Endocrinology Vol. 140, No. 9 3992-3998
Copyright © 1999 by The Endocrine Society
The Role of Amino Acids Surrounding Tyrosine 1062 in Ret in Specific Binding of the Shc Phosphotyrosine-Binding Domain1
Yoshihiro Ishiguro,
Toshihide Iwashita,
Hideki Murakami,
Naoya Asai,
Ken-ichi Iida,
Hidemi Goto,
Tetsuo Hayakawa and
Masahide Takahashi
Departments of Pathology (Y.I., T.I., H.M., N.A., K.I., M.T.) and
Internal Medicine II (Y.I., H.G., T.H.), Nagoya University School of
Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
Address all correspondence and requests for reprints to: Department of Pathology, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: mtakaha{at}med.nagoya-u.ac.jp
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Abstract
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We investigated the role of the I-E-N-K-L (amino acids 1057–1061)
sequence amino-terminal to Tyr1062 in Ret for binding of
the Shc phosphotyrosine-binding (PTB) domain. Substitution of Ser for
Ile1057 (I1057S), Ala for Asn1059 (N1059A), or
Pro for Leu1061 (L1061P) in this sequence significantly
decreased the transforming activity of Ret with the multiple endocrine
neoplasm type 2A (MEN2A) mutation as well as the binding affinity of
Shc to it in vivo and in vitro,
indicating that these three amino acids play a role in Shc binding. In
addition, as the RET protooncogene is translated as
three isoforms of 1114 amino acids (Ret 51), 1106 amino acids (Ret 43),
and 1072 amino acids (Ret 9) that differ from one another in the
sequence carboxyl-terminal to Tyr1062, we examined whether
these sequence differences influence the binding affinity of Shc to
Ret. As a result, we found that the transforming activity of Ret 43
isoform with the MEN2A mutation and the binding affinity of Shc to it
were very low, although the consensus sequence for the binding of the
Shc PTB domain is conserved in the Ret 43 isoform. This finding
suggested that the sequence carboxyl-terminal to Tyr1062 in
Ret could also influence the binding affinity to Shc.
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Introduction
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ACTIVATION of receptor tyrosine kinases by
their ligands induces receptor autophosphorylation, resulting in
binding of a variety of intracellular signaling molecules such as Shc
adaptor proteins. Shc has two functional domains, the
phosphotyrosine-binding (PTB) domain and the Src homology (SH) 2
domain, that recognize phosphotyrosine in the receptors (1). Bound Shc
is also phosphorylated on tyrosine by receptor tyrosine kinases and
interacts with the Grb2/Ras guanine nucleotide exchange factor SOS
complex, leading to activation of the Ras mitogen-activated protein
kinase signaling pathway (2, 3, 4). It is known that the binding
specificity of the PTB and SH2 domains of Shc is determined by residues
amino-terminal and carboxyl-terminal to phosphotyrosine in activated
receptors, respectively. The consensus sequence for binding of the Shc
PTB domain is N-P-X-pY, whereas the sequence for binding of the SH2
domain is pY-(I/E/Y/L)-X-(I/L/M) (1, 5, 6, 7).
The RET protooncogene codes for a receptor tyrosine kinase
whose ligands are members of the glial cell line-derived neurotropic
factor protein family, including glial cell line-derived neurotropic
factor, neurturin, persephin, and artemin (8, 9, 10, 11, 12, 13, 14). It was demonstrated
that germline mutations of RET are associated with the
development of multiple endocrine neoplasia (MEN) types 2A and 2B, and
Hirschsprung’s disease (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25). MEN2A and MEN2B are autosomal
dominant cancer syndromes characterized by the development of medullary
thyroid carcinoma and pheochromocytoma. MEN2B is distinguished from
MEN2A by a more complex phenotype, such as mucosal neuroma,
hyperganglionosis of the gastrointestinal tract, and marfanoid habitus.
Hirschsprung’s disease is a developmental disorder associated with the
absence of intrinsic ganglion cells in the large intestine. The MEN2A
and MEN2B mutations resulted in the constitutive activation of Ret
kinase, leading to transformation of NIH-3T3 cells (18, 19, 20, 21). On the
other hand, Hirschsprung’s mutations represent loss of function
mutations (22, 23, 24, 25).
We and others have recently demonstrated that Tyr1062 in
Ret represents a Shc binding site (26, 27, 28, 29). The sequence
(I-E-N-K-L) amino-terminal to Tyr1062 matches the
consensus sequence for binding of the Shc PTB domain (30). When this
tyrosine was replaced with phenylalanine, the transforming activity of
Ret with the MEN2A or MEN2B mutation markedly decreased (26),
suggesting that Shc binding to Ret is crucial for the transforming
activity of activated Ret. In the present study we investigated the
role of the I-E-N-K-L sequence amino-terminal to Tyr1062 in
Ret for the Shc binding by introducing several mutations into this
sequence. In addition, as Ret is known to be translated as three
isoforms that differ from one another in the sequence carboxyl-terminal
to Tyr1062, we examined whether these sequence differences
influence the binding ability of Shc to Ret.
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Materials and Methods
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Plasmid construction
Complementary DNA clones with the entire coding sequence of the
human RET protooncogene encoding three isoforms (1072, 1106,
or 1114 amino acids) were inserted into the APtag-1 vector containing
the Moloney murine leukemia virus long terminal repeat, provided by Dr.
P. Leder (Harvard Medical School, Cambridge, MA). Each mutation was
introduced by PCR as described previously (19).
Transfection
Plasmid DNAs containing mutant RET genes (0.1–0.5
µg) were transfected into NIH-3T3 cells (5 x 105
cells in a 60 mm-diameter dish) with 10 µg NIH-3T3 genomic DNA by the
calcium phosphate precipitation method as described previously (19, 20). Transfection experiments were carried out four times for each
plasmid. Transformed foci were scored on day 12 after transfection.
Then foci were picked up and grown into cell lines. Each cell line was
fed with DMEM supplement with 8% calf serum.
Antibodies
Anti-Ret rabbit polyclonal antibodies were developed against the
carboxyl-terminal 19 amino acids of three Ret isoforms. Anti-Ret mouse
monoclonal antibodies (Ret-R5) were developed against a peptide
corresponding to part of the Ret extracellular domain (31). Anti-Shc
polyclonal and anti-Grb2 monoclonal antibodies were purchased from
Transduction Laboratories (Lexington, KY), and antiphosphotyrosine
monoclonal (4G10) antibody was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY).
Immunoprecipitation and immunoblotting
Cells were lysed in radioimmunoprecipitation assay buffer [20
mM Tris-HCl (pH 7.5), 150 mM NaCl, 2
mM EDTA, and 1% Triton X-100] containing 1 mM
phenylmethylsulfonylfluoride and 0.5 mM sodium
orthovanadate. Equal amounts of the lysates, whose protein
concentrations were measured using the Bio-Rad protein assay kit
(Bio-Rad Laboratories, Inc., Hercules, CA), were clarified
by centrifugation (15,000 x g) for 1 h, incubated
with Sepharose beads conjugated with antibodies at 4 C overnight, and
washed with radioimmunoprecipitation assay buffer four times. The
resulting antigen-antibody complex was eluted by boiling in SDS-sample
buffer [20 mM Tris-HCl (pH 6.8), 2 mM EDTA,
2% SDS, 10% sucrose, and 20 µg/ml bromophenol blue] in the
presence of 80 mM dithiothreitol, subjected to SDS-PAGE,
and transferred to polyvinylidene difluoride membranes (Nihon,
Millipore Corp., Tokyo, Japan). After membranes were
reacted with antibodies, the reaction was examined by enhanced
chemiluminescence (Amersham Pharmacia Biotech, Tokyo,
Japan).
In vitro binding assay using glutathione-S-transferase (GST)-fused
proteins
The Shc PTB and SH2 domains were inserted into the pGEX-3X
vector (Amersham Pharmacia Biotech), and the resulting
recombinant plasmids were used to transform Escherichia
coli, JM109. Bacteria cultures were grown in Luria Bertonia
medium containing 100 µg/ml ampicillin and induced with 1
mM isopropyl-ß-D-thiogalactopyranoside for
6 h. The induced GST fusion proteins were purified using
glutathione-agarose beads. Cells expressing Ret were lysed in ice-cold
lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM
NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5 mM
sodium orthovanadate, and 10 µg/ml aprotinin] and clarified by
centrifugation. The supernatants were incubated with 5 µg immobilized
GST or GST fusion proteins at 4 C for 3 h and washed with lysis
buffer four times. The proteins bound to GST fusion proteins were
eluted by boiling in SDS-sample buffer, resolved by SDS-PAGE, and
immunoblotted with anti-Ret or antiphosphotyrosine antibody.
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Results
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Transforming activity of Ret with mutations in the Shc PTB domain
binding motif
Asn at the -3 position and a hydrophobic amino acid at the
-5 position amino-terminal to phosphotyrosine in receptor tyrosine
kinases are known to be important for binding of the Shc PTB domain (5, 7). We previously demonstrated that the sequence (IENKL) amino-terminal
to Tyr1062 in Ret matches this consensus sequence (Fig. 1
) and represents an Shc PTB domain
binding site (26, 30). To examine the importance of this sequence for
Shc binding to Ret, we changed Asn at the -3 position (codon 1059 in
Ret) to Ala and Ile at the -5 position (codon 1057) to Ser (Fig. 1B).
In addition, we changed Leu at the -1 position to Pro, as this
mutation was identified in a HSCR patient. These mutations were
introduced into the Ret long isoform complementary DNA with a MEN2A
(Cys634
Arg, C634R) mutation. Because the transforming
activity of Ret with the MEN2A mutation (Ret-MEN2A) appears to mainly
depend on the signal transduction arising from Tyr1062 that
is an Shc docking site (26), we investigated the correlation between
the transforming activity of mutant Ret proteins and their ability to
bind to Shc. As shown in Table 1, all of
the I1057S, N1059A, L1061P, and Y1062F mutations significantly impaired
the transforming activity of Ret-MEN2A, although the impairment by the
Y1062F mutation was more severe than other mutations. The activity of
Ret-MEN2A with the I1057S/N1059A double mutation was reduced to a
degree similar to that of Ret-MEN2A with the Y1062F mutation (Table 1).
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Figure 1. Schematic illustration of three Ret isoforms. A,
S, Signal sequence; CAD, cadherin-like domain; CYS, cysteine-rich
region; TM, transmembrane domain; TK, tyrosine kinase domain. A MEN2A
mutation (Cys634Arg) is indicated. Tyr1062
represents an Shc binding site. Three isoforms of Ret (Ret 9, Ret 43,
and Ret 51) are generated by alternative splicing in the 3'-region
downstream of Tyr1062. B, Amino acid sequences around
Tyr1062 in three isoforms. Ile (I), Asn (N), and Leu (L) at
-5, -3, and -1 positions relative to Tyr1062 were
replaced with Ser (S), Ala (A), and Pro (P), respectively.
Tyr1062 (Y) was replaced with Phe (F). The
underline and double underline indicate
the putative binding sites for the Shc PTB and SH2 domains,
respectively.
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NIH-3T3 cells expressing each mutant Ret protein were
established. Ret is expressed as the 155- and 175-kDa proteins that
correspond to the immature glycosylated form in the cytoplasm and the
mature glycosylated form at the plasma membrane, respectively. The
pattern and level of tyrosine phosphorylation of mutant Ret proteins in
these cell lines were similar despite the difference in their
transforming activity (Fig. 2). As we
have recently described (32), the level of Ret tyrosine phosphorylation
does not appear to directly correlate with the level of biological
activity.
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Figure 2. Expression and tyrosine phosphorylation of the
mutant Ret proteins in the transfectants. Total cell lysates (20 µg
proteins) were prepared from the designated cell lines, separated on
SDS-8% polyacrylamide gels under reducing conditions, and subjected to
immunoblotting with anti-Ret 51type or antiphosphotyrosine ( pTyr)
antibodies. Each mutation was introduced into Ret 51type with a MEN2A
(C634R) mutation. Protein concentrations of the lysates were measured
using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc.). The 155- and 175-kDa Ret proteins are indicated. IB,
Immunoblotting.
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Binding affinity of Shc to mutant Ret proteins
The lysates from the established cell lines expressing the
mutant Ret proteins were immunoprecipitated with anti-Shc antibody,
followed by immunoblotting with anti-Shc, anti-Ret, or
antiphosphotyrosine antibody. As shown in Fig. 3A, the amounts of Ret-MEN2A with the
I1057S, N1059A, I1057S/N1059A, L1061P, or Y1062F mutation
coprecipitated with Shc were markedly decreased compared with that of
the original Ret-MEN2A coprecipitated with Shc. In particular, in the
case of Ret-MEN2A with the I1057S/N1059A mutation, coprecipitation with
Shc was almost undetectable. The phosphorylation of the
immunoprecipitated 46- and 52-kDa Shc (66-kDa Shc was not visible in
this experiment) was significantly reduced in the NIH-3T3 cells
expressing each mutant Ret protein, except for the cells expressing
Ret-MEN2A with the I1057S mutation (Fig. 3B).
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Figure 3. Coprecipitation of mutant Ret proteins with Shc.
A, Equal amounts of cell lysates from the designated cells were
immunoprecipitated with anti-Shc antibody, separated on SDS-12%
polyacrylamide gels, and subjected to immunoblotting with anti-Ret
51type or anti-Shc antibody. B, The same immunoprecipitates were
immunoblotted with antiphosphotyrosine antibody. The 175- and 155-kDa
Ret proteins and 66-, 52-, and 46-kDa Shc proteins are indicated. IP,
Immunoprecipitation; IB, immunoblotting.
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To confirm the results obtained from in vivo analysis, we
investigated the ability of Shc to bind to these mutant Ret in
vitro using the GST fusion proteins. The binding of full-length
Shc as well as its PTB domain to Ret-MEN2A was markedly decreased by
the mutations examined, although the degree of the reduction by the
I1057S mutation was lower than that by other mutations (Fig. 4).
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Figure 4. Interaction of Ret with Shc PTB and SH2 domains
in vitro. Cell lysates from the designated cells were
incubated with GST-Shc (full-length), GST-Shc PTB, or GST-Shc SH2
fusion proteins immobilized on glutathione-agarose beads. Bound
proteins were separated on SDS-8% polyacrylamide gels and subjected to
immunoblotting with anti-Ret antibody.
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Transforming activity of three Ret isoforms and their ability to
bind to Shc
The RET protooncogene is translated as three
isoforms of 1114 amino acids (long isoform; designated Ret 51type),
1072 amino acids (short isoform; Ret 9type) and 1106 amino acids
(middle isoform; Ret 43type) that are generated by alternative splicing
in the 3'-region downstream of Tyr1062 (Fig. 1A
) (33, 34).
We developed the antibodies specific to each isoform and found that the
former two isoforms are expressed at high levels in medullary thyroid
carcinoma and neuroblastoma cell lines, whereas Ret 43type is a minor
product in them (data not shown). When the transforming activities of
three isoforms with the C634R mutation (designated Ret-MEN2A 9type,
43type, and 51type) were investigated, the activity of Ret-MEN2A 43type
was very low compared with those of Ret-MEN2A 9type and 51type (Table 2). However, Western blot analysis
revealed that the levels of tyrosine phosphorylation of three Ret
isoforms were comparable in the transfectants despite the different
transforming activities (Fig. 5).
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Figure 5. Expression and tyrosine phosphorylation of three
Ret isoforms. Total cell lysates (20 µg proteins) from the designated
cell lines were immunoblotted with anti-Ret (Ret-R5) or
antiphosphotyrosine antibodies. Expression of Ret 9type, Ret 43type,
and Ret 51type was confirmed by immunoblotting with the specific
antibodies developed against the carboxyl-terminal 19 amino acids of
each isoform (data not shown).
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We next examined the binding ability of each Ret isoform to Shc
in vivo or in vitro. The lysates from the
transfectants were immunoprecipitated with anti-Shc antibody, followed
by immunoblotting with anti-Ret, anti-Shc, anti-Grb2, or
antiphosphotyrosine antibody. As a result, Ret-MEN2A 9type and 51type
were efficiently coprecipitated with Shc, whereas coprecipitation of
Ret-MEN2A 43type was undetectable (Fig. 6A). Grb2 was also coprecipitated with
Shc from the lysates of the transfectants expressing Ret-MEN2A 9type or
51type (Fig. 6A). The level of tyrosine phosphorylation of Shc was
correlated with the transforming activity of each isoform (Fig. 6B).
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Figure 6. Coprecipitation of three Ret isoforms with Shc. A,
Cell lysates from the designated cells were immunoprecipitated with
anti-Shc antibody, followed by immunoblotting with anti-Ret (Ret-R5),
anti-Shc, or anti-Grb2 antibodies. B, The same immunoprecipitates were
immunoblotted with antiphosphotyrosine antibody. The 175- and 155-kDa
Ret proteins; the 66-, 52-, and 46-kDa Shc proteins, and the 24-kDa
Grb2 proteins are indicated.
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Using the GST fusion proteins, Ret-MEN2A 9type interacted with both the
Shc PTB and SH2 domains, whereas Ret-MEN2A 51type interacted only with
the Shc PTB domain (Fig. 7). This result
is consistent with the fact that Ret 9type contains the consensus
sequences for binding of both PTB and SH2 domains, and Ret 51type has
the consensus sequence for binding of the Shc PTB domain only (Fig. 1B). Although Ret 43type has the consensus sequence for binding of the
Shc PTB domain, the interaction of Ret-MEN2A 43type with the PTB domain
was undetectable (Fig. 7). These results suggested that the low
transforming activity of Ret-MEN2A 43type could be due to the reduction
of its binding ability to the Shc PTB domain.
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Figure 7. Interaction of three Ret isoforms with Shc PTB and
SH2 domains in vitro. Cell lysates from the designated
cells were incubated with GST-Shc (full-length), GST-Shc PTB, or
GST-Shc SH2 fusion proteins immobilized on glutathione-agarose beads.
Bound proteins were immunoblotted with anti-Ret antibody (Ret-R5).
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Discussion
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To date, the role of amino acids around phosphotyrosine in
the binding of Shc adaptor proteins has been investigated using several
receptor tyrosine kinases, such as epidermal growth factor receptor,
nerve growth factor receptor, and insulin receptor (7, 35, 36, 37, 38). These
studies demonstrated that Asn at the -3 position relative to
phosphotyrosine is important for binding of the Shc PTB domain in
vivo or in vitro. However, the role of amino acids at
-5 and -1 positions has not been well analyzed in vivo.
Our previous study demonstrated that Tyr1062 in Ret
represents an Shc binding site and is crucial for the transforming
activity of Ret with the MEN2A or MEN2B mutation (26, 30). Consistent
with this finding, the sequence (I-E-N-K-L) amino-terminal to
Tyr1062 in Ret matches the consensus sequence for the
binding of the Shc PTB domain. In the present study we introduced
mutations into amino acids at -5, -3, and -1 positions relative to
Tyr1062 and investigated their effects on the biological
activities of Ret. As a result, it turned out that all amino acid
substitutions at the -5, -3, and -1 positions significantly
decreased the transforming activity of the Ret-MEN2A mutant protein as
well as the binding affinity of Shc to it in vivo. The
reduction of Shc binding by these mutations was also confirmed by
in vitro binding assay using the GST-Shc fusion proteins,
suggesting that amino acids at the -5, -3, and -1 positions play a
role in Shc binding to Ret.
Mutations in the I-E-N-K-L sequence, however, did not completely
abolish the transforming activity of Ret-MEN2A or its ability to bind
to Shc as observed for Ret with the Y1062F mutation. This finding
suggested that other tyrosines present in the Ret intracellular domain
may represent minor binding sites for Shc that are responsible for the
low transforming activity of mutant Ret proteins. Alternatively, it is
possible that another signaling molecule recognizes tyrosine 1062 and
is crucial for their transforming activity. Recently, Durick et
al. (29) reported that Enigma binds to tyrosine 586 of Ret/ptc2, a
rearranged form of Ret identified in papillary thyroid carcinoma, that
corresponds to tyrosine 1062 of Ret-MEN2A. Both Shc and Enigma
interacted with the same site on Ret/ptc2 and were required for its
mitogenic signaling.
A variety of mutations affecting both the extracellular and
intracellular domains of Ret have been identified in Hirschsprung’s
disease (39). The mutations in the extracellular domain resulted in a
marked reduction of Ret cell surface expression, whereas most kinase
domain mutations appeared to impair its tyrosine kinase activity
(22, 23, 24, 25). Several Hirschsprung mutations, including the L1061P and
M1064T mutations, were also found in the carboxyl-terminal sequence of
Ret. Thus, it was interesting to investigate the mechanisms of Ret
dysfunction by these carboxyl-terminal mutations. Lorenzo et
al. (28) recently reported that the M1064T mutation specifically
causes a reduction in Shc PTB domain binding to Tyr1062in vitro as well as Ret-dependent Shc phosphorylation
in vivo. Interestingly, our present study also suggested
that the L1061P mutation could impair the intracellular signaling of
Ret by decreasing its binding affinity to the Shc PTB domain.
Differential splicing in the 3'-region downstream of
Tyr1062 generates three Ret isoforms: Ret 9, Ret 43, and
Ret 51 (33, 34). Ret 9 and Ret 51 isoforms are major products in human
neuroblastoma and medullary thyroid carcinoma cell lines (our
unpublished observation). The Ret 9 and Ret 51 isoforms with the MEN2A
mutation can efficiently transform NIH-3T3 cells and differentiate PC12
cells (40), whereas the biological activity of the Ret 43 isoform,
which is a minor product in human cell lines, has not been
investigated. The present study showed that the transforming activity
of Ret 43 isoform with the MEN2A mutation was very low. In addition, it
turned out that despite the presence of the consensus sequence for the
binding of the Shc PTB domain, Ret 43 isoform does not bind Shc with
high affinity. Thus, the low transforming activity of Ret 43 isoform
with the MEN2A mutation appeared to correlate to its low affinity to
Shc. Moreover, these findings suggested that the sequence
carboxyl-terminal to Tyr1062 in Ret may also influence the
transforming activity and the binding affinity to Shc and that Ret 43
isoform may not significantly contribute to the development of
medullary thyroid carcinoma and pheochromocytoma in MEN2A and MEN2B
patients.
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Acknowledgments
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We are grateful to K. Imaizumi, J. Aoki, K. Uchiyama, and M.
Kozuka for their technical assistance.
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Footnotes
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1 This work was supported in part by grants-in-aid for COE
research, scientific research, and cancer research from the Ministry of
Education, Science, Sports, and Culture of Japan and by a grant from
the Mitsubishi Foundation. 
Received March 15, 1999.
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Top
Abstract
Introduction
