analysis, showing bands of the
expected molecular size, as indicated by arrows. Ret/PTC
products appeared as doublets, probably due to the alternative splicing
reported to occur at the level of the C tail of Ret. In conclusion, all
of the previously characterized RET-positive thyroid cancers (45, 46)
scored positive at the analysis with anti-pY1062.
Finally, we used Ab-pY1062 in an immunohistochemical analysis
of the TT and TPC-1 cell lines. TPC-1 and TT cells reacted strongly
with Ab-pY1062; the signal was specific, as it was displaced when the
antibody was preincubated with a molar excess of the immunizing peptide
(Fig. 5). On the other hand, ARO cells
did not react with Ab-pY1062 (Fig. 5), consistent with the absence of
signal observed by Western analysis (Fig. 4B). Thus, it can be
concluded that Ret phosphorylation on tyrosines 1015 and 1062 is also
present in human tumoral cells harboring RET-derived oncogenes and that
phospho-Ret antibody can be used to detect an activated Ret in tumoral
cells by either immunoblot or immunohistochemical analyses.
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By binding specifically to the intracellular portion of Ret,
antiphospho-Ret antibodies were predicted to inhibit in vivo
Ret signaling. To test this hypothesis, we microinjected living
NIH-EGFr-Ret cells with Ab-pY1062 and with purified preimmune IgG as a
control. After microinjection, DNA synthesis was monitored by BrdU
incorporation with and without EGF and calf serum. Microinjection of
Ab-pY1062 significantly reduced the proliferative activity of the
EGF-stimulated cells; only 10% of the Ab-pY1062 microinjected cells,
on the average, incorporated BrdU, whereas preimmune IgG had no effect.
Microinjection of Ab-pY1062 did not affect serum-induced DNA synthesis,
demonstrating that the inhibitory effect was specifically directed
against EGFr-Ret (Fig. 6A, left
panel).
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A hallmark of the transformation process is the ability of neoplastic
cells to proliferate in the absence of exogenously added growth
factors. Thus, we assessed whether microinjection of Ab-pY1062 affected
the proliferation of TPC-1 cells, which harbor the RET/PTC1 oncogene.
We microinjected living TPC-1 cells with Ab-pY1062 or with purified
preimmune IgG as a control. After microinjection, DNA synthesis was
monitored by BrdU incorporation. Microinjection of Ab-pY1062
significantly reduced the proliferation of TPC-1 cells; only 10% of
the Ab-pY1062-microinjected cells, on the average, incorporated BrdU,
whereas preimmune IgG had no effect (Fig. 6). Furthermore,
microinjection of Ab-pY1062 did not affect DNA synthesis of NPA cells,
which derive from a Ret-negative papillary carcinoma (35),
demonstrating that the inhibitory effect of Ab-pY1062 iss specifically
directed against oncogenic Ret.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ret tyrosines 1015 and 1062 have been implicated in the Ret-mediated
transforming activity in cultured cells. Tyrosine 1015 is involved in
binding to phospholipase C, and tyrosine 1062 is the docking site
for Shc and Enigma proteins (28, 29, 30, 31, 32). To study the phosphorylation
state of these tyrosines in oncogenic Ret variants in vivo,
we have produced phosphorylation-specific antibodies. Here we
demonstrate that both residues are in vivo
autophosphorylation sites of the Ret receptor. We show that
phosphorylation of tyrosines 1015 and 1062 occurs early after Ret
activation, being detected as early as 2 min after Ret stimulation.
These findings lend weight to the hypothesis that tyrosine 1015 and
1062 are involved in the early phases of Ret signaling, concomitantly
with such a crucial early event as MAPK activation. Upon ligand
withdrawal, phosphorylation of the two tyrosines declined with slightly
different kinetics, which indicates that the pathways triggered by the
tyrosines may act for different periods of time after Ret
stimulation.
We demonstrate that regardless of the molecular mechanisms underlying Ret activation, different Ret-derived oncoproteins (Ret-MEN2A, Ret-MEN2B, and Ret/PTC) are constitutively phosphorylated on tyrosines 1015 and 1062. The finding that Ret-MEN2B is phosphorylated to a greater extent on Y1062 with respect to Ret-MEN2A deserves further investigation. In vitro phosphorylation studies can be biased by the abnormally high expression levels of the analyzed kinase. Here we show that tyrosines 1015 and 1062 are also in vivo phosphorylated in Ret oncoproteins expressed at endogenous levels in human tumoral cell lines and even in human surgical samples.
We also show that Ab-pY1062 specifically reacts with activated Ret when used in immunohistochemical or immunoblot studies conducted on samples as small as protein lysates of 50 µg. Thus, Ab-pY1062 can be used as a molecular marker of Ret activation in human neoplasms and possibly to identify neoplastic cells in fine needle bioptic specimens or in surgically removed lymph nodes. These antibodies may also be useful to study physiological Ret activation in adult and embryonic tissues.
The microinjection experiments reported herein indicate that Ab-pY1062 inhibits Ret activity in vivo. Using this approach, we have shown that RET/PTC activity is required for the proliferation of TPC1, a human thyroid carcinoma- derived cell line harboring the RET/PTC1 oncogene. The development of PTC in transgenic mice has demonstrated that RET/PTC oncogenes initiate a thyroid tumorigenic process in vivo (47, 48, 49, 50, 51). On the other hand, the data reported here suggest that continuous RET/PTC signaling may be required for the maintenance of neoplastic proliferation. This suggests that although it is likely that PTC, like most human tumors, are caused by multiple genetic alterations, inactivation of RET/PTC can lead to tumor regression. This opens the intriguing possibility that manipulation of the RET/PTC oncogene pathway may be useful in the treatment of RET/PTC-positive carcinomas.
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Acknowledgments |
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Footnotes |
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Received April 25, 2000.
Revised June 28, 2000.
Accepted July 7, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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of cyclic AMP-dependent protein kinase A. Mol Cell Biol. 13:358–366.. Mol Cell Biol. 16:2151–2163.[Medline]
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M. Coulpier, J. Anders, and C. F. Ibanez Coordinated Activation of Autophosphorylation Sites in the RET Receptor Tyrosine Kinase. IMPORTANCE OF TYROSINE 1062 FOR GDNF MEDIATED NEURONAL DIFFERENTIATION AND SURVIVAL J. Biol. Chem., January 11, 2002; 277(3): 1991 - 1999. [Abstract] [Full Text] [PDF]
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