A coordinated phosphorylation by Lats
and CK1 regulates YAP stability through
Bin Zhao,1Li Li,1,2Karen Tumaneng,1Cun-Yu Wang,3and Kun-Liang Guan1,4
1Department of Pharmacology and Moores Cancer Center, University of California at San Diego, La Jolla, California 92093-0815,
USA;2Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA;3Laboratory of Molecular
Signaling, Division of Oral Biology and Medicine, University of California at Los Angeles School of Dentistry, Los Angeles,
California 90095, USA
The Yes-associated protein (YAP) transcription coactivator is a key regulator of organ size and a candidate human
oncogene. YAP is inhibited by the Hippo pathway kinase cascade, at least in part via phosphorylation of Ser 127,
which results in YAP 14–3–3 binding and cytoplasmic retention. Here we report that YAP is phosphorylated by
Lats on all of the five consensus HXRXXS motifs. Phosphorylation of Ser 381 in one of them primes YAP for
subsequent phosphorylation by CK1d/e in a phosphodegron. The phosphorylated phosphodegron then recruits the
SCFb-TRCPE3 ubiquitin ligase, which catalyzes YAP ubiquitination, ultimately leading to YAP degradation. The
phosphodegron-mediated degradation and the Ser 127 phosphorylation-dependent translocation coordinately
suppress YAP oncogenic activity. Our study identified CK1d/e as new regulators of YAP and uncovered an intricate
mechanism of YAP regulation by the Hippo pathway via both S127 phosphorylation-mediated spatial regulation
(nuclear–cytoplasmic shuttling) and the phosphodegron-mediated temporal regulation (degradation).
[Keywords: YAP; Hippo pathway; CK1; SCF; Lats; phosphodegron]
Supplemental material is available at http://www.genesdev.org.
Received July 14, 2009; revised version accepted November 9, 2009.
The Yes-associated protein (YAP) transcription coactiva-
tor is a key regulator of organ size (Camargo et al. 2007;
Dong et al. 2007). Mutation of Yki, the Drosophila
homolog of YAP, results in dramatically reduced organ
size, while Yki overexpression causes tissue overgrowth
(Huang et al. 2005). Similarly, transgenic overexpression
of YAP in mice liver results in enlarged liver four times
the size of normal control and causes liver tumors
(Camargo et al. 2007; Dong et al. 2007). Consistently,
yap has been identified as a candidate oncogene in human
chromosome 11q22 amplicon and the equivalent geno-
mic amplicon in a mouse hepatocellular carcinoma
(HCC) model (Overholtzer et al. 2006; Zender et al.
2006). Besides the genomic amplification, YAP protein
levels and nuclear localization were also shown to be
elevated in multiple types of human cancers (Zender et al.
2006; Dong et al. 2007; Zhao et al. 2007; Steinhardt et al.
2008). YAP cooperates with the myc oncogene to stimu-
late tumor growth in vivo (Zender et al. 2006), and active
YAP mutants induce oncogenic cell transformation in
vitro (Zhao et al. 2009). These observations strongly
indicate the function of yap as an oncogene.
As shown above, the mechanism controlling YAP
activity is obviously a key question. Regulation of YAP/
Yki activity was first revealed by genetic studies of the
Hippo tumor suppressor pathway in Drosophila (Kango-
Singh et al. 2002; Tapon et al. 2002; Harvey et al. 2003; Jia
et al. 2003; Pantalacci et al. 2003; Udan et al. 2003; S Wu
et al. 2003; Lai et al. 2005). The Hippo pathway limits
organ size by inhibiting cell proliferation and promoting
apoptosis through inhibition of Yki (Huang et al. 2005).
Biochemical studies showed that Yki is directly phosphor-
ylated and inhibited by the Wts protein kinase, which is
phosphorylated and activated by the Hippo (Hpo) protein
kinase (Dong et al. 2007; Oh and Irvine 2009). Compo-
nents of the Hippo pathway are highly conserved in mam-
mals. Recent studies from our group and others (Zhao
et al. 2007; Hao et al. 2008; Oka et al. 2008; Zhang et al.
2008) have demonstrated that YAP is phosphorylated and
inhibited by the Lats tumor suppressor kinase, the mam-
malian homolog of Wts. YAP has five HXRXXS Lats phos-
phorylation consensus motifs, in which phosphorylation
of Ser 127 results in 14–3–3 binding and cytoplasmic
retention of YAP (Zhao et al. 2007). Therefore, YAP can
be inhibited by spatial separation from its nuclear target
transcription factors, such as TEAD family members
(Zhao et al. 2008). This mechanism of YAP regulation is
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implicated in cell contact inhibition and tissue growth
control (Zhao et al. 2007; Zeng and Hong 2008). However,
whether the other four HXRXXS sites in YAP are phos-
phorylated in vivo is unknown. Nevertheless, there is
evidence that at least some of these sites are important for
YAP regulation because the YAP-5SA mutant, in which
serine residues in all five HXRXXS motifs are mutated to
proliferation (Zhao et al. 2009). Furthermore, YAP-5SA
expression can transform NIH-3T3 cells, but YAP-S127A
cannot (Zhao et al. 2009). Therefore it is important to
know which HXRXXS site regulates YAP activity and
what the underlying mechanism is.
We previously reported cell density-dependent activa-
tion of the Hippo pathway and increase of YAP phosphor-
ylation (Zhao et al. 2007). We also observed a dramatic
cell density-dependent decline of YAP protein levels (this
study), indicating a possible role of the Hippo pathway in
regulating YAP protein stability. Ubiquitin-dependent
proteolysis regulates protein turnover and serves key
regulatory roles in various biological processes (Hershko
and Ciechanover 1998). Ubiquitination is mostly cata-
lyzed by the sequential action of three enzymes; namely,
enzymes), and E3 (ubiquitin ligase). E3 determines sub-
strate specificity, and its substrate binding is the primary
event controlling the stability of a particular protein
(Pickart 2001). The SCF is a four-subunit RING-type E3
consisting of the RING domain protein Rbx1, two scaf-
fold proteins (Cul1 and SKP1), and one of the many F-box
proteins (Deshaies 1999). The F-box proteins contain
various C-terminal protein–protein interaction domains
that determine substrate specificity (Deshaies 1999).
b–Transducin repeat-containing proteins (b-TRCP) be-
long to the F-box protein family with two paralogs in
mammals: b-TRCP1 and b-TRCP2. They play redun-
dant roles in the ubiquitination of well-established sub-
strates, including b-catenin (Hart et al. 1999; Latres et al.
1999) and IkB (Li and Verma 2002). b-TRCP recognizes a
DSGXXS motif in which the serine residues are phosphor-
ylated, and therefore this motif is called phosphodegron
(Fuchs et al. 2004). Phosphorylation of phosphodegron is
the major mechanism regulating b-TRCP substrate bind-
ing (Fuchs et al. 2004).
In this study, we describe a new mechanism of YAP
inhibition involving coordinated phosphorylation by
two kinases and subsequent SCFb-TRCP-mediated ubiqui-
tination and degradation. We show that YAP is phosphor-
ylated by Lats on Ser 381 in one of the HXRXXS motifs,
and this phosphorylation provides the priming signal
for CK1d/e to phosphorylate a phosphodegron in YAP.
The phosphorylated phosphodegron recruits b-TRCP,
leading to YAP ubiquitination and degradation under
conditions of elevated Hippo pathway activity, such as
cell contact inhibition. Hence, YAP is inhibited by
two mechanisms—the S127 phosphorylation-mediated
spatial regulation (nuclear–cytoplasmic shuttling) and
the S381 phosphorylation-mediated temporal regulation
(the phosphodegron-induced degradation)—that coordi-
nately suppress YAP oncogenic activity.
YAP is phosphorylated in vivo on multiple sites,
including the five Lats consensus HXRXXS motifs
YAP phosphorylation on S127 in the Lats consensus
HXRXXS motif increases with cell density, and inhibits
YAP activity by promoting YAP cytoplasmic retention
through interaction with 14–3–3 (Zhao et al. 2007).
Serines in the other four HXRXXS motifs in YAP could
all be phosphorylated by Lats in vitro (Zhao et al. 2009).
However, their phosphorylation in vivo has not been
shown, and the function of these Lats consensus sites is
unclear. To study the function of these candidate phos-
phorylation sites, we performed mass spectrometry anal-
ysis of Flag-SBP (streptavidin-binding peptide)-tagged
YAP protein isolated from cultured cells. As shown in
Figure 1A, 11 phosphopeptides were identified represent-
ing 10 unique phosphorylation sites, including all five
predicted Lats phosphorylation sites (S61, S109, S127,
S164, and S381). This indicates that the Last phosphory-
lation consensus sites are indeed phosphorylated in vivo.
Phosphorylation on several other residues was also iden-
tified, including T63, S138,S289, S351, and S384 (Fig. 1A).
To further confirm phosphorylation on the HXRXXS
motifs in vivo, we examined the electrophoretic mobility
of various YAP phosphorylation site mutants expressed in
HEK293 cells on gels containing Phos tag-conjugated
acrylamide, which specifically retards phosphorylated pro-
teins.YAPexpressed in HEK293cells migrated asmultiple
bands on Phos tag gel (Fig. 1B), indicating various phos-
phorylation states on multiple sites. However, with Mst
and Lats cotransfection, wild-type YAP mostly showed as
the slowest migrating form (Fig. 1B). This result suggests
that the differentially migrating forms of YAP resulted
mainly fromphosphorylationonthe Hippo pathwaytarget
sites. Consistent with the mass spectrometry data, muta-
tion of S61 alone or in combination with S127 mutation
increased YAP mobility with or without Mst and Lats
cotransfection (Fig. 1B), supporting that S61 of YAP is
phosphorylated by Lats in vivo. Similar effects on YAP
electrophoretic mobility were observed by mutating S109
or S164 alone or in combination with other mutations
(Fig. 1B), indicating that these two sites are also phosphor-
ylated in vivo. Under Mst/Lats cotransfection, YAP-5SA
exhibited a slightly faster mobility than YAP-4SA/S381
(Fig. 1B). However, in other conditions, mutation of S381
did not increase YAP mobility (Fig. 1B). This might result
from sequence context-dependent interference with Phos
tag binding. Nevertheless, the phosphorylation of S381
was confirmed by two different phosphorylation-specific
antibodies (Supplemental Figs. S1A, S4A).
Ser 127 and Ser 381 are key phosphorylation sites
in suppressing YAP oncogenic activity
We observed that YAP-5SA is more active than YAP-
S127A in promoting cell proliferation (Zhao et al. 2009),
indicating that the other HXRXXS motifs also contribute
to YAP regulation. There is also a dramatic difference
between YAP-5SA and YAP-S127A mutants in oncogenic
Regulation of YAP degradation by SCFb-TRCP
GENES & DEVELOPMENT73
transformation, as YAP-5SA can potently transform NIH-
3T3 cells, while YAP-S127A is inactive (Fig. 1C,D).
Therefore, at least one site among S61, S109, S164, and
S381 inhibits YAP oncogenic activity. To identify this
site, we restored individual serines in the YAP-5SA mu-
tant, resulting in YAP-4SA proteins retaining a single
phosphorylation site. As predicted, restoration of S127
(4SA/S127) largely inhibited colony formation (Fig. 1C).
Interestingly, restoration of S381 (4SA/S381) also abol-
ished colony formation activity, while restoration of S61
(4SA/S61), S109 (4SA/S109), and S164 (4SA/S164) did not
(Fig. 1C). Therefore, restoration of either S127 or S381 is
sufficient to suppress YAP oncogenic activity. As in the
case of S127, mutation of S381 alone to alanine was
insufficient to transform NIH-3T3 cells (Fig. 1D). In-
terestingly, the combination of S127 and S381 phosphor-
ylation-deficient mutations (S127/381A) was enough to
induce oncogenic transformation (Fig. 1D).
The importance of S127 and S381 in cellular trans-
formation was also demonstrated in MCF10A cells. As
shown in Figure 1E, YAP-S127/381A induced anchorage-
independent growth of MCF10A cells in soft agar, as
did YAP-5SA. In contrast, YAP wild type, YAP-S127A, or
YAP-S381A failed to induce anchorage-independent
growth. These results indicate that S127 and S381 are
the two key phosphorylation sites suppressing YAP trans-
Ser 381 regulates YAP protein stability
Ser 127 inhibits YAP by mediating YAP–14–3–3 binding
and cytoplasmic retention upon phosphorylation (Zhao
et al. 2007). However, mutation of S381 did not affect
YAP interaction with 14–3–3 (Supplemental Fig. S1D).
As shown in Figure 2A, YAP-S381A showed normal
cytoplasmic translocation upon cotransfection with
S381 inhibits YAP oncogenic activity. (A)
YAP is phosphorylated in vivo on all
five Lats phosphorylation consensus sites
(S61, S109, S127, S164, and S381). YAP
expressed in MCF10A cells was tandem
affinity-purified and analyzed by LC-MS/
MS. Identified YAP phosphopeptides were
shown. Phosphorylated residues are high-
lighted in bold. (M9) Methionine oxidation.
In the phosphopeptide containing S163 and
S164, the mass spectronomy data could not
determine which one of them is phosphor-
ylated. However, S164 phosphorylation
was confirmed by later experiments and
therefore is highlighted here. (B) Mutations
of S61, S109, S127, and S164 to alanine
YAP wild type and mutants were expressed
in HEK293 cells with or without Mst and
Lats cotransfection as indicated. Cell ly-
sates were resolved on SDS-PAGE gels
containing 25 mM Phos tag-conjugated
acrylamide followed by standard Western
blotting with anti-Flag antibody. (C) Resto-
ration of Ser 127 or Ser 381 is sufficient to
inhibit YAP-5SA transformation activity.
NIH-3T3 cell colony formation assays were
performed using vector control or indicated
YAP constructs. Colonies were visualized
with crystal violet staining 2 wk after
transfection. Expression of YAP wild type
or mutants 24 h after transfection was
shown by Western blot (Supplemental Fig.
S1B). (D) Mutation of both Ser 127 and Ser
381 is required to activate YAP transform-
ing activity. Experiments were similar to
those in C with indicated YAP constructs.
Expression of YAP wild type or mutants 24
h after transfection was shown by Western
blot (Supplemental Fig. S1C). (E) Transfor-
mation of MCF10A cells by YAP-S127/381A
Phosphorylation of S127 and
and YAP-5SA. Indicated MCF10A stable cells were cultured in soft agar for 16 d before pictures were taken. Colonies were then visualized
by crystal violet staining and colonies observable by the naked eye were counted and are shown in the right panel.
Zhao et al.
74 GENES & DEVELOPMENT
Mst2/Lats2. In contrast, mutation of S127 abrogated YAP
cytoplasmic translocation. We also observed that, when
expressed at similar levels, YAP-S381A or YAP-S127/
381A did not exhibit significantly higher activity than
wild-type or YAP-S127A protein to activate Gal4-TEAD4
in luciferase assays (Supplemental Fig. S1E). This result
argues against the possibility that S381 phosphorylation
directly inhibits YAP transactivation activity or TEAD
binding. We set out to find out how S381 phosphorylation
regulates YAP function.
We reported previously that the Hippo pathway activ-
ity increases with cell density in NIH-3T3 cell culture,
which results in increased YAP phosphorylation and
cytoplasmic retention (Zhao et al. 2007). We also noticed
that, as cell density increases, YAP protein level declined
dramatically (Fig. 2B). We ruled out the possibility that
this observed decrease of YAP protein was due to inter-
ference of antibody recognition by YAP phosphorylation.
In fact, the total amount of S381- or S127-phosphorylated
YAP was also decreased at high density (Fig. 2C). How-
ever, when normalized to YAP protein levels, YAP S127
phosphorylation was increased, while YAP S381 phos-
phorylation was decreased upon cell density (Fig. 2C).
Since both S127 and S381 are phosphorylated by the same
kinase (Lats), a plausible explanation is that the S381-
phosphorylated YAP is unstable, and therefore could not
YAP protein stability. (A) Mutation of S381
does not affect YAP subcellular localiza-
tion. YAP wild type or mutants were ex-
pressed in HeLa cells without (left panels)
or with (right panels) Mst2/ Lats2 cotrans-
fection. YAP localization was visualized by
immunofluorescence staining with anti-
Flag antibody (red). HA-Mst2 and HA-Lats2
were shown by anti-HA staining (green).
DNA was stained with DAPI (blue). (B)
YAP protein level decreases with increas-
ing cell density. NIH-3T3 and mouse em-
bryonic fibroblast cells were cultured at
different densities from sparse to conflu-
ent. Endogenous YAP protein levels were
determined by Western blot with anti-
tubulin as a loading control. Relative YAP
protein levels were quantified by the ratio
between YAP and tubulin, which was arbi-
trarily set to 1 at the lowest cell density. (C)
Effect of cell density on YAP phosphoryla-
tion. NIH-3T3 cells were cultured at differ-
ent densities from sparse to confluent.
Endogenous YAP protein levels and YAP-
S381 and YAP-S127 phosphorylations were
determined by Western blot with specific
antibodies. Anti-tubulin was used as a load-
ing control. Relative YAP phosphorylation
levels were quantified by the ratio between
pYAP and total YAP protein, which were
arbitrarily set to 1 at the lowest cell den-
sity. (D) YAP is unstable in high-density
cell culture. NIH-3T3 cells were seeded at
1 3 106per 10-cm dish (low density) or 3 3
106per well (high density) in six-well plates
and cultured for 24 h. Protein synthesis
was blocked by treatment with 50 mg/mL
Mutation of Ser 381 increases
CHX for the indicated time. Relative YAP protein levels were quantified by YAP/tubulin ratio, which was arbitrarily set to 1 at the 0
time point. When the same samples were resolved for a longer time, YAP showed a shift as we reported previously (Supplemental Fig.
S1F) that was eliminated by l phosphatase treatment (Supplemental Fig. S1G). (E) YAP is ubiquitinated at high cell density. NIH-3T3
cells cultured at low or high density were treated with 25 mM MG132 for 5 h before harvest. Endogenous YAP was immunoprecipitated
and Western blot was done with anti-YAP or anti-Ub antibodies. (S) Shorter exposure; (L) longer exposure. (F) Mutation of S381 to
alanine stabilizes YAP. NIH-3T3 cells were transfected with indicated YAP constructs. After 24 h, cells were plated into six-well plates
and cultured to confluence. Protein synthesis was blocked by treatment with 50 mg/mL CHX for the indicated time. Transfected YAP
protein levels were shown by anti-Flag Western blot, and anti-tubulin Western blot was used as a loading control. Relative YAP protein
levels were quantified by the ratio between Flag-YAP and tubulin, which was arbitrarily set to 1 at the 0 time point. The dot in the last
lane of Flag-YAP-S127AWestern blot, as indicated by the asterisk, was due to an artifact on the membrane, and this lane is therefore not
Regulation of YAP degradation by SCFb-TRCP
GENES & DEVELOPMENT75
be accumulated. The decreased YAP protein level is due,
in large part, to YAP destabilization, because when pro-
tein synthesis was blocked by cycloheximide (CHX), YAP
was stable at low cell density, but unstable at high cell
density (Fig. 2D; Supplemental Fig. S1F). Proteasome-
mediated degradation of ubiquitinated protein is a com-
mon method for protein stability regulation (Hershko and
tion is affected by cell density. As shown in Supple-
mental Figure S1H, YAP immunoprecipitated from pro-
HeLa cells did exhibit incorporation of ubiquitin. Con-
sistent with YAP destabilization at high cell density,
under MG132 treatment, ubiquitinated YAP was accu-
mulated in cells cultured at high cell density (Fig. 2E).
These results indicate that activation of the Hippo
pathway by cell density inhibits YAP not only by retain-
ing YAP in cytoplasm, but also by stimulating YAP
To examine if S381 is involved in YAP degradation, we
tested YAP mutant stability by CHX chase experiments.
As shown in Figure 2F, similar to endogenous YAP, Flag-
YAP wild-type protein expressed in NIH-3T3 cells was
degraded quickly at high cell density. Mutation of S127 to
alanine did not stabilize YAP. Although mutation of S381
to alanine alone did not substantially stabilize YAP (Fig.
2F), when S381 was mutated in the S127A mutant
background, it dramatically stabilized YAP. Similarly,
the YAP-5SA mutant was also stabilized. When those
YAP mutants were tested in similar experiments at low
cell density, they were all stable (Supplemental Fig. S1I).
These results indicate that destabilization of YAP
through S381 phosphorylation is an important mecha-
nism of YAP inhibition by the Hippo pathway, and this
mechanism of YAP regulation is cell density-dependent.
Ser 381 and the phosphodegron are required
for YAP–b-TRCP interaction
To elucidate the mechanism of S381 in the regulation
of YAP stability, we carefully examined the sequence
around S381 and found a DSGXS motif, similar to the
canonical phosphodegron DpSGXXpS recognized by
b-TRCP, an F-box-containing substrate recognition sub-
unit in the SCF E3. Phosphorylation of the two serine
residues in the phosphodegron is the key mechanism
regulating b-TRCP binding and target ubiquitination
(Fuchs et al. 2004). Alignment of the sequences around
YAP S381 to b-catenin phosphodegron is shown in Figure
3A. In the case of b-catenin, S45 phosphorylation by
CK1a primes phosphorylation by GSK3 on T41, S37, and
S33 sequentially (Liu et al. 2002; Wu and He 2006). Based
on the sequence similarity between YAP and b-catenin
and the function of S381 in YAP stability, we hypothesize
that Lats phosphorylates S381 and primes YAP for phos-
phorylation by another kinase on S384 and possibly S387
in a phosphorylation relay mechanism similar to that
observed in b-catenin. This sequential phosphorylation
activates phosphodegron and destabilizes YAP when the
Hippo pathway is activated at high cell density. It is
worth noting that YAP S384 phosphorylation was also
identified by mass spectrometry (Fig. 1A). Furthermore,
S387 phosphorylation has also been reported by another
study (Dephoure et al. 2008).
To test the above hypothesis, we first sought evi-
dence for YAP–b-TRCP interaction. Interestingly, in our
required for YAP–b-TRCP interaction. (A)
Alignment of YAP sequence around S381 with
the b-catenin phosphodegron sequence. The
phosphodegron is underlined. Positions of
phosphorylation residues are labeled. Kinases
responsible (or hypothesized to be) for phos-
phorylation of each residue are shown. Dashed
arrows indicate priming relationship. (B) YAP
and b-TRCP coimmunoprecipitated with each
other. Indicated plasmids were transfected into
HEK293 cells. YAP or b-TRCP was immuno-
precipitated with anti-HA or Flag antibody.
Western blots were done to detect specific
proteins as indicated on the left sides of each
panel. (IP) Immunoprecipitation. (C) S381 and
S384 in YAP are required for b-TRCP interac-
tion. Indicated plasmids were transfected into
HEK293 cells. b-TRCP was immunoprecipi-
tated with anti-Flag antibody. Coimmunopre-
cipitated YAP was shown by anti-HA Western
blot. (S) Shorter exposure; (L) longer exposure.
(D) S387 is important for YAP–b-TRCP inter-
action. Experiments were similar to those in C.
(E) D383 and G385 in the phosphodegron are
required for YAP–b-TRCP interaction. Experi-
ments were similar to those in C.
Ser 381 and the phosphodegron are
Zhao et al.
76 GENES & DEVELOPMENT
LC-MS/MS analysis of YAP tandem affinity purification
products, we did detected a b-TRCP peptide, VLE-
GHEELVR (peptide prophet score of 0.97), suggesting that
YAP interacts with b-TRCP. This interaction was con-
firmed in HEK293 cells by reciprocal coimmunoprecipi-
tation (co-IP) of HA-YAP and Flag-b-TRCP (Fig. 3B).
If S381 phosphorylation is required for YAP–b-TRCP
interaction, phosphorylation-deficient mutation of this
residue should abolish this interaction. As predicted, the
YAP-S381A mutant lost b-TRCP binding, while the phos-
phomimetic mutant S381D partially retained the inter-
action (Fig. 3C). This result supports that YAP-S381
phosphorylation is a prerequisite for YAP–b-TRCP bind-
ing. Similarly, mutation of S384 to alanine also abolished
YAP–b-TRCP interaction (Fig. 3C). The function of phos-
phorylated S384 could not be mimicked by S384D (Fig.
3C), which is not unexpected because the corresponding
phosphate group in S33-phosphorylated b-catenin di-
rectly forms hydrogen bonds with b-TRCP (G Wu et al.
2003), and therefore an acidic residue at this position may
not substitute for the function of a phosphate group.
A classical phosphodegron has three or more residues
between the two phosphorylated serines (Fuchs et al.
2004). In the case of YAP, there are only two residues
between S384 and S387 (Fig. 3A). To test whether S387
phosphorylation is also important for YAP–b-TRCP bind-
ing, we examined co-IP of S387A and S387D mutants
with b-TRCP. Mutation of S387 to alanine significantly
reduced YAP–b-TRCP interaction (Fig. 3D), indicating
the importance of this residue. Moreover, the S387D
mutant largely retained the ability of b-TRCP binding
(Fig. 3D), suggesting that S387 phosphorylation contrib-
utes to b-TRCP interaction. In the case of b-catenin,
mutation of the conserved D and G residues in the phos-
phodegron found in cancer patients attenuates b-TRCP
interaction and ubiquitination (Provost et al. 2005). The
equivalent YAP mutants, D383A and G385V, also lost
YAP–b-TRCP interaction (Fig. 3E). The above results
strongly suggest that YAP contains a phosphodegron.
The interaction between YAP and b-TRCP depends on
phosphorylation of the serine residues 381, 384, and 387,
and also the intact DSG motif in the phosphodegron.
The Hippo pathway stimulates YAP-S381/384
phosphorylation and b-TRCP interaction
To study the regulation of YAP-S381 and YAP-S384
phosphorylation and the relationship with YAP–b-TRCP
interaction, we generated a phosphorylation-specific
antibody against pYAP (S381/384). As shown in Figure
4A, this antibody has only minimal recognition of phos-
phorylation-deficient YAP-S381A or YAP-S384A mutants
compared with YAP wild type. Furthermore, l protein
phosphatase treatment abolishedrecognition of wild-type
YAP by this antibody, thus confirming its specificity (Fig.
4A). Using this antibody, we showed that coexpression of
Mst2 and Lats2 enhanced phosphorylation on these res-
idues, while coexpression of Mst2 and Lats2 dominant-
negative mutants inhibited YAP phosphorylation (Fig.
4B). Regulation of S381/384 phosphorylation by the
b-TRCP interaction. (A) pYAP (S381/384)
antibody preferentially recognizes S381/
384-phosphorylated YAP. YAP wild type
or mutants were coexpressed with Mst2
and Lats2 in HEK293 cells. Flag-YAP was
then immunoprecipitated and one set of
the immunoprecipitates was treated with
l protein phosphatase as indicated. The
immunoprecipitated YAP phosphorylation
and protein levels were then detected by
anti-pYAP (S381/384) antibody and anti-
Flag antibody, respectively. (B) Mst2 and
Lats2 induce YAP-S381/384 phosphoryla-
tion. Indicated plasmids were cotransfected
into HEK293 cells. (KR)Kinase-inactive
mutants. YAP phosphorylation was shown
by a pYAP (S381/384)-specific antibody. (C)
Lats knockdown significantly attenuates
YAP-S381/384 phosphorylation. HA-YAP
The Hippo pathway induces
phosphorylation S381 andYAP–
was expressed in HEK293 cells. Lats1 and Lats2 knockdown was achieved by two rounds of siRNA transfection with 24-h intervals.
YAP phosphorylation was shown by a pYAP (S381/384)-specific antibody. Anti-HA Western blot indicates YAP protein level. Lats
knockdown efficiency was shown by an antibody against both Lats1 and Lats2. (D) Mst2 and Lats2 stimulate YAP–b-TRCP interaction.
Indicated plasmids were transfected into HEK293 cells. b-TRCP was immunoprecipitated with anti-Flag antibody, and coimmunopre-
cipitated YAP was shown by anti-HA Western blot. (E) Dominant-negative Mst2 and Lats2 inhibit YAP–b-TRCP interaction. Indicated
plasmids were transfected into HEK293 cells. (KR) Kinase-inactive mutants. YAP was immunoprecipitated with anti-Flag antibody, and
coimmunoprecipitated b-TRCP was shown by anti-HA Western blot. (F) Lats knockdown inhibits YAP–b-TRCP interaction. Indicated
plasmids were cotransfected with or without Lats1/2 siRNA. Lats1/2 siRNA transfection was repeated once after 24 h. b-TRCP was
immunoprecipitated with anti-Flag antibody, and coimmunoprecipitated YAP was shown by anti-HA Western blot. Lats knockdown
efficiency was shown by an antibody against both Lats1 and Lats2.
Regulation of YAP degradation by SCFb-TRCP
GENES & DEVELOPMENT77
Hippo pathway was further confirmed by a significant
decrease of YAP-S381/384 phosphorylation upon Lats1/2
knockdown (Fig. 4C).
Consistent with the up-regulation of S381/384 phos-
phorylation, Mst2 and Lats2 cotransfection also dramat-
ically enhanced YAP–b-TRCP interaction, as shown by
co-IP experiments (Fig. 4D). The effect of Mst2 and Lats2
was also seen by a reciprocal co-IP (Fig. 4E). Moreover,
expression of dominant-negative Mst2 and Lats2 in-
hibited YAP–b-TRCP interaction (Fig. 4E). Finally, knock-
down of Lats1/2 also strongly inhibited YAP–b-TRCP
binding (Fig. 4F), while this inhibition was rescued by
expressing RNAi-resistant mouse Lats2 wild-type pro-
tein, but not the kinase-inactive form (Supplemental Fig.
S2), supporting a role of endogenous Lats in regulating
YAP interaction with b-TRCP. The above results confirm
our hypothesis that the Hippo pathway promotes S381/
384 phosphorylation, and therefore YAP–b-TRCP inter-
action. S381 in the Lats phosphorylation consensus is
directly phosphorylated by Lats, while S384 is likely to be
phosphorylated by another kinase.
S381 phosphorylation by Lats primes YAP
for a sequential phosphorylation by CK1d/e
in the phosphodegron and subsequent b-TRCP binding
According to our hypothesis, the kinase responsible for
S384 phosphorylation may require priming phosphoryla-
tion on ?3 position serine (S381), and the same kinase
may be responsible for S387 phosphorylation. Interest-
ingly, the CK1 family kinases are known to have a canon-
ical consensus of pS/T-X1–2-S/T (Knippschild et al. 2005).
We tested whether CK1 is involved in S384 phosphory-
lation by using a general CK1 inhibitor (D4476) and
a CK1d/e-specific inhibitor (IC261). Both inhibitors
strongly decreased YAP interaction with b-TRCP (Sup-
plemental Fig. S3A), and, as shown in Figure 5A, IC261
blocked YAP phosphorylation as detected by the pYAP
(S381/384)-specific antibody, supporting a role of CK1d/e
in S384 phosphorylation. This was further confirmed by
coexpression of CK1e with YAP. Wild-type but not the
kinase-inactive CK1e increased recognition by anti-pYAP
(S381/384) antibody by 40% (Fig. 5B).
To confirm the relationship between Lats phosphory-
lation on S381 and the subsequent CK1d/e phosphoryla-
tion on serine residues 384 and 387, we performed a two-
step phosphorylation reaction in vitro. GST-YAP sub-
strates were first subjected to Lats2 kinase assay reaction
with cold ATP followed by removal of Lats2. The Lats2-
pretreatedGST-YAP was subsequently phosphorylated by
CK1e kinase in the presence of
without CK1e treatment, no32P incorporation was de-
tected in GST-YAP wild type (Fig. 5C). After the two-step
kinase reaction, GST-YAP was efficiently phosphorylated
by CK1e (as indicated by the incorporation of32P). The
32P-ATP. As expected,
it for S384 phosphorylation by CK1d/e and
stimulates b-TRCP binding. (A) YAP phos-
phorylation is inhibited by CK1d/e inhibi-
tor. HA-YAP was transfected into HEK293
cells. Cells were treated with CK1d/e inhib-
itor IC261, and phosphorylation of S381
and S384 was detected in Western blot by
a phosphospecific antibody. (B) CK1e in-
activity-dependent manner. HEK293 cells
were transfected and Western blot was done
as indicated. (KR) Kinase-inactive form of
CK1e. Quantification of the ratio between
pYAP (S381/384) and
a 40% increase of the phosphorylation by
coexpression of CK1e. (C) In vitro phosphor-
ylation of YAP by CK1e requires S381 prim-
ing phosphorylation by Lats. Flag-Lats2 and
HA-CK1e were expressed in HEK293 cells
with resin conjugated with anti-Flag or anti-
HA antibody. GST-YAP substrates were
expressed and purified from Escherichia coli
and were first subjected to Lats kinase assay
with cold ATP as indicated. Aliquots of the
YAP-S381 phosphorylation primes
reaction mixture supernatant (without the resin-bound Lats kinase) were taken to the second-step kinase assay with immunoprecipitated
HA-CK1e and32P-ATP. Resin after the first-step reaction was prepared for Western blot for Lats protein level. All autoradiography and
other Western blots were from samples after the second-step reaction. (D) CK1d/e inhibitor dose-dependently inhibited the interaction
between YAP and b-TRCP. HA-YAP and Flag-b-TRCP were expressed in HEK293 cells as indicated. Cells were treated with the indicated
concentration of IC261 for 1 h before harvest. Flag-b-TRCP was immunoprecipitated, and coimmunoprecipitated YAP was shown by anti-
HA Western blot. (E) CK1d/e increases YAP–b-TRCP interaction in a kinase activity-dependent manner. HEK293 cells were transfected
with indicated plasmids and were treated with IC261 as indicated. Flag-b-TRCP was immunoprecipitated, and coimmunoprecipitated
YAP was shown by anti-HA Western blot.
Zhao et al.
78 GENES & DEVELOPMENT
kinase-inactive CK1e-KR mutant failed to phosphorylate
GST-YAP, supporting a direct role of CK1e in GST-YAP
phosphorylation. Furthermore, without the pretreatment
with Lats2, GST-YAP was minimally phosphorylated by
CK1e. Together, these data demonstrate the importance
of Lats priming phosphorylation for CK1e to phosphory-
To further test the requirement of S381 for YAP phos-
phorylation by CK1e, various YAP mutants were tested.
As expected, phosphorylation of GST-YAP by CK1e was
largely diminished if the YAP-5SA mutant was used as
a substrate (Fig. 5C), and was not rescued by restoration of
the S127 site (4SA/S127) (Fig. 5C; Supplemental Fig. S3B).
In contrast, restoration of S381 in the 5SA mutant (4SA/
S381) completely rescued YAP phosphorylation by CK1e.
The above results further support our model that phos-
phorylation of S381 by Lats is a prerequisite for YAP-S384
and possibly YAP-S387 phosphorylation by CK1e.
We then examined the function of CK1d/e on YAP–
b-TRCP interaction. Consistent with the inhibition of
S384 phosphorylation (Fig. 5A), IC261 dose-dependently
inhibited the interaction between YAP and b-TRCP (Fig.
5D). To rule out the possibility that IC261 inhibits YAP–
b-TRCP interaction by nonspecifically inhibiting the
Hippo pathway, we examined the effect of IC261 on the
interaction of b-TRCP with YAP mutant 4SA/S381 or
S381D. When four out of the five Lats phosphorylation
sites, except S381, were mutated in YAP, its interaction
with b-TRCP was similarly inhibited by IC261 (Supple-
mental Fig. S3C), indicating the effect of IC261 is not me-
diated by the other four HXRXXS sites. When S381 was
mutated to a phosphomimetic aspartic residue (S381D),
IC261 still inhibited the interaction between YAP (S381D)
and b-TRCP (Supplemental Fig. S3C). Moreover, the in-
hibition of YAP–b-TRCP interaction by IC261 was also
observed when the Hippo pathway was activated by
coexpression of Mst2 and Lats2 (Supplemental Fig. S3D).
The above results ruled out the possibility that the
inhibitory effect of IC261 on the interaction between
YAP and b-TRCP is due to inhibition of the Hippo path-
way, thus supporting our model that CK1d/e phosphory-
lation of S384 is required for YAP–b-TRCP binding.
Consistently, increase of YAP–b-TRCP interaction was
observed by coexpression of CK1d or CK1e (Supplemental
Fig. S3E). This stimulation of YAP–b-TRCP interaction
depends on CK1d/e kinase activity, as the effect of CK1d/e
was blocked by IC261 treatment and the kinase-inactive
CK1d/e hadlittle effect(Fig.5E).Theaboveresultssupport
our model that, following S381 phosphorylation by Lats,
CK1d/e phosphorylate YAP on S384 and possibly S387 to
activate the phosphodegron and permit b-TRCP binding.
YAP ubiquitination depends on phosphorylation
of S381 and phosphodegron
YAP ubiquitination was elevated when cells were cul-
Hippo pathway is activated. We tested if YAP ubiquitina-
tion is stimulated by the Hippo pathway kinases and
SCFb-TRCP. Indeed, expression of Mst2 and Lats2 induced
Myc-Ub incorporation and a ladder pattern of YAP pro-
tein when proteasome function was inhibited by MG132
(Fig. 6A). Coexpression of the SCFb-TRCPcomplex compo-
nents (b-TRCP, SKP1, and Cul1) further increased YAP
ubiquitination in a manner dependent on Mst/Lats co-
expression. These results support the functional impor-
tance and collaboration of Mst/Lats and SCFb-TRCPin
stimulating YAP ubiquitination.
We next examined the function of S381 and phospho-
degron in YAP ubiquitination. As shown in Figure 6B, the
phosphorylation-deficient S381A mutant largely lost its
tated to a phosphomimetic S381D, which partially re-
stored b-TRCP binding (Fig. 3C), YAP ubiquitination was
also partially rescued (Fig. 6B). The above results con-
firm the importance of S381 phosphorylation in YAP
ubiquitination. When D383 and S384, two key residues
in the phosphodegron, were mutated to alanine, YAP
ubiquitination was abolished, highlighting the essential
role of the phosphodegron in YAP ubiquitination. Muta-
tion of S387 to alanine (S387A) showed a lower degree in
reduction of YAP ubiquitination, while S387D mutation
rescued YAP ubiquitination (Fig. 6B). The behavior of
these two mutants is completely consistent with their
effect on YAP–b-TRCP binding (Fig. 3D), further support-
ing that S387 is important but not essential for YAP
ubiquitination. We also tested the function of CK1d/e on
YAP ubiquitination by using the inhibitor IC261. As
shown in Figure 6C, IC261 inhibited Myc-Ub incorpora-
tion into YAP, supporting an important function of
endogenous CK1d/e in YAP ubiquitination.
SCFb-TRCP-mediated YAP degradation is stimulated
by the Hippo pathway and CK1d/e
To confirm that YAP stability is regulated by the
SCFb-TRCP, we examined the effect of blocking SCFb-TRCP
function by a dominant-negative Cul1. As shown by
CHX chase, YAP is unstable at high cell density. How-
ever, YAP was stabilized when dominant-negative Cul1
was coexpressed (Fig. 7A). This supports that the
SCFb-TRCPpromotes YAP degradation. At low cell den-
sity, the Hippo pathway is inactive and YAP is stable (no
significant degradation under CHX chase up to 6 h) (Fig.
7B). However, if the Hippo pathway was activated by
expressing Mst2 and Lats2 in low-density cells, YAP was
destabilized (Fig. 7B). Similarly, expression of CK1e also
destabilized YAP. However, the S127/381A mutant was
not significantly destabilized by Mst2/Lats2 or CK1e (Fig.
7B). These observations indicate a role of Mst/Lats and
CK1d/e in promoting YAP degradation.
We observed that mutation of the Lats phosphorylation
site S381 stabilized YAP, especially in the S127A back-
ground (Fig. 2F), and the mutant was resistant to Mst2-,
Lats2-, and CK1e-induced degradation (Fig. 7B). We also
examined the CK1d/e phosphorylation site S384 andfound
that S384A mutation in the S127A background dramati-
cally stabilized YAP (Fig. 7C). Similar observation was
made with another key phosphodegron residue mutant,
D383A (Fig. 7C). If a protein is actively degraded by
Regulation of YAP degradation by SCFb-TRCP
GENES & DEVELOPMENT79
proteasome, blocking proteasome function should result
in accumulation of this protein. Indeed, MG132 treatment
induced a steady accumulation of YAP protein when cells
the Hippo pathway activity was inhibited by dominant-
negative Mst2 and Lats2, the MG132-induced accumula-
tion of YAP was not observed, indicating that the Hippo
pathway is required for YAP degradation. Similarly, muta-
tion of the Lats phosphorylation site S381, the CK1d/e
phosphorylation site S384, or a key phosphodegron residue
D383 on the S127A background diminished MG132-
induced YAP accumulation (Fig. 7D). Together, the above
results strongly support the regulation of YAP stability by
the Hippo pathway, CK1d/e, and the phosphodegron.
The phosphodegron suppresses YAP oncogenic
Finally, we investigated the function of the CK1d/e phos-
phorylation site S384 andthephosphodegron in regulating
YAP oncogenic activity. As shown in Figure 7E, YAP with
a single-site mutation of S127, S381, S384, S387, or D383
was unable to transform NIH-3T3 cells. However, similar
to the S127/381A double mutant, the S127/384A and
S127/D383A double mutants could transform NIH-3T3
cells as potently as the 5SA protein (Fig. 7E). The S127/
387A double mutant could also transform NIH3T3 cells,
though less actively, consistent with the less stringent
requirementof S387 for b-TRCP binding. Collectively, our
data support the importance of S381 priming phosphory-
lation by Lats, the subsequent S384 and S387 phosphory-
lation by CK1d/e, and the phosphodegron recognition by
SCFb-TRCPin repression of YAP oncogenic activity.
Spatial and temporal regulation of YAP
Accumulating evidence supports the role of YAP as a key
controller of organ size and as a human oncogene.
pathway components and SCF stimulate YAP ubiquitination. Flag-YAP, Myc-Ub, the Hippo pathway components Mst2 and Lats2, and
the SCF complex components b-TRCP, SKP1, and CUL1 were cotransfected into HEK293 cells as indicated. Cells were treated with
MG132 as indicated before harvest. Flag-YAP was immunoprecipitated, and Western blots were done with specific antibodies. (L) longer
exposure for Flag-YAP; (S) shorter exposure for Flag-YAP. (B) YAP ubiquitination is dependent on S381, 384, and 387 phosphorylation
and on intact phosphodegron. YAP wild type and mutants were cotransfected with other plasmids into HEK293 cells as indicated. Cells
were treated with 25 mM MG132 for 5 h before harvest. Flag-YAP was immunoprecipitated, and Western blots were done with
indicated antibodies. (C) YAP ubiquitination is inhibited by the CK1d/e inhibitor IC261. HEK293 cells were transfected as indicated.
Cells were pretreated with 10 mM IC261 for 1 h and then treated with MG132 as indicated before harvest. Flag-YAP was
immunoprecipitated, and Western blots were done with indicated antibodies.
SCF, Mst/Lats, and CK1d/e promote YAP ubiquitination through S381 and phosphodegron phosphorylation. (A) The Hippo
Zhao et al.
80GENES & DEVELOPMENT
Elucidating the mechanisms regulating YAP activity will
have implications in the normal physiology of organ size
regulation and pathogenesis of human cancer. The Hippo
pathway is the only inhibitor of YAP known to date. It
has been shown to play a key role in limiting organ size
in Drosophila (Pan 2007), and deregulation of several
components of this pathway, such as NF2 mutation
(McClatchey and Giovannini 2005), has been implicated
in human cancer. Previously, we showed that the Hippo
pathway inhibits YAP by S127 phosphorylation-mediated
14–3–3 binding and cytoplasmic retention (Zhao et al.
2007), therefore providing a mechanism of spatial sepa-
ration of YAP from its nuclear target transcription factors,
such as TEAD (Fig. 8).
YAP has been shown recently to be ubiquitinated,
although the mechanism was unknown (Lapi et al. 2008).
The data we presented in this study elucidated another
layer of YAP regulation. By phosphorylation on S381, the
and subsequent ubiquitination and degradation (Fig. 8).
This provides a mechanism of temporal regulation of YAP
protein levels upon activation of the Hippo pathway.
Under physiological conditions like high cell density, the
S381 phosphorylation-mediated degradation might be the
major cause for YAP degradation. As shown in Figure 2C,
relative S381 phosphorylation dropped dramatically when
cell density increased, although relative S127 phosphory-
lation of YAP was increased, indicating that the S381-
phosphorylated YAP could not be accumulated, possibly
a S127 phosphorylation-dependentfail-safemechanism for
YAP destabilization when S381-mediated degradation is
notworkingproperly.Suchamechanism may explainwhy
both S127 and S381 mutations are required for YAP
stabilization. Our study reveals that inhibition of YAP by
the Hippo pathway is more complex than expected, with
both spatial and temporal mechanisms. We speculate that
the spatial regulation could provide a reversibleshort-term
inhibition of YAP, while the temporal regulation through
YAP degradation may provide an irreversible long-term
inhibition. Dysregulation of both mechanisms could lead
to oncogenic transformation.
It is worth noting that the S381-initiated degradation
of YAP is not conserved in Drosophila Yki, because
this phosphorylation site and the phosphodegron are
not present in Yki, although they are conserved through
vertebrates. However, this does not exclude the possibil-
ity that Yki protein stability is controlled by the Hippo
formation ability through SCFb-TRCP-mediated
YAP degradation depends on the Hippo path-
way, CK1d/e, and the phosphodegron. (A) YAP
degradation is inhibited by dominant-negative
Cul1. Flag-YAP was transfected into NIH-3T3
cells at low expression level alone (left
panels) or together with dominant-negative
Cul1 (right panels), and cells were cultured to
confluence. Cells were treated with 50 mg/mL
CHX for the indicated time. Anti-Flag West-
ern blot was used to show YAP protein levels
and anti-tubulin Western blot was used as
a loading control. (B) The Hippo pathway and
CK1e destabilize YAP in a S381-dependent
manner. Flag-YAP wild type or S127/381A
mutant were transfected alone or together
with the Hippo pathway components Mst2/
Lats2, or CK1e into NIH-3T3 cells. Cells were
cultured at low density so that most cells
do not have contact with each other. CHX
chase and Western blots were done as in
A. (C) Mutation of S384 or D383 in the
phosphodegron stabilizes YAP. YAP wild type
or mutants were transfected into NIH-3T3
cells. Cells were cultured to confluence, and
CHX chase and Western blots were done as in
A. (D). Regulation of YAP stability by protea-
some is dependent on functional Hippo path-
way, as well as S381 and the phosphodegron
of YAP. Flag-YAP wild type or mutants were
transfected alone or together with kinase-
inactive Mst2-KR and Lats2-KR as indicated.
Cells were cultured to confluence and were
treated with 25 mM MG132 for the indicated
Inhibition of YAP oncogenic trans-
time before harvest. Western blots were done as in A. (E) Mutation of the phosphodegron together with S127 is sufficient for YAP to
transform NIH-3T3 cells. NIH-3T3 cell colony formation assays were performed using indicated YAP constructs. Colonies were
visualized with crystal violet staining.
Regulation of YAP degradation by SCFb-TRCP
GENES & DEVELOPMENT81
pathway through other mechanisms. The phosphodegron
is conserved in TAZ, a YAP paralog, and also modulates
TAZ stability in a similar manner (QYL and KL Guan,
Are there additional mechanisms of YAP regulation by
the Hippo pathway? The possibility exists. Our studies
confirmed three other Lats phosphorylation sites in YAP,
but their functions are unknown. Although these sites do
not seem to play an obvious role in controlling the on-
cogenic activity of YAP, as indicated by NIH-3T3 cell
Lessons from b-catenin for the regulation of YAP
The similarity between YAP and b-catenin is quite in-
cated in malignant transformation. Without Wnt signal-
ing, b-catenin is constantly degraded through SCFb-TRCP-
mediated ubiquitination (Clevers 2006). Similar to YAP,
b-catenin binding with SCFb-TRCPdepends strictly on
multistep phosphorylation of the phosphodegron involv-
ing CK1a and GSK-3 (Liu et al. 2002). Perturbation of this
process leads to b-catenin accumulation in colorectal
cancer, HCCs, and malignant melanomas (Frescas and
Pagano 2008). There are similarities between YAP and
b-catenin in many aspects, including their function as
transcription coactivators with growth-promoting activ-
ity and as latent oncogenes. They are both subject to
multistep phosphorylation and phosphodegron-dependent
ubiquitination by SCFb-TRCP, and deregulation of the deg-
radation leads to oncogenic transformation.
Extensive studies have been done to analyze mutations
leading to b-catenin stabilization, which should shed
light on future studies of YAP. In the case of b-catenin,
its stabilization in cancer is frequently due to failure to
recruit GSK3 as a result of inactivating mutations of
adenomatous polyposis coli (APC) or axin (Rubinfeld
et al. 1996; Liu et al. 2000). In some cases, stabilization
of b-catenin also results from mutation in the phospho-
degron and its priming phosphorylation sites (Morin et al.
1997). Interestingly, elevated YAP protein levels have
been observed in some cancers (Zender et al. 2006; Dong
et al. 2007; Zhao et al. 2007; Steinhardt et al. 2008). It will
be interesting to survey possible YAP mutations in can-
cer samples and identify proteins regulating YAP phos-
phodegron phosphorylation. It will also be important to
examine deregulation of YAP protein levels as a result of
Hippo pathway component mutations in cancer.
CK1d/e as new players in the Hippo–YAP pathway
CK1 is a family of multifunctional kinases with unique
substrate specificity as pS/T-X1–2-S/T (Knippschild et al.
2005). Phosphorylation by CK1 requires preceding phos-
phorylation of residue at the ?2 or ?3 position of the
target residue. This requirement of a priming phosphor-
ylation by another kinase provides a possible mechanism
of signal integration in complex biological processes. For
example, in the case of YAP destabilization, the require-
ment of CK1d/e phosphorylation following Lats phos-
phorylation may integrate other signals besides the Hippo
pathway to regulate YAP. CK1 is often referred to as
constitutively active kinase. However, it has also been
reported that CK1 is regulated by subcellular localization
and inhibitory autophosphorylation by stimuli such as g
irradiation and Wnt signaling (Knippschild et al. 2005). At
high cell density, we observed a clear drop of relative
YAP-S381 phosphorylation and an increase of relative
YAP-S127 phosphorylation. The fact that both sites are
phosphorylated by Lats kinase suggests that phosphory-
lation of S384 might induce YAP degradation. It will be
interesting to investigate if cell density increases CK1
In Drosophila, the CK1d/e homolog discs overgrown
(dco) has been positioned in the Hippo pathway upstream
of dachs by its regulation of the Hippo pathway down-
stream target genes and by genetic epistasis experiments
(Cho et al. 2006). Recently, dco has further been shown to
phosphorylate Fat (Sopko et al. 2009), although it has not
been determined if this phosphorylation directly affects
Fat function and the Hippo pathway activity. However,
the function of CK1d/e in regulating YAP–b-TRCP in-
teraction is not due to inhibition of the Hippo pathway, as
both YAP-4SA/S381 and YAP-S381D mutants are still in-
hibited by IC261. Conversely, the mechanism of CK1d/e
in regulating YAP stability is unlikely to be conserved in
CK1. When the Hippo pathway is activated, YAP is phosphor-
ylated on both S127 and S381 in the HXRXXS motifs. Phos-
phorylation of S127 results in 14–3–3 binding and cytoplasmic
retention of YAP. Therefore, YAP can be inhibited by spatial
separation from its nuclear target transcription factors, such as
TEAD. This mechanism of regulation is reversible. Phosphory-
lation of S381 primes YAP phosphorylation by CK1d/e, resulting
in activation of a phosphodegron, which then recruits the
SCFb-TRCPE3 ubiquitin ligase, leading to YAP degradation. This
mechanism of regulation is irreversible. Through these two
mechanisms, YAP activity is under both spatial and temporal
control, which coordinately inhibit YAP oncogenic activity.
A model of YAP inhibition by the Hippo pathway and
Zhao et al.
82 GENES & DEVELOPMENT
dco, as the phosphodegron is not conserved in Yki.
Nevertheless, the function of dco/CK1d/e in inhibiting
Yki/YAP is conserved between Drosophila and mam-
mals, although different mechanisms may be employed.
The phosphodegron of YAP
YAP contains a phosphodegron, DSGXS, that is highly
similar to but does not exactly match the canonical
DSGXXS phosphodegron. However, the requirement of
the second serine residue for b-TRCP binding is less
stringent compared with the first one. In the reported
phosphodegron variants, some of them require the second
serine to be further away from the DSG (Frescas and
Pagano 2008), and, in certain cases like CDC25A, the
second S is not even required (Jin et al. 2003). In the case
of YAP, the second serine (S387) is not absolutely re-
quired, but contributes to YAP interaction with b-TRCP
and YAP ubiquitination. This was shown by the residual
binding between b-TRCP and the phosphorylation-
deficient S387A, and the largely normal binding between
b-TRCP and the phosphomimetic S387D (Fig. 3D).
The exact YAP sequence S(?3)TDS(0)G, where S(?3)
(S381) serves as a priming phosphorylation site for S(0)
(S384), is conserved in some other b-TRCP substrates like
CDC25A, which contains S(?6)XXS(?3)TDS(0)G (Supple-
mental Fig. S4). In this case, the ?6 position serine phos-
phorylation by Chk1 is shown to be required for CDC25A
binding with b-TRCP and subsequent degradation in vivo
(Jin et al. 2003). However, in an in vitro binding assay,
a peptide with phosphorylation on the S(0)but not S(?3)
showed a strong binding to b-TRCP, which was not
further enhanced by phosphorylation on S(?3)(Jin et al.
2003). This in vitro binding assay using peptides sharing
similar phosphodegron structure with YAP helps us to
exclude the function of YAP-S381 as an integral part of
the phosphodegron directly involved in b-TRCP binding,
but rather supports S381 as a priming phosphorylation
site for S384 phosphorylation by CK1d/e. Compared
with YAP, we speculate that the main function of the
S(?3)in the CDC25A phosphodegron might be a phos-
position to the 0 position instead of being directly in-
volved in b-TRCP binding. Phosphodegron with a phos-
phorylated ?3 position serine also exists in other known
SCFb-TRCPsubstrates, such as RE-1 silencing transcrip-
tion factor (REST) (Supplemental Fig. S4; Westbrook et al.
2008). Together with YAP, they may represent a class of
SCFb-TRCPsubstrates containing a SXDSG phosphode-
gron, in which the first serine serves as a priming phos-
phorylation site. In the case of CDC25A and REST, the
kinase responsible for phosphorylating the second serine
residue is unknown. The CK1 family kinases are at-
tractive candidates for this function because of their pS/
T-X1–2-S/T target consensus. We speculate that there may
be a broader role for the CK1 family in SCFb-TRCP-
mediated protein ubiquitination and degradation.
In close proximity with the YAP phosphodegron, there
is a tyrosine residue (Y391) reported to be phosphorylated
by c-Abl in response to DNA damage, which results in
YAP stabilization (Levy et al. 2008). Future studies are
needed to test if the Y391 phosphorylation modulates
SCFb-TRCP-mediated YAP ubiquitination and degradation.
Materials and methods
Cells were cultured on coverslips to the appropriate density.
Cells were fixed with 4% paraformaldehyde for 15 min and then
permeabilized with 0.1% Triton X-100. After blocking in 3%
bovine serum albumin (BSA) for 30 min, slides were incubated
with first antibody diluted in 1% BSA for 1.5 h. After washing
with PBS, slides were incubated with Alexa Fluor 488- or 594-
conjugated secondary antibodies (1:1000 dilution) for 1.5 h. The
slides were then washed and mounted.
Immunoprecipitation and kinase assay
For the Lats2 and CK1e two-step kinase assays, HEK293
cells were transfected with Flag-Lats2 or HA-CK1e. Forty-eight
hours post-transfection, cells were lysed with lysis buffer
(50 mM HEPES at pH 7.5, 150 mM NaCl, 1 mM EDTA, 1%
NP-40, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM
NaF, 1.5 mM Na3VO4, protease inhibitor cocktail [Roche], 1 mM
DTT, 1 mM PMSF), and immunoprecipitated with anti-Flag or
anti-HA antibody-conjugated resin. The immunoprecipitates
were washed three times with lysis buffer, and then once with
wash buffer (40 mM HEPES, 200 mM NaCl) and once with
kinase assay buffer (30 mM HEPES, 50 mM potassium acetate,
5 mM MgCl2). In each reaction, 1 mg of GST-YAP2, which was
expressed and purified from Escherichia coli, was first subjected
to Lats2 kinase assay in the presence of the immunoprecipitated
Lats2 and 500 mM cold ATP. The reaction mixtures were
incubated for 30 min at 30°C, and then briefly incubated on ice
followed by centrifugation and removal of an aliquot of the
supernatant to the next-step reaction. The leftover with the resin
was supplemented with SDS loading buffer and cooked to exam-
ine the Lats2 protein level. Supernatants from the first step were
then supplemented with immunoprecipitated HA-CK1e and 10
mCi (g-32P) ATP. The mixtures were incubated for 30 min at
30°C, and the reactions were terminated with SDS sample buffer
and subjected to SDS-PAGE and autoradiography.
Smart pool siRNA oligonucleotides toward human Lats1 or
Lats2 and control siRNA toward firefly luciferase were pur-
chased from Dharmacon. siRNAs were transfected into HEK293
cells twice with a 24-h interval. Cell lysate was made 48 h post-
Colony formation assay
Colony formation assay was performed as described briefly
below. NIH-3T3 fibroblasts were seeded on six-well plates at
a density of 105cells per well and then transfected with YAP wild
type or mutants using FuGene6 (Roche) according to the man-
ufacturer’s instructions. After 2 d, cells were trypsinized and an
aliquot of cells were lysed for YAP expression; other cells were
replated onto a 10-cm dish and were maintained in DMEM
supplemented with 5% fetal bovine serum for 2–3 wk until foci
were evident. Cells were fixed with 10% acetic acid and 10%
methanol, and then colonies were stained with 1% crystal violet
Regulation of YAP degradation by SCFb-TRCP
GENES & DEVELOPMENT 83
We thank Drs. Philip Gafken at the Fred Hutchinson Cancer
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Cul1, pcDNA3-SKP1, and PCW7-Myc-His-Ub plasmids; Stephen
J. Elledge for DN-Cul1 plasmid; and Xi He for CK1d and CK1e
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