, 1043 (2007);
et al. Michael B. Major,
Regulates WNT/ß-Catenin Signaling
Wilms Tumor Suppressor WTX Negatively
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gene amplification of a kinase that is not a direct
or downstream target of gefitinib or erlotinib.
Moreover, MET has not previously been shown
to signal through ERBB3. These findings may
have important clinical implications for NSCLC
patients who develop acquired resistance to
gefitinib or erlotinib. Our findings also suggest
that irreversible EGFR inhibitors, which are
currently under clinical development as treat-
ments for patients whose tumors have developed
be ineffectiveinthesubsetof tumorswithaMET
amplification even if they contain an EGFR
T790M mutation. Therefore, combination thera-
pies with MET kinase inhibitors, which are in
early-stage clinical trials, and irreversible EGFR
inhibitors shouldbeconsidered for patientswhose
tumors have become resistant to gefitinib or
erlotinib. Notably, a small percentage of NSCLCs
from EGFR TKI–naïve patients have been re-
ported to contain both an EGFR-activating muta-
is analogous to the observation that untreated
NSCLCs occasionally have an EGFR T790M.
These concurrent genetic alterations may help
explain why some NSCLCs with EGFR-activating
mutations fail to respond when initially treated
with gefitinib (22).
It will continue to be important to study
NSCLC primary tumors and cell lines with ac-
quired resistance to EGFR inhibitors for insights
into additional resistance mechanisms. Our find-
ings illustrate the value of studying genetic alter-
in the presence of gefitinib rather than focusing
solely on mutations in the EGFR gene itself. It
amplification contributes to resistance in other
EGFR-dependent cancers such as glioblastoma
multiforme, head and neck cancer, and colorectal
cancer after treatment with EGFR-directed
therapies. Finally, since ERBB2-amplified breast
cancers also activate PI3K/Akt signaling through
ERBB3, it will be interesting to explore whether
MET amplification also occurs in breast cancers
such as trastuzumab and lapatinib (9, 23).
References and Notes
1. B. J. Druker et al., N. Engl. J. Med. 344, 1038 (2001).
2. G. D. Demetri et al., N. Engl. J. Med. 347, 472 (2002).
3. J. G. Paez et al., Science 304, 1497 (2004).
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8. J. A. Engelman et al., J. Clin. Invest. 116, 2695 (2006).
9. N. V. Sergina et al., Nature 445, 437 (2007).
10. Materials and methods are available as supporting
material on Science Online.
11. G. A. Smolen et al., Proc. Natl. Acad. Sci. U.S.A. 103,
12. C. T. Miller et al., Oncogene 25, 409 (2006).
13. J. G. Christensen et al., Cancer Res. 63, 7345 (2003).
14. K. M. Weidner et al., Nature 384, 173 (1996).
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16. M. C. Heinrich et al., J. Clin. Oncol. 24, 4764 (2006).
17. M. Debiec-Rychter et al., Gastroenterology 128, 270
18. A. Hochhaus et al., Leukemia 16, 2190 (2002).
19. N. J. Donato et al., Blood 101, 690 (2003).
20. T. Kosaka et al., Cancer Res. 64, 8919 (2004).
21. T. Shibata et al., Clin. Cancer Res. 11, 6177 (2005).
22. A. Inoue et al., J. Clin. Oncol. 24, 3340 (2006).
23. F. M. Yakes et al., Cancer Res. 62, 4132 (2002).
24. We thank M. Begley for providing the lentiviral
expression vector, E. Fox for MET sequencing, and
K. Cichowski and D. E. Fisher for helpful discussions. This
work was supported by grants from the National Institutes
of Health 1K12CA87723-01 (P.A.J.), RO1CA114465-01
(B.E.J. and P.A.J.), RO1-CA111560 (C.L.), NIH GM41890
(L.C.C.) and PO1 CA089021 (L.C.C.), the National Cancer
Institute K08CA120060-01 (J.A.E.), the National Cancer
Institute Lung SPORE P20CA90578-02 (B.E.J.), the
American Cancer Society RSG-06-102-01 (P.A.J. and J.A.E.),
and by the American Association for Cancer Research, the
International Association for the Study of Lung Cancer
(J.A.E.) and the Italian Association for Cancer Research
(F.C.). P.A.J. and B.E.J. are part of a pending patent
application on EGFR mutations.
Supporting Online Material
Materials and Methods
Figs. S1 to S7
Tables S1 to S4
20 February 2007; accepted 11 April 2007
Published online 26 April 2007;
Include this information when citing this paper.
Wilms Tumor Suppressor
WTX Negatively Regulates
Michael B. Major,1,2,3Nathan D. Camp,1,2,3Jason D. Berndt,1,2,3XianHua Yi,4
Seth J. Goldenberg,2Charlotte Hubbert,1,2,3Travis L. Biechele,1,2,3Anne-Claude Gingras,5
Ning Zheng,2Michael J. MacCoss,4Stephane Angers,1,2,6Randall T. Moon1,2,3*
Aberrant WNT signal transduction is involved in many diseases. In colorectal cancer and melanoma,
mutational disruption of proteins involved in the degradation of b-catenin, the key effector of the
WNT signaling pathway, results in stabilization of b-catenin and, in turn, activation of transcription.
We have used tandem-affinity protein purification and mass spectrometry to define the protein
interaction network of the b-catenin destruction complex. This assay revealed that WTX, a protein
encoded by a gene mutated in Wilms tumors, forms a complex with b-catenin, AXIN1, b-TrCP2
(b-transducin repeat–containing protein 2), and APC (adenomatous polyposis coli). Functional
analyses in cultured cells, Xenopus, and zebrafish demonstrate that WTX promotes b-catenin
ubiquitination and degradation, which antagonize WNT/b-catenin signaling. These data provide a
possible mechanistic explanation for the tumor suppressor activity of WTX.
and subsequent proteosomal clearance. A com-
plex of proteins including adenomatous polypo-
sis coli (APC), AXIN, casein kinase 1a (CK1a),
and glycogen synthase kinase 3 (GSK3) phospho-
rylates N-terminal serine residues in b-catenin,
n the absence of WNT ligands, cytosolic
b-catenin is constitutively degraded through
by the Skp1, Cullin1, F-box protein b-TrCP
(SCFbTrCP) ubiquitin ligase (1). The engagement
ly,b-catenin levelsincrease inthenucleus,where
it functions as a transcriptional coactivator for
members of the TCF-LEF family of transcription
factors (2, 3). Although mutations in APC are
common in colorectal cancer, many human ma-
lignancies harboring active WNT/b-catenin sig-
naling have no identified causative mutation(s)
To identify proteins associated with the
b-catenin destruction complex, we performed a
tandem-affinity purification (TAP) of b-catenin(SA),
AXIN1, APC (amino acids 1 to 1060), b-TrCP1,
and b-TrCP2 in mammalian cells (6). The
b-catenin(SA)mutant has alanine substituted for
serine at codon 37. Specifically, cDNA for each
vector encoding a dual-affinity tag containing
streptavidin-binding protein (SBP), calmodulin-
binding protein (CBP), and the hemagglutinin
(HA) epitope (7). Lines of human embryonic
each of the tagged-bait fusion proteins were
generated, then detergent-solubilized, subjected
1Howard Hughes Medical Institute, University of Washing-
ton School of Medicine, Box 357370, Seattle, WA 98195,
ton School of Medicine, Seattle, WA 98195, USA.3Institute
for Stem Cell and Regenerative Medicine, University of
Washington School of Medicine, Seattle, WA 98195, USA.
4Department of Genome Sciences, University of Washington
School of Medicine, Seattle, WA 98195, USA.
Lunenfeld Research Institute, 983-600 University Avenue,
Toronto, Ontario, Canada M5G 1X5.6Leslie Dan Faculty of
Pharmacy, University of Toronto, Ontario, Canada, M5S 3M2.
*To whom correspondence should be addressed. E-mail:
2Department of Pharmacology, University of Washing-
VOL 31618 MAY 2007
on May 18, 2007
Fig. 1. The b-catenin protein interaction network.
Green circles represent proteins used as bait in the
tandem affinity purification, blue circles represent
known interactors, and red circles represent novel
interactors. The arrows indicate directionality for the
bait-interactor discovery, and the single asterisks (*)
show interactions that were confirmed in secondary
assays. **The protein interaction networks for b-TrCP1
and b-TrCP2 are not yet complete.
Fig. 2. WTXdirectlybindstheb-catenindestructioncomplex.(A)WTXassociateswithectopically
expressed in HEK293T cells stably expressing SBP-HA-WTX. Protein lysates were subjected to
streptavidin affinity pull-down followed by Western blot analysis. (B). WTX associates with
endogenous b-catenin and b-TrCP1. Parental HEK293T cells or HEK293T cells stably expressing
N-terminal or C-terminal pGlue-WTX were treated with WNT3a-conditioned medium (CM) for 2
hours before lysis, streptavidin-affinity pull-down assay, and Western blot analysis (ABC,
active b-catenin). (C) WTX directly binds b-catenin and b-TrCP1. GST–vesicular stomatitis virus
(VSV)–WTX recombinant protein was incubated with recombinant Cul1, b-catenin, or b-TrCP at
equal molar ratios. After GST affinity purification, protein complexes were washed with buffered
350 mM NaCl before associated proteins were resolved by Western blot. (D) WTX protein
sequences C-terminal to the region mutated in Wilms tumors bind b-catenin. (Top) The cartoon
illustrates the location of missense mutations found in Wilms tumors, as well as the N-terminal
and C-terminal WTX expression constructs used to create HEK293T stably expressing cell lines.
WNT3a CM treatment, affinity pull-down assay, and Western blotting were performed as in (B).
18 MAY 2007VOL 316
on May 18, 2007
and analyzed by liquid chromatography–tandem
mass spectrometry (LC-MS/MS). The resulting
data for all bait proteins were integrated to yield
the protein-protein interaction network of the
b-catenin destruction complex (Fig. 1 and table
S1). This proteomic analysis confirmed the pres-
ence of all the core proteins identified in previous
screens (1), including b-catenin, APC, AXIN1,
AXIN2, protein phosphatase PP2A, GSK3a,
GSK3b, and CK1a. In addition, 13 new proteins
were found to associate with known components
of the destruction complex.
We further explored WTX (FLJ39287/
FAM123B) because it copurified with each of the
baits examined. The WTX gene was recently dis-
covered to be mutated in ~30% of Wilms tumors,
activation of WNT/b-catenin signaling is common
in Wilms tumors; ~10% of tumors harbor acti-
vating mutations in b-catenin (9), and nuclear
b-cateninis observed in~50% of tumors lacking
and b-catenin mutations were mutually exclusive
in the tumor samples examined (8).
To test the hypothesis that WTX negatively
regulates WNT/b-catenin signaling in normal
kidney, we generated HEK293Tcells that stably
express pGlue-WTX (supporting online text).
(Fig. 1 and table S1). b-Catenin and b-TrCP
were among the most abundant WTX-interacting
actions of b-catenin(SA)–WTX and b-TrCP2–
WTX. To validate the WTX protein interaction
tagged fusion proteins in cells stably expressing
pGlue-WTX, isolated WTX by streptavidin af-
finitychromatography,and detectedbound FLAG-
tagged fusion proteins by Western blot (Fig. 2A).
The reverse pull-down strategy yielded iden-
tical results (fig. S1). These data demonstrate that
WTX binds both wild type b-catenin and the
stabilized b-catenin(SA)mutant (Fig. 2A and
or C-terminal tagged WTX, we next investigated
whether endogenous proteins within the destruc-
tion complex bound WTX. Streptavidin affinity
purification of WTX revealed that it associates
with endogenous b-catenin and b-TrCP (Fig. 2B
and supporting online text). Additionally, using
purified recombinant protein in vitro, we found
that WTX directly binds b-catenin and b-TrCP1,
but not the Cullin1 scaffold within the E3 ligase
complex (Fig. 2C). These results show that post-
translational modifications are not required for
WTX binding to b-catenin or b-TrCP1.
Although deletion of the WTX gene was more
commonly found in Wilms tumor samples, five
truncating mutations were identified in tumors
within the amino-terminal half of the protein (8).
As such, these mutations are consistent with the
existence of a putative tumor suppressor motif
within the C terminus of WTX. If WTX regulates
Fig. 3. WTXpromotesb-cateninubiquitinationanddegradation.(A) Acell-free
function of time. In vitro transcribed and translated35S-labeled b-catenin was
added to Xenopus egg extracts in the presence of methylated ubiquitin (MeUb)
and either purified GST orGST-WTXprotein.Measuringtheextentof35S-labeled
b-catenin ubiquitination was followed by SDS–polyacrymamide gel electropho-
resis (SDS-PAGE) and autoradiography. As a measure of specificity, LiCl (10 mM)
was added to inhibit b-catenin phosphorylation and subsequent ubiquitination.
(B) Quantification of nonubiquitinated35S-labeled b-catenin levels from (A). (C)
Recombinant GST-WTX and myelin basic protein (MBP)–AXIN1 synergize to
35S-labeled b-catenin degradation as a function of time; note absence of
methylated ubiquitin (meUb) in this experiment, as well as difference in
time scale. (D and E) WTX silencing synergizes with WNT3a CM to activate a
b-catenin–responsive luciferase reporter (pBAR) in mammalian cells. HEK293T
luciferase values were normalized to Renilla and plotted. Error bars represent
standard deviation from the mean. Data are representative of 4 independent
WTX silencing stabilizes b-catenin. RKO cells were transfected with siRNAs
targeting the indicated mRNAs. Two days after transfection, cells lysates were
subjected to Western blot analysis for the indicated proteins. IkBa, inhibitor of
nuclear factor kB and a b-TrCP substrate induced by tumor necrosis factor–a
stimulation, as well as b-tubulin, demonstrate equal protein loading in the blots.
VOL 316 18 MAY 2007
on May 18, 2007
kidney biology through negative regulation of Download full-text
WNT/b-catenin signaling, then we should be able
to ascribe a WNT-related function to the C ter-
minus of WTX. Therefore, we mapped the do-
mainof WTXthat interacts with b-catenin and
found that b-catenin purified with full-length
but interacted poorly with the N-terminal half
(WTX-N) (Fig. 2D and fig. S2 and supporting
online text). As additional confirmation, we used
our TAP–LC-MS/MS analysis on cells express-
ing pGlue–WTX-C and found both b-TrCP and
b-catenin within the protein complex (table S1).
Thus, mutational alteration of WTX in Wilms
The direct binding of WTX to both b-catenin
and to its E3 ubiquitin ligase adaptor, b-TrCP,
suggests that WTX regulates b-catenin degrada-
tion. We tested this hypothesis using cell-free
Xenopus egg extracts, an experimental system
that allows quantitative monitoring of b-catenin
of recombinant glutathione S-transferase (GST)
in complex with WTX protein increased the rate
of b-catenin ubiquitination, but GST control did
not (Fig. 3, A and B, and fig. S3). Inhibition of
GSK3 by lithium chloride (LiCl) suppressed
b-catenin ubiquitination in the presence of GST
and GST-WTX. As a scaffold protein, AXIN nu-
turnover in Xenopus extracts (11). When WTX
and AXIN1 were added to the extracts individ-
ually, each increased the rate of b-catenin degra-
dation (Fig. 3C). When WTX and AXIN1 were
added together, the rate of b-catenin degradation
was more rapid than observed with either alone.
These data suggest that WTX negatively regu-
lates WNT signaling by promoting b-catenin
If WTX promotes b-catenin degradation,
WNT/b-catenin signaling in mammalian cells.
To test this prediction, we measured the activity
of a b-catenin–dependent transcriptional reporter
after small interfering RNA (siRNA)-mediated
silencing of WTX. Specifically, HEK293T hu-
man embryonic kidney cells and RKO human
colon carcinoma cells were transduced with
lentiviruses encoding a firefly luciferase–based
b-catenin–activated reporter (pBAR), along with
of the constitutively active thymidine kinase
promoter for normalization. To validate the
dynamic range of this reporter system, stably
transduced cell lines were treated with WNT3a-
conditioned medium, which activated the re-
porterby afactorof 100 to300(Fig.3,DandE).
As a control, we showed that siRNAs directed
against b-catenin abolished this WNT3a-induced
reporter activity in both cell lines (fig. S4 and
supporting online text). Using this assay system,
we found that two different siRNAs targeting
WTX produced an increase in WNT3a-induced
reporter activity in both cell types. Furthermore,
in RKO-pBAR/Renilla cells, siRNA-mediated
2 synergized with a GSK3 inhibitor, (2′Z,3′E)-6-
bromoindirubin-3′-oxime, to activate the pBAR
reporter (fig. S4). These data suggest that WTX is
a negative regulator of WNT/b-catenin signal
transduction in mammalian cells.
We next tested whether silencing of WTX with
siRNAsincreases b-cateninlevelsin cells.In RKO
cells, b-catenin does not localize to the plasma
membrane, whereas in other cell types, such as
HEK293T cells, it resides with a relatively long
half-life at the inner surface of the plasma mem-
brane. Thus, in the absence of membrane-
in RKO cells are very low, which allows study of
cytoplasmic and nuclear b-catenin stability in re-
ly transfected RKO cells with siRNAs targeting
WTX, b-catenin, AXIN1 and 2, or b-TrCP1 and
2. Silencing of WTX, AXIN1 and 2, or b-TrCP1
and 2, but not b-catenin, was found to increase
b-catenin levels, as determined by immunoblot
cells as a negative regulator of both b-catenin pro-
To extend these experiments to organisms,
we performed gain-of-function experiments in
Xenopus embryos and loss-of-function experi-
ments in zebrafish (supporting online text).
Ectopic activation of WNT/b-catenin signaling
embryo ventral blastomeres induced duplication
of the embryonic axis, yielding two-headed
tadpoles (fig. S5). Injection of WTX mRNA
blocked Xenopus Wnt8–induced axis dupli-
cation. In developing zebrafish embryos, ectopic
activation of WNT/b-catenin signaling leads to
anterior truncations. When we silenced endoge-
nous zebrafish wtx expression, we observed
anterior truncations and the activation of a
WNT/b-catenin reporter gene (fig. S5). These
results suggest that WTX is a negative regulator
of WNT/b-catenin signaling in vivo.
In summary, these data establish that the
cancer-associated WTX protein is a required
component of the b-catenin destruction complex.
Furthermore, our data underscore the power of
proteomic approaches for identifying new com-
ponents of cellular signal transduction pathways
that may ultimately provide important mechanis-
tic insights into human disease.
References and Notes
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2. C. Y. Logan, R. Nusse, Annu. Rev. Cell Dev. Biol. 20, 781
3. K. Willert, K. A. Jones, Genes Dev. 20, 1394 (2006).
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Nat. Rev. Genet. 5, 691 (2004).
5. H. Clevers, Cell 127, 469 (2006).
6. Materials and methods are available as supporting
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9. R. Koesters et al., Cancer Res. 59, 3880 (1999).
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12. Purified b-catenin was a kind gift from W. Xu, University
of Washington, Seattle. C.H. is supported by a post-
doctoral F32 NIH National Research Service Award
Supporting Online Material
Materials and Methods
Figs. S1 to S6
Tables S1 and S2
20 February 2007; accepted 30 March 2007
Revisiting the Role of the Mother
Centriole in Centriole Biogenesis
A. Rodrigues-Martins,1,2M. Riparbelli,3G. Callaini,3D. M. Glover,2* M. Bettencourt-Dias1,2*
Centrioles duplicate once in each cell division cycle through so-called templated or canonical
duplication. SAK, also called PLK4 (SAK/PLK4), a kinase implicated in tumor development, is an
upstream regulator of canonical biogenesis necessary for centriole formation. We found that
overexpression of SAK/PLK4 could induce amplification of centrioles in Drosophila embryos and
their de novo formation in unfertilized eggs. Both processes required the activity of DSAS-6 and
DSAS-4, two molecules required for canonical duplication. Thus, centriole biogenesis is a template-
free self-assembly process triggered and regulated by molecules that ordinarily associate with the
existing centriole. The mother centriole is not a bona fide template but a platform for a set of
regulatory molecules that catalyzes and regulates daughter centriole assembly.
centrioles duplicate in coordination with the cell
entrioles are essential for the formation
of cilia and flagella and for the organi-
zation of the centrosome (1). Normally,
onally to each old one, the mother (1), in S
phase. This led to the idea that the mother cen-
triole templates the formation of the daughter
(2, 3). However, daughter centrioles do not incor-
porate a substantial proportion of the mother (4),
18 MAY 2007VOL 316
on May 18, 2007