1 Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston,
MA 02115, USA
2 Centre de Biochimie, UMR6543/CNRS, Faculté des Sciences, 06108 Nice,
France
3 Division of Cancer Genetics, deCODE Genetics, Sturlugata 8, 108 Reykjavik,
Iceland
4 Department of Molecular and Cell Biology, University of California, Berkeley,
CA 94720-3200, USA
5 Division of Signal Transduction, Beth Israel Deaconess Medical Center, 330
Brookline Avenue, Boston, MA 02215, USA
6 UC Davis Cancer Center, 4645 2nd Avenue, Sacramento, CA 95817, USA
7 Howard Hughes Medical Institute, Harvard Medical School, 200 Longwood Avenue,
Boston, MA 02115, USA
Authors for correspondence (e-mail:
perrimon{at}rascal.med.harvard.edu
and
klcarraway{at}ucdavis.edu)
Accepted 20 May 2003
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SUMMARY |
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Key words: kek1, EGFR, Negative feedback loop, LRR domains, Mammary tumor
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INTRODUCTION |
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Two mechanisms for ErbB receptor activation in human tumors have been
described. First, it is known that the overexpression of receptors results in
their constitutive activation, either by facilitating the formation of the
active dimeric form of the receptors or by swamping phosphatases that keep
basal tyrosine kinase activity in check. In fact, overexpression of one of the
ErbB family members, ErbB2, correlates with a poor prognosis of individuals
with breast cancer (Slamon et al.,
1989). ErbB activation in tumors also arises from autocrine
activation mechanisms where cells produce and secrete EGF-like growth factors
(Normanno et al., 1994
). For
both mechanisms it is thought that receptor activation leads to receptor
autophosphorylation on tyrosine residues, which in turn triggers cascades of
intracellular events culminating in tumor cell growth. An effective
ErbB-directed anti-tumor agent would suppress ErbB signalling arising from
both overexpression and autocrine stimulation mechanisms.
Like its vertebrate homologs, the Drosophila EGF Receptor (DER)
mediates various inductive signalling events in many developmental processes
to regulate proper cell specification and tissue patterning
(Ray and Schüpbach, 1996;
Perrimon and Perkins, 1997
;
Schweitzer and Shilo, 1997
).
During developmental processes, DER signalling activity is precisely
controlled by the carefully orchestrated deployment of the activating and
inhibiting ligands (for reviews, see
Perrimon and McMahon, 1999
;
Freeman, 2000
). So far, four
activating ligands have been identified: Vein (Vn)
(Schnepp et al., 1996
), Spitz
(Spi) (Rutledge et al., 1992
),
Gurken (Grk) (Neuman-Silberberg and
Schüpbach, 1993
) and Keren
(Reich and Shilo, 2002
;
Urban et al., 2002
), each of
which possesses an EGF repeat similar to that of transforming growth factor
(TGF
), a known ligand of the vertebrate EGFR. In addition, DER
signalling can be regulated by negative factors such as Argos (Aos)
(Schweitzer et al., 1995
),
Sprouty (Spry) (Casci et al.,
1999
) and Kekkon 1 (Kek1)
(Ghiglione et al., 1999
).
kek1 was isolated in a screen for genes whose expression overlaps
that of activated DER during oogenesis
(Musacchio and Perrimon, 1996;
Ghiglione et al., 1999
). It
was shown to be transcriptionally regulated by DER and the Ras/MAPK pathway in
follicle cells (Ghiglione et al.,
1999
). In developmental assays, the loss of kek1 activity
was associated with an increase in DER activity, whereas ectopic
overexpression of the gene suppressed receptor activation, strongly suggesting
that the Kek1 protein acts as a feedback negative regulator of DER activity.
Consistent with this model, epistasis studies placed the function of Kek1
upstream of DER (Ghiglione et al.,
1999
). This, together with the observation that Kek1 encodes a
single-pass transmembrane protein containing six contiguous Leucine-Rich
Repeats (LRR) and one Immunoglobulin (Ig)-like domain
(Musacchio and Perrimon,
1996
), suggests that Kek1 acts at the cell surface to suppress DER
signalling. Indeed, our previous studies indicate that Kek1 is capable of
physically interacting with DER.
Despite the crucial role of Kek1 in controlling the level of DER activity during oogenesis, little is known about the precise mechanism by which Kek1 antagonizes this RTK. The purpose of the present study was to better understand the Kek1 inhibitory mechanism and to ascertain whether this regulation is tissue specific. In addition, we wanted to determine whether Kek1 expression could antagonize the growth of human and mouse mammary tumor cells through the suppression of ErbB receptors signalling. We show that as is the case during oogenesis, Kek1 antagonizes DER activity in the wing and eye imaginal discs through a negative feedback loop. Furthermore, we show that Kek1 and DER/ErbB form heterodimers, preventing activating ligands to bind to these receptors, and that this interferes with autophosphorylation and signal transduction of the receptors. Finally, we show that Kek1 may be employed as an inhibitor of mammalian ErbB receptors.
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MATERIALS AND METHODS |
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Construction of kek1 plasmids and generation of transgenic
lines
Cloning details for making these deletion constructs are available under
request. After mutagenesis by PCR, the coding regions were subcloned into the
P-element vector pUAST (Brand and Perrimon,
1993) or pUASP (Rorth,
1998
). All these kek1 constructs contain two consecutive
Myc epitope tags in frame to the C terminus.
NT,
NT+LRR,
NT+LRR+Ig,
LRR,
Ig are deletions in the kek1
extracellular domain from amino acids 23 to 88, 23 to 277, 23 to 430, 126 to
277, 329 to 430, respectively. kek1sec was made by introducing a stop
codon immediately before the TM domain (at amino acid 446).
The kek1/DER chimeric constructs were made by fusing the extracellular and TM domains of different kek1 deletions described above (amino acids 1 to 473) to the DER intracellular domain (at amino acids 744, immediately after the TM domain).
The UAS constructs were introduced into w1118 flies by
standard methods of P-element-mediated germline transformation
(Spradling, 1986). For each
construct, several independent transgenic lines were generated and tested.
Antibody staining
Embryos and third instar wing imaginal discs were collected, fixed and
stained (Bilder and Perrimon,
2000), using the monoclonal mouse anti-Myc antibody (Ab-1,
Calbiochem). Tissues were co-stained with anti-FasIII to reveal embryonic
basolateral membranes, and rhodamine-phalloidin to reveal the apical surface
of imaginal discs. Confocal images were collected on a Leica TCS confocal
microscope.
For detection of the ß-galactosidase activity, imaginal discs were dissected in PBS and fixed in 1% glutaraldehyde for 20 minutes. Imaginal discs were then stained with 1 mg/ml X-Gal in X-Gal staining buffer at 37°C for 2 hours.
Sf9 cells on cover slips were co-infected at an M.O.I. of 0.2 with baculovirus encoding EGFR and Kek1. Cells were then fixed in methanol, incubated with rabbit anti-EGFR (1005, Santa Cruz) and mouse anti-Myc Ab-2 antibodies, and stained with FITC-goat-anti-mouse IgG and rhodamine-goat-anti-rabbit IgG (Jackson Immunochemicals).
Sf9 insect cells experiments
Recombinant baculoviruses encoding DER and Kek1 have been described
previously (Ghiglione et al.,
1999). The Kek1 versions
LRR and
Ig described above
were subcloned into the baculovirus transfer vector pVL1392 (Pharmingen) and
recombinant baculoviruses were produced as described previously
(Ghiglione et al., 1999
).
For co-immunoprecipitation studies between DER and Kek1LRR or
Kek1
Ig, we proceeded as described previously
(Ghiglione et al., 1999
).
Immunoprecipitations from lysates were carried out using anti-Myc Ab-2 or
anti-DER (a generous gift from M. Freeman) antibodies. Precipitates were
blotted with anti-DER. Filters were then stripped and reprobed with
anti-Myc.
For [125I]EGF crosslinking experiments, insect cells were infected as described above and incubated for 5 minutes at room temperature with 0.5 µCi [125I]EGF (Amersham) in the absence and presence of excess cold EGF (30 nM). Bis-sulfosuccinimidyl suberate (BS3) was added to 1 mM and the incubation was continued for another 30 minutes. Cells were then lysed and immunoprecipitated as above, and the radioactive bands were visualized using a Molecular Dynamics Storm Phosphorimaging system.
Mammalian cell lines
Mouse mammary tumor cell lines IJ9921 (MMTV-heregulin/NDF), NF-639
(MMTV-neu) and AC-816 (MMTV-v-Ha-ras) were derived from mammary tumors from
transgenic mice and have been described previously
(Krane and Leder, 1996;
Muller et al., 1988
;
Sinn et al., 1987
). MDA-MB-468
human mammary tumor cells were from ATCC, and HEK293-Ecr human embryonic
kidney cells expressing the ecdysone receptor for inducible protein expression
were obtained from Invitrogen. Cells were routinely grown in DMEM supplemented
with 10% bovine calf serum (Gibco-BRL), 4 mM glutamine (Bio-Whittaker),
penicillin (50 U/ml) and streptomycin (50 mg/ml) (Sigma).
Plasmids and transfections
The kek1 cDNA was subcloned into pcDNA3.1 and pIND expression
vectors (Invitrogen) adding either a Myc or a HA epitope tag in frame to the C
terminus. The Kozak sequence was also changed from the native
Drosophila sequence to the consensus mammalian sequence CCACCAUGG to
achieve optimal expression in mammalian cells
(Kozak, 1987). Stable
transfectants were generated by lipofection (Lipofectamine PlusTM,
Gibco-BRL) and selection in G418 for 2-4 weeks. Kek1 expression in picked
clones was verified by western blotting and/or immunoprecipitation using
anti-Myc (Ab2, NeoMarkers) or anti-HA epitope (Ab Y11, Santa Cruz
Biotechnology) antibodies.
Cell growth and transformation assays
Growth assays were performed by seeding 500-2000 cells in 24-well plates
with DMEM media/10% FBS. Time points were taken at days 2, 4, 6 and 8 by
trypsinization and counting. Anchorage-independent growth assays were
performed by suspending 104 cells in 0.36% Bactoagar (Difco) over a
0.6% agar base layer in DMEM/10% FCS in 35 mm dishes. Every four days,
300 µl of media was added to each plate. After 2-3 weeks, colonies
were stained overnight with 0.5 mg/ml nitrobluetetrazolium (NBT, Sigma) in PBS
and counted. Each experiment was performed in triplicate and repeated at least
two times. In vivo transformation was measured by injecting 106
cells subcutaneously behind each front leg of nude mice. Tumors were excised
3-4 weeks later and weighed. Each experiment was performed at least three
times.
293 cell co-immunoprecipitation and immunoblotting experiments
HEK293-Ecr cells expressing HA-tagged Kek1 were treated without or with 5
µM Ponasterone A (Invitrogen) for 20-24 hours to induce Kek1 expression.
For EGF stimulation, experiments cells were serum-starved for another 4 hours
and then treated without or with 50 ng/ml EGF (Sigma) for 5 minutes at
37°C. Equivalent protein amounts of cleared lysates were
immunoprecipitated with 1.5 µg anti-phosphotyrosine (4G10, Upstate
Biotechnology), anti-EGFR (Ab-1, NeoMarkers) or anti-HA epitope. Precipitates
were resolved by SDS-PAGE, transferred to nitrocellulose and blotted with
anti-HA or anti-EGFR (1005, Santa Cruz) antibodies. Blotted proteins were
detected using horseradish peroxidase-coupled secondary antibody followed by
enhanced chemiluminescence. Erk1/2 activation was measured by blotting lysates
with an anti-phospho-Erk1/2 (Thr202/Tyr204) antibody according to the
instructions of the manufacturer (New England Biolabs), and levels were
correlated with total Erk2 protein detected with an anti-Erk2 antibody
(sc-1647, Santa Cruz).
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RESULTS |
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The LRR domains of Kek1 are critical for its function
Both Ig-like and LRR domains are present in the extracellular domain of
Kek1. To determine the contribution of these domains in Kek1 activity, we
generated a series of deletion constructs that removed some of these regions
(Fig. 3A). These Kek1 truncated
proteins retained the signal peptide, and their ability to inhibit DER
signalling was assessed in overexpression assays in the follicle cells and in
the wing discs using CY2-Gal4 or MS1096-Gal4, respectively.
|
We previously showed in Sf9 cells that Kek1TM, but not the Kek1
intracellular domain, is able to bind to DER as efficiently as Kek1
(Ghiglione et al., 1999). To
define this interaction more precisely, we tested which different truncated
Kek1 proteins were able to bind to DER. After co-expression of a Myc-tagged
version of these truncated proteins and DER in Sf9 cells, Kek1 was
immunoprecipitated from the cell lysates using an anti-Myc antibody.
Coprecipitations between Kek1
Ig or Kek1 and DER were observed by
probing the resulting blot with the anti-DER antibody
(Fig. 3B, lane 3 and 4,
respectively). However, Kek1
LRR was not able to co-precipitate DER
(Fig. 3B, lane 2), suggesting a
selectivity of the Kek1 LRR domains for binding to DER. Altogether, our
structure-function analysis demonstrates the importance of the LRR domains for
Kek1 function. These domains allow Kek1 to bind to DER, and this physical
interaction is necessary for the inhibition of the receptor.
Kek1 and DER form heterodimers in vivo
To determine whether the physical association between Kek1 and DER occurs
in vivo, we generated a series of Kek1-DER chimeras in which the whole
extracellular and transmembrane part of the receptor is replaced with the
corresponding regions of Kek1 (Fig.
4A). Indeed, if Kek1 interacts with DER, then the chimera should
promote heterodimerization with the endogenous receptor, hence its signalling
activation. Overexpression of the Kek1-DER chimera in follicle cells was
associated with hyperactivation of the DER pathway because the derived eggs
were strongly dorsalized (Fig.
4C). A similar phenotype was obtained after overexpressing the
Kek1Ig-DER chimera (Fig.
4E) but not Kek1
LRR-DER
(Fig. 4D), thus confirming the
importance of the LRR domains for Kek1 function.
|
Altogether, we conclude that Kek1 is able to form heterodimers with DER in vivo, and that this association inhibits DER activity.
Kek1 subcellular localization
Deletion of its cytoplasmic domain strikingly decreases the ability of Kek1
to inhibit DER (Fig. 3A)
(Ghiglione et al., 1999). As
the Kek1 cytoplasmic domain is not implicated in the association with DER, we
reasoned that it could possibly play a role in Kek1 subcellular
localization.
To test this hypothesis, UAS-kek1-myc and UAS-kek1TM-myc
were expressed in embryonic and imaginal wing disc epithelia using the en-Gal4
driver. First, we observed a complete and a weak inhibition of DER signalling
by Kek1-Myc and Kek1TM-Myc, respectively
(Fig. 5E,F), a result that is
consistent with our previous results using untagged proteins. This indicates
that the Myc epitopes do not interfere with Kek1 function. Interestingly, the
subcellular localization of these proteins, visualized using an anti-Myc
antibody, was strikingly different. Although expression of Kek1-Myc is clearly
apical (Fig. 5A,C), expression
of Kek1TM-Myc is basolateral (Fig.
5B,D), indicating that the intracellular domain of Kek1 is
required for its correct subcellular localization. As DER has been shown to be
apically located (Sapir et al.,
1998), the aberrant localization of Kek1TM provides a likely
explanation for its inability to inhibit the receptor efficiently when
compared with the wild-type Kek1 protein.
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|
In Fig. 6C, we examined the
stimulation of the Erk1 and Erk2 serine/threonine kinases by probing lysates
from treated cells with an antibody that recognizes the phosphorylated
(activated) forms of these proteins. We observed that Kek1-HA expression
inhibited the activation of Erks in response to EGF by 75% in the
HEK293-Ecr cells. These observations indicate that, consistent with its
activity in flies, Kek1 interacts with the mammalian EGFR to suppress receptor
activation and signalling through the MAPK cascade.
Kek1 inhibits ligand binding
To examine the mechanistic details underlying Kek1 suppression of EGFR
activity, we used the baculovirus/Sf9 insect cell expression system. This
system was employed because the viral infection allows tight control of both
the relative levels of proteins expressed in each cell and the number of
protein expressing cells.
In the experiment depicted in Fig. 7A, we tested the role of Kek1 on ligand binding and activation of the EGFR. Kek1-Myc or human EGFR were expressed alone, or the two proteins were co-expressed with EGFR in excess. Cells were treated without or with EGF, and lysates were immunoprecipitated with antibodies directed to either Myc epitope (lanes 1-4) or EGFR (lanes 5-9). When precipitates were blotted with anti-phosphotyrosine, we observed a strong stimulation of receptor autophosphorylation by the growth factor in anti-receptor precipitates (upper panel, lanes 6-9), indicating that the total EGFR population responded strongly to ligand treatment. However, although the presence of EGFR was apparent in the anti-Myc precipitates (middle panel, lanes 2 and 3), no stimulation of the tyrosine phosphorylation of this Kek1-associated population of receptors was observed (upper panel, lanes 2 and 3). Moreover, EGFR in anti-Myc precipitates were not capable of interacting with [125I]EGF (lower panel, compare lane 2 with lanes 6 and 8).
One possible explanation for the Kek1-mediated suppression of ligand binding and activation is that Kek1 becomes trapped in an intracellular compartment and retains a population of the EGFR. To examine this possibility we looked at the localization of Kek1-Myc and EGFR or DER by immunofluorescence in Sf9 cells and in egg chambers and found that the two proteins co-localize at the cell surface of the co-expressing cells (Fig. 7B; data not shown).
One additional line of evidence that Kek1 is acting at the cell surface comes from the fact that Kek1, after ectopic expression in the germline, is able to weakly inhibit DER that is expressed in the overlying follicle cells (Fig. 7C). This `trans-inhibition' is greatly enhanced after removal one copy of top or one copy of the germline-specific ligand grk (Fig. 7D; data not shown).
Altogether, our results indicate that Kek1 directly interacts with EGFR/DER at the cell surface to inhibit ligand binding. These results are also consistent with other results that we have obtained in the fly assays showing that although Kek1 inhibits the ability of Grk and Spi to activate DER in various tissues, Kek1 does not physically associate with these ligands (data not shown).
Kek1 inhibits the growth of mammary cell lines with activated ErbB
receptors
Our results demonstrate that the Drosophila Kek1 protein can act
as a potent inhibitor of EGFR/ErbB in tissue culture cells. To extend this
observation, we asked whether Kek1 could act as a suppressor of mammalian
ErbB-mediated mammary tumor cell growth. We constructed a series of mammalian
cell lines stably transfected with Kek1 and then compared the growth
properties of Kek1 transfectants with control cells stably transfected with
vector alone. Two human cell lines were examined: HEK293-Ecr transfectants, a
human embryonic kidney cell line where Kek1 was expressed in an inducible
manner, and MDA-MB-468 cells, a mammary epithelial cell line that
overexpresses the EGFR. More importantly, the impact of Kek1 expression on a
series of cell lines derived from oncogene-induced mouse mammary tumors from
transgenic mice was also examined. IJ9921 cells were derived from expression
of the EGF-like growth factor neuregulin 1
(Krane and Leder, 1996),
NF-639 cells were from an activated form of ErbB2
(Muller et al., 1988
), and
AC-816 cells were derived from tumors induced by activated Ras
(Sinn et al., 1987
).
Kek1 transfectants exhibited a reproducibly slower growth rate than controls in four of the cell lines examined (Fig. 8A). Turning on Kek1 expression in HEK293 cells with the addition of PonA was sufficient to slow their growth, suggesting that differences in cellular growth rates in all lines is probably not a result of clonal variation. Moreover, these four Kek1 transfectants also exhibited a much lower tendency than controls to grow in soft agar and to grow as tumors when introduced into animals (Fig. 8B). The exception to this trend was the AC-816 cell line, which exhibited similar growth properties whether or not Kek1 was expressed.
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DISCUSSION |
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Kek1 acts in a negative feedback loop to modulate DER activity in
diverse tissues
kek1 was originally identified as a negative regulator of DER
signalling in follicle cells (Ghiglione et
al., 1999). We have extended these observations to two additional
tissues, the eye and wing imaginal discs. We show that kek1 is
expressed in cells where DER activity is required and that kek1
expression is lost in the absence of DER activity. Furthermore, we found that
more cells express kek1 following DER hyperactivation. Finally, we
showed that, in a sensitized genetic background, Kek1 acts as a negative
regulator of DER activity. These studies extend our previous findings and
strengthen the functional relationship between Kek1 and DER.
Results from both biochemical experiments and in vivo tests revealed that
the LRR domains of Kek1 are crucial for the association between DER and Kek1,
and DER inhibition. Furthermore, the Kek1 cytoplasmic domain, which has
previously been shown to play a role in the overall efficiency of DER
inhibition, appears to be critical for the proper apical subcellular
localization of Kek1 in epithelial cells. Interestingly, the Kek1 C terminus
contains a concensus sequence for a PDZ domain-binding site that we have shown
can bind the PDZ domains of proteins such as Disc-Large or Scribble (data not
shown). Because these proteins are crucial for the organization of apicobasal
cell polarity (Bilder et al.,
2003), it is possible that Kek1 localization depends on these
factors or related polarity cues. Interestingly, subcellular localization of
Kek1 to the apical side may be coordinated with DER/ErbB subcellular
localization as well, as PDZ-containing proteins have also been implicated in
ErbB subcellular localization (reviewed by
Carraway and Sweeney, 2001
).
Further characterization of these interactions will be needed to clarify how
subcellular localization of Kek1 and DER is regulated.
Mechanism of DER/ErbB binding and inhibition
Epistasis studies placed the action of Kek1 upstream of DER. As Kek1 is
expressed in the same cell as DER, these observations suggest that Kek1
interacts with either the receptor to suppress its signalling function or with
the ligand to sequester its activity. Our observations indicate that Kek1 can
be co-immunoprecipitated with DER
(Ghiglione et al., 1999) (this
study) but not with its ligands (data not shown) suggesting that Kek1
interacts directly with receptors to interfere with ligand binding
activity.
These findings are consistent with the biochemical interaction of Kek1 with
all four mammalian ErbB receptor family members. When reconstituted in Sf9
insect cells, Kek1 blocked the binding of radiolabeled EGF to the population
of EGFR associated with Kek1, but not the total receptor pool. Likewise,
EGF-stimulated autophosphorylation of the Kek1-associated receptor population
was blocked, but autophosphorylation of the total receptor pool was not. These
observations suggest that Kek1 acts to suppress receptor signalling at least
in part by physically interfering with ligand binding. However, other effects
on receptor activation cannot be ruled out. We observed that Kek1 suppressed
the growth properties of the NF-639 mouse mammary tumor cells, obtained from
an activating point mutation in the transmembrane region of the ErbB2
receptor. As this mutation is thought to generate constitutive receptor
tyrosine kinase activity via a ligand-independent mechanism
(Bargmann and Weinberg, 1988),
it is likely that Kek1 also acts to interfere with receptor dimerization or
other events necessary for its activity.
Our studies suggest that Kek1 is functionally similar to another
Drosophila suppressor of DER signalling called Argos. Argos is also a
transcriptional target of activated DER in developing tissues
(Golembo et al., 1996;
Wasserman and Freeman, 1998
),
and it has been demonstrated that Argos binds directly to DER to inhibit the
binding of the natural ligand Spitz (Jin
et al., 2000
). However, the sequences of the two inhibitors are
very distinct. Although Kek1 contains a series of LRR and Ig domains in its
extracellular region, Argos contains an imperfect EGF-like domain
(Freeman et al., 1992
). Given
that at least two proteins in the Drosophila genome are dedicated to
a similar purpose, it seems likely that ErbB antagonists are also present in
higher organisms.
Kek1-related genes?
Our previous studies indicated that the extracellular and TM region of Kek1
was sufficient to mediate its biological activity as well as its interaction
with DER (Ghiglione et al.,
1999). The present study indicate that the LRR domains of the
extracellular region are necessary for the suppression of DER-mediated
developmental events in flies. These results suggest that Kek1/receptor
interactions are mediated by the LRR domains, pointing to LRR-containing
extracellular proteins as candidates for mammalian Kek1 homologs. Numerous
mammalian LRR proteins have been described and several have arrangements of
subdomains similar to Kek1, including the Trk receptor tyrosine kinases
(Shelton et al., 1995
), LIG-1
(Suzuki et al., 1996
) and a
number of proteins of unknown function. The role of such proteins in
ErbB-mediated developmental processes and tumor cell growth remains to be
explored.
Particularly noteworthy is the small leucine-rich proteoglycan decorin,
which has been shown to directly bind to human EGFR
(Iozzo et al., 1999). However,
although decorin is also a potent suppressor of tumor cell growth, its
mechanism of action appears to differ from that of Kek1. Treatment of cells
with soluble decorin induces the immediate tyrosine phosphorylation of the
EGFR and subsequent signalling events
(Moscatello et al., 1998
;
Patel et al., 1998
), and
sustained expression of decorin suppresses EGFR levels without affecting
ligand binding activity. These results indicate that decorin is not
functionally identical to Kek1. However, taken with our observations these
data suggest that some LRR-containing extracellular proteins are capable of
interacting with ErbB receptors to modulate their activities by multiple
mechanisms.
Direct modulation of growth factor signalling
In a broader context, proteins such as Kek1 and decorin may be thought of
as direct modulators of ErbB receptors that could assist in the integration of
extracellular events with growth factor signalling. Numerous studies suggest
that signalling through integrins, cell adhesion molecules and other cell
surface proteins impact ErbB receptor signalling pathways, largely by
influencing the extent to which various intracellular signalling pathways
respond to receptor activation (Giancotti
and Ruoslahti, 1999; Moghal
and Sternberg, 1999
). These examples represent indirect modulation
of growth factor signalling through crosstalk between downstream components.
We propose that LRR-containing proteins such as Kek1 and decorin are members
of a larger functionally related class of glycoproteins that directly modulate
growth factor signalling pathways by interacting with and influencing the
properties of the receptors themselves
(Carraway and Sweeney,
2001
).
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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