www.landesbioscience.com Cell Cycle 285
Cell Cycle 10:2, 285-292; January 15, 2011; © 2011 Landes Bioscience
Nuclear translocation of Skp2 facilitates
its destruction in response to TGFβ signaling
*Correspondence to: Yong Wan; Email: email@example.com
Submitted: 09/28/10; Revised: 12/13/10; Accepted: 12/16/10
Transforming Growth Factor β (TGFβ) signaling regulates a
variety of cellular processes, including cell proliferation, differ-
entiation, apoptosis and fate specification during embryogenesis.
Transduction of the complex signaling starts on the cell sur-
face where TGFβ binding induces the formation of type I and
II receptor complex. Type II receptor phosphorylates the GS
domain of Type I receptor, resulting in kinase activation. Type
I receptor then propagates the signal through phosphorylation
of Smad2 or Smad3 (receptor-regulated Smad, R-Smad) on the
carboxy-terminal SXS motif. Upon phosphorylation, Smad2 or
Smad3 releases from SARA (Smad anchor for receptor activa-
tion), forms oligomeric complex with Smad4 (co-mediator Smad,
Co-Smad) and translocates to the nucleus where they regulate
target gene transcription in collaboration with DNA-binding co-
factors such as forkhead family member FOXH1, co-activators
such as p300 or co-repressors such as Ski.1,2
TGFβ signaling plays a critical role in tumorigenesis of sev-
eral types of epithelia, paradoxically switching from a role as a
tumor suppressor to a promoter of invasiveness and metastasis
during tumor progression.3 The cytostatic effect of TGFβ in
Dong Hu,† Weijun Liu,† George Wu and Yong Wan*
Department of Cell Biology and Physiology; Hillman Cancer Center; University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute; Pittsburgh, PA USA
†These authors contributed equally to this work.
Key words: Skp2, nuclear translocation, ubiquitylation, TGFβ
Abbreviation: Skp2, S-phase kinase-associated protein 2; TGFβ, transforming growth factor β; SCF, SKP1-CUL1-F-box protein;
APC, anaphase promoting complex; R-smad, receptor-regulated smad; SARA, smad anchor for receptor activation;
Co-Smad, co-mediator smad; CDKI, cyclin-dependent kinase inhibitors
early stages of tumorigenesis primarily involves activated tran-
scription of the cyclin-dependent kinase inhibitors (CDKI)
p15,4 p21,5 and p57,6 and repressed transcription of the growth
promoting transcription factors c-Myc and Id. Repression of
c-Myc and Id facilitates the induction of p15 and p21, which
is a critical prerequisite for the execution of the full cytostatic
response of epithelial cells to TGFβ.7-11 Increased expression of
p15 facilitates its binding to CDK4 and CDK6 and displaces p27
from these kinases to cyclin E-CDK2, causing CDK2 inhibi-
tion. Concomitantly, p21 is induced and binds to CDK2 as well,
ensuring a maximal suppression of CDK2 activity and cell cycle
arrest at G1/S transition.12 In addition to transcriptional control,
the proteolytic regulation of CDKIs is also involved in cell cycle
arrest, as mediated by TGFβ. Our recent studies demonstrate
that p27, a short-lived regulatory protein, is stabilized in response
to TGFβ stimulation through Skp2SCF degradation targeted by
Cdh1/APC.13 Since p27 is constitutively degraded by Skp2SCF,
the stabilization of p27 by TGFβ facilitates its complete and suc-
cessful redistribution from CDK4 and CDK6 to cyclin E-CDK
to achieve arrest of cell growth.
Although our findings indicate that TGFβ-induced Skp2
degradation is governed by Cdh1/APC, the mechanism by which
Skp2, a F-box protein that determines the substrate specificity for SCF ubiquitin ligase, has recently been demonstrated
to be degraded by Cdh1/APC in response to TGFβ signaling. The TGFβ-induced Skp2 proteolysis results in the stabilization
of p27 that is necessary to facilitate TGFβ cytostatic effect. Previous observation from immunocytochemistry indicates
that Cdh1 principally localizes in the nucleus while Skp2 mainly localizes in the cytosol, which leaves us a puzzle on how
Skp2 is recognized and then ubiquitylated by Cdh1/APC in response to TGFβ stimulation. Here, we report that Skp2 is
rapidly translocated from the cytosol to the nucleus upon the cellular stimulation with TGFβ. Using a combinatorial
approach of immunocytochemistry, biochemical-fraction-coupled immunoprecipitation, mutagenesis as well as
protein degradation assay, we have demonstrated that the TGFβ-induced Skp2 nucleus translocation is critical for TGFβ
cytostatic effect that allows physical interaction between Cdh1 and Skp2 and in turn facilitates the Skp2 ubquitylation
by Cdh1/APC. Disruption of nuclear localization motifs on Skp2 stabilizes Skp2 in the presence of TGFβ signaling, which
attenuates TGFβ-induced p27 accumulation and antagonizes TGFβ-induced growth inhibition. Our finding reveals a
cellular mechanism that facilitates Skp2 ubiquitylation by Cdh1/APC in response to TGFβ.
286 Cell Cycle Volume 10 Issue 2
Skp2 protein levels is due to altered transcriptional regulation,
we measured Skp2 mRNA levels after stimulation with TGFβ
by RT-PCR. As shown in Figure 1A, Skp2 mRNA remains con-
stant, suggesting that the drop in Skp2 protein levels is due to
Previous study indicated that Skp2 protein abundance is regu-
lated by Cdh1/APC during cell cycle.18,19 We previously demon-
strated that Cdh1/APC activity is enhanced in response to TGFβ
stimulation.20 Thus, we tested the possibility that Cdh1/APC is
a putative E3 ligase that governs the TGFβ-induced Skp2 deg-
radation. As shown in Figure 1D and E, depletion of Cdh1 by
RNA interference in Mv1Lu results in significant attenuation of
Skp2 degradation in response to TGFβ, while Skp2 is drastically
destroyed in response to TGFβ in the wild-type cells.
To evaluate the biological significance of Skp2 degradation
upon TGFβ stimulation, we constructed a stable Skp2 with
deletion of its destruction box (ubiquitin-targeting-degron) and
further engineered a Mv1Lu cell that stably expresses stabilized
Skp2 (D box deletion) (Fig. 1F). As shown in Figure 1F and G,
failure of Skp2 proteolysis in response to TGFβ stimulation sig-
nificantly antagonizes TGFβ-induced growth inhibition. Taken
all together, our results suggest an important role for Skp2 prote-
olysis by Cdh1/APC in TGFβ-mediated growth inhibition.
Nuclear localization of Skp2 induced by TGFβ promotes its
recognition and degradation by Cdh1/APC. Previous work sug-
gested that the cellular presence of Cdh1 is in the nucleus.21,22
Localization of Skp2 has been variable depending on cell type.23-
27 To assess the localization of Cdh1 and Skp2 in Mv1Lu cell, we
performed immunocytochemistry in the presence and absence
of TGFβ. Localization of Cdh1 was measured by using mono-
clonal antibody against Cdh1 and FITC conjugated anti-mouse
secondary antibody. As show in Figure 2A, expression of Cdh1
is localized in nucleus, which is consistent with previous observa-
tion. To test the localization of Skp2, we used rabbit antibody
against Skp2 coupled with Texas Red conjugated secondary
antibody. As shown in Figure 2A, the majority of Skp2 in the
absence of TGFβ is localized in the cytosol, which is consistent
with known previous Skp2 localization in prostate, melanoma,
colon, lymphoma and breast cancer tissues.23-27 To examine the
potential effect of TGFβ stimulation on cellular translocation of
Cdh1 and Skp2, a similar study was performed as described in
the above. As shown in Figure 2A, TGFβ stimulation results in
significant translocation of Skp2 from the cytosol to the nucleus,
while no obvious redistribution of Cdh1 was observed.28 The
finding of TGFβ-induced Skp2 nuclear translocation provides
APC is regulated in response to TGFβ signaling and how Skp2 is
ubiquitylated for destruction by Cdh1/APC still remain unclear.
Result from our recent immunocytochemistry provided us an
unexpected indication that Skp2 resides mainly in the cytosol of
Mv1Lu mink epithelial cells in the absence of TGFβ. Given the
notion that Cdh1 is principally located in the nucleus, we face
a puzzle on how Cdh1/APC and Skp2 communicate with each
other given that they are in different cellular compartments not
to permit recognition and ubiquitylation of Skp2 by its E3 ligase
Cdh1/APC. Using a combinatorial approach encompassing cell
biology and biochemistry, we have revealed that Skp2 cellular
localization is quite dynamic in response to TGFβ signaling,
while Cdh1 localization is relatively stable. Our present results
show, upon stimulation with TGFβ, Skp2 is rapidly translocated
from the cytosol to the nucleus. The measurement of TGFβ-
induced nuclear localization of Skp2 by immunocytochemistry
and biochemical cellular fractionation further suggests that Skp2
translocation is a critical condition precedent for the ubiquitin
protein ligase Cdh1/APC to physically interact with its substrate
Skp2. Characterization of Skp2 translocation by altering its NLS
(nuclear localization signal) results in the stabilization of Skp2 in
the presence of TGFβ signaling, which consequentially results in
a failure to accumulate p27 that is necessary for TGFβ-induced
growth inhibition. This present work fills a knowledge gap on
how Skp2 is regulated by Cdh1/APC in response to TGFβ for
growth inhibition, which further advances our understanding of
the molecular basis of TGFβ signaling pathway.
Targeted degradation of Skp2 by Cdh1/APC is involved in
TGFβ-induced growth inhibition. Recent studies have demon-
strated that TGFβ signaling pathway is tightly regulated by the
UPS (ubiquitin-proteasome system),14 where UPS targets various
components of the TGFβ pathway including cytoplasmic second
messengers, transmembrane bound receptors and accumulated
nuclear proteins.15-17 Our endeavor to search for TGFβ-induced
fast turnover proteins led us to identify Skp2 as a rapidly degraded
protein in response to TGFβ signaling. As shown in Figure
1A–C, Skp2 is rapidly degraded in response to TGFβ stimula-
tion, which in turn results in accumulation of p27. The half-
life of Skp2 in response to TGFβ signaling is approximately 60
minutes (Fig. 1B). Furthermore, Skp2 degradation is blocked by
incubating cells with 50 μM of a proteasome inhibitor, MG-132
(Fig. 1C). To exclude the possibility that the observed change in
Figure 1 (See opposite page). Targeting Skp2 by Cdh1/APC for destruction is involved in TGFβ-mediated growth inhibition. (A) Skp2 protein levels
drop in response to TGFβ stimulation. Skp2 and actin (control) mRNA levels were monitored by RT-PCR analysis. (B and C) Skp2 protein chase analysis
in response to TGFβ stimulation. Mink lung epithelia cells (Mv1Lu) cells were treated with 20 μM cycloheximide. Skp2 protein turnover was measured
by immunoblotting. The half-life of Skp2 in response to TGFβ signaling is about 60 minutes while half-life for SnoN is about 30 minutes. p27 protein
levels gradually increase while Skp2 protein levels decrease. Mv1Lu were treated with TGFβ (100 pM). Skp2 degradation is further blocked by protea-
somal inhibitor, MG-132 (50 μM). Protein levels were measured by immunoblotting. Equal amounts of total protein were subjected to immunoblot
analysis, as evidenced by the equal concentration of tubulin. (D) TGFβ-induced Skp2 degradation is mediated via Cdh1/APC. Depletion of Cdh1 blocks
Skp2 degradation in response to TGFβ stimulation, which in turn results in lower levels of p27. (E) Summary of TGFβ-induced alteration of Skp2 and
p27 protein levels in wild-type and Cdh1 knockdown cells. (F) Disruption of destruction box in Skp2 stabilizes Skp2 protein in the presence of TGFβ.
Upper part shows construction of retroviral vector harboring wild-type or non-degradable Skp2. (G) Stabilization of Skp2 antagonizes TGFβ-induced
growth inhibition. Mv1Lu cells stably expressing Skp2 or Skp2Δdb respectively were incubated for 4 days with various concentration of TGFβ1 as indi-
cated. The growth of cells was quantified by cell counting and compared with the growth of unstimulated cells.
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288 Cell Cycle Volume 10 Issue 2
ubiquitin conjugates were visualized in the cytosol fraction in
both absence and presence of TGFβ. Taken together, the above
results demonstrated Skp2 localization is tightly regulated in
response TGFβ, where TGFβ-induced Skp2 nuclear transloca-
tion permits recognition of Skp2 by Cdh1/APC and its ubiquity-
lation by Cdh1/APC.
Identification of nuclear localization signal (NLS) respond-
ing to Skp2 translocation in response to TGFβ. To elucidate
the mechanism by which Skp2 translocates into the nucleus in
response to TGFβ, we analyzed the Skp2 sequence for a nuclear
localization signal (NLS). The search revealed a conserved NLS
motif (Fig. 3A) (cubic.bioc.columbia.edu/newwebsite/services/
predict NLS/), a sequence necessary and sufficient for nuclear
import of other host proteins.30,31 To test whether the putative
NLS in Skp2 mediates its nuclear transport, we deleted the NLS.
HA-tagged wild-type Skp2 or NLS mutant Skp2 was trans-
fected into Mv1Lu cells and extracts were prepared from cells
exposed (or not exposed) to TGFβ. The fractionated cytosol and
nuclear preparations were further examined by immunoblotting.
us an important clue to explain the possible mechanism of how
Skp2 is recognized and catalyzed by Cdh1/APC for ubiquity-
lation and degradation in response to TGFβ signaling.
To biochemically confirm the observation from our immuno-
staining, we measured the interaction between Cdh1 and Skp2
in the absence and presence of TGFβ by immunoprecipitation.
As shown in Figure 2B, coimunoprecipitation of Cdh1 and
Skp2 were detected in nuclear fraction after stimulation with
TGFβ. In contrast, no obvious interaction of Cdh1 and Skp2
was observed by co-IP in the cytosol in both absence and pres-
ence of TGFβ.
To determine where Cdh1 interacts with Skp2 and catalyzes
the ubiquitylation of Skp2, we performed an ubiquitylation assay
by using either cytosolic or nuclear fraction. Skp2 was pulled
down with antibody against Skp2. The ubiquitin conjugated
Skp2 was measured by immunoblotting of Skp2 IP complex with
antibody against ubiquitin.13,29 As shown in Figure 2C, Skp2
ubiquitin conjugates were significantly detected in the nuclear
fraction after stimulation with TGFβ, while no obvious Skp2
Figure 2. Skp2 translocation facilitates recognition of Skp2 by Cdh1/APC that ensures the TGFβ-induced Skp2 degradation. (A) TGFβ-induced translo-
cation of Skp2 generates compartmental interaction for Skp2 and Cdh1. In the absence of TGFβ, Cdh1 is mainly localized in the nucleus, while Skp2 is
principally expressed in the cytosol in Mv1Lu cells indicating by immuno-staining using antibodies against Cdh1 and Skp2. In the presence of TGFβ,
Skp2 are translocated into the nucleus and therefore meet with Cdh1, measuring between 30–60 minutes after stimulation with TGFβ. (B) Time-
dependent interaction between Skp2 and Cdh1 in response to TGFβ signaling. Mv1Lu cell were treated with TGFβ and collected at various time points.
Cytosol and nuclear lysates were prepared for immunoprecipitation. Cdh1 IP complexes from cytosol and nucleus were immunoblotted with antibody
against Skp2. Transient interaction of Cdh1 and Skp2 was only observed in the nucleus but not in the cytosol. (C) Skp2 forms ubiquitin-conjugates in
the nucleus in response to TGFβ signaling. Skp2 ubiquitin-conjugates were pulled down by immunoprecipitation followed by immunoblotting using
antibody against ubiquitin.
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Consistent with the data from the immuno-staining assay, the
majority of Skp2 was observed in the cytosol in the absence of
TGFβ but redistributed to the nucleus in response to TGFβ
stimulation (Fig. 3B). As predicted, deletion of the NLS on Skp2
completely blocked TGFβ-induced Skp2 nuclear translocation
(Fig. 3C and D). To ask whether nuclear translocation is coupled
to Skp2 degradation, we measured the change in Skp2 levels in
response to TGFβ. As shown in Figure 3E and F, deletion of the
NLS significantly attenuated the Skp2 degradation in response to
Figure 3. Identification of a nuclear localization signal (NLS) in Skp2 that facilitates Skp2 translocation and degradation in response to TGFβ signaling.
(A) Skp2 contains a conserved nuclear localization signal (NLS) motif. The alignment was performed using the CLUSTA W method. (B) Translocation of
endogenous Skp2 in response to TGFβ signaling. Cells were collected 30 minutes after the stimulation with TGFβ. (C) Translocation of HA tagged Skp2
in response to TGFβ signaling. (D) Disruption of NLS in Skp2 abrogates Skp2 translocation in response to TGFβ signaling.
TGFβ. These results suggest that the NLS in Skp2 is required for
TGFβ-induced Skp2 translocation and destruction.
Impaired Skp2 translocation inhibits its degradation and
antagonizes TGFβ-induced growth inhibition. Impaired Skp2
translocation could disturb the recognition and ubiquitylation
of Skp2 by Cdh1/APC and cause the stabilization of Skp2 and
proteolysis of p27. Failure to degrade Skp2 and the resulting
stabilization of p27 in response to TGFβ should antagonize
the TGFβ response. To test this hypothesis, wild-type Skp2
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APC, which helps to maintain p27 protein levels to inhibit
Previous studies from us and others have implicated a role for
Cdh1/APC in TGFβ signaling pathway.20,33 Elevated activity for
Cdh1/APC was measured in the presence of TGFβ. The TGFβ-
induced activation of APC was thought to remove SnoN, a tran-
scriptional co-suppressor, for the induction of TGFβ-responsive
genes and to degrade Skp2 to stabilize p27 for TGFβ cytostatic
effect.13,32,34 However, how Cdh1/APC targets Skp2 for degra-
dation remains a puzzle since both components are localized in
different cellular compartments in the testing model cell line-
Mv1Lu. In the present work, finding that Skp2 translocates to
the nucleus in response to TGFβ and the further identification of
or NLS-deleted Skp2 was introduced stably into Mv1Lu cells
by retroviral infection (Fig. 4A).13,32 Subsequently, the pools
of infected cells were measured for their ability to respond to
TGFβ-induced growth inhibition.13 As shown in Figure 4A
and B, stably expressed Skp2 degraded in response to TGFβ
stimulation while NLS-deleted Skp2 was quite stable. Growth
inhibition analysis showed that expression of wild-type Skp2
only moderately blocked the ability of cells to undergo TGFβ-
induced cell cycle arrest. The reason for this may be because
the wild-type Skp2 was unstable in response to TGFβ stimu-
lation and thus failed to accumulate (Fig. 4C). In contrast,
NLS-deleted Skp2 markedly attenuated TGFβ-induced growth
inhibition (Fig. 4C). Therefore, failure of Skp2 translocation in
response to TGFβ impairs the TGFβ-induced growth inhibi-
tory effect. These results support the hypothesis that TGFβ-
induced Skp2 translocation facilitates its degradation by Cdh1/
Figure 4. Disruption of Skp2 translocation attenuates its degradation in response to TGFβ, which in turn antagonizes TGFβ-induced growth inhibition.
(A) Deletion of NLS in Skp2 impairs Skp2 degradation and p27 accumulation induced by TGFβ. Upper part indicates the retro-viral expression vector of
wild-type and NLS Skp2 mutant. (B) Summary of protein degradation for wild-type and NLS Skp2 mutant in response to TGFβ signaling.
(C) Impaired Skp2 translocation leads to attenuation of TGFβ-induced growth inhibition. Mv1Lu cells stably expressing Skp2 or Skp2DNLS respectively
were incubated for four days with various concentration of TGFβ1 as indicated. The growth of cells was quantified by cell counting and compared with
the growth of unstimulated cells.
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Skp2 cytosol-nucleus trafficking. Ubiquitylation assay indi-
cated that in the presence of TGFβ signaling, Skp2 translocates
to the nucleus and is ubiquitylated by Cdh1/APC for degrada-
tion. Deletion of the nuclear localization signal (NLS) sequence
impairs Skp2 nuclear translocation and inhibits its degradation,
which in turn antagonizes growth inhibition by TGFβ. This
work implicates that subcellular distribution of Skp2 is an impor-
tant cellular marker that reflects the status of cell proliferation.
Regulation of Skp2 translocation could be a valuable therapeutic
strategy to control cell growth. Moreover, cytoplasmic Skp2 has
been suggested to promote cell migration and thus has a potential
function in tumor metastasis.27 Given the critical role of TGFβ
in epithelial-mesenchymal transition (EMT), which correlates to
increased invasiveness and metastasis, it would be important to
investigate the possible link between TGFβ-induced transloca-
tion and degradation of Skp2 and cell motility in the context of
Materials and Methods
Plasmids and constructs. Skp2ΔNLS (nuclear localization signal
deleted Skp2) was constructed by PCR using following primers
and then cloned into pCS2-HA or pREX-IRES-CD2, a mam-
malian expression vector:
5'-AAA ATC GAT ATG CAC GTA TTT AAA ACT CCC
5'-AAA GTC TTT GTC ACT CCC TTT GGG GCT CTC
CGG GTG GCC CAG GTT-3'
5'-AAA GGG AGT GAC AAA GAC TTT GTG-3'
5'-TTG GCG CGC CTA GAC AAC TGG GCT TTT GCA
pREX-IRES-CD2 is a gift from Dr. Xuedong Liu (University
of Colorado-Boulder). pREX-HA-Skp2-IRES-CD2 and pREX-
HA-Skp2ΔNLS-IRES-CD2 were generated by PCR using fol-
5'-AAA AGT CGA CCC ACA GGA AGC ACC TCC AGG
5'-TTG CGG CCG CTC ATA GAC AAC TGG GCT TTT
Antibodies and immunoprecipitation. Western blot analy-
sis was performed using the anti-Skp2 (Santa Cruz, sc-7164),
anti-p27 (Santa Cruz, sc-776), anti-Cdh1 (Abcam, DCS-266),
anti-PCNA (Santa Cruz, sc-56), anti-HA (Santa Cruz, sc-7392),
anti-Myc (Santa Cruz, sc-789), anti-Flag (Sigma, F3165),
anti-SnoN (Cascade BioScience, ABM-3002), Ubiquitin (BD
bioscience, 550944) and HRP-conjugated goat-anti-mouse
(Promega, W4021) or anti-rabbit secondary antibody (Promega,
W4011) with ECL detection kit (Amersham, RPN2106). Semi-
quantification of data was performed using NIH image.
For immunoprecipitation assay, cell lysate was incubated with
anti-Cdh1 antibody overnight at 4° on a rotator, following by
addition of UltraLink Immobilized Protein A/G (Pierce, 53133).
IP-complex was resolved by SDS-PAGE. Western blotting was
conducted by using antibody against Skp2.
Growth inhibition assay. For growth inhibition assay, 5 x
103 Mv1Lu cells were incubated with various concentrations of
Skp2’s nuclear translocation motifs have unveiled the mystery of
how Skp2 is recognized for ubiquitylation by Cdh1/APC in the
presence of TGFβ signaling. This work elucidates the mechanism
by which Skp2 is regulated by Cdh1/APC in TGFβ signaling.
Skp2 was first identified as a cyclinA-CDK2 S-phase kinase-
associated protein35 and subsequently characterized as a rate-
limiting component of the SCF machinery that ubiquitylates
p27 for degradation, which in turn releases cyclin E-CDK2
and promotes entry into S phase.36,37 p27 functions as a tumor
suppressor and its inactivation predisposes mice to tumorigen-
esis. It has been proposed that Skp2 confers oncogenic function
through accelerated degradation of p27 and other tumor sup-
pressors, such as CDKIs p21 and p57; RASSF1 (Ras association
domain family 1); and RBL2 (retinoblastoma-like, also known
as p130).38 Indeed, increased levels of Skp2 proteins are often
observed in many human cancers where Skp2 overexpression is
usually inversely correlated with reduced p27 expression, and
positively correlated with tumor malignancy and poor progno-
sis in terms of cancer treatment.39,40 Mechanistic studies show
that, in addition to gene amplification and mRNA accumula-
tion, deregulated proteolysis is also involved in elevated Skp2
expression in human cancers. We and other groups have dem-
onstrated that in normal cell cycle, Cdh1/APC targets Skp2 for
degradation, thus preventing premature entry into S phase.18,19
Our recent work further reveals that the reduced expression of
Cdh1 and inverse elevation of Skp2 are evident in various human
cancers. Moreover, the deregulated Cdh1/Skp2/p27 cascade is
implicated in the development of breast and colorectal cancer.41-43
The tumor suppressor potential of Cdh1 is further illustrated in
knockout mice. Cdh1 heterozygous mice show increased suscep-
tibility to spontaneous tumors.44 Given the pivotal role of TGFβ
signaling in tumorigenesis and the notion that Cdh1/APC medi-
ates TGFβ-induced cell growth inhibition as demonstrated here
and by other studies,13,34 abrogation of Cdh1/APC function in
TGFβ signaling could be one mechanism for the initiation of
Besides aberrant Cdh1 activity, Skp2 cytoplasmic localization
might also be another mechanism for the abnormal accumula-
tion of Skp2 protein in human cancers, which allows Skp2 to
escape from Cdh1-mediated degradation. Cytoplasmic Skp2 has
been observed in many clinical tumor samples and correlated
with aggressive malignancy. Recent mechanistic studies suggest
that Akt-dependent phosphorylation of Skp2 at Ser 72 is respon-
sible for Skp2 cytoplasmic translocation.27,28 In this study, both
immunofluorescence staining and compartmental immunopre-
cipitation assay shows that Skp2 resides mainly in the cytosol of
Mv1Lu mink lung epithelial cells, and translocates to nucleus
in response to TGFβ signaling. Identification and characteriza-
tion of translocation motifs on Skp2 advanced the knowledge
about the Cdh1-Skp2-p27-cyclin E/CDC2 axis in TGFβ signal-
ing. Nevertheless, the mechanism of how Skp2 translocation is
orchestrated in response to TGFβ is still unknown. Given the
report of Skp2 regulation by Akt, it would be interesting to
address the possible connection between Akt and the TGFβ-
induced localization of Skp2.27,28 It would be helpful to explore
how whether TGFβ signaling alters the activity of Akt to control
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115-095-146) and Texas Red conjugated anti-rabbit (Jackson
Lab, 111-075-045) secondary antibodies.
We thank Drs. X. Liu, W. Malcolm, W. Kaelin, A. Weissman
and D. Zhang for cDNA clones. We are grateful to members of
our laboratory for critical reading of the manuscript. This work
is supported by NIH grants CA115943. Y. Wan is a scholar of
American Cancer Society and V Cancer Research Foundation.
TGFβ1 (R&D, 40-B) for 3–4 days. The growth of cells was
determined by counting compared with that of unstimulated
Immunofluorescence microscopy. Cells were washed and
fixed in 4% paraformaldehyde solution for 10 min at room tem-
perature and permeabilized with 0.1% Triton X-100 in PBS. Cells
with and without stimulation by TGFβ1 were incubated with
a rabbit anti-Skp2 and a mouse anti-Cdh1 antibodies followed
by incubating with FITC conjugated anti-mouse (Jackson Lab,