From the
Phosphorylation of two newly identified epidermal growth factor
(EGF) receptor substrates, eps8 and eps15, which do
not possess Src homology (SH2) domains, was investigated using EGF
receptor mutants of the autophosphorylation sites and deletion mutants
of the carboxyl-terminal region. Two mutants, F5, in which all five
tyrosine autophosphorylation sites substituted by phenylalanine, and Dc
123F, in which four tyrosines were removed by deletion and the fifth
(Tyr-992) was mutated into phenylalanine, phosphorylated eps8 and eps15 as efficiently as the wild-type receptor. In
contrast, SH2-containing substrates, phospholipase C
The receptor for epidermal growth factor (EGF-R),
In some instances, association of
an SH2-containing substrate (e.g. PLC
Recently, novel non-SH2-containing substrates of
the EGF receptor have been identified (25) and termed EGF
receptor pathway substrates (eps) clone 8(26) , clone
15(27) , and ezrin(28) . Overexpression of eps15 by itself is sufficient to induce transformation of NIH
3T3(27) , and overexpression of eps8 potentiates the
mitogenic activity of the EGF receptor(26) , suggesting that
both proteins are involved in receptor mitogenic pathway. These
substrates do not possess typical SH2 motifs, and only eps8 has been shown to be a direct substrate of activated EGF receptor
by in vitro analysis with purified receptor (26). However, eps15 seems to be specific to the EGF receptor pathway,
because its phosphorylation is not induced by the ErbB-2
kinase(27) , whereas eps8 and ezrin are phosphorylated
by several growth factor receptors(26, 27, 34) .
Because eps8 and eps15 do not possess SH2 domains,
it is important to establish how they are phosphorylated by the EGF
receptor and what region in the receptor is important to allow their
phosphorylation. In this study we have examined the requirement of the
receptor COOH terminus and autophosphorylation sites for eps8 and eps15 phosphorylation.
NIH 3T3
cells, which contain about 3,000 receptors/cell, were used for
transfections. Transfections, G418 selection, foci formations, and
growth in agar were performed as described previously(5) . As
determined by
To determine whether tyrosine phosphorylation of the EGF
receptor is necessary for phosphorylation of eps8 and eps15 proteins, NIH 3T3 cells expressing wild-type or mutant
receptors were used. Fig. 1A schematically represents
the EGF receptor mutants used in this study. In the F5 mutant, all five
autophosphorylation sites were mutated into phenylalanine, whereas in
the Dc 123F, four autophosphorylation sites were removed by deletion,
and the fifth site, Tyr-992, was mutated to
phenylalanine(20, 21) . In addition, we used mutants
with progressive deletions of the COOH terminus, creating receptors
lacking the final 123, 165, 196, and 214 COOH-terminal amino
acids(20, 21) . Clones of NIH 3T3 cells expressing EGF
receptor mutants were chosen to express comparable levels of receptor,
as shown in Fig. 1B and confirmed by
The behavior of eps8 phosphorylation resembles that of two other EGF receptor SH2
substrates, GAP (21, 22) and
SCH(22, 23) , which can be efficiently phosphorylated by
the Dc 214 mutant, although they are unable to form an association
complex with the mutant receptor. In contrast to GAP and SHC, however,
Dc 214 and Dc 196 phosphorylated eps8 to a much greater extent
than wild-type receptor, possibly because of the reduced interaction
and phosphorylation of some of the other SH2-containing substrates,
like PLC
Co-immunoprecipitation experiments,
performed by immunoprecipitation with EGF-R antibodies and Western
blotting with eps8 antibodies, indicated that, although very
little association could be detected with the wild-type receptor, the
Dc 196 and Dc 214 receptor mutants were able to associate directly with eps8, mostly with the 68-kDa form, (Fig. 4). In
addition, eps8 associated constitutively with the Dc 196 and
Dc 214 mutant, whereas, under the same conditions, association with the
wild-type receptor could be detected only in the presence of EGF.
The phosphorylation properties of eps15 were similar to those of PLC
In conclusion, the EGF receptor COOH-terminal tail has
multiple complex functions that can be mapped to different regions. The
extreme COOH terminus with the autophosphorylation sites positively
affects receptor biological activity and binding to SH2 substrates; the
upstream region contains the EGF-R internalization
domains(8, 20, 36) . Finally, a small
intermediate region of 30 amino acids negatively influences EGF-R
biological and transforming ability and affects phosphorylation of the
two newly identified substrates eps8 and eps15.
Equal amounts of cell lysates (3 mg of
proteins) were immunoprecipitated with EGF-R antibodies. Aliquots of
the immunoprecipitates were washed and incubated with 100 nM EGF for 30 min at 20 °C and then incubated with 10 µM [
We thank Dr. Graham Carpenter (Department of
Biochemistry, Vanderbilt University) for many helpful suggestions, for
careful revision of the manuscript, and for providing anti-PLC
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, the
GTPase-activating protein of Ras, the p85 subunit of
phosphatidylinositol 3 kinase, and the Src and collagen homology
protein, are not phosphorylated by the F5 and Dc 123F mutants. A longer
EGF receptor deletion mutant, Dc 214, lacking all five
autophosphorylation sites, was unable to phosphorylate eps15 but phosphorylated eps8 13-fold more than the wild-type
receptor. To determine the EGF receptor region important for
phosphorylation of eps8 and eps15, progressive
deletion mutants lacking the final 123, 165, 196, and 214 COOH-terminal
residues were used. eps8 phosphorylation was progressively
increased in Dc 165, Dc 196, and Dc 214 EGF receptor mutants,
indicating that removal of the final 214 COOH-terminal residues
increases the phosphorylation of this substrate by the EGF receptor. In
contrast, eps15 was phosphorylated by Dc 123 and Dc 165 EGF
receptor mutants but not by Dc 196 and Dc 214 mutants. This indicates
that a region of 30 residues located between Dc 165 and Dc 196 is
essential for eps15 phosphorylation. This is the first
demonstration of structural requirements in the EGF receptor COOH
terminus for efficient phosphorylation of non-SH2-containing
substrates. In addition, enhanced eps8 phosphorylation
correlates well with the increased transforming potential of EGF
receptor deletion mutants Dc 196 and Dc 214, suggesting that this
substrate may be involved in mitogenic signaling.
(
)a 175-kDa transmembrane glycoprotein, is a member of
the protein-tyrosine kinase family(1, 2) . EGF binding
triggers the activation of receptor tyrosine kinase activity, which is
essential to induce all responses to EGF(3, 4) .
Receptor tyrosine kinase activity leads to autophosphorylation and to
tyrosine phosphorylation of specific cellular
substrates(1, 2) . Autophosphorylation regulates the
biological activity of the EGF-R by influencing receptor kinase
activity (5, 6) and, by creating binding sites for
substrates. A number of these substrates, such as phospholipase C
(PLC
), GTPase-activating protein of Ras (GAP), the regulatory
subunit of phosphatidylinositol 3 kinase (p85), Src and collagen
homology protein (SHC) (for review see Refs. 1 and 7), phosphotyrosine
phosphatase Syp (9) and D1(10) , contain sequence motifs
called Src homology (SH2) domains.
) with activated
growth factor receptors is considered essential for tyrosine
phosphorylation and activation(7) . Mutation of single
autophosphorylation sites in the platelet-derived growth
factor(11, 12, 13, 14) , the
colony-stimulating factor(15) , the fibroblast growth
factor(16, 17) , and the nerve growth factor(18, 19) receptors prevents association and tyrosine phosphorylation
of the various substrates. In these examples, specificity depends on
the sequence motifs surrounding each autophosphorylation site and on
the structure of individual SH2 domain. Data with the EGF receptor,
however, indicate that autophosphorylation sites can compensate for
each other and do not stringently define association motifs for SH2
substrates(20, 21, 22, 23, 24) .
The COOH terminus of the EGF-R contains all five autophosphorylation
sites (Tyr-1173, Tyr-1148, Tyr-1086, Tyr-1068, and Tyr-992), and
substitution or removal of all five is required to completely abolish
association and/or phosphorylation of PLC
, GAP, and the p85
subunit of phosphatidylinositol 3
kinase(20, 21, 22) . The requirement for SHC and
Grb-2 interaction is more stringent because mutation of both Tyr-1173
and Tyr-1148 reduces the binding of SHC to very low
levels(22, 23) . Mutation of Tyr-1068, Tyr-1086, and
Tyr-1148 abolishes interaction with Grb-2(24) .
(
)
Materials
EGF was from Promega, and I-EGF and
I-protein A were from Amersham
Corp. Aprotinin, phenylmethylsulfonyl fluoride, and polylysine were
from Sigma. Protein G-Sepharose was from Pharmacia. Nitrocellulose
membranes were from Schleicher & Schuell. G418, transferrin, cell
culture medium, and serum were from Life Technologies, Inc. Monoclonal
antibodies against phosphotyrosine and the extracellular domain of the
EGF-R were from Upstate Biotechnology Incorporated. Polyclonal
antibodies against eps8, eps15, and PLC
were
kindly provided by Dr. Francesca Fazioli (Dipartimento di Ricerca
Biologica e Tecnologica, Milano) and Graham Carpenter (Vanderbilt
University, Nashville, TN), respectively, and were previously
described(21, 26, 27) .
EGF Receptor Mutants and Cell Culture
The human
EGF-R mutant F5 was obtained by site-directed mutagenesis of single
tyrosine residues with phenylalanine as described
previously(21) . Dc 123F was obtained by deletion of the last
123 amino acids and site-directed mutagenesis of Tyr-992 with
phenylalanine(20) , and Dc 214 was obtained by deletion of the
final 214 residues(20) . Mutants Dc 196 and Dc 165 were obtained
by deletion of the last 196 and 165 carboxyl-terminal residues by
site-directed mutagenesis of residue 992 or 1021, respectively, into
stop codons using the following oligonucleotides:
5`-GATGCCGACGAGTAGTACTTCCCACAGCAG-3` and
5`-CACCCCAGCAACAATATAGACCGTGTCCTTGCAT-3`. Mutagenesis was performed in
M13mp18 encoding the AccII-HincI EGF-R cDNA
fragment(3113-3625). A single-stranded template was prepared, and
mutagenesis was performed as described by Taylor et al.(32) and confirmed by dideoxy sequencing(33) . The
mutated fragments were cloned back into the pMMTV-EGF-R vector, and the
mutated EGF-R cDNAs (SacII-XhoI) were subcloned into
the pCO11 vector (5) to produce pDc 165 and pDc 196.
I-EGF binding and Scatchard analysis, most
mutant receptors were expressed at 3-5
10
receptors/cell, whereas F5 mutant had about 2
10
receptor/cell(5) . As control, a NIH 3T3 line (Cl 17)
expressing 4
10
human wild-type EGF-R/cell was
used(29) . Cells were maintained in Dulbecco's modified
Eagle's medium (DMEM) containing 10% newborn calf serum with
penicillin, streptomycin, and glutamine.
Growth Factor Treatment and Cell Lysate
Preparation
Transfected cells were grown to about 90% confluence
in 150-mm polylysine-coated dishes in DMEM containing 10% newborn calf
serum. Subsequently cells were incubated for 16 h in DMEM containing
0.5% newborn calf serum, 5 µg/ml transferrin, and 10M sodium selenite. Experiments were initiated with or
without 100 ng/ml EGF for 60 min at 4 °C in DMEM supplemented with
20 mM Hepes, pH. 7.4, and 0.1% bovine serum albumin. The
capacity of the EGF-R to autophosphorylate, phosphorylate, and
associate with cellular substrates is not altered at 4
°C(21, 22) , and preliminary experiments showed that
the maximal level of phosphorylation was achieved under these
conditions. Identical results were obtained when lysates were prepared
after cell incubation with EGF at 37 °C for 10 min. Lysates were
prepared by washing the monolayers with ice-cold phosphate-buffered
saline and scraping the cells in ice-cold lysis buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 20 µM Na
pyrophosphate, 10 mM orthovanadate, 4 µM phenylmethylsulfonyl fluoride, and 1 mg/ml aprotinin). Lysates
were centrifuged at 14,000
g for 15 min at 4° C.
Total proteins were measured by the Bio-Rad method.
Immunoprecipitation and Western Blot
Analysis
Total cellular lysates (3.5 mg of proteins) were
immunoprecipitated with agarose-conjugated anti-Tyr(P) or with protein
G-Sepharose coupled with eps8 and eps15 antibodies.
The immunoprecipitates were washed five times with HNTG buffer (20
mM Hepes, pH 7.4, 150 mM NaCl, 10% glycerol, and 0.1%
Triton X-100) and boiled in Laemmli buffer. Samples were run on
SDS-polyacrylamide gels and transferred to nitrocellulose membranes.
Membranes were then incubated for 2 h with primary antibodies
(anti-eps8, anti-eps15, or anti-Tyr(P) Ab),
extensively washed and incubated with 0.2 µCi/ml I-protein A, washed extensively, and exposed for
autoradiography. Specific bands were quantitated using a PhosphorImager
(Molecular Dynamics). Quantitation of phosphotyrosine recovered from eps8 and eps15 was performed as described
previously(21) . To examine tyrosine phosphorylation of total
cellular proteins in response to EGF, an aliquot of cell lysates
(50-100 µg) was mixed with 2
Laemmli buffer, loaded
on a 7.5% SDS-polyacrylamide gel, transferred to nitrocellulose
membrane, and blotted with anti-Tyr(P) Ab.
In Vitro Kinase Activity
The EGF receptor was
immunoprecipitated from cellular extracts, as described above.
Immunoprecipitated receptors were incubated with 100 ng/ml EGF for 10
min at room temperature and then incubated for 2 min at 4° C with
15 mM MgCl, 20 mM MnCl
, 10
µCi of [
-
P]ATP and various
concentrations of a dodecapeptide corresponding to the sequence
surrounding Tyr-1173, ranging from 0.0065 to 0.65 µM or
various concentrations of ATP from 0.003 to 100 µM. The
peptide NH
-AENAEYLRVAPQ-COOH was kindly provided by Dr.
Ettore Appella (Laboratory of Cellular Biology, NIH). Reactions were
terminated by the addition of 7.5 µl of 5
SDS Laemmli
buffer. A 4-µl aliquot of the samples was then loaded on high
density SDS-polyacrylamide gels, run with the Phast system, dried, and
exposed at -80° C for autoradiogaphy. Quantitation was
performed using a PhosphorImager.
I-EGF
binding (3-5
10
receptors/cell) (data not
shown). The F5 mutant receptor was expressed at a slightly lower level
(about 50%) than wild-type receptor.
Figure 1:
Schematic
representation and EGF receptor levels of EGF COOH-terminal receptor
mutants. A, the COOH-terminal domain of the EGF receptor is
shown with the five autophosphorylation sites, Tyr-992, Tyr-1068,
Tyr-1086, Tyr-1148, and Tyr-1173. F5 has all five tyrosines mutated to
phenylalanines (F). Dc 123F has a deletion of the 123
COOH-terminal amino acids and substitution of Tyr-992 with
phenylalanine. Dc 165, Dc 196, and Dc 214 have a deletion of the
COOH-terminal 165, 196, and 214 amino acids, respectively. F5, Dc 123F,
and Dc 214 were previously published (20, 21). The numbers of EGF
binding sites were calculated from Scatchard plot analysis of I-EGF binding data (5). Y, Tyr. B,
equal amounts of total lysates (150 µg of proteins) from NIH 3T3
expressing wild-type (WT) EGF-R (Cl 17, 4
10
EGF-R/cell, lane 5) and mutant cells, F5 (lane
1), Dc 123F (lane 2), Dc 165 (lane 3), Dc 196 (lane 4), and Dc 214 (lane 6) were run on a 7.5%
SDS-polyacrylamide gel and transferred to nitrocellulose, and
anti-EGF-R mAb was used to detect the EGF-R. Exposure of the
autoradiogram was for 15 h.
Phosphorylation of eps15 and eps15 by EGF Receptors Lacking the
Autophosphorylation Sites
To study eps8 and eps15 phosphorylation, lysates of EGF-stimulated cells were
immunoprecipitated with antiphosphotyrosine antibodies and analyzed for
the presence of eps8 and eps15 by Western blotting.
As previously observed, eps8 and eps15 were
phosphorylated only in EGF-stimulated receptor transfected cells, and
no phosphorylation was detected in EGF-treated, untransfected NIH 3T3
cells (data not shown; Refs. 26 and 27). A long EGF-R deletion mutant
lacking almost the entire COOH terminus was investigated in order to
address the role of the COOH terminus in eps8 and eps15 phosphorylation. As shown in Fig. 2, eps8 phosphorylation was dramatically increased in cells expressing the
Dc 214 mutant, whereas eps15 phosphorylation was almost
abolished (Fig. 2A, lane 6). Quantitation
indicates that phosphorylation by Dc 214 was increased 13-fold for eps8 and decreased by 80% for eps15 (). eps8 appeared as two bands of 92 and 68 kDa, the latter
representing a degradation product(26) . To test the role of the
EGF receptor autophosphorylation sites in tyrosine phosphorylation of eps8 and eps15, the F5 receptor mutant was used. Fig. 2shows that phosphorylation of eps8 and eps15 was equally effective by the wild-type and F5 mutant receptors (Fig. 2A, lane 2). Quantitation of eps8 and eps15 phosphorylation by the F5 mutant and wild-type
receptor normalized to the amount of receptor expressed indicated that eps8 was equally phosphorylated, whereas eps15 was
slightly more phosphorylated (about 2-fold) by the F5 than by wild-type
receptors. Control blots indicated that the levels of eps8 and eps15 were identical in all unstimulated and EGF-treated cells (Fig. 2B). Phosphorylation of eps8 (Fig. 3, lane 2) and eps15 (see Fig. 5, lane 2) by the Dc 123F mutant was similar to
that of the wild-type receptor. eps15 appeared mainly as a
single band of 142 kDa in unstimulated cells and as two bands of 142
and 155 kDa in EGF-treated cells, both of which contained
phosphotyrosine. The appearance of the 155-kDa band as a predominant
form in EGF-treated cells suggests that the more slowly migrating form
is due to phosphorylation. eps15 has been reported to be
phosphorylated on Tyr and Ser/Thr residues(25) . However, it is
not known whether this slower migration is due to tyrosine
phosphorylation or also to serine/threonine phosphorylation induced by
EGF. In addition, identical results were obtained when eps8 and eps15 were first immunoprecipitated with their
specific antibodies and then blotted with phosphotyrosine antibodies
(data not shown). These data, therefore, indicate that the five EGF-R
autophosphorylation sites are not necessary for efficient tyrosine
phosphorylation of these two substrates that lack SH2 domains. These
results contrast with the inability of the F5 and Dc 123F mutants to
phosphorylate and/or associate with SH2-containing substrates, such as
PLC, GAP, the p85 subunit of phosphatidylinositol 3 kinase, SHC,
and Grb-2(20, 21, 22) . Indeed, control
experiments showed that phosphorylation of PLC
was undetectable
with the F5 and Dc 123F mutants (data not shown), as described
previously(21, 22) .
Figure 2:
Phosphorylation of eps8 and eps15 substrates by F5 and Dc 214 EGF receptor mutants. A, cells were treated with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5)
100 ng/ml EGF for 60 min at 4 °C, and the lysates were prepared.
Equal amounts of lysates (3 mg of proteins) from F5 (lanes 1 and 2), wild-type (lanes 3 and 4), and
Dc 214 (lanes 5 and 6) expressing cells were
immunoprecipitated with anti-Ptyr mAb, run on 7.5% SDS-polyacrylamide
gels, transferred to nitrocellulose filters, and then reacted with
anti-eps8 Ab (left) or anti-eps15 Ab (right). Exposure of the blots was for 7 and 13 h,
respectively. B, to compare levels of expression of eps8 and eps15, equal amounts of lysates (150 µg of
proteins) were directly blotted with anti-eps8 Ab (left) or anti-eps15 Ab (right).
Quantitation of the autoradiograms was performed with the
PhosphorImager. Exposure of the blots was for 5 and 12 h, respectively. w.t., wild type.
Figure 3:
Phosphorylation of eps8 by EGF
receptor deletion mutants. A, cells were treated with (lanes 2, 4, 6, 8, and 10)
or without (lanes 1, 3, 5, 7, and 9) 100 ng/ml EGF for 60 min at 4 °C, and the lysates were
prepared. Equal amounts of lysates (3 mg of proteins) from Dc 123F (lanes 1 and 2), Dc 165 (lanes3 and 4), Dc 196 (lanes5 and 6), wild-type (lanes 7 and 8), and Dc 214 (lanes 9 and 10) expressing cells were
immunoprecipitated with anti-Ptyr mAb, run on 10% SDS-polyacrylamide
gels, transferred to nitrocellulose filters, and then reacted with
anti-eps8 Ab. Exposure of the autoradiogram was for 18 h. B, to compare levels of expression of eps8, equal
amounts of lysates (150 µg of proteins) were directly blotted with
anti-eps8 Ab (lanes 1-10). Exposure of the
autoradiogram was for 20 h. w.t., wild
type.
Figure 5:
Phosphorylation of eps15 by EGF
receptor deletion mutants. A, cells were treated with (lanes 2, 4, 6, 8, and 10)
or without (lanes 1, 3, 5, 7, and 9) 100 ng/ml EGF for 60 min at 4 °C, and the lysates were
prepared. Equal amounts of lysates (3 mg of proteins) from Dc 123F (lanes 1 and 2), Dc 165 (lanes 3 and 4), Dc 196 (lanes 5 and 6), wild-type (lanes 7 and 8), and Dc 214 (lanes 9 and 10) expressing cells were immunoprecipitated with anti-Ptyr
mAb, run on 7.5% SDS-polyacrylamide gels, transferred to nitrocellulose
filters, and reacted with anti-eps15 Ab. Exposure of the
autoradiogram was for 24 h. B, to compare levels of expression
of eps15, equal amounts of lysates (150 µg of proteins)
were directly blotted with anti-eps15 Ab (lanes
1-10). Exposure of the autoradiogram was for 22
h.
Phosphorylation of eps8 by EGF Receptor Deletion
Mutants
Shorter EGF receptor deletion mutants were used to
better define the region of the EGF-R carboxyl terminus responsible for
modulating eps8 and eps15 phosphorylation. As shown
in Fig. 3, eps8 was similarly phosphorylated by the
wild-type, Dc 123F, and Dc 165 receptors. However, its phosphorylation
was greatly increased by the Dc 196 and Dc 214 mutants. Quantitation
relative to wild-type phosphorylation indicated that the Dc 196 and Dc
214 mutants had 9- and 13-fold increases, respectively, in eps8 phosphorylation (Tables I and II).
, p85, and Grb-2.
Figure 4:
Co-immunoprecipitation of EGF receptor Dc
196 and Dc 214 mutants with eps8.A, cells were
treated with (lane 2, 4, and 6) or without (lanes 1, 3, and 5) 100 ng/ml EGF for 60 min
at 4 °C, and the lysates were prepared. Equal amounts of lysates (5
mg of proteins) from wild-type (lanes 1 and 2), Dc
196 (lanes 3 and 4), and Dc 214 (lanes 5 and 6) expressing cells were immunoprecipitated with anti-EGF-R
mAb, run on 10% SDS-polyacrylamide gels, transferred to nitrocellulose
filters, and then reacted with anti-eps8 Ab. Exposure of the
autoradiogram was for 72 h. eps8 indicates the 96- and 68-kDa
forms of eps8. B, to compare levels of expression of eps8, equal amounts of lysates (150 µg of proteins) were
directly blotted with anti-eps8 Ab (lanes 1-6).
Exposure of the autoradiogram was for 20 h. eps8 indicates the
96- and 68-kDa forms of eps8. C, to compare levels of
EGF receptor immunoprecipitated, 1/40 of the immunoprecipitation (lanes 3-6) and 1/20 (lane 1 and 2)
were directly blotted with anti-EGF-R Ab (lanes 1-6).
Exposure of the autoradiogram was for 12 h.
Phosphorylation of eps15 by EGF Receptor Deletion
Mutants
The same EGF receptor deletion mutants were also tested
for their ability to phosphorylate eps15. As shown in Fig. 5, Dc 123F and Dc 165 phosphorylated eps15 similarly to the wild-type EGF receptor, whereas Dc 196 and Dc 214
were essentially unable to phosphorylate eps15. Quantitation
indicated that phosphorylation of eps15 by Dc 196 and Dc 214
was decreased to 10 and 20%, respectively, of the wild-type receptor
level ().
, which could not be
phosphorylated by either the Dc 196 or the Dc 214 deletion mutants. In
the case of PLC
, the mechanism is fairly evident, because these
two mutants lack all five autophosphorylation sites. On the contrary,
the reason for the decreased capacity of phosphorylation of eps15 is not evident. Our results delimit a region of the EGF receptor,
encompassing residues 991-1021 (between Dc 165 and Dc 196), which seems
critical for eps15 phosphorylation. The slightly higher
phosphorylation of eps15 by the F5 and Dc 123F mutants
compared with wild-type receptor (less than 2-fold) may be due to the
lack of binding and activation of a tyrosine phosphatase. In
particular, the major EGF receptor binding site for Tyr PTPase 1B is
Tyr-992(31) , which is absent in the F5 and Dc 123F mutants.
Because it is not known whether eps15 is a direct EGF receptor
substrate because its association with the receptor cannot be detected
or whether it is phosphorylated by another tyrosine kinase activated by
EGF receptor, it is difficult at the moment to interpret the role of
these 30 amino acids in the EGF receptor tail. One possibility is that
both eps15 and the EGF receptor bind an adaptor protein
independently of the autophosphorylation sites, which would bring eps15 in contact with the EGF receptor. Alternatively, this
region may be important in allowing EGF receptor activation of another
tyrosine kinase(35) . Finally, it is also possible that the lack
of eps15 phosphorylation is due to a change in conformation of
the two EGF receptor mutants that does not allow eps15 phosphorylation.
In Vitro Kinase Activity of EGF Receptors Lacking the
Autophosphorylation Sites
To test the kinase activity of the the
F5 and Dc 123F mutant receptors, in vitro kinase activity
toward a synthetic peptide corresponding to the amino acids surrounding
Tyr-1173 was measured. As shown in , F5 and Dc 123F mutant
receptors showed a 3-fold higher K for
the peptide than the wild-type EGF receptor. Therefore, in vitro kinase activity of an EGF receptor lacking the autophosphorylation
sites was lower than that of the wild-type but not totally abolished.
Also, tyrosine phosphorylation of total cellular proteins in vivo is known to be drastically decreased but not completely absent in
the F5 and Dc 123F mutant receptor (Ref. 21; data not shown), possibly
due to the presence of a compensatory autophosphorylation site.
Transforming Activity of EGF Receptor Deletion Mutants
and eps8 Phosphorylation
Finally, phosphorylation of eps8 correlates well with the high transforming ability of the Dc 196
and Dc 214 mutants. As shown in , EGF-dependent
transforming ability of the F5, Dc 123F(5, 21) , and Dc
165 mutants is very low (about 10% of that of the wild-type EGF
receptor), whereas in longer deletions the wild-type transformation
level is recovered (100 and 120%, respectively, in Dc 196 and Dc 214).
In addition, Dc 196 and Dc 214 possessed low transforming activity even
in the absence of EGF (15 and 20% of EGF-dependent wild-type EGF-R,
respectively). Transformation in the absence of EGF has never been
observed with the wild-type or any other EGF receptor
mutant(5, 27, 29) . These data were confirmed
when the ability to grow in agar was estimated (data not shown). The
high transforming activity of Dc 214 and Dc 196 also correlates well
with higher tyrosine phosphorylation of endogenous proteins observed
using phosphotyrosine blots of total cellular proteins (Ref. 21; data
not shown).
Table: In vitro
kinase activity of wild-type and mutant EGF receptors lacking all five
autophosphorylation sites
P]ATP, and various concentrations of
Tyr-1173 peptide ranging from 0.0065 to 0.65 mM. Reactions
were terminated by the addition of 2
Laemmli buffer, and
4-µl aliquots were run on Phast gels, dried, and exposed for
autoradiography. The Tyr(P) peptide bands were scanned and quantified
with the PhosphorImager. K
values are in
µM; V
values are in µmol/min.
Table: Comparison of transforming activity and
tyrosine phosphorylation of eps8 and eps15 by different EGF receptor
mutants
, phospholipase C
; GAP,
GTPase-activating protein of Ras; SHC, Src and collagen homology
proteins; SH2, Src homology; DMEM, Dulbecco's modified
Eagle's medium; Ab, antibody; mAb, monoclonal antibody.
Ab;
Dr. Francesca Fazioli (Laboratory of Molecular Genetics, Dipartimento
di Ricerca Biologica e Tecnologica, Milano) and Dr. PierPaolo Di Fiore
(Laboratory of Molecular and Cellular Biology, NIH) for helpful
discussions and for anti-eps8 and anti-eps-15 Ab; Dr.
Ettore Appella (Laboratory of Cellular Biology, NIH) for the precious
gift of the 1173 peptide; Dr. Maria Mazzotti for great help with tissue
culture; Dr. Paola Castagnino for help with anti-EGF-R blots
(Laboratory of Molecular and Cellular Biology, NIH); and Dr. Claudio de
Santis (Dipartimento di Ricerca Biologica e Tecnologica, Milano) for
help with the Phast gel system.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.