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Endocrinology |
Centro di Endocrinologia ed Oncologia Sperimentale del CNR, c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare [D. S., R. M. M., C. M., R. V., G. V., M. S.], and Dipartimento di Endocrinologia ed Oncologia Molecolare e Clinica, Facoltà di Medicina e Chirurgia [G. F.], Università di Napoli "Federico II," Naples, Italy; and Dipartimento di Medicina Sperimentale e Clinica, Facoltà di Medicina e Chirurgia di Catanzaro, Università di Catanzaro, Catanzaro [A. F], Italy
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ABSTRACT |
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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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1–4, that in turn induce dimerization and activation of Ret
kinase (2)
.
Germ-line point mutations that cause oncogenic activation of Ret are
responsible for the inheritance of MEN2A and MEN2B and FMTC. Each
disease has a distinct phenotype: MEN2A is associated with MTC,
pheochromocytoma, and parathyroid hyperplasia. The MEN2B phenotype is
more severe, being characterized by an earlier occurrence of more
aggressive MTC. Unlike MEN2A patients, MEN2B patients develop multiple
mucosal neuromas and several skeletal abnormalities. Finally, FMTC
consists only of an inherited predisposition to MTC (3)
.
Involvement of different tissues corresponds to differences in the
nature of the underlying RET gene mutation. In most MEN2A
cases, RET mutations consist in the loss of one of six
cysteines localized in the extracellular domain. This causes
constitutive dimerization mediated by the formation of disulfide bonds
and activation of Ret kinase (4
, 5)
. The same mutations
have been found in various FMTC patients (3)
. In >80% of
patients, MEN2B is caused by substitution of methionine 918 with a
threonine (M918T; Ref. 6
). Interestingly, the same
mutation at a somatic level has been identified in 
30% of sporadic
MTC, whereas MEN2A-like mutations are very rare in sporadic tumors
(7)
.
The MEN2B (M918T) mutation occurs in the "activation loop" of Ret kinase, a domain that is critical not only for kinase activation but also for substrate selection (6) . The MEN2B mutation induces constitutive activation of Ret kinase; however, the intrinsic kinase activity of Ret-MEN2B is not more potent than that of Ret-MEN2A (4 , 5 , 8) . It is believed that Ret-MEN2B is not simply an activated Ret kinase. Maps of phosphorylated Ret peptides and two-dimensional gel electrophoresis experiments have demonstrated that the MEN2B mutation alters Ret autophosphorylation sites as well as the pattern of intracellular proteins undergoing tyrosine phosphorylation with respect to wild-type ligand-triggered Ret and to Ret-MEN2A (4 , 9) . Thus, the shift of Ret autophosphorylation sites and of Ret intracellular substrates, rather than the simple potentiation of Ret kinase activity, may be crucial for the oncogenic activity of Ret-MEN2B alleles.
The biological effects of RTKs are exerted mainly through
autophosphorylation of tyrosine residues. The substrates of the RTK
protein are characterized by docking sites, i.e., PTB or
Src-homology 2 domains. By recognizing receptor
phosphotyrosines, these docking sites provide a means whereby enzyme
and substrate can physically interact to induce the phosphotransfer
reaction and activation of intracellular signal transduction (10
, 11)
. The cytoplasmic domain of Ret contains 14 tyrosine
residues; a longer form (1114 residues long), which arises from
alternative splicing, contains 16. Ret phosphorylated tyrosines 905 and
1015 are docking sites for the Grb7/Grb10 adapters (12
, 13)
and for phospholipase C
(14)
, respectively.
Tyrosine 1062 (Y1062) is essential for Ret signaling. Mutation of Y1062
impairs neoplastic transformation mediated by Ret-derived oncogenes
(Ret-MEN2A and Ret-MEN2B; Refs.15
, 16
) and
Ret-mediated PC12 cell survival (17)
. Y1062 is necessary
for the binding of Shc (15
, 18
, 19)
and Enigma
(20)
to Ret. Shc is a docking protein involved in the
coupling of several receptors to the Ras/MAPK pathway
(21)
. Enigma is a PDZ- and LIM-domain containing protein
whose function is relatively obscure; however, evidence suggests that
Enigma is involved in recruitment and clustering of Ret protein
products at membrane level (20)
. Tyrosine 1062 is
essential for Ret-mediated activation of the Ras/MAPK cascade
(17)
, for the binding of Ret to the p85 regulatory subunit
of PI3K, and for activation of PI3K and of the serine-threonine kinase
Akt(PKB), a well-known PI3K effector (16
, 17)
.
Neither the signaling pathways triggered by the MEN2B mutation nor the specific Ret sites whose autophosphorylation is altered by the MEN2B mutation, are known. Phosphorylation of Y1096, a residue present only in the 1114-amino acid-long Ret isoform and involved in Grb2 binding to Ret, is reduced in the Ret MEN2B protein (9 , 22) . Furthermore, the M918T mutation modifies the pattern of tyrosine phosphorylated proteins that bind to the Nck and Crk adapters. However, the identities and roles of these molecules in Ret signal transduction have yet to be demonstrated (23) .
We have used phosphorylated-Ret specific antibodies directed against Y1015 and Y1062 (24) to analyze the phosphorylation states of Ret-MEN2B and Ret-MEN2A. Here we show that Y1062 and Y1015 are constitutively phosphorylated in Ret-MEN2A and Ret-MEN2B; however, phosphorylation of Y1062, but not of Y1015, is greatly increased in Ret-MEN2B compared with Ret-MEN2A. In vitro assay confirmed the more potent autokinase activity toward Y1062 resulting from the MEN2B mutation. Consistent with the increased phosphorylation of Y1062, the Ret-MEN2B oncoprotein is more active than Ret-MEN2A in recruiting the Shc adapter and in inducing activation of the Ras/MAPK and PI3K/Akt cascades. We conclude that selective increase of Y1062 autophosphorylation and of coupling to the Shc/Ras/MAPK and PI3K/Akt pathways is part of the specific pathogenetic mechanism of the MEN2B syndrome.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells and Transfection Experiment.
PC12 cells were grown in RPMI medium 1640 supplemented with 10%
horse serum and 5% FCS. NIH-3T3 and COS-7 cells were grown in DMEM
(Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FCS
and were transfected with 1 µg of epitope (AU5)-tagged Ras construct
(V12) as a positive control of the Ras activation assay, using the
calcium phosphate precipitation method as described previously
(27)
.
Protein Studies and Calf Intestinal Phosphatase Treatment.
Cells were lysed in a buffer containing 50 mM HEPES
(pH 7.5), 1% (v/v) Triton X-100, 50 mM NaCl, 5
mM EGTA, 50 mM NaF, 20 mM
sodium PPi, 1 mM sodium vanadate, 2
mM phenylmethylsulfonyl fluoride, 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 for immunoprecipitation. Protein
concentrations were measured with a modified Bradford assay (Bio-Rad,
Munich, Germany). Immunocomplexes were revealed with an enhanced
chemiluminescence detection kit (ECL; Amersham, Bucks., England). The
signal was quantified with scanner densitometry of the films.
For the phosphatase treatment, lysates were dialyzed twice against phosphatase reaction buffer [05 mM Tris-Cl (pH 8.0), 10 mM MgCl2, 150 mM NaCl] supplemented with 0.1% Triton X-100, 0.05% SDS, and 2 mM phenylmethylsulfonyl fluoride, suspended in 20 µl of phosphatase reaction buffer containing 1% SDS, 1% 2-mercaptoethanol, and 2 mM phenylmethylsulfonyl fluoride, and heated (60°C for 3 min). Samples were then divided in half (10 µl), diluted with 40 µl of phosphatase reaction buffer, and incubated for 3 h at 37°C with or without 3 units of molecular biology-grade calf intestinal alkaline phosphatase (Boehringer Mannheim, 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.
Detection of Activated Ras.
Gst-Raf(RBD) encodes amino acids 1–149 of c-Raf-1 fused to GST
(28)
. Bacterial cultures were induced with 1
mM isopropyl ß-D-thiogalactopyranoside for
3 h. The induced bacteria were lysed by sonication in PBS
containing 10 µg/ml aprotinin, and GST-Raf(RBD) protein was purified
using glutathione-Sepharose (Pharmacia). The beads were washed in a
solution containing 20 mM HEPES (pH 7.5), 120
mM NaCl, 10% glycerol, 0.5% NP-40; 2 mM EDTA,
and 10 µg/ml aprotinin and used within 2–3 days after preparation.
Cells were lysed, and the clarified extract (1 mg) was incubated with
15 µg of immobilized GST-Raf(RBD) protein for 1 h at 4°C with
rocking. Protein complexes were resolved by SDS-PAGE (10% acrylamide),
transferred to polyvinylidene difluoride (Immobilon-P) membranes, and
stained with anti-Ras antibodies.
Pull-Down Experiments
Cell lysates were subjected to "pull-down" binding assays with
purified GST-Shc(PTB) recombinant protein immobilized on agarose beads.
The GST fusion protein of the PTB domain of Shc (29)
was
expressed in bacteria and purified with glutathione-conjugated
Sepharose beads (Sigma, St. Louis, MO) by standard procedures. The
protein complexes were washed several times with the cell lysis buffer,
eluted, and resolved by SDS-PAGE. Western blotting with specific
antibodies and enhanced chemiluminescence (ECL; Amersham) were used for
immunodetection of proteins in the complex.
Kinase Assay.
ERK activity was measured in PC12 cells that express Ret-MEN2A and
Ret-MEN2B. Protein lysates (1 mg) were immunoprecipitated with
anti-ERK; 20 µg of myelin basic protein was used as a substrate for
the kinase reaction, as described previously (30)
. Ret
kinase activity was assayed in PC12 Ret-MEN2A and Ret-MEN2B cells,
essentially as described elsewhere (4)
. A GST fusion
protein containing the COOH-terminal 72 residues of Ret,
GST-Ret(C-tail), which include Y1015 and Y1062 cloned in the pGEX2T
vector (Amersham Pharmacia Biotech), served as substrate. Protein
lysates (1 mg) were immunoprecipitated with anti-Ret; 15 µg of
GST-Ret(C-tail) was used as substrate for the kinase reaction.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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phosphorylation levels, we used antibodies that detect the
phosphorylation of two critical autophosphorylation sites of human Ret
(Ab-pY1015 or Ab-pY1062). Protein extracts were
obtained from stably transfected NIH-3T3 and PC12 cells and subjected
to immunoblotting with the anti-phospho-Ret antibodies. The
oncoproteins of the two cell lines appeared as a doublet of 155 and 170
kDa and had similar Y1015 phosphorylation levels (Fig. 1B)
.
In contrast, in both cell types, the phosphorylation levels of Y1062
were much higher (5-fold) in the Ret-MEN2B protein than in the
Ret-MEN2A protein (Fig. 1B)
.
|
, the signal
detected in NIH-2A was not present in the untransfected NIH-3T3 cells
(Fig. 1C
, Lane C) as well as in the
phosphatase-treated NIH-2A (Fig. 1C
, Lane
2A-CIP), indicating that the phosphorylated tyrosine residue is
required for antibody recognition.
Ret oncoproteins bind the PTB domain of Shc through phosphotyrosine
1062 (15
, 18
, 19)
. Consequently, an independent way to
determine the extent of Y1062 phosphorylation is to measure the
Shc(PTB) interaction. Protein extracts from PC12-MEN2A and -MEN2B cells
were subjected to a pull-down assay with a recombinant Shc(PTB) domain
expressed in Escherichia coli as a GST fusion protein.
Protein extracts from parental PC12 cells served as a control. Proteins
interacting with GST-Shc(PTB) were separated by SDS-PAGE and
immunoblotted with anti-Ret. As shown in Fig. 2A
, higher amounts of Ret-MEN2B interacted with GST-Shc(PTB)
than with Ret-MEN2A. This shows that Ret-MEN2B contains higher levels
of phosphotyrosine 1062 than Ret-MEN2A. An immunoblot stained with
anti-Ret antibody demonstrated equal expression levels of the Ret
mutants (Fig. 2B)
.
|
(bottom), Ret-MEN2B kinase was clearly more
potent than Ret-MEN2A kinase in phosphorylating GST-Ret(C-tail) on
Y1062. Consistently, the autophosphorylation of Ret-MEN2B on Y1062 was
also clearly more potent than Ret-MEN2A (Fig. 3A
,
top). We know that most of the phosphorylation detected on
the Ret oncoproteins occurred during the in vitro assay
because cells were harvested in the absence of phosphatase inhibitors,
thus minimizing the amount of phosphorylation occurred in
vivo before collecting cells (data not shown). On the other hand,
the two kinases phosphorylated GST-Ret(C-tail) on Y1015 to a
similar extent. Immunoblotting demonstrated that equal amounts of Ret
kinases were used for the assay (Fig. 3B)
.
|
, the 66-, 52-, and 46-kDa Shc proteins were tyrosine-phosphorylated in
PC12 cells expressing oncogenic Ret alleles with respect to parental
cells. More importantly, Shc phosphorylation was at least 3-fold more
pronounced in Ret-MEN2B- than in Ret-MEN2A-expressing cells (Fig. 4A)
. Immunoblot analysis with Ab-Shc, as a loading control,
showed comparable levels of Shc proteins in the tested cell lines.
|
, Ras
activation induced by Ret-MEN2B was 2-fold stronger than that caused
by Ret-MEN2A. Ras expression levels were comparable in MEN2A and MEN2B
cells and were slightly elevated versus untransfected cells
(Fig. 4B)
. The V12 oncogenic Ras mutant transiently
expressed in COS-7 cells was used as a positive control for the assay. Y1062 is also essential for recruitment of PI3K to the Ret receptor (16 , 17) . PI3K activation results in the triggering of multiple downstream effectors including the serine-threonine kinase Akt(PKB) (31) . On the other hand, activation of the Ras cascade leads to a series of events culminating in activation of the MAPKs ERK1 and ERK2 and also contributes to the activation of PI3K/Akt pathways. Thus, we determined ERK and Akt activation as a measure of activation of the Ras and of the PI3K signaling pathways in cells expressing Ret-MEN2A or Ret-MEN2B.
We used two techniques to evaluate ERK activation. ERK2 protein was
recovered by immunoprecipitation from PC12 cells expressing Ret-MEN2A
or Ret-MEN2B. The immunoprecipitates were subjected to an in
vitro kinase assay using myelin basic protein as substrate.
Untransfected PC12 cells had very low MAPK activity, whereas Ret-MEN2A
expression promptly induced MAPK activity (Fig. 5A)
. Activation of MAPK by Ret-MEN2B was 2-fold higher than
that caused by Ret-MEN2A. These findings were confirmed by an
independent assay. Protein lysates from the same cells were analyzed by
immunoblot with an antibody specific for the phosphorylated
(i.e., active), ERK proteins. Again, ERK phosphorylation
levels in Ret-MEN2B cells was 2–3-fold higher than in Ret-MEN2A cells.
An immunoblot with an anti-ERK antibody was performed for normalization
(Fig. 5A)
.
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6-fold) in
Ret-MEN2B- than in Ret-MEN2A-expressing cells. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oncogenic activation of RTKs is mediated by constitutive activation of their enzymatic activity. In most cases, this catalytic activation is caused by mutations that induce constitutive dimerization (32) . This is the case of most of the Ret-MEN2A and Ret-FMTC mutants (4 , 33) . MEN2B is an exception to this paradigm. The M918T mutation does not cause constitutive dimerization, and the overall activation levels of the Ret kinase it induces can hardly account for its high oncogenic potential (4) . Thus, Ret-MEN2B is probably more than simply an active Ret kinase, and qualitative changes in Ret kinase activity may be responsible for its specific neoplastic phenotype. M918 is highly conserved in RTKs, and it maps in a loop of the kinase domain that is predicted to interact with the protein substrate. A threonine is found at the equivalent position in cytosolic PTKs, and the two kinase classes (RTK and PTK) have different signaling specificities (34) . Accordingly, the MEN2B mutation causes a shift of the optimal peptide substrate from that preferentially phosphorylated by RTKs to that selected by PTKs (34) . The change in substrate specificity can affect Ret-mediated phosphorylation of intracellular proteins as well as the pattern of Ret autophosphorylation sites. Both possibilities have been experimentally proven. The pattern of phosphorylated intracellular proteins differs in Ret-MEN2B- and Ret-MEN2A-expressing cells (4) . Moreover, phosphopeptide mapping has shown that Ret-MEN2B autophosphorylation sites differ dramatically from those of wild-type Ret and of Ret-MEN2A (4 , 9) .
We have produced phosphorylation-specific antibodies directed against Ret Y1015 and Y1062 (24) . Here we demonstrate that although both residues are constitutively phosphorylated in Ret-MEN2A and Ret-MEN2B, Y1062 autophosphorylation is significantly increased by the MEN2B mutation with respect to the MEN2A mutation. In agreement with this finding, a MEN2B kinase phosphorylated Y1062 more efficiently than did a MEN2A kinase in an in vitro kinase assay, We believe that the increased phosphorylation of Y1062 depends on the altered specificity of the Ret-MEN2B kinase. Indeed, the Ret sequence that flanks Y1062 (Leu-Tyr-Gly-Arg-Ile) corresponds to the optimal substrate (Ile/Leu/Val-Tyr-Gly-X-hydrophobic) of PTK (to which Ret-MEN2B functionally belongs; see Ref. 35 ) and thus, Y1062 has the structural requisites for efficient phosphorylation by a Ret-MEN2B kinase.
Several lines of evidence suggest that the high phosphorylation of Y1062 has significant biological effects. Residue Y1062 is essential for oncogenic and survival Ret signaling and for activation of Ras/MAPK and PI3K/Akt. Parallel to the increased phosphorylation of Y1062, we show that the MEN2B mutation potentiates recruitment of Shc and activation of Ras/MAPK and of Akt with respect to Ret-MEN2A. Given the susceptibility of Ret-MEN2B to ligand triggering (36) , the above-mentioned differences may be more pronounced in vivo in the presence of ligands.
In conclusion, we propose that the high level of Y1062 phosphorylation caused by the MEN2B mutation can be involved in determining the MEN2B phenotype. This suggests the intriguing possibility of using residue Y1062 as a target for therapeutic approaches to MEN2B as well as to sporadic MTC neoplasias.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by the Associazione
Italiana per la Ricerca sul Cancro (AIRC), by European Community
Grant BMH4-CT96-0814, by the Progetto Biotecnologie "5%" of the
Consiglio Nazionale delle Ricerche (CNR), and by the Project MURST
"Terapie antineoplastiche innovative." This paper was written while
G. Vecchio was a Scholar-in-Residence at the Fogarty International
Center for Advanced Study in the Health Sciences, NIH, Bethesda, MD 
2 To whom requests for reprints should be
addressed, at Centro di Endocrinologia ed Oncologia Sperimentale del
CNR, Universitá degli Studi di Napoli, via S. Pansini 5, 80131
Naples, Italy. Phone: 39-081-7463056; Fax: 39-081-7463037; E-mail: masantor{at}unina.it
3 The abbreviations used are: GDNF, glial cell
line-derived neurotrophic factor; MEN2A and MEN2B, multiple endocrine
neoplasia type 2A and type 2B; FMTC, familial medullary thyroid
carcinoma; MTC, medullary thyroid carcinoma; RTK, receptor tyrosine
kinase; PTB, phosphotyrosine binding; MAPK, mitogen-activated protein
kinase; PI3K, phosphatidyl-inositol-3 kinase; ERK, extracellular
signal-regulated kinase; GST, glutathione-S-transferase;
RBD, Ras-binding domain; PTK, protein tyrosine kinase.
Received 7/31/00. Accepted 12/13/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.. Mol. Cell. Biol., 16: 2151-2163, 1996.[Medline]
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