| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, July 28, 1995; and in revised form, October 2, 1995) From the
Insulin and insulin-like growth factor (IGF-I) receptors are
heterotetrameric proteins consisting of two
The multiple physiological actions, including cell growth and
differentiation of the insulin-like growth factors (IGFs) ( Whereas
the function of the triple tyrosine cluster in the tyrosine kinase
domain of the insulin receptor has been well characterized (11, 12, 13) , much less is known about the
corresponding tyrosines of the IGF-I receptor. Single substitutions of
tyrosine 1131 or 1135 have relatively small effects on receptor and
endogenous substrate phosphorylation or on cell
proliferation(14, 15) . In contrast, substitution of
tyrosine 1136 apparently has an inhibitory effect on those
functions(14) . In addition, it has been demonstrated, at least
in the case of the insulin receptor, that in intact cells, bis
phosphorylation of the kinase domain at Tyr-1158 and either Tyr-1162 or
Tyr-1163 comprises 80% of phosphorylated
receptors(7, 16, 17, 18) . Thus, a
study involving the substitution of combinations of double tyrosines
may be more instructive. To further characterize the role of these
tyrosines in IGF-I receptor function, we have performed substitutions
of combinations of two tyrosines in the kinase domain of the IGF-I
receptor. We transfected NIH-3T3 cells to study receptor
autophosphorylation and post-receptor signaling pathways as well as
biological functions of the receptor including cell growth and
tumorigenicity.
Figure 1:
IGF-I stimulation of receptor
autophosphorylation in intact cells. Cells were stimulated with 100
ng/ml IGF-I for 1 min at 37 °C as described under
``Experimental Procedures.'' Similar amounts of receptors
were separated by 7.5% SDS-PAGE, transferred to nitrocellulose filters,
and blotted with a monoclonal anti-phosphotyrosine antibody (4G10). The
position of protein molecular weight standards as well as the
Figure 2:
IRS-1 signal transduction pathway.
Confluent monolayers of cells were made quiescent in serum-free medium
overnight and then stimulated with 100 ng/ml IGF-I for 1 min at 37
°C. Cells were lysed and IRS-1 immunoprecipitated as described
under ``Experimental Procedures.'' IRS-1 immunoprecipitates
were separated by 9% SDS-PAGE, transferred to a nitrocellulose filter,
and blotted with the appropriate antibodies. Panel A,
immunoblot with polyclonal anti-phosphotyrosine antibody RC20H. Panel B, immunoblot with monoclonal anti-PTP1D antibody. Panel C, immunoblot with monoclonal anti-Grb2 antibody. The
positions of protein molecular weight standards are indicated. WT, wild type.
To evaluate
Shc pathway activation, cells were stimulated with 100 ng/ml IGF-I for
5 min at 37 °C as described above, and Shc immunoprecipitates were
assayed for Shc phosphorylation and Grb2 association. Similar amounts
of Shc proteins were detected when the membranes were reblotted with a
polyclonal anti-Shc antibody (data not shown). A typical experiment is
shown in Fig. 3, A and B, respectively. IGF-I
stimulation of tyrosine phosphorylation of the 52- and 46-kDa isoforms
of Shc protein (Fig. 3A) was observed in wild-type
clones, whereas cells overexpressing the double tyrosine mutant
receptors presented very low levels of Shc phosphorylation. Shc-Grb2
association following IGF-I stimulation was similarly diminished in
mutant clones with respect to the wild-type clones (Fig. 3B). These results suggest that activation of
both the IRS-1 and Shc pathways by IGF-I are at least partially blocked
in cells overexpressing double tyrosine mutant receptors as compared
with cells overexpressing a similar number of wild-type receptors.
Figure 3:
Shc signal transduction pathway. Confluent
monolayers of cells were made quiescent in serum-free medium overnight
and then stimulated with 100 ng/ml IGF-I for 5 min at 37 °C. Cells
were lysed, and Shc proteins were immunoprecipitated as described under
``Experimental Procedures.'' Shc immunoprecipitates were
separated by 9% SDS-PAGE, transferred to a nitrocellulose filter, and
blotted with the appropriate antibodies. Panel A, immunoblot
with monoclonal anti-phosphotyrosine antibody RC20H. Panel B,
immunoblot with monoclonal anti-Grb2 antibody. The positions of protein
molecular weight standards are indicated. WT, wild
type.
Figure 4:
Cell
growth stimulation by IGF-I in culture. Cells were plated at 3
Figure 5:
Tumorigenicity of NIH-3T3 cells
transfected with the double tyrosine mutants and wild-type IGF-I
receptor. 1
The earliest post-binding event following the interaction of
insulin and IGF-I with their specific receptors is the
autophosphorylation of the triple tyrosine cluster within the
Although IGF-I and insulin receptors are structurally
similar, their in vivo biological actions are different. At
which level this divergence of function occurs is not yet defined.
Thus, analyses of the structural and functional aspects of the IGF-I
receptor is of considerable scientific interest. Studies using
mutational analyses of the IGF-I receptor are considerably less
complete than of the insulin receptor. We and others have reported
previously that substitution of the triple tyrosine cluster with
phenylalanine has similar effects as seen with the insulin receptor, i.e. essentially all of the functions of the IGF-I receptor
were abrogated(9, 10) . A single substitution of
tyrosine residue 1131 reduces autophosphorylation and receptor
internalization, whereas IRS-1 phosphorylation, thymidine
incorporation, and cell proliferation were unaffected(15) . Li et al.(14) showed that single substitutions of
tyrosines 1131 or 1135 did not affect mitogenicity and only slightly
reduced autophosphorylation. In contrast, they reported that
substitution of tyrosine 1136 abrogated autophosphorylation and cell
growth. In the present study, we show that substitutions of
tyrosines 1131 and 1135 reduced Of particular
interest in our study is the finding that all the cells expressing
double tyrosine substitutions failed to respond mitogenically to IGF-I
stimulation, and tumor formation was reduced compared to cells
overexpressing wild-type IGF-I receptors, despite the fact that
substitution of tyrosines 1131/1136 or 1135/1136 only slightly reduced
receptor autophosphorylation. These findings suggest that perhaps a
relatively high threshold of autophosphorylation is required to fully
activate the signaling pathways. The inability of all double mutant
receptors to mediate biological activities could be explained by the
absence of IGF-I-induced tyrosine phosphorylation of the two major
IGF-I signaling pathways, IRS-1 and Shc. IRS-1 is considered an adapter
protein between the insulin and the IGF-I receptors and the network of
their signaling
pathways(26, 27, 28, 29) . IRS-1 is
phosphorylated on multiple tyrosine residues upon receptor
stimulation(30) . This provides multiple sites of interaction
for proteins with SH2 (src-homology 2) domains(31) .
Several SH2-containing proteins have been shown to associate with
IRS-1: PI3-kinase(32, 33, 34) ,
Nck(35) ,
Grb2(36, 37, 38, 39, 40, 41) ,
and PTP1D(30, 39) . PTP1D is a tyrosine phosphatase
that binds to IRS-1 at tyrosine 1172; this binding provides a potential
mechanism for its activation(40) . Evidence suggests that PTP1D
is involved in stimulation of mitogenesis, and regulation of Ras and
mitogen-activated protein kinase activation (41, 42) .
Grb2 is thought to stimulate p21 In summary, we have demonstrated that the presence of both tyrosines
1131 and 1135 of the IGF-I receptor are necessary for full
IGF-I-stimulated autophosphorylation of the
Volume 270,
Number 49,
Issue of December 8, 1995 pp. 29176-29181
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
- and two
-subunits and members of the transmembrane tyrosine kinase
receptors. Specific ligand binding to the receptor triggers a cascade
of intracellular events, which begins with autophosphorylation of
several tyrosine residues of the -subunit of the receptor. The
triple cluster in the tyrosine kinase domain of the -subunit is
the earliest and major autophosphorylation site. Previous studies have
shown that substitutions of these three tyrosines by phenylalanines of
both insulin and IGF-I receptors practically abolish any activation of
cellular signaling pathways. We have studied the effect of double
tyrosine mutations on IGF-I-induced receptor autophosphorylation,
activation of Shc and IRS-1 pathways, and cell proliferation and
tumorigenicity. Substitution of tyrosines 1131/1135 blocks any
detectable autophosphorylation, whereas substitution of tyrosines
1131/1136 or 1135/1136 only reduces autophosphorylation levels in some
clones by 50%. Nevertheless, all the cells expressing IGF-I
receptors with double tyrosine substitutions demonstrated markedly
reduced signaling through Shc and IRS-1 pathways. In addition, they
were unable to respond to IGF-I-stimulated cell growth in culture, and
tumor formation in nude mice was abrogated. These data suggest that the
presence of tyrosine 1131 or 1135 essential for receptor
autophosphorylation, whereas the presence of each of these tyrosines is
necessary for a fully functional receptor.
)are mediated by the IGF-I receptor. While the IGF-I
receptor and the structurally related insulin receptor are members of
the type II receptor tyrosine kinase family, their in vivo biological functions are quite separate. Both the IGF-I and
insulin receptors are heterotetrameric proteins composed of two
extracellular -subunits and two membrane-spanning -subunits
linked by disulfide bonds(1, 2, 3) .
Sequences found in the -subunits of each receptor are important
for determining ligand specificity. The amino-terminal and
carboxyl-terminal portions of the subunit of the insulin receptor
are critical for high affinity insulin binding, while the cysteine-rich
domain of the IGF-I receptor determines high affinity IGF-I
binding(4, 5, 6) . Likewise, the
-subunits contain a number of structurally distinct domains
including the extracellular, transmembrane, juxtamembrane, tyrosine
kinase, and carboxyl-terminal regions. Binding of ligand to the
-subunit activates the tyrosine kinase activity of the
-subunit resulting in autophosphorylation on distinct tyrosine
residues. The triple tyrosine cluster within the kinase domain (1131,
1135, and 1136 tyrosines in the IGF-I receptor and the equivalent
residues in the insulin receptor; numbering system of Ullrich et
al.(2) ) is the earliest and major site of
autophosphorylation. Phosphorylation of these three tyrosine residues
is necessary for activation of the kinase toward other
substrates(7, 8) . When the triple tyrosine cluster is
substituted by phenylalanine residues, the receptor loses all
ligand-induced biological actions(9, 10) .
Materials
Restriction endonucleases
were purchased from New England Biolabs, Boehringer Mannheim, and Life
Technologies, Inc. Cell culture media and reagents were purchased from
Biofluids, Inc. (Rockville, MD) and Advanced Biotechnologies (Columbia,
MD). Insulin-free bovine serum albumin (fraction V) was obtained from
Armour (Kankakee, IL). Monoclonal antiphosphotyrosine antibody (clone
4G10) was purchased from Upstate Biotechnology, Inc. Recombinant
antiphosphotyrosine RC20H horseradish peroxidase-conjugated, polyclonal
anti-Shc, monoclonal anti-Grb2, and monoclonal anti-PTP1D antibodies
were purchased from Transduction Laboratories (Lexington, KY).
Recombinant human IGF-I, fetal bovine serum (FBS), monoiodinated I-IGF-I, horseradish peroxidase-conjugated anti-mouse
immunoglobulin, and the ECL detection kit were purchased from Amersham
Corp. Prestained high molecular weight protein standards and
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) reagent were purchased from Sigma. Protein A-Sepharose was
purchased from Pharmacia Biotech (Uppsala, Sweden). Siddle 1-2 antibody
was a gift from Ken Siddle (Cambridge, United Kingdom). Anti-IRS-1
polyclonal antibody was a gift from Jacylyn Pierce (NCI, National
Institutes of Health).
Construction of the Mutant IGF-I Receptor
DNA
The wild-type human IGF-I receptor expression vector
has been previously described(19) . Mutation of the human IGF-I
receptor cDNA at amino acid residues 1131, 1135, and 1136 (numbering
system is that of Ullrich et al.(2) ) was performed by in vitro site-directed mutagenesis using the Double Take
Mutagenesis Kit (Stratagene) and a pBluescript II plasmid containing an EcoRI-BamHI fragment of the human IGF-I receptor
(previously described by Kato et al.(19) ). The
tyrosines at positions 1131, 1135, and 1136 were designated as being
the first(1) , second(2) , or third (3) in the
triple tyrosine cluster. The following double tyrosine mutations were
generated: DYF12 (tyrosines 1131 and 1135 mutated to phenylalanines
1131 and 1135), DYF13 (tyrosines 1131 and 1136 mutated to
phenylalanines) and DYF23 (tyrosines 1135 and 1136 mutated to
phenylalanines). The sequence of the bridging primer was
5`-GCCGCCACCGCGGTGGAGCTCCAATTCGCC-3` (the SacI site used for
mutagenesis is underlined), and the sequence of the extension primer
was 5`-AGCTCCACCGCGGTGGCGGCCGCT-3`. The sequence of the mutagenic
primer to generate DYF12 was
5`-CTTTCCGGTAAAAGTCTGTTTCGAAGATATCTCGCGT-3`. The sequence of the
mutagenic primer to generate DYF13 was
5`-CTCCTTTCCGGAAATAGTCTGTCTCAAAGATATCTC-3`. The sequence of the
mutagenic primer to generate DYF23 was
5`-TGCCTCCTTTCCGGAAAAAGTCTGTCTCATA-3`. In the mutagenic primers,
codons mutated from tyrosine to phenylalanine are underlined.
Nucleotide sequences mutated to generate a restriction enzyme site are
presented in bold type. A new SfuI site was introduced in the
cDNA for DYF12. A new AccIII site was introduced in the cDNA
for both DYF13 and DYF23. The cDNA sequences of all the mutations were
confirmed by dideoxy sequencing. The mutated cDNAs in pBluescript II
were excised with SalI and NotI and cloned into a
bovine papilloma virus-derived mammalian expression vector (pBPV;
Pharmacia Biotech Inc.) that had been linearized with XhoI and NotI.Cell Culture and Transfection
All NIH-3T3
cell lines were cultured in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml
penicillin, 100 µg/ml streptomycin, and 2 mM glutamine in
a humidified atmosphere of 95% air and 5% CO
at 37 °C.
NIH-3T3 cells were cotransfected with 20 µg of wild-type or mutant
expression vector or insert-less pBPV plus 1 µg of pMCINeo
(Clontech) in Lipofectamine reagent (Life Technologies, Inc.).
Selection was carried out as described previously(19) . Clones
overexpressing IGF-I receptors were selected based on results of IGF-I
binding assays as described previously(19) . Stably transfected
cells were maintained in the media described above supplemented with
500 µg/ml G418 (Geneticin, Life Technologies, Inc.). Serum-free
medium containing 0.1% bovine serum albumin, 20 mM Hepes, pH
7.5, and antibiotics was used in phosphorylation assays of the receptor
and cellular substrates.Receptor Autophosphorylation
Confluent
cells in 100-mm plates were serum-starved overnight and then incubated
either with or without IGF-I (100 ng/ml) for 1 min at 37 °C. The
cells were washed rapidly with ice-cold phosphate-buffered saline and
lysed in the presence of 50 nM Hepes, pH 7.9, 100 mM NaCl, 10 mM EDTA, 1% Triton X-100, 4 mM sodium
pyrophosphate, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 mM sodium fluoride.
Cell lysates were cleared by centrifugation. Protein content was
determined by the method of Bradford using a protein assay kit
(Bio-Rad). Equal amounts of protein (up to 40 µg) were reduced by
-mercaptoethanol and fractionated by 7.5% SDS-PAGE. Resolved
proteins were electrophoretically transferred to nitrocellulose
membrane (Hybond, ECL, Amersham). The amount of IGF-I receptor present
was determined by immunoblotting with monoclonal anti-IGF-I receptor
-subunit antibody (Siddle 1-2) at a 1:500 dilution at 4 °C
overnight; this antibody was detected with horseradish
peroxidase-conjugated anti-mouse immunoglobulin (1:5000 dilution). The
blots were then developed with the ECL system. Based on these
immunoblots, equal amounts of receptors were fractionated by SDS-PAGE
as described above. Blots for tyrosine phosphorylation were
immunoblotted with monoclonal antiphosphotyrosine antibody (clone 4G10)
(1:1000 dilution) and detected with horseradish peroxidase-conjugated
anti-mouse immunoglobulin (1:5000 dilution) using the ECL system. The
level of phosphorylation of the -subunits was quantitated by
digitalizing the signal from the x-ray film and analyzing the signal
using NIH image version 1.55 software and calculated according to
receptor density assessed using the Siddle 1-2 antibody(15) .IRS-1 and Shc
Immunoprecipitation
Confluent cells in 100-mm plates were
serum-starved overnight and then incubated either with or without IGF-I
(100 ng/ml) at 37 °C. Cleared cell lysates were prepared as
described above. For immunoprecipitation, 600 µg of protein were
incubated either with 3 µg of anti-Shc antibody or 3 µg of
anti-IRS-1 antibody at 4 °C overnight. Then, 50 µl of 10%
protein A-Sepharose in Tris-HCl buffer, pH 7, was added and incubated
at 4 °C for 3 h. The precipitates were washed three times with
immunoprecipitation buffer (20 mM Tris-HCl, pH 7.4, 300 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.4 mM
Na
VO
, 0.4 mM phenylmethylsulfonyl
fluoride, 1% Triton, 1% Nonident P-40) and fractionated by 9% SDS-PAGE.
Resolved proteins were electrophoretically transferred to
nitrocellulose membrane. Blots were incubated with anti-phosphotyrosine
RC20H antibody (1:2500 dilution) for phosphorylation studies or with
anti-Grb-2 (1:1000 dilution) or anti-PTP1D (1:1000 dilution) for
coimmunoprecipitation studies. Anti-Grb-2 and anti-PTP1D were detected
by horseradish peroxidase-conjugated anti-mouse immunoglobulin (1:5000
dilution).MTT Cellular Proliferation Assay
3
10 cells were plated in each well of 96 multi-well
plates and allowed to recover overnight in DMEM plus 10% FBS.
Thereafter, cell growth was continued in either 1% FBS or 1% FBS
supplemented with 100 ng/ml IGF-I for 7 days. Media were replenished at
72 h of growth. The cells were processed at varying time points as
described previously(20) , and the cellular number was
calculated daily.Tumorigenicity Assay
The ability of
various cell lines to form tumors was determined by injecting 1
10
cells subcutaneously in the lower dorsal region of nude
mice. Mice were housed in accordance with the NIH Guide for the Care
and Use of Laboratory Animals. After 4 weeks, mice were examined weekly
to monitor tumor formation, and measurements were taken by using a
caliber. Tumor volume was calculated by the following formula: length
width/2 (V = a b/2).Statistics
Statistical significance
between groups was tested using the Student's t test.
Expression of Wild-type and Mutant
Receptors
Two separate clones overexpressing each mutant or
wild-type IGF-I receptor were used in this study. DYF12 designates
substitution of the tyrosine residues with phenylalanine at positions
1131 and 1135. Substitution of tyrosine residues with phenylalanine at
positions 1131 and 1136 is designated as DYF13, and substitution of
tyrosine residues at positions 1135 and 1136 is designated as DYF23.
WT43 and WT50 are clones overexpressing wild-type human IGF-I
receptor(15) . pNeo is a clone cotransfected with a
neomycin-resistant plasmid and the pBPV vector (15) . All
clones overexpressing IGF-I receptor had similar numbers of cell
surface IGF-I receptors as determined by Scatchard analysis (Table 1)(21) .
Receptor Autophosphorylation
One of the
first events after IGF-I binding to the IGF-I receptor is the
autophosphorylation of tyrosine residues of the -subunit of the
receptor. The triple tyrosine cluster in the tyrosine kinase domain is
considered to be the major autophosphorylation site of the IGF-I
receptor(10) . To study the autophosphorylation capability of
the mutant receptors, cells were stimulated with 100 ng/ml IGF-I for 1
min at 37 °C. As shown in Fig. 1, IGF-I-induced receptor
autophosphorylation is dramatic in cells overexpressing wild-type
receptors. In contrast, cells overexpressing a similar number of
receptors in which tyrosines 1131 and 1135 have been substituted (DYF12
clones 4 and 10) have undetectable levels of autophosphorylation. Cells
overexpressing receptors with tyrosines 1131 and 1136 substitutions
(DYF13 clones 33 and 42) or 1135 and 1136 (DYF23 clones 1 and 46)
demonstrated clonal variation. Clones 42 (DYF 13) and 46 (DYF 23)
showed no significant reduction in autophosphorylation compared with
cells expressing wild-type receptors. However, clones 33 (DYF 13) and 1
(DYF 23) demonstrated less than a 50% reduction in autophosphorylation
levels. These results suggest a differential role for these three
tyrosines in the IGF-I-induced autophosphorylation of the receptor. The
presence of either tyrosine 1131 or 1135 is critically important to
have detectable levels of autophosphorylation, whereas the presence of
tyrosine 1136 is not critical for autophosphorylation.
-subunit of the IGF-I receptor are indicated. The results shown
are representative of at least three individual experiments. WT, wild type.
Phosphorylation of Endogenous
Substrates
The two major signal transduction pathways
following IGF-I receptor stimulation characterized thus far are those
mediated by IRS-1 and Shc. We therefore studied the activation of IRS-1
and Shc pathways by IGF-I in wild-type and double tyrosine mutants. To
evaluate IRS-1 pathway activation, cells were stimulated with 100 ng/ml
IGF-I for 1 min at 37 °C, and IRS-1 immunoprecipitates were assayed
for tyrosine phosphorylation of IRS-1 and Grb2 and PTP1D association.
Stripping and reblotting the membranes with a polyclonal anti-IRS-1
antibody confirmed that similar amounts of IRS-1 protein were
immunoprecipitated in all samples (data not shown). A typical result of
these experiments is presented in Fig. 2. Panel A shows
IRS-1 phosphorylation immunoblotted with a phosphotyrosine antibody
RC20H. The ability of IGF-I to stimulate IRS-1 tyrosine phosphorylation
was reduced in cells expressing the double mutant receptors as compared
to wild-type clones. In addition, the IGF-I-stimulated association of
PTP1D and Grb2 with IRS-1 occurred to a much lower extent in double
tyrosine mutants when compared with stimulated levels in wild-type
clones (panels B and C, respectively). This effect
was most dramatic in studies of IRS-1/PTP1D association.
In Vitro Cell Growth
To determine whether
the differential receptor autophosphorylation levels in IGF-I receptor
double tyrosine mutants resulted in alterations in IGF-I-induced
biological effects, we studied the growth rate of both wild-type and
mutant cell lines in culture using the MTT assay. Cells were cultured
either in DMEM with 1% FBS or DMEM plus 1% FBS supplemented with 100
ng/ml IGF-I. The results of a typical assay are shown in Fig. 4A. The mean and standard deviations of three
separate experiments are plotted for the 120-h time point in Fig. 4B. Both wild-type and mutant clones failed to
grow in DMEM with 1% FBS (data not shown). In contrast, wild-type
clones grew well when stimulated with 100 ng/ml IGF-I. All mutant
clones demonstrated a flat growth response to IGF-I and were not
different than the pNeo clone (p > 0.05), whereas their
growth response was significantly less than the wild-type clones (p < 0.01).
10 cells per well in 96-well plates in DMEM with 10% FBS.
After 24 h, medium was changed to DMEM with 1% FBS and supplemented
with 100 ng/ml IGF-I. All assays were carried out in triplicate. Panel A shows the results representative of three individual
experiments. Panel B shows the mean of three independent
experiments ± S.D., and the data are expressed as percentage of
WT43 cell number at 120 h.
Tumor Formation
Overexpression of
wild-type IGF-I receptors in NIH-3T3 cells confers
tumorigenicity(22) . We therefore tested the double tyrosine
mutants for tumor formation in nude mice. 1 10 cells of each clone were injected per mouse, and tumor
measurements were performed as described under ``Experimental
Procedures.'' Cells overexpressing wild-type receptors typically
started to form measurable tumors five and a half weeks after injection (Fig. 5). The size of these tumors increased progressively until
the animals died or had to be sacrificed. In contrast, none of the
cells expressing the double tyrosine mutants formed large tumors. Only
the DYF23 (no. 1) clone gave rise to some tumors that were very small
in comparison to the wild-type clones.
10 cells were injected subcutaneously
in nude mice. The animals were examined for tumor formation on the
dorsal region, and tumor volume was calculated as described under
``Experimental Procedures.'' Each point represents the mean
of tumor volume measured in a group of four (DYF12(10) and DYF13(42)),
five (DYF12(4), DYF12(33), DYF23(1), DYF23(46), and WT43), or six
(WT50) mice.
-subunit tyrosine kinase domain(7, 8) .
Mutational analysis of the insulin receptor tyrosine kinase domain has
provided interesting, and at the same time, controversial information
about the regulatory role of these three tyrosines in receptor kinase
activity. The most dramatic effect has been shown with insulin
receptors where these three tyrosine residues (tyrosines 1158, 1162,
and 1163) have been mutated to phenylalanine. Insulin-stimulated
autophosphorylation and cell signaling by these mutant receptors are
impaired(13, 23) . Single substitutions of any one of
these tyrosine residues with phenylalanine (i.e. Y1158F,
Y1162F, or Y1163F) may reduce in vitro autophosphorylation of
the receptor -subunit on the remaining tyrosines and reduce
tyrosine kinase activity, although, in contrast, Zhang and Roth (6) reported that the Y1158F mutation had no effect on
autophosphorylation or on insulin-stimulated exogenous tyrosine kinase
activity. Data on thymidine incorporation is also controversial. In
general, however, substitutions of any single tyrosine have only slight
effects on insulin-stimulated thymidine
incorporation(13, 23) . A more substantial reduction
in insulin-induced tyrosine kinase activity was observed when double
tyrosine substitutions were studied(23) . Generally, the triple
tyrosine substitution results in even more reduction in thymidine
incorporation, although HTC cells expressing the triple tyrosine mutant
receptor exhibit normal thymidine incorporation(24) . Despite
these discrepancies, the data overall are consistent with the idea that
autophosphorylation of tyrosines 1158, 1162, and 1163 is essential in
autoactivation of the insulin receptor tyrosine kinase toward other
substrates.-subunit autophosphorylation to
levels below the level of detectability by our antibody, suggesting
that the presence of either tyrosine 1131 or 1135 is required for full
-subunit autophosphorylation. Substitutions of 1131/1136 and
1135/1136 affect autophosphorylation to a lesser degree, suggesting
that tyrosine 1136 is not crucial for IGF-I-stimulated
autophosphorylation. The crystal structure of the tyrosine kinase
domain of the human insulin receptor has recently been
characterized(25) . Apparently, the phosphorylation of tyrosine
1162 (the second of the three tyrosines) is a key step in the receptor
kinase activation. The apo structure shows that the hydroxyl group of
tyrosine 1162 is bound in the active site autoinhibiting the tyrosine
kinase of the receptor. This finding suggests that phosphorylation of
this tyrosine would disengage it from the active site. Our results, in
the present study, suggest an important role not only for tyrosine 1135
(equivalent to tyrosine 1162 in insulin receptor) but also for tyrosine
1131 in autophosphorylation of the IGF-I receptor. activity through a
monovalent interaction with p21
GDP-GTP exchange factor,
mSOS, resulting in stimulation of a serine/threonine phosphorylation
cascade leading to activation of the mitogen-activated protein kinase
pathway implicated in cell growth and metabolism. Thus, the binding of
the Grb2-mSOS complex to IRS-1 after insulin and IGF-I stimulation
links insulin and IGF-I receptor tyrosine kinases and p21
signaling pathways. An alternative and possible redundant pathway
that links insulin and IGF-I signaling with p21
activation is through
Shc(43, 44, 45, 46, 47) .
Shc is tyrosine phosphorylated upon insulin and IGF-I
stimulation(44, 45, 46) . The phosphorylation
of Shc provides a binding site for Grb2, resulting in the formation of
the Shc-Grb2-mSOS complex and probably leading to activation of the
mitogen-activated protein kinase pathway. Therefore, diminished
stimulation of the IRS-1/PTP1D and IRS-1/Grb2 as well as Shc-Grb2
pathways by IGF-I in our cells expressing receptors with double
tyrosine substitutions is associated with decreased IGF-I-stimulated
cell growth response of cells expressing these receptors. We cannot
exclude the possible involvement of other signaling pathways, such as
Crk, that are also involved in IGF-I-induced signaling(48) .
-subunit. However, the
absence of any of the tyrosine residues of the triple tyrosine cluster
significantly reduced signal transduction through IRS-1 and Shc
activation, thus abrogating IGF-I-stimulated cell growth and IGF-I
receptor-mediated tumor formation of transfected fibroblasts.
)
We thank Dr. Hisanori Kato for advice on creating the
mutations and Drs. Carol Renfew Haft and Dana Beitner-Johnson for
critical review of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Li, Z. He, S. Ermakova, D. Zheng, F. Tang, Y.-Y. Cho, F. Zhu, W.-Y. Ma, Y. Sham, E. A. Rogozin, et al. Direct Inhibition of Insulin-Like Growth Factor-I Receptor Kinase Activity by (-)-Epigallocatechin-3-Gallate Regulates Cell Transformation Cancer Epidemiol. Biomarkers Prev., March 1, 2007; 16(3): 598 - 605. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Li and W. T. Miller Role of the Activation Loop Tyrosines in Regulation of the Insulin-like Growth Factor I Receptor-tyrosine Kinase J. Biol. Chem., August 18, 2006; 281(33): 23785 - 23791. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Fowlkes, D. M. Serra, R. C. Bunn, K. M. Thrailkill, J. J. Enghild, and H. Nagase Regulation of Insulin-Like Growth Factor (IGF)-I Action by Matrix Metalloproteinase-3 Involves Selective Disruption of IGF-I/IGF-Binding Protein-3 Complexes Endocrinology, February 1, 2004; 145(2): 620 - 626. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Tajinda, J. Carroll, and C. T. Roberts Jr. Regulation of Insulin-Like Growth Factor I Receptor Promoter Activity by Wild-Type and Mutant Versions of the WT1 Tumor Suppressor Endocrinology, October 1, 1999; 140(10): 4713 - 4724. [Abstract] [Full Text]
|
|
|
|
 |
|
 W. Li, Y.-X. Jiang, J. Zhang, L. Soon, L. Flechner, V. Kapoor, J. H. Pierce, and L.-H. Wang Protein Kinase C-delta Is an Important Signaling Molecule in Insulin-Like Growth Factor I Receptor-Mediated Cell Transformation Mol. Cell. Biol., October 1, 1998; 18(10): 5888 - 5898. [Abstract] [Full Text]
|
|
|
|
|
|
R. Foncea, M. Andersson, A. Ketterman, V. Blakesley, M. Sapag-Hagar, P. H. Sugden, D. LeRoith, and S. Lavandero Insulin-like Growth Factor-I Rapidly Activates Multiple Signal Transduction Pathways in Cultured Rat Cardiac Myocytes J. Biol. Chem., August 1, 1997; 272(31): 19115 - 19124. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Esposito, V. A. Blakesley, A. P. Koval, A. G. Scrimgeour, and D. LeRoith Tyrosine Residues in the C-Terminal Domain of the Insulin-Like Growth Factor-I Receptor Mediate Mitogenic and Tumorigenic Signals Endocrinology, July 1, 1997; 138(7): 2979 - 2988. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. G. Scrimgeour, V. A. Blakesley, B. S. Stannard, and D. LeRoith Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase Pathways Are Not Sufficient for Insulin-Like Growth Factor I-Induced Mitogenesis and Tumorigenesis Endocrinology, June 1, 1997; 138(6): 2552 - 2558. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Cunningham, R. M. Stephens, D. R. Kaplan, and L. A. Greene Autophosphorylation of Activation Loop Tyrosines Regulates Signaling by the TRK Nerve Growth Factor Receptor J. Biol. Chem., April 18, 1997; 272(16): 10957 - 10967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Parrizas, A. Gazit, A. Levitzki, E. Wertheimer, and D. LeRoith Specific Inhibition of Insulin-Like Growth Factor-1 and Insulin Receptor Tyrosine Kinase Activity and Biological Function by Tyrphostins Endocrinology, April 1, 1997; 138(4): 1427 - 1433. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. R. D'Mello, K. Borodezt, and S. P. Soltoff Insulin-Like Growth Factor and Potassium Depolarization Maintain Neuronal Survival by Distinct Pathways: Possible Involvement of PI 3-Kinase in IGF-1 Signaling J. Neurosci., March 1, 1997; 17(5): 1548 - 1560. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Ling, D. Templeton, and H.-J. Kung Identification of the Major Autophosphorylation Sites of Nyk/Mer, an NCAM-related Receptor Tyrosine Kinase J. Biol. Chem., August 2, 1996; 271(31): 18355 - 18362. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. F. Kuemmerle and K. S. Murthy Coupling of the Insulin-like Growth Factor-I Receptor Tyrosine Kinase to Gi2 in Human Intestinal Smooth Muscle. Gbeta gamma -DEPENDENT MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION AND GROWTH J. Biol. Chem., March 2, 2001; 276(10): 7187 - 7194. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Yam, T. Hyun, and W. Li Characterization of Insulin-like Growth Factor I (IGF-I) Receptor Mutants for Their Effects on IGF-I- and Interleukin 4-mediated DNA Synthesis of 32D Cells J. Biol. Chem., June 22, 2001; 276(26): 24409 - 24413. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Brodt, L. Fallavollita, A.-M. Khatib, A. A. Samani, and D. Zhang Cooperative Regulation of the Invasive and Metastatic Phenotypes by Different Domains of the Type I Insulin-like Growth Factor Receptor beta Subunit J. Biol. Chem., August 31, 2001; 276(36): 33608 - 33615. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
