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Cell regulation by the Apc protein
Apc as master regulator of epithelia
Brooke M McCartney1 and Inke S Näthke2
The adenomatous polyposis coli (Apc) protein participates in
many of the fundamental cellular processes that govern
epithelial tissues: Apc is directly involved in regulating the
availability of b-catenin for transcriptional de-repression of Tcf/
LEF transcription factors, it contributes to the stability of
microtubules in interphase and mitosis, and has an impact on
the dynamics of F-actin. Thus Apc contributes directly and/or
indirectly to proliferation, differentiation, migration, and
apoptosis. This particular multifunctionality can explain why
disruption of Apc is especially detrimental for the epithelium of
the gut, where Apc mutations are common in most cancers. We
summarise recent data that shed light on the molecular
mechanisms involved in the different functions of Apc.
Addresses
1
Department of Biological Sciences, Carnegie Mellon University, 4400
5th Avenue, Pittsburgh, PA, USA
2
College of Life Sciences, Cell & Dev. Biology, University of Dundee,
Dow Street, Dundee, Scotland, UK
Corresponding author: Näthke, Inke S ([email protected])
Current Opinion in Cell Biology 2008, 20:186–193
This review comes from a themed issue on
Cell regulation
Edited by Alan Hall and Joan Massagué
Available online 24th March 2008
0955-0674/$ – see front matter
Published by Elsevier Ltd.
DOI 10.1016/j.ceb.2008.02.001
The initial identification of the adenomatous polyposis coli
gene as the site of mutations in the familial form of colon
cancer, familial adenomatous polyposis (FAP), was
described in 1992 [1,2]. A causal relationship between
Apc mutations and intestinal tract tumours was confirmed
three years later with the establishment of the Min mouse
model [3]. These mice are heterozygous for Apc and
develop numerous intestinal tumours that mimic FAP.
Subsequently, Apc has emerged as the most commonly
mutated gene in colorectal cancer with reports of between
50 and >80% of sporadic tumours carrying such
mutations. The search for how mutations in Apc initiate
and/or support progression of tumours in the intestinal
tract revealed that Apc is a multifunctional participant in a
diverse array of cellular functions. In this review, we
describe these diverse functions and their relationship
to epithelial biology, and discuss recent significant
Current Opinion in Cell Biology 2008, 20:186–193
advances in our understanding of their underlying molecular mechanisms.
Wnt pathway regulation
The first recognised function of Apc was its role in Wnt
signalling [4,5]. This function is one of the driving
forces for how mutations in Apc ensure that cells remain
proliferative. Many of the molecular details of this pathway have been described extensively in many reviews [6].
Apc negatively regulates Wnt signalling by participating
in the destruction complex, a complex that targets the key
effector b-catenin for degradation (reviewed in [7]).
However, the precise role of Apc in the destruction
complex has not been clear. A number of recent papers
employing largely biochemical and structural approaches
have shed significant light on this question. The central
region of the Apc protein contains three distinct types of
repeats that are essential to its role in the negative
regulation of Wnt signalling (Figure 1). Seven 20 amino
acid repeats (20R) and three 15 amino acid repeats (15R)
function in the binding and degradation of b-catenin,
while three SAMP repeats bind to the RGS domain of
Axin, a partner in the destruction complex (reviewed [7]).
It has long been appreciated that phosphorylation of this
central repeat region of APC significantly enhances its
affinity for b-catenin [8]. The degree of conservation
between repeats of the same type suggested that they
might be functionally equivalent. However, recent work
has clearly demonstrated that individual 15R and 20R
differ significantly in their binding affinities for b-catenin
[9]. This difference seems to be achieved in part by the
N-terminal flanking regions of the repeats [9]. The 20R
with the highest binding affinity to b-catenin (R3, 4 and 5)
also contain a typical Tcf-like nine amino acid b-cateninbinding motif in their N-terminal flanking regions. The
Tcf/LEF family of transcription factors partner with bcatenin in the nucleus to activate Wnt target genes.
Changing a key aspartate residue in the flanking region
to alanine resulted in a 30-fold reduction in the binding
affinity for b-catenin. When the repeats are phosphorylated by the cooperative action of CK1 and GSK3b, the
binding interaction is significantly altered and enhanced.
Phosphorylation of any of the 20R, even those with no
detectable binding to b-catenin in a non-phosphorylated
state, increased their affinity for b-catenin dramatically.
When phosphorylated, binding of the 20R to b-catenin
was no longer modulated by the key aspartate in the Nterminal flanking region. Although the phosphorylation
sites of Apc have not been identified in vivo, the fact that
CK1 and GSK3b are endogenous components of the
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Regulation of cellular functions by the Apc protein McCartney and Näthke 187
Figure 1
Linear sequence schematic of Apc.The human Apc protein with 2843 amino acids is shown with the location of different functional sites.
destruction complex suggests that the CK1 and GSK3b
phosphorylation sites identified in vitro may be physiologically relevant [9].
These data significantly extend recently reported structural studies of the interaction between b-catenin and the
20R of Apc [10,11]. These studies demonstrated that
when phosphorylated, 20R3 and its N-terminal and Cterminal flanking sequences make contact with the entire
structural-binding groove of b-catenin. This contrasted
significantly with binding of unphosphorylated 20R3
[10]. Phosphorylated Apc can compete with Axin for
binding to b-catenin [10,12], but these proteins can
bind simultaneously when Apc is not phosphoryated
[10]. These observations suggest a model where phosphorylation of Apc is important for the recruitment and
turnover of b-catenin in the destruction complex. Phosphorylation of Apc binds Axin and b-catenin simultaneously, helping to bring these components together
(Figure 2). Once complexed, dephosphorylation of Apc
reduces its affinity for b-catenin, and b-catenin is transferred to Axin, but retains a reduced interaction with Apc.
Figure 2
Cycling of Apc phosphorylation may drive destruction complex function.(1) Apc with phosphorylated 20Rs has high affinity for b-catenin and may
facilitate the entry of b-catenin into the complex (2). (3) Dephosphorylation of Apc, perhaps by PP2A, drives the hand-off of b-catenin to Axin where it
can be phosphorylated by GSK3b. (4) P-b-catenin is identified by b-TrCP, the F-box subunit of the E3 ubiquitin ligase, ubiquitinated and degraded by
the proteosome. Modified from [7].
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Current Opinion in Cell Biology 2008, 20:186–193
188 Cell regulation
b-catenin phosphorylated by Axin-associated GSK3b is
targeted for ubiquitination and degradation by the proteosome. It is tempting to speculate that the phosphatase
responsible for dephosphorylating Apc is PP2A. This
phosphatase associates with Axin [13–15] and its regulatory subunit B56 interacts with both Axin and Apc through
its Arm repeats [16,17]. Consistent with this model,
mutations that disrupt the Arm repeats of Drosophila
APC2 also disrupt the function of the destruction complex [18]. Ha et al. tested this model by exposing the
phosphorylated-Apc/b-catenin complex to PP1, a biochemically tractable phosphatase similar to PP2A.
Although PP1 could effectively dephosphorylate Apc
alone, Apc complexed with b-catenin was resistant to
its activity [10]. Although PP2A within the destruction
complex may function differently, the mechanisms
for dephosphorylating Apc within the complex clearly
require more careful examination. Cycling of Apc
phosphorylation has also been suggested to affect the
release of b-catenin from the complex [12]. However,
the presence of Apc in a complex with b-catenin and the
ubiquitin ligase subunit b-TrCP [19] suggests that complete release from the destruction complex may not be
necessary to hand over phosphorylated b-catenin to the
proteosome [10] (Figure 2).
Although many details of this pathway remain unknown,
it is clear that phosphorylation of the 20R is a key step in
regulating the binding of Apc to b-catenin. Furthermore,
the different binding properties of each repeat suggest
that they may play different roles in distinct cellular
contexts [9]. In the unphosphorylated state, the binding affinities of any of the 20R except 20R3 are likely too
low to be relevant. The different binding affinities in the
phosphorylated state may be important to fine tune
the Apc/b-catenin interactions that occur not only in
the destruction complex, but also at other sites in the
cell including the nucleus (see below), the cortex [20,21],
and at the centrosome [22]. Further dissection of the
repeats using a wide range of functional assays will be an
essential step to understand this interaction. In the Wnt
signalling context, identification of 20R3 as the highest
affinity b-catenin binding repeat provides a more
detailed explanation for the observation that the
mutation cluster region (between amino acids 1250
and 1450 (Figure 1)) is associated with human colon
cancer. Truncated proteins resulting from these
mutations lack all of the Axin-binding SAMP repeats
as well as 20R3–7, retaining only two of the weakest
binding repeats 20R1-2.
Nuclear Apc
Phosphorylation of Apc is emerging as a key mechanism
for regulating Apc function in the canonical destruction
complex as well as in the regulation of Apc’s subcellular
distribution. As part of its role as a negative regulator of
Wnt signalling, Apc proteins shuttle in and out of the
Current Opinion in Cell Biology 2008, 20:186–193
nucleus where they interact with b-catenin, CtBP, and
other proteins involved in transcription (reviewed in
[23]). Consistent with these observations, Apc contains
two classic monopartite basic nuclear localisation signals
in the C-terminal half of the protein and an additional
domain within the Arm repeats that facilitates nuclear
import (Figure 1). The regulation of Apc’s nuclear import
is complex and involves a number of factors including the
activity of several NESs (Figure 1), sequestration of Apc
at non-nuclear sites by interactions with other protein
partners, and regulation of NLS activity (reviewed in
[23]). Regulation of NLS activity involves, at least in
part, the phosphorylation of NLS flanking sites where
phosphorylation by casein kinase 2 (CK2) may enhance
NLS activity, while phosphorylation by protein kinase A
(PKA) inhibits NLS activity [24]. Recent evidence
suggests that p38 MAPK and its regulator Gadd45a act
to enhance the nuclear import of Apc by enhancing the
activity of CK2 and suppressing the activity of PKA [25].
The relationship between Apc and CK2 is complicated
not only does CK2 appear to phosphorylate Apc but the
direct binding of Apc to CK2 inhibits CK2 activity [26],
suggesting that Apc imposes negative feedback on its own
nuclear localisation.
What is the function of nuclear Apc? Mounting evidence
favours the hypothesis that Apc sequesters nuclear bcatenin. This is supported by careful observations of
nucleo-cytoplasmic shuttling of b-catenin using live cell
microscopy and fluorescence recovery after photobleaching (FRAP) [27], which revealed that none of the factors
that bind b-catenin in the nucleus, including positive
effectors TCF4, BCL9/Pygopus and negative regulators
Apc and Axin influence the rate of b-catenin nuclear
import and export. Rather, the data suggest that these
proteins affect the compartmentalisation of b-catenin by
sequestering it at specific sites. Taken together, these
data support a model whereby p38 MAPK signalling
activates CK2 to phosphorylate Apc, promoting its
nuclear import where it acts to sequester b-catenin,
perhaps in cooperation with CtBP [28], and so downregulates b-catenin activity. An interesting twist to this
model was revealed by the finding that CK2 phosphorylates LEF1, and is required for the assembly and turnover of b-catenin/LEF1 nuclear complexes [29,30].
However, nuclear Apc may not be the only pool of Apc
that regulates nuclear b-catenin. A recent study of C.
elegans Apc, APR-1, suggests that cortical APR-1 functions
to regulate the Wnt pathway primarily by facilitating the
nuclear export of WRM1, a worm b-catenin [31]. Furthermore, although there has been significant progress in
understanding the mechanisms that regulate nuclear
Apc, its function in this compartment is not clear. There
are many ideas that have not been explored in detail,
particularly the possibilities that Apc itself may interact
with DNA [32], and components of the base excision
repair pathway [33].
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Regulation of cellular functions by the Apc protein McCartney and Näthke 189
Figure 3
Apc in intestinal tissue.(a) In wild-type intestinal tissue, stem cells near the base of the crypt (red), give rise to transit amplifying cells (yellow) that
differentiate into absorptive epithelial cells (green), neuroendocrine (brown), Paneth (blue), or Goblet (pink) cells. Differentiation is accompanied by
active migration along the crypt-villus axis. Near the top of villi in the small intestine (or in the collar of the crypt in the colon, where villi are not present),
cells are extruded and shed into the gut lumen while undergoing apoptosis (dark green). Apc contributes to this tissue organisation by supporting
differentiation, thus keeping proliferation restricted to the appropriate compartment, and migration to ensure continuous efflux of cells from the
proliferative compartment. It also contributes to apoptosis, and can enhance or inhibit cell death depending on the cellular context (position, tissue
type etc.). (b) In the absence of Apc, or when only mutated Apc is present, cells do not differentiate normally; they remain proliferative and retain some
stem cell characteristics (red-yellow). They also fail to migrate normally. Thus cells accumulate and form pre-malignant polyps that can deteriorate into
adenoma and carcinoma. The top of villi retains normal enterocytes, because each villus is supplied with new cells by many crypts and usually only one
crypt becomes malignant.
Apc in apoptosis
Mutations in Apc may also promote changes in apoptosis,
a crucial component of epithelial biology in the intestinal
tract. When cells reach the luminal collar of the colonic
crypt or the tip of the intestinal villus they are extruded
from the tissue while undergoing apoptosis and are shed
into the lumen of the gut (Figure 3) [34]. Although the
molecular mechanism for a role for Apc in apoptosis has
not been identified, evidence for a role of Apc in the
normal execution of an apoptotic program is accumulating
(reviewed in [35]). Whether components of the cell death
pathway are direct targets of Wnt signalling has yet to be
elucidated, though screens for Wnt target genes have
detected changes in genes encoding proteins directly
involved in the execution of apoptosis [36,37]. However,
other changes induced by Apc mutations (see below) may
also contribute [38]. Importantly, the overall balance of
pro-apoptotic and anti-apoptotic changes induced by Apc
loss may be highly context- and tissue-specific and may
cause increased or decreased apoptosis, depending on
tissue type, tissue compartment, developmental stages,
etc.
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Apc as a cytoskeletal regulator important in
migration, orientation and division
The transcriptional changes induced by mutant Apc provide the background against which the other cellular
functions of Apc become particularly important in the
digestive tract epithelium. In addition to its function in
the b-catenin destruction complex, Apc can also directly
contribute to the regulation of cytoskeletal proteins: it
binds directly and indirectly to microtubules and also
interacts with a number of actin-regulatory proteins
(Figures 1 and 4). So it is not surprising that loss of Apc
leads to changes in cell migration, cell orientation, polarity
and division [38,39,40,41]. Transcriptional changes
induced by Apc mutations also contribute to these defects;
nonetheless, direct interactions of Apc with cytoskeletal
elements contribute significantly and directly.
The stabilising effect on microtubules by Apc has been
established biochemically [42,43] and in cells [39,44]
and may be affected by the interaction of Apc with the
microtubule plus tip-binding protein EB1 [45]. The ability of C-terminal fragments of Apc to nucleate the assembly
Current Opinion in Cell Biology 2008, 20:186–193
190 Cell regulation
Figure 4
Figure 5
Apc complexes in migrating cellular protrusions. A number of proteins
support the linkage between Apc and microtubules, but also F-actin in
migrating cellular protrusions near the plasma membrane. Whether all of
these interactions can occur at the same time, and how they affect each
other is not known.
Apc in mitotic spindles.Apc (red dot) localises to spindle microtubules,
concentrates at kinetochores, centrosomes and at sites of cortical
attachment. At these different sites Apc may co-operate with the
indicated proteins to support microtubule attachment and dynamics at
both kinetochores and cortical sites, but also at centrosomes.
of F-actin in vitro [46] together with the ability of Apc to
interact with a number of actin-regulatory proteins including ASEF, an exchange factor for CDC42 and Rac,
and IQGAP, suggests that Apc also contributes to the
regulation of the cellular F-actin network [47,48]. The
idea that Apc helps to co-ordinate the F-actin and microtubule cytoskeletons is further supported by the interaction of Apc with mDia, a protein that can affect both
actin and microtubules [49] (Figure 4). How these interactions are co-ordinated, whether they occur simultaneously or whether these proteins compete with each
other, and how they contribute individually or together to
the localised recruitment of Apc clusters in migrating
cellular protrusion is not at all clear and will require careful
biochemical and functional experiments (Figure 4).
function of the microtubule-based mitotic spindle
(Figure 5). Loss of Apc leads to defects in spindle
alignment [51], and the mitotic spindle checkpoint
[38], and to the development of anaphase bridges
[52]. An immediate consequence of these changes is
the accumulation of tetraploid cells, not only in culture,
but also in intestinal tissue depleted of Apc [38]. Similar
defects are observed in cells expressing N-terminal fragments of Apc [53] and intestinal tissue from Min mice
displays failures in cytokinesis [54]. The expression of Nterminal fragments of Apc, similar to those present in
human tumours, may disrupt normal Apc function by
forming complexes with wild-type Apc and interfering
with its normal interactions [55]. One possible interaction
that may be affected by such Apc heterodimeric complexes is the binding of Apc to EB1 [56].
Nonetheless, it has been clearly established that these
interactions contribute to a number of cellular processes
that are crucial for gut epithelium maintenance, most
prominently in cell migration. Differentiating epithelial
cells in the gut actively migrate upwards from the crypts
and loss of Apc leads to a decrease in this migration [50]
(Figure 3). Similarly, depleting Apc from cultured cells
leads to a decrease in the ability to migrate in a directed
fashion [39] that is accompanied by a decrease in overall
microtubule stability and loss of polarisation of acetylated
microtubules towards the leading edge [39,44]. Similar
to migrating epithelial cells, highly polarised migrating
astrocytes also accumulate Apc at their leading edge and
loss of Apc causes a loss of migration in these cells [40].
The cytoskeletal interactions of Apc not only affect cells
in interphase but also contribute significantly to the
Current Opinion in Cell Biology 2008, 20:186–193
The interaction between Apc and EB1 and thus dynein/
dynactin, which binds to EB1 directly, may also contribute to the ability of Apc to support anchoring of astral
microtubules at cortical sites (Figure 5) [54,57–59]. Similarly this interaction may contribute to the function(s) of
Apc at the centrosome [22]. Together these functions of
Apc in the mitotic spindle could explain the spindle
positions defects in Apc mutant cells [53].
Apc in whole tissue
Much of the evidence for the functions of Apc is based on
data obtained in cultured cells. However, important
insights into how these functions contribute to tissue
homeostasis and tumourigenesis have been gained using
animal models. Mice with constitutive or conditional
mutations in Apc confirmed its contribution to differenwww.sciencedirect.com
Regulation of cellular functions by the Apc protein McCartney and Näthke 191
tiation, migration, division and genetic stability: both Min
mice and PIRC rats are heterozygous for Apc mutations,
similar to FAP patients, and both develop tumours in the
digestive tract similar to those in human FAP patients
[60]. Conditional depletion of Apc specifically in the
intestine results in major changes in cell differentiation,
proliferation, ploidy and apoptosis consistent with the
findings in cell culture systems [50]. Importantly, the
defects induced by acute loss of Apc in gut tissue do not
occur when c-myc is also missing [61]. Cells lacking both
of these genes are selectively depleted from intestinal
tissue within a few weeks. It is possible that a downstream
target of c-myc that is upregulated when Apc is lost is
responsible for the migration defect observed in Apcdepleted cells. One difficulty in measuring a change in
migration in c-myc-depleted cells in this context is that
these cells do not survive and are out-competed rapidly
by wild-type cells in this tissue. This means that mixed
populations of cells, plus and minus c-myc, co-exist and it
is impossible to resolve how mixing between such cells
affects migration and general cell behaviour. However,
during the lifetime of these double-mutant cells in intestinal tissue, b-catenin is nuclear suggesting that it is
acting as a transcriptional activator [61]. These data
suggest that upregulation of c-myc is absolutely required
for Apc-deficient cells to survive.
Conclusion
Many of the defects that result from Apc-deficiency can
be overcome by overexpressing other components of
the relevant pathways: overexpression of Axin can rescue b-catenin regulation in Apc-deficient cells [62], cell
cycle checkpoint deficiency can be rescued by overexpressing Bub1 [38]. Furthermore, Apc is not absolutely required for any of the processes discussed above
and most of them continue to proceed, albeit less
efficiently in its absence: cells still move, but more
slowly and not as directed; cells divide, but make more
mistakes in this process; apoptotic pathways still function, but are not initiated appropriately in response to
certain checkpoints [38]. In support of the notion that
Apc is not essential for these processes, Drosophila
completely lacking Apc do not exhibit detectable
defects in mitosis, cytokinesis, cell polarity, or adhesion,
but lethality appears to result primarily from excessive
Wnt signalling [18]. A role for Apc in ensuring
accuracy and efficiency of many cellular processes
explains why Apc mutations are particularly detrimental
for tissues that rely on the fidelity of these processes.
The cumulative destabilisation that results from Apc
disruption makes Apc a perfect tumour suppressor,
particularly in gut epithelial tissues: loss of Apc leads
to a large increase in mistakes in the processes that
govern the overall homeostasis of this tissue and these
mistakes are not corrected, instead they are tolerated
allowing these oncogenic changes to propagate.
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References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
of special interest
of outstanding interest
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tion that when phosphorylated not only does the 20R itself interact with bcatenin, but N-terminal and C-terminal flanking regions make contact as
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Current Opinion in Cell Biology 2008, 20:186–193
This paper provided key evidence that the NLS activity of Apc is regulated
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export. Instead, these binding partners may act to sequester b-catenin in
specific cellular compartments. This elegant study strongly supports the
model that one function of Apc in the nucleus is to sequester b-catenin
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This paper along with [29] provide evidence for additional roles for casein
kinase 2 (CK2) in Wnt signalling. Not only does CK2 appear to regulate the
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31. Mizumoto K, Sawa H: Cortical beta-catenin and APC regulate
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www.sciencedirect.com
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39. Kroboth K, Newton IP, Kita K, Dikovskaya D, Zumbrunn J,
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This paper shows that Apc status correlates with over microtubule
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40. Etienne-Manneville S, Hall A: Cdc42 regulates GSK-3beta and
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This paper provides important link between small GTPases and Apc in
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41. Etienne-Manneville S, Manneville JB, Nicholls S, Ferenczi MA,
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42. Munemitsu S, Souza B, Müller O, Albert I, Rubinfeld B, Polakis P:
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This is the first demonstration that Apc can bind to microtubules.
43. Zumbrunn J, Inoshita K, Hyman AA, Näthke IS: Binding of the
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and in cells and provides evidence that this function can be regulated by
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44. Kita K, Wittmann T, Näthke IS, Waterman-Storer CM: APC on
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This paper demonstrates direct link between endogenous Apc protein
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This paper identifies EB1 as an Apc binding partner and thus brings Apc
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46. Moseley JB, Bartolini F, Okada K, Wen Y, Gundersen GG,
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Identifies Apc as a regulator for the activity of an exchange factor for small
GTP-ases thus establishing a functional link between Apc and actin.
48. Watanabe T, Wang S, Noritake J, Sato K, Fukata M, Takefuji M,
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49. Wen Y, Eng CH, Schmoranzer J, Cabrera-Poch N, Morris EJ,
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www.sciencedirect.com
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Shows that conditionally inactivating Apc in the gut immediately leads to
changes that underlie tumourigenesis.
51. Draviam VM, Shapiro I, Aldridge B, Sorger PK: Misorientation and
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This paper demonstrates a dominant effect of N-terminal Apc fragments
in mitosis.
54. Caldwell CM, Green RA, Kaplan KB: APC mutations lead to
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59. McCartney BM, McEwen DG, Grevengoed E, Maddox P,
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60. Amos-Landgraf JM, Kwong LN, Kendziorski CM, Reichelderfer M,
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modeling of familial human colon cancer. Proc Natl Acad Sci U
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This paper introduces a novel rat model for Apc-mediated tumourigenesis
that mimics the human disease more accurately.
61. Sansom OJ, Meniel VS, Muncan V, Phesse TJ, Wilkins JA,
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This paper demonstrates that Myc is absolutely required for Apc-deficient
cells to survive in intestinal tissue and form tumours and that de-regulated, nuclear b-catenin is not sufficient.
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Current Opinion in Cell Biology 2008, 20:186–193