Institute for Molecular Biology and Biotechnology, Department of Biology, McMaster University, Hamilton, Ontario L8S 4K1, Canada
Author for correspondence (e-mail:
william.muller{at}mcgill.ca)
Accepted 31 August 2004
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SUMMARY |
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Key words: Erbb2, Tyrosine Phosphorylation, Knock-in, Hypomorph
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Introduction |
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Despite the lack of a direct soluble ligand for Erbb2, the tyrosine kinase
activity of Erbb2 can be stimulated by other Egfr ligands through the
formation of specific heterodimers with other members of the Egfr family
(Goldman et al., 1990;
Karunagaran et al., 1996
;
Pinkas-Kramarski et al.,
1996a
; Pinkas-Kramarski et
al., 1996b
; Tzahar et al.,
1996
). For example, stimulation of cells with Egf can result in
transphosphorylation of Erbb2 through the formation of Egfr-Erbb2 heterodimers
(King et al., 1988
;
Stern and Kamps, 1988
).
Similarly, stimulation of cells with heregulin can result in the
transphosphorylation of Erbb2 through the formation of specific heterodimers
of Erbb2 and Erbb4 or heterodimers of Erbb2 and Erbb3
(Goldman et al., 1990
;
Karunagaran et al., 1996
;
Pinkas-Kramarski et al.,
1998a
; Pinkas-Kramarski et
al., 1996a
; Pinkas-Kramarski
et al., 1998b
;
Pinkas-Kramarski et al.,
1996b
; Sliwkowski et al.,
1994
; Stern and Kamps,
1988
; Tzahar et al.,
1997
; Tzahar et al.,
1996
). Transphosphorylation of Erbb3 is absolutely dependent on
its capacity to form specific heterodimers with other members of the Egfr
family since it lacks intrinsic tyrosine kinase activity
(Guy et al., 1994
). Taken
together, these observations argue that the formation of specific heterodimers
play an important biological role in the function of the Egfr family.
Members of the Egfr family share structural similarities including an
extracellular ligand-binding domain, a single-pass transmembrane domain, a
highly conserved tyrosine kinase domain, and a regulatory carboxyl-terminal
tail containing several tyrosine autophosphorylation sites
(Hynes and Stern, 1994).
Ligand-mediated activation results in strong mitogenic signals from the
receptors, leading to cellular growth and differentiation. Not surprisingly,
members of the Egfr family are collectively involved in both development and
disease. Whereas amplification and aberrant overexpression of Erbb2 has been
implicated in various cancers, most notably in breast cancer
(Simpson et al., 1995
;
Slamon et al., 1987
;
Slamon et al., 1989
), the loss
of Erbb2, Erbb3 or Erbb4 expression in knock-out mice has deleterious
effects on the developing embryo (Britsch
et al., 1998
; Gassmann et al.,
1995
; Lee et al.,
1995
; Riethmacher et al.,
1997
). For example, Erbb2- and Erbb3-deficient
animals share similar hypoplastic development of the sympathetic nervous
system (Britsch et al., 1998
),
and Erbb2- and Erbb4-deficient animals exhibit defective
formation of cardiac ventricular trabecules
(Gassmann et al., 1995
;
Lee et al., 1995
).
Upon receptor activation, specific tyrosine residues in the terminal tail
of the receptor are autophosphorylated
(Akiyama et al., 1991;
Hazan et al., 1990
), and then
serve as potential binding sites for intracellular signaling proteins
harboring phosphotyrosine binding (PTB)
(Kavanaugh et al., 1995
) or
Src homology 2 (SH2) (Pawson,
1995
) domains. In a series of studies by Dankort et al.
(Dankort et al., 1997
), the
five major tyrosine autophosphorylation sites (Y1028, Y1144, Y1201, Y1227,
Y1253) were evaluated systematically for their roles in constitutively
activated Erbb2-mediated transformation of fibroblast cells. Although
simultaneous ablation of all five sites in the tyrosine
phosphorylation-deficient (NYPD) mutant drastically impaired
transformation, independent tyrosine-tophenylalanine mutations at four of five
sites only modestly reduced the transforming ability. Substitution at the one
remaining site (Y1028) resulted in a consistent increase in
transformation. Conversely, restoration of individual tyrosine residues to the
NYPD mutant at one of four sites (`add-back' mutants) was not only
able to fully restore the transforming ability, but was also able to transform
with a modest increase over the fully functional receptor. The Y1028
add-back mutant had the opposite effect and completely ablated the residual
transforming abilities of the NYPD mutant. These observations
suggested that while four of the five tyrosine sites positively mediate Erbb2
signaling, Y1028 negatively modulates Erbb2 activity.
We describe here the introduction of wild-type and phosphotyrosine mutant Erbb2 cDNAs into the endogenous mouse Erbb2 locus to examine their physiological roles, in vivo. Although mice derived from the different knock-in alleles were viable, examination of the levels of Erbb2 expression revealed that the knock-in strains expressed only 10% of the expected Erbb2 protein. A further reduction of Erbb2 protein, achieved by intercrossing the Erbb2 knock-in mutants with Erbb2 null mice, resulted in perinatal lethality. Thus, we established a minimum threshold level of Erbb2 required for viability. In contrast, expression of the Y1028F mutation in knock-in animals genetically rescued this perinatal lethality. Biochemical analyses revealed that these animals expressed higher levels of Erbb2 above the threshold. We further identify that Y1028 mediates the negative regulation of Erbb2 signaling by influencing the turnover rate of the receptor.
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Materials and methods |
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Immunoblot analysis of embryo lysates
Embryos were lysed in modified TNE lysis buffer (50 mM Tris-HCl pH 7.6, 150
mM NaCl, 1% NP-40, 10 mM NaF, 2 mM EDTA) supplemented with 10 µg/ml
leupeptin, 10 µg/ml aprotinin and 1 mM sodium orthovanadate. For
immunoblotting, the membranes were incubated with an anti-Erbb2 monoclonal
(Ab-3, Oncogene Research) or anti-Grb2 polyclonal (1:2500; C-23, Santa Cruz)
antibody overnight at 4°C. Subsequently, the membranes were treated with
anti-mouseHRP or anti-rabbitHRP secondary antibodies for 1 hour at room
temperature, washed three times in TBS and visualized by enhanced
chemiluminescence (Amersham), as specified by the manufacturer.
Whole-mount in situ hybridization
For whole-mount in situ hybridization, embryos were fixed in 4%
paraformaldehyde/0.2% glutaraldehyde, dehydrated through a graded series of
methanol/PBT (PBS+0.1% Tween-20) baths and stored in 100% MeOH at
20°C until needed. Whole-mount in situ hybridization was carried
out as previously described (Wilkinson and
Nieto, 1993).
Immunohistochemistry
E12.5 embryos were fixed in fresh 4% paraformaldehyde, washed several times
with PBS and then post-fixed and bleached in Dent's fixative (methanol:DMSO,
4:1)+5% H2O2. After extensive washes, the embryos were
incubated in blocking solution (5% goat serum+1% DMSO in TBS) overnight at
room temperature. Incubation in anti-NF150 (1:1000, Chemicon) diluted in
TBS+1% DMSO was performed for 2 days at room temperature. After 5x1 hour
washes in TBS-T (TBS+1% Tween20), the embryos were incubated overnight in goat
anti-rabbitHRP (1:1000 in TBS-T). DAB+ kit (DAKO, catalogue no. K3468) was
used to visualize the neurofilament staining. Whole-mount immunohistochemistry
was performed on diaphragm dissected from E18.5 embryos and fixed in 4% PFA.
They were first treated in 0.1 M glycine in PBS, washed several times, then
incubated in PBT (PBS+0.5% Triton-X-100) for 5 minutes. Diaphragms were
incubated overnight with anti-NF150 diluted 1:1000 in PBT+5% goat serum. After
extensive washes in PBT, they were incubated overnight with goat
anti-rabbit-Alexa488 (Molecular Probes) diluted 1:1000 in PBT and
-bungarotoxin-Alexa594 (Molecular Probes). Diaphragms were visualized
using the Zeiss LSM 510Meta confocal microscope.
Receptor turnover assay
Rat-1 cells stably expressing the oncogenic Erbb2 tyrosine phosphorylation
site mutants were seeded (13x106 cells) onto 60-mm plates and
pulse labeled using 0.1 mCi/ml of 35S-EXPRESS Protein Labeling mix
(NEG772, NEN Life Science Products). The cells were then washed and chased
with unlabeled medium for the indicated times. Protein lysates from the
labeled cells were immunoprecipitated using an anti-Erbb2 antibody (Ab4,
Oncogene Science) and resolved on an 8% SDS-PAGE. The gel was dried and
exposed to a PhosphoImager screen (Molecular Dynamics) and scanned using the
Typhoon8600 scanner (Amersham). Image analyses were performed using ImageQuant
software (Molecular Dynamics).
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Results |
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The use of a rat Erbb2 cDNA instead of a mouse cDNA was necessary
to distinguish expression of the knock-in allele from the endogenous gene and
allow for the validation of the knock-in strategy. The mouse and rat
Erbb2 share a greater than 93% sequence similarity at both the
nucleic acid and amino acid levels. Importantly, the tyrosine
autophosphorylation sites and the sequences surrounding these sites in the
carboxyl-terminus are conserved. To confirm that rescue of the embryonic
lethality associated with disruption and inactivation of the mouse
Erbb2 gene (Morris et al.,
1999) was attributed solely to expression of the knock-in rat
Erbb2 cDNA, RT-PCR was performed on total RNA isolated from wild-type
and knock-in animals. Analysis of the RT-PCR product suggested that homozygous
animals indeed only expressed the rat Erbb2 cDNA (data not
shown).
Hypomorphic expression of Erbb2 protein resulted in neonatal lethality
Since specific expression of the knock-in allele was validated, we next
performed immunoblot analyses on protein lysates prepared from E12.5
wild-type, heterozygous, and homozygous mutant embryos
(Fig. 1E) to determine whether
the knock-in Erbb2 allele expressed wild-type levels of Erbb2
protein. Surprisingly, the levels of Erbb2 expressed in the
Erbb2Erbb2/Erbb2 embryos
(Fig. 1E, lanes 4-5) were
dramatically reduced relative to the expression of endogenous Erbb2 in
wild-type littermates (Fig. 1E,
lanes 1 and 2). However, in spite of the considerably reduced levels of Erbb2
protein, homozygous Erbb2Erbb2/Erbb2 knock-in animals did
not display any obvious phenotype and appeared generally healthy.
Although mice bearing the knock-in rat Erbb2 cDNA appeared
phenotypically normal, we have previously demonstrated that they indeed
exhibited subtle defects. In particular, our lab showed that rat
Erbb2 cDNA knock-in animals possessed only 10% of the number of
muscle spindles found in wild-type animals
(Andrechek et al., 2002).
Consequently, we investigated the effects of further reducing the expression
of Erbb2 by interbreeding heterozygous knock-in
(Erbb2wt/Erbb2) animals with heterozygous Erbb2
knock-out (Erbb2wt/ko) animals. This strategy allowed us
to express a single Erbb2 knock-in allele in an
Erbb2-deficient background to generate hemizygous
Erbb2Erbb2/ko animals. Significantly, no
Erbb2Erbb2/ko animals were found at weaning age
(three-weeks old) when the animals were genotyped
(Table 1A).
To determine the precise reason that the Erbb2Erbb2/ko animals were dying, we interbred homozygous Erbb2Erbb2/Erbb2 knock-in animals with heterozygous Erbb2wt/ko animals and assessed whether we could detect viable hemizygous Erbb2Erbb2/ko animals at birth. Although the expected number of animals were present at birth, 18 of the 38 newborn pups were either stillborn or started dying immediately after birth (Table 1B). They could not breathe independently, despite the ability to open their mouths, and they became cyanotic and died within a few minutes. Indeed, all of the dead pups were genotyped to be Erbb2Erbb2/ko animals. Subsequent postmortem histological analyses of the lungs confirmed that the hemizygous Erbb2Erbb2/ko pups were unable to inflate and expand their lungs, despite being vascularized and structurally intact (data not shown). These observations suggest that a critical minimal threshold level of Erbb2 protein is required to maintain viability.
Expression of the Y1028F mutation rescues the hypomorph
Previous in vitro analyses of the tyrosine phosphorylation mutants
identified tyrosine 1028 as a negative regulator of Erbb2-induced
transformation via an undetermined mechanism. Accordingly, we next asked
whether we could genetically rescue the perinatal lethality observed with the
hemizygous Erbb2Erbb2/ko animals, by removing the putative
negative regulatory tyrosine residue in Erbb2. This was accomplished by
similarly crossing the heterozygous Erbb2wt/Y1028F
knock-in animals with Erbb2wt/ko animals to generate mice
expressing a single Y1028F cDNA knock-in allele in an
Erbb2-deficient background. Interestingly, in contrast to the
hemizygous Erbb2Erbb2/ko pups, no perinatal lethality was
observed with the hemizygous Erbb2Y1028F/ko animals
(Table 1A). In fact, all of the
progeny survived and the adult Erbb2Y1028F/ko animals
appeared normal and healthy and were fertile. To preclude any possibility that
the phenotypic differences observed in the Erbb2-Y1028F knock-in
animals versus the control Erbb2 knock-in animals were not by chance
and that it is specifically due to this particular point mutation, mice
expressing either the Y1144F or the Y1227F phosphotyrosine mutant Erbb2
receptor in an Erbb2-deficient background were also generated. As
indicated in Table 1B, none of
the hemizygous Erbb2Y1144F/ko or
Erbb2Y1227F/ko animals survived, and similar to the
Erbb2wt/ko animals, they died shortly after birth
struggling unsuccessfully to breathe.
To further elucidate the nature of the defect resulting in the inability of
the animals to inflate their lungs, the neuromuscular junctions in the
diaphragm muscles of E18.5 embryos were examined by whole-mount immunostaining
with a neurofilament-150 antibody and with -bungarotoxin. In the
control animals, Erbb2wt/wt and
Erbb2Erbb2/Erbb2, the central region of the diaphragm
muscles was innervated by the relatively large main phrenic nerve with smaller
intramuscular branches (Fig.
2A,B). Accordingly, innervation of the diaphragm from the
genetically rescued embryos, Erbb2Y1028F/ko was similar to
that in the control animals (Fig.
2C). In contrast, mutant diaphragms from
Erbb2Erbb2/ko, Erbb2Y1144F/ko,
Erbb2Y1227F/ko embryos were very poorly innervated, which
probably resulted in the perinatal respiratory failure. As shown in
Fig. 2D-F, the innervations
were thin, disorganized, and discontinuous or incomplete and they lacked an
apparent main nerve trunk from which smaller presynaptic branches originate.
However, labeling of postsynaptic acetylcholine receptor (AChR) clusters with
-bungarotoxin did not reveal any significant differences in the number,
density or shape of AChR clusters (Fig.
2G-L,G'-L').
|
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|
In order to assess whether the variation in Erbb2 protein levels observed in the Erbb2 or Y1028F knock-in alleles were occurring at a transcriptional or post-transcriptional level, Erbb2 transcripts in E12.5 embryos were detected using an RNase protection assay (Fig. 4D). The results showed that samples harboring either the Erbb2 or the Y1028F knock-in allele expressed identical levels of Erbb2 transcripts (Fig. 4D, lanes 4-5 versus lanes 6-8). Note that either knock-in alleles express significantly lower levels of Erbb2 transcripts compared to wild-type animals. This phenomenon is probably a result of using a cDNA knock-in strategy and explains the overall lower Erbb2 expression levels in the knock-in animals. Regardless of this, our observations argue that specifically expressing the Y1028F knock-in allele results in increased Erbb2 protein levels without affecting transcriptional activity, when compared to the Erbb2 knock-in allele.
Since the function and activity of the ErbB family of receptors are closely related, it is plausible that other family members may be affected by the low Erbb2 levels or may be elevated to compensate for the loss Erbb2 activity. In this regard, the expression level of other ErbB receptor family members was measured to determine if there were corresponding differences in expression. Interestingly, Egfr levels were modestly affected depending on the level of Erbb2 in the knock-in animals, which may augment the defects observed. No significant differences in ErbB3 or ErbB4 levels were noted in the wild-type and knock-in animals that would suggest any kind of compensatory role for these receptors.
Y1028 influences Erbb2 receptor turnover rate
Since many receptor tyrosine kinases are downregulated by endocytosis and
subsequently targeted for degradation
(Katzmann et al., 2002), we
examined whether Y1028 affected Erbb2 receptor turnover rate
(Fig. 5A,B). Rat-1 cell lines
stably expressing oncogenic Erbb2 (V664E mutation) or its mutant tyrosine
phosphorylation site derivatives were established. Specifically, we compared
the turnover rate of the oncogenic Erbb2 receptor versus the Y1028F
mutant receptor. Conversely, we also assessed the effects of restoring
Y1028 to the Y1144 add-back mutant to generate the
Y1028/Y1144 double add-back mutant Erbb2 receptor. Add-back mutants
are derived from an Erbb2 receptor stripped of the five major tyrosine
autophosphorylation sites by tyrosine-to-phenylalanine mutations and then
individual mutant sites are `added-back' or reverted to tyrosine residues.
|
Based on the above results, we next examined whether the E3-ubiquitin
ligase, c-Cbl, was responsible for mediating the negative regulatory effect of
Y1028 on Erbb2 activity since c-Cbl has been shown to mediate the
downregulation of the Egfr and other receptor tyrosine kinases
(Thien and Langdon, 2001). Our
data suggest that c-Cbl is able to associate with Erbb2 through other specific
phosphotyrosine residues, and that Y1028 is not responsible for the
recruitment of c-Cbl to Erbb2, nor is it required for the ubiquitylation of
Erbb2 (Fig. 5D). Taken
together, these observations suggest that Y1028 modulates Erbb2 protein levels
in a c-Cbl- and ubiquitylation-independent manner.
Differential Erbb2 signaling requirements in development
Although knock-in animals expressing a single Erbb2 receptor tyrosine
phosphorylation site mutation were grossly normal, it was conceivable that
there may be subtle defects as a result of ablating a particular
Erbb2-initiated signaling pathway. Thus, whole embryos (E12.5) were
immunostained with anti-neurofilament (Fig.
6) to examine the appearance of sensory cutaneous nerves, which
were abnormally defasciculated in the cardiac rescued Erbb2 mutants
and in Erbb3-deficient mutants
(Woldeyesus et al., 1999).
Interestingly, in comparison with Erbb2wt/wt embryos
(Fig. 6A), there was a striking
defect in the morphology of the developing cutaneous nerves in the thoracic
body wall of Erbb2Y1227F/Y1227F embryos
(Fig. 6D) that was not observed
in Erbb2Y1028F/Y1028F or
Erbb2Y1144F/Y1144F knock-in embryos
(Fig. 6B,C). The trajectories
of the sensory nerves in Erbb2Y1227F/Y1227F mutants
appeared intact but were highly disorganized and severely defasciculated,
similar to the defects in the cardiac-rescued Erbb2 mutants
(Woldeyesus et al., 1999
).
These results suggest that the signal transduction pathways initiated
specifically by Erbb2-Y1227 are absolutely required for the proper development
of the cutaneous sensory nerves in the thoracic body wall.
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Discussion |
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Nonetheless, it is intriguing that the animals express significantly higher
levels of Erbb2 but only require about 10% for development. Since Erbb2 acts
primarily as a co-receptor and its activation is dependent on the
ligand-mediated activation of its ErbB counterparts, the high levels of Erbb2
may suggest its excess availability to engage in a dimer signaling unit. Thus,
the threshold that we have identified may also reflect the threshold activity
of the other ErbB receptors. When the Erbb2 knock-in animals were
inter-crossed with Erbb2-deficient animals to generate hemizygous
knock-in animals (Erbb2Erbb2/ko), this additional twofold
reduction in Erbb2 levels fell below the threshold level required and resulted
in perinatal lethality due to acute respiratory distress. The inability of
newborn Erbb2Erbb2/ko pups to inflate their lungs is
remarkably similar in phenotype to the cardiac-specific rescue of
Erbb2-deficient mutants (Morris
et al., 1999; Woldeyesus et
al., 1999
).
Evaluation of the diaphragm muscles of E18.5 embryos revealed an obvious
defect in the phrenic nerve of mutant embryos. In the cardiac-rescued
Erbb2 mutants, Woldeyesus et al.
(Woldeyesus et al., 1999)
reported that the phrenic nerve was thin and poorly fasciculated at E14.5 but
only remnants were detected at later stages. Observations by Morris et al.
(Morris et al., 1999
) showed
that diaphragms of E18.5 mutant embryos were completely devoid of
innervations, and AChR clusters were more dispersed and rounded in shape.
Interestingly, the diaphragm muscles of our Erbb2Erbb2/ko
embryos at E18.5 (Fig. 2) were
innervated, although a main nerve trunk was absent and any nerves that were
present were very thin, highly disorganized and fragmented. We also did not
observe any differences in the density or shape of AChR receptor clusters
compared to those of wild-type embryos. These results support the notion of
progressive threshold sensitivities to the level of Erbb2 signaling in the
developing peripheral nervous system. Although the low levels of Erbb2
prevented or impeded the complete degeneration of motor neurons in the
diaphragm muscle and maintained apparently normal clustering of AChR at the
neuromuscular junctions, this was not sufficient for the diaphragm to respond
and function. Also note that the low levels of Erbb2 were sufficient to bypass
the cardiac trabeculation defects in Erbb2 null embryos, suggesting
that the cardiac system is not as sensitive as the peripheral nervous system
to this threshold level of Erbb2.
Conversely, we were able to genetically rescue the perinatal lethality in hemizygous Erbb2Erbb2/ko embryos by similarly introducing the Y1028F knock-in allele into an Erbb2-deficient background to generate Erbb2Y1028F2/ko animals. Tyrosine 1028 is a negative regulatory signal that affects the stability of the receptor (discussed below). All Erbb2 knock-in mice bearing the Y1028F allele expressed higher levels of Erbb2 protein than in the Erbb2 knock-in mice. Thus, this genetic manipulation ablating a negative regulatory phosphotyrosine site in the carboxyl-terminus increased the level of Erbb2 above the minimal threshold required for the phrenic nerve in the diaphragm to develop normally. Erbb2Y1028F2/ko animals survived and were phenotypically healthy. In contrast, similar experiments performed with the Y1144F or the Y1227F knock-in alleles also resulted in perinatal lethality with similar defects in the innervation of the diaphragm muscle. These two mutations, of course, did not affect the level of Erbb2 expressed in the knock-in animals.
Although previous in vitro experiments concluded that Y1028 negatively regulated oncogenic Erbb2-mediated transformation, we were also interested in determining whether Y1028 would also have this suppressive effect in vivo in the context of a non-oncogenic Erbb2 receptor. As described above, the results of the in vivo experiments with the Y1028F knock-in animals are consistent with a negative regulatory effect of Y1028 on Erbb2 activity. Moreover, here we have also identified the biochemical basis for this effect, which was previously unknown. Phosphotyrosine 1028 modulates Erbb2 protein levels by promoting the downregulation and turnover of the receptor in a c-Cbl- and ubiquitin-independent manner. Conversely, loss of Y1028 resulted in an increase in Erbb2 protein stability. Thus, the stabilization of Erbb2 levels above the minimum threshold in the hemizygous Erbb2Y1028F2/ko animals allowed these particular strains to develop normally.
Sequence alignment of Erbb2 with the Egfr shows that Erbb2-Y1028 and its
surrounding sequences share a high degree of similarity to the region
surrounding the Egfr-Y992 (Fig.
1D). Despite the identity and function of Egfr-Y992 suggested in
1989 (Chen et al., 1989), the
mechanism of its function has not been clearly elucidated. Carboxyl-terminal
truncation of the Egfr has suggested that an 18 amino acid region surrounding
Y992 conforms to an `internalization' domain and is required for Egf-dependent
receptor internalization. However, a subsequent study showed that point
mutation of Y992 did not affect Egfr internalization
(Sorkin et al., 1992
). In
another study, the Y992F mutation actually increased the rate of Egfr
internalization and it was suggested that the increase in negative charge
associated with phosphorylation of Y992 would reduce the rate of
ligand-induced endocytosis (Holbrook et
al., 1999
). The nature of these discrepancies may be due to
artefactual differences in cell types, the level of ectopic Egfr expression in
the cells, or the nature of the mutation used in their analyses. Our results
showing the effect of Y1028, in a physiologically relevant context, to promote
the turnover rate of the Erbb2 receptor from the cell surface should lead to
clarification of these conflicting reports concerning the role of Y992 in the
Egfr.
Homozygous knock-in animals expressing Erbb2 phosphotyrosine
mutations where the Grb2 site (Y1144) or the Shc-binding site (Y1227) were
ablated had little impact on the gross development of mice, even at tenfold
less expression. As discussed above, previous studies in our lab revealed a
significant reduction in the number of muscle spindle cells in
Erbb2Erbb2/Erbb2 animals without any obvious phenotypic
consequences (Andrechek et al.,
2002). Therefore, it is conceivable that other specific cell or
tissue types may be more sensitive to the altered Erbb2 signaling. Indeed, the
cutaneous sensory nerves in the thoracic body wall of
Erbb2Y1227F/1227F mutants were thin and defasciculated,
whereas Erbb2Y1144F/Y1144F embryos were similar to wild
type. Since Erbb2-Y1227 binds to the Shc adapter protein, these results
suggest that the Shc signaling pathway may play an important and unique role
downstream of Erbb2 in the development of these sensory nerves. Furthermore,
it is interesting to note that Shc can recruit phosphatidylinositol 3-kinase
(PI3K) (Gu et al., 2000
). PI3K
is a major signaling pathway downstream of ErbB3, which also display a
strikingly similar abnormal sensory nerve phenotype in
Erbb3-deficient animals
(Woldeyesus et al., 1999
).
Thus, it is conceivable that the Erbb2 and ErbB3 signaling pathways converge
downstream at the level of PI3K, such that PI3K plays a key role in the
development of sensory nerves. In general, this raises the idea that signaling
pathways downstream of the individual receptor phosphotyrosine sites can
function differentially and independently in the development of specific
tissues.
Unlike with Erbb2, where loss of the Grb2 binding site (Y1144) did not
result in any discernable phenotype, uncoupling of Grb2 from the Met receptor
in a knock-in model resulted in severe defects in muscle development
(Maina et al., 1996). Taken
together, these observations suggest that the remaining Erbb2
autophosphorylation sites are able to functionally substitute for the
inability of these Erbb2 mutants to recruit Grb2. Alternatively, it
is possible that the Erbb2 heterodimerization partners such as Egfr, ErbB3 or
ErbB4 can compensate for the lack of Grb2-binding sites on Erbb2. Indeed, it
has been demonstrated that Egfr or ErbB3 can independently bind Shc and Grb2
(Batzer et al., 1994
;
Carraway and Cantley, 1994
;
Okutani et al., 1994
;
Prigent and Gullick, 1994
). It
is also possible that there may be other more subtle developmental defects in
different tissues of the Erbb2Y1144F knock-in mutants. In
this regard, transgenic mice expressing constitutively activated
Erbb2 mutants coupled to either Grb2 or Shc specifically in the
mammary epithelium develop morphologically distinct mammary tumors that
possess inherently different metastatic properties
(Dankort et al., 2001
).
In summary, using a series of unique Erbb2 knock-in animals and genetic manipulation, we have established that a minimum threshold level of Erbb2 receptor expression is required during proper development. This critical threshold is only 5-10% of normal Erbb2 levels in wild-type embryos. We have also identified an important in vivo function of the intrinsic negative regulatory site in Erbb2 (Y1028) to modulate the stability/turnover rate of the receptor throughout development. In addition, our data suggest that individual Erbb2 signaling pathways are not redundant, but rather play unique roles in specific tissues throughout development.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Samuel Lunenfeld Research Institute, Mount Sinai Hospital,
University of Toronto, Toronto, Ontario, Canada
Present address: University of California San Francisco, Comprehensive
Cancer Center, San Francisco, CA, USA
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REFERENCES |
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---|
Akiyama, T., Matsuda, S., Namba, Y., Saito, T., Toyoshima, K. and Yamamoto, T. (1991). The transforming potential of the c-erbB-2 protein is regulated by its autophosphorylation at the carboxyl-terminal domain. Mol. Cell. Biol. 11,833 -842.[Medline]
Andrechek, E. R., Hardy, W. R., Girgis-Gabardo, A. A., Perry, R.
L., Butler, R., Graham, F. L., Kahn, R. C., Rudnicki, M. A. and Muller,
W. J. (2002). ErbB2 is required for muscle spindle and
myoblast cell survival. Mol. Cell. Biol.
22,4714
-4722.
Batzer, A. G., Rotin, D., Urena, J. M., Skolnik, E. Y. and Schlessinger, J. (1994). Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor. Mol. Cell. Biol. 14,5192 -5201.[Abstract]
Britsch, S., Li, L., Kirchhoff, S., Theuring, F., Brinkmann, V.,
Birchmeier, C. and Riethmacher, D. (1998). The ErbB2
and ErbB3 receptors and their ligand, neuregulin-1, are essential for
development of the sympathetic nervous system. Genes
Dev. 12,1825
-1836.
Carraway, K. L., 3rd and Cantley, L. C. (1994). A neu acquaintance for erbB3 and erbB4: a role for receptor heterodimerization in growth signaling. Cell 78, 5-8.[Medline]
Carraway, K. L., 3rd, Sliwkowski, M. X., Akita, R., Platko, J.
V., Guy, P. M., Nuijens, A., Diamonti, A. J., Vandlen, R. L., Cantley,
L. C. and Cerione, R. A. (1994). The erbB3 gene product is a
receptor for heregulin. J. Biol. Chem.
269,14303
-14306.
Carraway, K. L., 3rd, Rossi, E. A., Komatsu, M., Price-Schiavi,
S. A., Huang, D., Guy, P. M., Carvajal, M. E., Fregien, N., Carraway,
C. A. and Carraway, K. L. (1999). An intramembrane modulator
of the ErbB2 receptor tyrosine kinase that potentiates neuregulin signaling.
J. Biol. Chem. 274,5263
-5266.
Chan, R., Hardy, W. R., Laing, M. A., Hardy, S. E. and Muller,
W. J. (2002). The catalytic activity of the ErbB-2 receptor
tyrosine kinase is essential for embryonic development. Mol. Cell.
Biol. 22,1073
-1078.
Chen, W. S., Lazar, C. S., Lund, K. A., Welsh, J. B., Chang, C. P., Walton, G. M., Der, C. J., Wiley, H. S., Gill, G. N. and Rosenfeld, M. G. (1989). Functional independence of the epidermal growth factor receptor from a domain required for ligand-induced internalization and calcium regulation. Cell 59, 33-43.[Medline]
Dankort, D. L., Wang, Z., Blackmore, V., Moran, M. F. and Muller, W. J. (1997). Distinct tyrosine autophosphorylation sites negatively and positively modulate neu-mediated transformation. Mol. Cell. Biol. 17,5410 -5425.[Abstract]
Dankort, D., Jeyabalan, N., Jones, N., Dumont, D. J. and Muller,
W. J. (2001). Multiple ErbB-2/Neu Phosphorylation Sites
Mediate Transformation through Distinct Effector Proteins. J. Biol.
Chem. 276,38921
-38928.
Gassmann, M., Casagranda, F., Orioli, D., Simon, H., Lai, C., Klein, R. and Lemke, G. (1995). Aberrant neural and cardiac development in mice lacking the ErbB4 neuregulin receptor. Nature 378,390 -394.[CrossRef][Medline]
Goldman, R., Levy, R. B., Peles, E. and Yarden, Y. (1990). Heterodimerization of the erbB-1 and erbB-2 receptors in human breast carcinoma cells: a mechanism for receptor transregulation. Biochemistry 29,11024 -11028.[Medline]
Gu, H., Maeda, H., Moon, J. J., Lord, J. D., Yoakim, M., Nelson,
B. H. and Neel, B. G. (2000). New role for Shc in activation
of the phosphatidylinositol 3-kinase/Akt pathway. Mol. Cell.
Biol. 20,7109
-7120.
Guy, P. M., Platko, J. V., Cantley, L. C., Cerione, R. A. and Carraway, K. L., 3rd (1994). Insect cell-expressed p180erbB3 possesses an impaired tyrosine kinase activity. Proc. Natl. Acad. Sci. USA 91,8132 -8136.[Abstract]
Hazan, R., Margolis, B., Dombalagian, M., Ullrich, A., Zilberstein, A. and Schlessinger, J. (1990). Identification of autophosphorylation sites of HER2/neu. Cell Growth Differ. 1,3 -7.[Abstract]
Holbrook, M. R., O'Donnell, J. B., Jr, Slakey, L. L. and Gross, D. J. (1999). Epidermal growth factor receptor internalization rate is regulated by negative charges near the SH2 binding site Tyr992. Biochemistry 38,9348 -9356.[CrossRef][Medline]
Hynes, N. E. and Stern, D. F. (1994). The biology of erbB-2/neu/HER-2 and its role in cancer. Biochim. Biophys. Acta 1198,165 -184.[CrossRef][Medline]
Karunagaran, D., Tzahar, E., Beerli, R. R., Chen, X., Graus-Porta, D., Ratzkin, B. J., Seger, R., Hynes, N. E. and Yarden, Y. (1996). ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer. EMBO J. 15,254 -264.[Abstract]
Katzmann, D. J., Odorizzi, G. and Emr, S. D. (2002). Receptor downregulation and multivesicular-body sorting. Nat. Rev. Mol. Cell Biol. 3, 893-905.[CrossRef][Medline]
Kavanaugh, W. M., Turck, C. W. and Williams, L. T. (1995). PTB domain binding to signaling proteins through a sequence motif containing phosphotyrosine. Science 268,1177 -1179.[Medline]
King, C. R., Borrello, I., Bellot, F., Comoglio, P. and Schlessinger, J. (1988). Egf binding to its receptor triggers a rapid tyrosine phosphorylation of the erbB-2 protein in the mammary tumor cell line SK-BR-3. EMBO J. 7,1647 -1651.[Abstract]
Lee, K. F., Simon, H., Chen, H., Bates, B., Hung, M. C. and Hauser, C. (1995). Requirement for neuregulin receptor erbB2 in neural and cardiac development. Nature 378,394 -398.[CrossRef][Medline]
Maina, F., Casagranda, F., Audero, E., Simeone, A., Comoglio, P. M., Klein, R. and Ponzetto, C. (1996). Uncoupling of Grb2 from the Met receptor in vivo reveals complex roles in muscle development. Cell 87,531 -542.[Medline]
Morris, J. K., Lin, W., Hauser, C., Marchuk, Y., Getman, D. and Lee, K. F. (1999). Rescue of the cardiac defect in ErbB2 mutant mice reveals essential roles of ErbB2 in peripheral nervous system development. Neuron 23,273 -283.[Medline]
Okutani, T., Okabayashi, Y., Kido, Y., Sugimoto, Y., Sakaguchi,
K., Matuoka, K., Takenawa, T. and Kasuga, M. (1994).
Grb2/Ash binds directly to tyrosines 1068 and 1086 and indirectly to tyrosine
1148 of activated human epidermal growth factor receptors in intact cells.
J. Biol. Chem. 269,31310
-31314.
Olayioye, M. A., Neve, R. M., Lane, H. A. and Hynes, N. E.
(2000). The ErbB signaling network: receptor heterodimerization
in development and cancer. EMBO J.
19,3159
-3167.
Pawson, T. (1995). Protein modules and signalling networks. Nature 373,573 -580.[CrossRef][Medline]
Peles, E., Ben-Levy, R., Tzahar, E., Liu, N., Wen, D. and Yarden, Y. (1993). Cell-type specific interaction of Neu differentiation factor (NDF/heregulin) with Neu/HER-2 suggests complex ligand-receptor relationships. EMBO J. 12,961 -971.[Abstract]
Pinkas-Kramarski, R., Shelly, M., Glathe, S., Ratzkin, B. J. and
Yarden, Y. (1996a). Neu differentiation
factor/neuregulin isoforms activate distinct receptor combinations.
J. Biol. Chem. 271,19029
-19032.
Pinkas-Kramarski, R., Soussan, L., Waterman, H., Levkowitz, G., Alroy, I., Klapper, L., Lavi, S., Seger, R., Ratzkin, B. J., Sela, M. et al. (1996b). Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions. EMBO J. 15,2452 -2467.[Abstract]
Pinkas-Kramarski, R., Lenferink, A. E., Bacus, S. S., Lyass, L., van de Poll, M. L., Klapper, L. N., Tzahar, E., Sela, M., van Zoelen, E. J. and Yarden, Y. (1998a). The oncogenic ErbB-2/ErbB-3 heterodimer is a surrogate receptor of the epidermal growth factor and betacellulin. Oncogene 16,1249 -1258.[CrossRef][Medline]
Pinkas-Kramarski, R., Shelly, M., Guarino, B. C., Wang, L. M.,
Lyass, L., Alroy, I., Alimandi, M., Kuo, A., Moyer, J. D., Lavi, S. et
al. (1998b). ErbB tyrosine kinases and the two neuregulin
families constitute a ligand-receptor network. Mol. Cell.
Biol. 18,6090
-6101.
Prigent, S. A. and Gullick, W. J. (1994). Identification of c-erbB-3 binding sites for phosphatidylinositol 3'-kinase and SHC using an EGF receptor/cerbB-3 chimera. EMBO J. 13,2831 -2841.[Abstract]
Riethmacher, D., Sonnenberg-Riethmacher, E., Brinkmann, V., Yamaai, T., Lewin, G. R. and Birchmeier, C. (1997). Severe neuropathies in mice with targeted mutations in the ErbB3 receptor. Nature 389,725 -730.[CrossRef][Medline]
Simpson, B. J., Phillips, H. A., Lessells, A. M., Langdon, S. P. and Miller, W. R. (1995). c-erbB growth-factor-receptor proteins in ovarian tumours. Int. J. Cancer 64,202 -206.[Medline]
Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A. and McGuire, W. L. (1987). Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235,177 -182.[Medline]
Slamon, D. J., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S. G., Keith, D. E., Levin, W. J., Stuart, S. G., Udove, J., Ullrich, A. et al. (1989). Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244,707 -712.[Medline]
Sliwkowski, M. X., Schaefer, G., Akita, R. W., Lofgren, J. A.,
Fitzpatrick, V. D., Nuijens, A., Fendly, B. M., Cerione, R. A.,
Vandlen, R. L. and Carraway, K. L., 3rd (1994). Coexpression
of erbB2 and erbB3 proteins reconstitutes a high affinity receptor for
heregulin. J. Biol. Chem.
269,14661
-14665.
Sorkin, A., Helin, K., Waters, C. M., Carpenter, G. and
Beguinot, L. (1992). Multiple autophosphorylation sites of
the epidermal growth factor receptor are essential for receptor kinase
activity and internalization. Contrasting significance of tyrosine 992 in the
native and truncated receptors. J. Biol. Chem.
267,8672
-8678.
Stern, D. F. and Kamps, M. P. (1988). EGF-stimulated tyrosine phosphorylation of p185neu: a potential model for receptor interactions. EMBO J. 7,995 -1001.[Abstract]
Thien, C. B. and Langdon, W. Y. (2001). Cbl: many adaptations to regulate protein tyrosine kinases. Nat. Rev. Mol. Cell Biol. 2,294 -307.[CrossRef][Medline]
Tzahar, E., Waterman, H., Chen, X., Levkowitz, G., Karunagaran, D., Lavi, S., Ratzkin, B. J. and Yarden, Y. (1996). A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor. Mol. Cell. Biol. 16,5276 -5287.[Abstract]
Tzahar, E., Pinkas-Kramarski, R., Moyer, J. D., Klapper, L. N.,
Alroy, I., Levkowitz, G., Shelly, M., Henis, S., Eisenstein, M.,
Ratzkin, B. J. et al. (1997). Bivalence of EGF-like ligands
drives the ErbB signaling network. EMBO J.
16,4938
-4950.
Wilkinson, D. G. and Nieto, M. A. (1993). Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts. Methods Enzymol. 225,361 -373.[Medline]
Woldeyesus, M. T., Britsch, S., Riethmacher, D., Xu, L.,
Sonnenberg-Riethmacher, E., Abou-Rebyeh, F., Harvey, R., Caroni, P. and
Birchmeier, C. (1999). Peripheral nervous system defects in
erbB2 mutants following genetic rescue of heart development. Genes
Dev. 13,2538
-2548.
Yarden, Y. and Sliwkowski, M. X. (2001). Untangling the ErbB signalling network. Nat. Rev. Mol. Cell Biol. 2,127 -137.[CrossRef][Medline]