Kibra Is a Regulator of the Salvador/Warts/Hippo Signaling Network

Apoptosis and Proliferation Control Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.
Developmental Cell (Impact Factor: 9.71). 02/2010; 18(2):300-8. DOI: 10.1016/j.devcel.2009.12.011
Source: PubMed
ABSTRACT
The Salvador (Sav)/Warts (Wts)/Hippo (Hpo) (SWH) network controls tissue growth by inhibiting cell proliferation and promoting apoptosis. The core of the pathway consists of a MST and LATS family kinase cascade that ultimately phosphorylates and inactivates the YAP/Yorkie (Yki) transcription coactivator. The FERM domain proteins Merlin (Mer) and Expanded (Ex) represent one mode of upstream regulation controlling pathway activity. Here, we identify Kibra as a member of the SWH network. Kibra, which colocalizes and associates with Mer and Ex, also promotes the Mer/Ex association. Furthermore, the Kibra/Mer association is conserved in human cells. Finally, Kibra complexes with Wts and kibra depletion in tissue culture cells induces a marked reduction in Yki phosphorylation without affecting the Yki/Wts interaction. We suggest that Kibra is part of an apical scaffold that promotes SWH pathway activity.

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Available from: Michael C Wehr, Oct 23, 2014
Developmental Cell
Short Article
Kibra Is a Regulator
of the Salvador/Warts/Hippo Signaling Network
Alice Genevet,
1
Michael C. Wehr,
1
Ruth Brain,
2
Barry J. Thompson,
2,
*
and Nicolas Tapon
1,
*
1
Apoptosis and Proliferation Control Laboratory
2
Epithelial Biology Laboratory
Cancer Research UK, London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK
*Correspondence: barry.thompson@cancer.org.uk (B.J.T.), nicolas.tapon@cancer.org.uk (N.T.)
DOI 10.1016/j.devcel.2009.12.011
SUMMARY
The Salvador (Sav)/Warts (Wts)/Hippo (Hpo) (SWH)
network controls tissue growth by inhibiting cell
proliferation and promoting apoptosis. The core of
the pathway consists of a MST and LATS family
kinase cascade that ultimately phosphorylates and
inactivates the YAP/Yorkie (Yki) transcription coacti-
vator. The FERM domain proteins Merlin (Mer) and
Expanded (Ex) represent one mode of upstream
regulation controlling pathway activity. Here, we
identify Kibra as a member of the SWH network. Ki-
bra, which colocalizes and associates with Mer and
Ex, also promotes the Mer/Ex association. Further-
more, the Kibra/Mer association is conserved in
human cells. Finally, Kibra complexes with Wts and
kibra depletion in tissue culture cells induces
a marked reduction in Yki phosphorylation without
affecting the Yki/Wts interaction. We suggest that Ki-
bra is part of an apical scaffold that promotes SWH
pathway activity.
INTRODUCTION
Growth regulation is a critical developmental process whose
dysfunction can lead to many diseases, including cancer (Conlon
and Raff, 1999). The Salvador (Sav)/Warts (Wts)/Hippo (Hpo)
(SWH) network, identified in Drosophila and conserved in
mammals, plays a major role in limiting growth by inhibiting cell
proliferation and promoting apoptosis (Harvey and Tapon, 2007;
Reddy and Irvine, 2008). Activation of the upstream kinase Hpo
allows it to phosphorylate the downstream kinase Wts, which in
turn phosphorylates and inhibits the transcription coactivator
Yorkie (Yki). Scaffold proteins, such as Salvador (Sav) and Mats,
potentiatethe activity of the Hpo/Wts complex (Harvey and Tapon,
2007). One upstream input of the pathway is mediated via Merlin
(Mer) and Expanded (Ex), two FERM (Four point one, Ezrin, Moe-
sin, Radixin) domain proteins (Hamaratoglu et al., 2006). Recently,
Ex was shown to bind Yki, and new experiments hint at the exis-
tence of an Ex/Hpo/Wts-containing apical complex anchoring
Yki at the cortex (Badouel et al., 2009; Oh et al., 2009). These find-
ings underline the importance of scaffold proteins in the regulation
of the pathway. However, though some upstream members are
known, how the SWH network is activated remains unclear.
Here, we identify the WW-domain-containing protein Kibra as
a regulator of the SWH network. Human KIBRA (Kremerskothen
et al., 2003) is known to be phosphorylated by Protein Kinase C z
(PKCz)(Buther et al., 2004) and has recently been reported to
have a role in cell migration (Duning et al., 2008; Rosse et al.,
2009). In Drosophila, Kibra had previously been recovered as a
minor hit in several screens for growth regulators (Boutros
et al., 2004; Muller et al., 2005; Ringrose et al., 2003; Tseng
and Hariharan, 2002) but has not been further studied. Our
experiments show that Kibra associates with Mer, Ex, and Wts
and stabilizes the Mer/Ex interaction. This suggests that Kibra
is a component of an apical scaffold that controls SWH pathway
activation.
RESULTS
kibra and wts RNAi Depletion Induce Similar
Overgrowth Phenotypes
We performed an in vivo screen in the fly wing in order to identify
genes implicated in growth control. Transgenic flies bearing
RNA interference (RNAi) constructs generated by the Vienna
Drosophila RNAi Centre (VDRC) (Dietzl et al., 2007) were crossed
to the hedgehog-GAL4 (hh-GAL4) driver, leading to target gene
silencing in the posterior compartment of the wing. We screened
a collection of 12,000 lines targeting genes conserved between
Drosophila and mammals. The results of this screen will be
described elsewhere.
In this context, expressing an RNAi line directed against kibra
induced overgrowth of the posterior wing compartment
(Figure 1B) compared to control flies (Figure 1A). This phenotype
was also observed upon wts depletion (Figure 1C). Driving the
same kibra RNAi line in the eye also led to increased organ
size (Figures 1D and 1E), similarly to a wts RNAi line (Figure 1F).
To exclude off-target effects, we generated a transgenic line
expressing a
nonoverlapping RNAi construct and observed
identical overgrowth phenotypes (data not shown). Furthermore,
adult eye sections revealed that kibra knockdown retinas present
an excess of interommatidial cells (IOCs) (Figures 1G and 1H).
The IOCs, the last population of cells to differentiate in the eye
primordium, give rise to the secondary and tertiary pigment cells
that optically isolate the ommatidia in the compound eye from
each other. Extra IOCs are produced during normal develop-
ment but are then eliminated by apoptosis at the pupal stage
to give rise to the adult lattice (Wolff and Ready, 1993). The pres-
ence of extra IOCs is a hallmark of SWH network loss of function
(Kango-Singh et al., 2002; Tapon et al., 2002), which reduces
300 Developmental Cell 18, 300–308, February 16, 2010 ª2010 Elsevier Inc.
Page 1
retinal apoptosis, as seen in wts RNAi adult eye sections
(Figure 1I). Thus, depletion of kibra elicits a similar phenotype
to SWH network mutants, suggesting a potential role for Kibra
in Hpo signaling.
kibra Loss of Function Causes Excess Proliferation
and a Reduced Apoptotic Rate, Similarly
to SWH Network Loss of Function
To study kibra loss of function, we generated the kibra
D32
allele
by imprecise excision of the EP747 transposon (see Figure 1J
and Experimental Procedures). This deletion allele, which re-
moves the translation initiation site, is homozygous lethal and
may be a null allele for kibra. kibra
D32
FLP/FRT mutant clones
in 40 hr after-puparium-formation (APF) retinas present extra
IOCs (Figure 1K–1M), similarly to what was observed in adult
eyes with kibra knockdown (Figures 1G–1I). Duplication of bris-
tles or missing bristles can also be observed. We determined
apoptotic indexes (Colombani et al., 2006) during the retinal
apoptosis wave (28 hr APF) in pupal retinas containing kibra
mutant clones stained with an anti-active Caspase-3 antibody.
kibra mutant tissue presents a reduced apoptotic index
compared with wild-type (WT) areas in the same retinas (Figures
1N–1P; see Figures S1A–S1A
00 0
available online). Thus, extra
IOCs persist in kibra mutant clones as a result of decreased
developmental apoptosis.
We assessed the proliferation rate of kibra mutant cells in
imaginal discs, the larval precursors to the adult appendages.
By using the FLP/FRT system under the control of the heat-shock
promoter, kibra mutant cells and their WT sister clones were
generated through single recombination events from heterozy-
gous mother cells (Brumby and Richardson, 2005). After several
rounds of divisions, the sizes of mutant clones (no GFP) and WT
twin spots (two copies of GFP) were compared, allowing us to
estimate the relative proliferation rates of mutant versus WT
cells. The total kibra clone area is 1.57 (±0.12)-fold larger than
the control twin spot area, compared to a ratio of 0.98 (±0.09)
when both clones and twin spots are WT (Figures 1Q–1S), indi-
cating that kibra
D32
mutant cells grow 1.6 times faster than WT
cells.
In addition to cell cycle rates, the timing of cell cycle exit can
readily be measured in the eye disc, where cell divisions follow
a spatially determined pattern (Wolff and Ready, 1993). During
the third larval instar, the morphogenetic furrow, a wave of
Figure 1. Kibra Loss of Function Induces a Phenotype Similar to
SWH Network Loss of Function
(A–C) Control wings (A) and wings where the kibra (B) or wts (C) genes were
silenced by RNAi in the posterior compartment (red dotted line).
(D–I) Control fly eyes (D and G) and eyes expressing the same RNAi lines
against kibra (E and H) or wts (F and I). In adult eye sections (G–I), interomma-
tidial cells (IOCs) are in red and photoreceptors in blue.
(J) Schematic of the kibra locus showing the localization of the kibra
D32
allele,
generated by excision of the EP747 P element (triangle). The coding sequence
is in red; 5
0
- and 3
0
-UTRs are in blue. In black is the peptide recognized by the
Kib18 antibody.
(K–M) 40 hr APF retinas containing kibra mutant clones. (K and L) Cell outlines
are visualized by anti-Arm staining (scale bar = 10 mm). (M) Quantification of the
IOCs. The p value from a Mann-Whitney test is shown.
(N–P) Apoptotic index in 28 hr APF retinas containing kibra mutant clones
(absence of GFP). A retina stained for activated Caspase-3 (Cas3) merged
with GFP is shown in (N). The Cas3 staining for the whole retina is shown in
(O). (P) Quantification of the apoptotic index. The p value from a Mann-Whitney
test is shown.
(Q–S) Wing disc (Q) and eye disc (R) containing kibra clones (lack of GFP).
Scale bars = 20 mm. (S) Quantification of the proliferation advantage of WT
or kibra mutant cells by calculating the ratio between total clonal area (no
GFP) and total twin spot area (two copies of GFP) for discs containing either
WT or kibra clones. The p value from a Mann-Whitney test is shown.
(T–V) EdU labeling (shown in gray or red) of WT discs (T) and of discs containing
kibra clones (U and V). Posterior is to the right, and the SMW is indicated by
a red arrowhead. See also Figure S1.
Error bars represent standard deviations.
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Hippo Signaling and Kibra
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differentiation, sweeps the eye disc from posterior to anterior.
Anterior to the furrow cells still proliferate asynchronously, while
in the furrow cells synchronize in G1. Immediately posterior to
the furrow, cells enter a final round of synchronous S phases,
the second mitotic wave (SMW). Posterior to the SMW, most
cells permanently exit the cell cycle. Thus, in WT discs, no S
phases (marked by EdU incorporation) can be observed poste-
rior to the SMW (Figure 1T). As expected, hpo mutant cells fail
to exit from the cell cycle in a timely manner and present ectopic
EdU-positive staining posterior to the SWM (Figures S1B and
S1B
0
). kibra mutant cells exhibit a less pronounced but similar
phenotype (Figures 1U and 1V). Thus, kibra mutant tissues have
a proliferative advantage and an apoptosis defect, consistent
with an involvement in the SWH network. The overgrowth defect
appears more subtle than that of core pathway members such as
wts and is more akin to upstream regulators (e.g., ex and mer).
Kibra Regulates SWH Network Targets
in Ovarian Posterior Follicle Cells
Several transcriptional targets of the SWH network have been
identified, such as the Drosophila Inhibitor of Apoptosis 1
(DIAP1) gene, the cell cycle regulator cycE, the miRNA bantam,
as well as ex (Harvey and Tapon, 2007; Saucedo and Edgar,
2007). In kibra mutant wing or eye discs, we could not detect
a strong change in DIAP1, CycE, or ex-lacZ reporter levels
(data not shown). Since overgrowth of kibra mutant cells in the
wing is subtle compared to wts mutants, it is possible that
Kibra plays a relatively minor role in SWH signaling in the wing.
Accordingly, using an anti-Kibra antibody we generated (Figures
S2A–S2C), we noted that Kibra staining in the wing disc is weak
and consists of a punctate apical staining which can clearly be
observed when kibra is overexpressed in a stripe of cells.
Thus, the extent to which Kibra is required may vary in different
tissues.
We and others have previously reported that ovarian posterior
follicle cells (PFCs) are particularly sensitive to SWH loss of func-
tion (Meignin et al., 2007; Polesello and Tapon, 2007; Yu et al.,
2008), leading us to study the kibra
D32
phenotype in the ovary.
First, we noted that Kibra protein levels are higher in follicle cells
than in the wing discs (Figure S2D–S2E
0
). Kibra staining is mainly
apical and is severely reduced in kibra
D32
clones. Similarly to hpo
or wts loss of function, kibra loss of function in the PFCs induces
an upregulation of the ex-lacZ reporter (Figures 2A–2B
00
,
compare with Figures 2C–2C
00
). hpo or wts mutant PFCs also
show a misregulation of the Notch (N) pathway and ectopic
cell divisions (Meignin et al., 2007; Polesello and Tapon, 2007;
Yu et al., 2008). The N target Hindsight (Hnt) is normally
repressed in all follicle cells up to stage 6 and switched on
from stage 7 to stage 10B (Figures 2D–2E
00
)(Poulton and Deng,
2007). Cut, which is repressed by Hnt, presents an opposite
pattern of expression (Figures 2H–2I
00
). In kibra mutant PFCs
from stage 7–10B egg chambers, Hnt expression is lost (Figures
2D–2D
00
and 2F–2G
00
), while Cut is ectopically expressed (Figures
2J–2K
00
). This indicates that N signaling is downregulated in kibra
mutant PFCs. Loss of kibra also leads to perturbation of epithelial
integrity, as mutant PFCs show an accumulation of the apical
polarity protein aPKC and the N receptor (Figures S2F–S2G
00
)
as well as multilayering of the follicular epithelium (Figures
2I–2I
00
and 2K–2K
00
). Ectopic mitotic divisions are also observed
in PFCs clones after stage 6, as detected by phospho-histone
H3 (PH3) staining (Figures S2H–S2H
00
). Together, these pheno-
types are identical to those observed in hpo or wts loss of func-
tion, suggesting that Kibra is indeed a member of the SWH
network.
Genetic Experiments Place kibra Upstream of the
Core SWH Kinase Cassette
To further explore the role of Kibra in the SWH network, genetic
interaction and epistasis experiments were performed. Overex-
pressing kibra in the eye under the GMR (Glass Multimer
Reporter) promoter elicits the formation of a small rough eye
with frequent ommatidial fusions (Figures S3A–S3B
0
). This
phenotype can be partially rescued by removing one copy of
the hpo gene (Figures S3C and S3C
0
). In contrast, overexpress-
ing kibra could not rescue the hpo-like overgrowth phenotype
induced by yki overexpression (Figures S3D and S3E), suggest-
ing that Kibra may be an upstream regulator of the pathway.
To conduct epistasis experiments between kibra and yki,we
used the MARCM system to generate clones of mutant cells
while simultaneously overexpressing or depleting other pathway
components (Lee and Luo, 1999). MARCM clones expressing yki
RNAi generated with eyFLP lead to the formation of a normal eye,
because yki-depleted cells are eliminated by apoptosis (data not
shown) and replaced by WT cells (Figure S3F). As expected,
eyFLP kibra MARCM clones cause eye overgrowth (Figure S3G).
This overgrowth is rescued by yki depletion in the mutant cells
(Figure S3H), indicating that the kibra overgrowth phenotype is
yki dependent. Furthermore, overexpressing kibra in the eye
under the GMR promoter induces apoptosis in third instar eye
discs, which is suppressed by loss of hpo (Figures 3A–3A
00
).
Together, these epistasis experiments are consistent with
Kibra being a member of the SWH network acting upstream of
Yki and Hpo.
Genetic interactions between kibra, mer, and ex, upstream
members of the SWH network, were then investigated. Express-
ing a kibra,anex, or a mer RNAi line in the eye under the GMR
promoter induces eye overgrowth (Figures S3I–S3L). Combined
depletion of either Ex/Kibra or Mer/Kibra shows stronger pheno-
types than individual depletion of these proteins (Figures S3M
and S3N). We used the MARCM technique to evaluate epistatic
relationships between those three genes. hsFLP MARCM clones
of various genotypes were generated and scored according to
the severity of the wing overgrowth phenotypes, with type 0 rep-
resenting normal wings and type 4 the strongest overgrowth
(Figures 3B and 3C). Overexpressing ex or mer in kibra mutant
clones significantly rescues the overgrowth of kibra mutant
clones (p < 0.0001 for both genotypes). Reciprocally, kibra
overexpression was also able to suppress the ex overgrowth
phenotype (p < 0.0001). Thus, we could not determine a strict
epistatic relationship between kibra, ex, and mer, consistent
with a model whereby kibra, ex, and mer cooperate to control
SWH pathway activity.
Kibra Is a Transcriptional Target of the SWH Network
and Colocalizes with Mer and Ex
As well as being an upstream regulator of the SWH network, ex
is also one of its transcriptional targets (Hamaratoglu et al.,
2006), as are other upstream regulators (e.g., mer, four-jointed,
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Figure 2. Kibra Regulates SWH Pathway Targets in Ovarian Posterior Follicle Cells
Egg chambers containing heat-shock-induced kibra mutant clones (absence of GFP). Yellow arrows point to kibra mutant cells in the PFC region while white arrow-
heads point to kibra cells in lateral follicle cells. Scale bars = 20 mm. (A–C
0
) Stage 10B egg chambers stained for b-galactosidase (gray or red), which monitors the
activity of the ex-lacZ reporter. (B–B
00
) Close-up of the PFC region in (A)–(A
00
). (D–K
00
) Egg chambers stained for the N target Hindsight (Hnt) (D–G
00
) or for Cut (H–K
00
).
Nuclei are shown in blue (Hoechst). (E)–(E
00
), (G)–(G
00
), (I)–(I
00
), and (K)–(K
00
) are close-ups of the PFC region of stage 10B egg chambers. (D–D
00
) The arrow points to
a stage 8 clone; the arrowhead points to a stage 9 clone. (F–F
00
and J–J
00
) Arrows and arrowheads point to clones in stage 9 egg chambers. See also Figure S2.
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Hippo Signaling and Kibra
Developmental Cell 18, 300–308, February 16, 2010 ª2010 Elsevier Inc. 303
Page 4
dachsous). Since epistasis experiments place Kibra at the level
of Mer and Ex, we wished to test whether this is also the case
for kibra. Kibra levels were highly upregulated in mer;ex or hpo
clones (Figures 3D–3E
0
), showing an apical localization (Figures
3F–3G
0
). The same is true in hpo clones in follicle cells (Figures
3H–3I
00
). Similarly, hpo-depleted cultured Drosophila S2R+ cells
have increased Kibra levels (Figure 3J). To determine whether
kibra is a transcriptional SWH network target, quantitative
RT-PCR experiments were performed on yki-overexpressing
and control wing imaginal discs (Figure 3K). As expected, ex
mRNA levels were increased (2.97 ± 0.25-fold) in yki-expressing
discs compared to control discs. Interestingly, kibra mRNA
levels were also upregulated in yki-expressing discs (6.24 ±
2.12-fold), confirming that kibra is a Yki transcriptional target
and suggesting the existence of a possible negative feedback
loop regulating Kibra expression.
Because hpo clones present increased levels of Kibra as well
as Mer and Ex, these constitute a good system to evaluate the
colocalization of those proteins. Indeed, Kibra colocalizes with
Mer in the wing disc (Figures 3L–3M
00
). As expected, Mer and
Ex also colocalize (Figures 3N–3O
00
). Thus, Kibra, Mer, and Ex
colocalize apically in imaginal disc cells, but are dispensable
for each other’s apical sorting, because Kibra is still apical in
mer;ex clones and Mer/Ex are normally localized in kibra clones
(Figures 3D, 3D
0
, 3F, and 3F
0
and data not shown).
Kibra Associates with Ex and Mer, and the Kibra/Mer
Complex Is Conserved in Human Cells
Because Kibra colocalizes with Mer/Ex, a possible association
between those proteins was examined by conducting coimmu-
noprecipitation (co-IP) assays in S2R+ cells. Kibra was found
to co-IP with Ex and Mer, but not with Hpo or with the negative
Figure 3. kibra Is Epistatic to hpo and Is a Transcriptional Target of the SWH Network
(A–A
00
) Third instar eye imaginal disc expressing kibra under the GMR promoter and containing hpo mutant cells (absence of GFP). Apoptosis is visualized by an
anti-activated Caspase-3 staining. Posterior is to the bottom.
(B and C) Epistatic relationships between Kibra, Mer and Ex. Examples of the 4 phenotypic classes used to score are shown in (B). The percentages of each class
for each genotype are summarized in (C).
(D–G
0
) Third instar imaginal discs containing clones (marked by absence of GFP) of cells mutant for mer;ex (D, D
0
, F, and F
0
)orhpo (E, E
0
, G, and G
0
). (D)–(E
0
) are XY
sections while (F)–(G
0
) are XZ transverse sections (apical is to the top).
(H–I
00
) Stage 10A egg chamber containing a hpo clone (marked by absence of GFP) in the PFCs and stained for Kibra (red). (I)–(I
00
) show a higher magnification of
the PFC region in (H). Nuclei are stained with Hoechst (blue). Scale bars = 20 mm (A–A
00
, D–E
0
, and H), 10 mm (F–G
0
and I–I
00
).
(J) Lysates from S2R+ cells treated with lacZ RNAi or hpo RNAi and probed for Kibra and Tubulin.
(K) A graph presenting a comparison of kibra and ex mRNA levels between yki overexpressing and WT wing discs, as measured by qRT-PCR, is shown. p values
from Mann-Whitney tests are shown. Error bars represent standard deviations.
(L–O
00
) XY and transverse sections of third instar imaginal wing discs containing hpo mutant clones (labeled by absence of b gal) and stained for Kibra (L–L
00
and
N–N
00
), Ex (M–M
00
and O–O
00
), and Mer (L–O
00
). See also Figure S3.
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304 Developmental Cell 18, 300–308, February 16, 2010 ª2010 Elsevier Inc.
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regulator of Hpo, dRASSF (Polesello et al., 2006)(Figure 4A).
Interestingly, Kibra was reported to interact with Mer in a large-
scale yeast two-hybrid screening study (Formstecher et al.,
2005). Kibra possesses two WW domains, which are predicted
to mediate protein-protein interactions by binding to PPXY
motifs. Furthermore, the first WW domain of human KIBRA
was shown to recognize the consensus motif RXPPXY in vitro
(Kremerskothen et al., 2003). In flies, Mer does not contain any
PPXY sites, while Ex has two PPXY sites (P
786
PPY and
P
1203
PPY) and an RXPPXY site (R
842
DPPPY). We therefore
further investigated the association between Kibra and Ex by
mutating amino acids that are known to be required for WW
domains and PPXY sites to interact (Kremerskothen et al.,
2003; Otte et al., 2003). A Kibra protein mutant for its first WW
domain (P85A) could no longer co-IP WT Ex. Reciprocally, WT
Kibra could not co-IP an Ex protein deficient for its RXPPXY
site (P845A) (Figure 4B). Thus, Kibra associates with Ex through
its first WW domain and the Ex RXPPXY motif.
In contrast, mutating either one or both Kibra WW domains
does not affect Kibra/Mer association (Figure S4A). Further
assays reveal that both Kibra N- and C-terminal fragments are
sufficient for the association with Mer, but a central stretch (aa
484–857) is not (Figure S4A). Because the WW motifs, which are
required for the Ex/Kibra association, are located in the N-terminal
fragment, this suggests that Mer can complex with Kibra both
through Ex and independently of Ex. To further test this possibility,
coimmunoprecipitation assays between Mer and Kibra were
performed in ex-depleted cells (Figure S4B). The Kibra/Mer
immunoprecipitation is not affected by ex depletion, suggesting
that Ex is not required for the Kibra/Mer association.
Because the Hpo pathway is highly conserved from
Drosophila to humans, we tested for potential interactions
Figure 4. Kibra Associates with Mer and Ex, and kibra Depletion Strongly Reduces Yki Phosphorylation without Interfering with the Yki/Wts
Interaction
(A and B) Western blots of coimmunoprecipitation assays between Myc-Kibra and HA-tagged members of the SWH network are shown.
(C) Western blots of co-IP assays between Mer-HA and Ex-Flag in presence or absence of Kibra are shown.
(D) Lysates of S2R+ cells treated with RNAi against different members of the SWH network and probed for P-Ser168 Yki (P-Yki), pan-Yki, Kibra, and Tubulin are
shown (the anti-Kibra blot was performed on a parallel run of the same samples). The bottom panel shows efficiency of the ex dsRNAi treatment.
(E) Western blots of co-IP assays between Wts-Flag and different versions of Myc-tagged Kibra are shown.
(F) Endogenous co-IP assays between Yki and Wts in control S2 cells and in cells treated with ex and/or kibra RNAi are shown. The anti-Wts input blot was
performed on a parallel run of the same samples. See also Figure S4.
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Hippo Signaling and Kibra
Developmental Cell 18, 300–308, February 16, 2010 ª2010 Elsevier Inc. 305
Page 6
between human KIBRA and the human orthologs of Ex (FRMD6),
Mer (NF2/MER), Hpo (MST2), and dRASSF (RASSF6). We used
split-TEV as readout, which is based on TEV protease comple-
mentation and represents a sensitive method for detecting
interactions between membrane-associated proteins (Wehr
et al., 2006, 2008). We found that KIBRA associates with NF2/
MER but did not interact with FRMD6, MST2, or RASSF6
(Figure S4C). Interestingly, FRMD6 contains only an N-terminally
conserved sequence of Ex but lacks the entire C-terminal part,
which in Ex harbors the PPXY motifs. Thus, the missing PPXY
motifs and the generally limited level of sequence conservation
in FRMD6 likely explain the absence of interaction between
KIBRA and FRMD6. These results imply that the ability to asso-
ciate with Kibra evolved in an ancestral Ex/Mer-like FERM
domain protein and was later lost in FRMD6 but retained in
NF2/MER. Alternatively, an Ex functional homolog other than
FRMD6 may exist.
As Kibra complexes with both Ex and Mer and Ex/Mer have
been reported to directly interact (McCartney et al., 2000), we
tested the possibility that Kibra could affect the Mer/Ex interac-
tion. We performed co-IP assays between Mer and Ex in cells
expressing different levels of Kibra protein (Figure 4C). The
Mer/Ex interaction is reduced in kibra-depleted cells compared
to WT cells, whereas the interaction is strengthened in cells
that express a Myc-Kibra construct. Thus, the presence of Kibra
is required to fine-tune the stability of the Mer/Ex interaction.
Kibra Associates with Wts and Is Required
for Yki Phosphorylation, but Not for Yki/Wts Binding
Because Kibra complexes with Ex and a Yki/Ex interaction has
recently been described (Badouel et al., 2009), we sought to
determine whether Kibra can affect Yki activity. S2R+ cells
were treated with RNAi against several SWH pathway compo-
nents, and Yki phophorylation on Ser168 was monitored by
western blotting (Figure 4D). The phosphorylation of Yki by Wts
at Ser168 leads to Yki inactivation and sequestration in the cyto-
plasm, where it has been reported to bind Ex, Wts, Hpo, and
14.3.3 (Badouel et al., 2009; Huang et al., 2005; Oh and Irvine,
2008; Oh et al., 2009). lacZ RNAi-treated cells show a high basal
level of phospho-Yki (P-Yki). As expected, Yki phosphorylation is
abolished when Wts is depleted, and mildly reduced when the
Wts cofactor Mats (Lai et al., 2005) is depleted. In wts treated
RNAi cells, a Yki downward shift can also be observed using a
pan-Yki antibody (Figure 4D, second row). ex RNAi treatment
has only a mild effect on P-Yki levels. Interestingly, kibra deple-
tion leads to a marked reduction in P-Yki. When depleted in
conjunction with ex, the P-Yki signal becomes even further
reduced.
This suggests that Kibra and Ex are required for Wts activity
on Yki, which prompted us to investigate whether Kibra could
associate with Wts. Co-IP assays reveal that Kibra interacts
with Wts (Figure 4E). Wts does not seem to compete with Ex
for Kibra association, because it could still complex with a
form of Kibra mutant for its first WW domain. Because Kibra
associates with Wts and Ex interacts with Yki, we investigated
whether Wts requires Kibra/Ex to bind Yki. Endogenous IPs
between Yki and Wts were performed in S2 cells treated with
various dsRNAs (Figure 4F). In these conditions, the effect of
kibra and ex depletion on Yki phosphorylation can also be
observed (see input). In control cells, Wts binding to Yki is
detected after immunoprecipitating Yki. This endogenous inter-
action is unaffected by the individual or combined depletion of
ex and kibra. These results suggest that Ex and Kibra are
required to activate the SWH pathway by nucleating an active
Hpo/Wts kinase cassette, rather than promoting the Wts/Yki
interaction.
DISCUSSION
Our data identify Kibra as a regulator of the SWH network that
associates with Ex and Mer, with which it is colocalized apically
and transcriptionally coregulated. Given that the apical surface
of epithelial cells is instrumental in both cell-cell signaling and
tissue morphogenesis, we speculate that Kibra may cooperate
with Ex and Mer to transduce an extracellular signal, or relay
information about epithelial architecture, via the SWH network,
to control tissue growth and morphogenesis.
Recent data have suggested that an apical scaffold machinery
containing Hpo, Wts, and Ex recruits Yki to the apical membrane,
facilitating its inhibitory phosphorylation by Wts (Badouel et al.,
2009; Oh et al., 2009). Since
Kibra associates with Ex and is
also apically localized, we can hypothesize that Kibra is also
part of this scaffold and participates in nucleating an active
Hpo/Wts complex and recruiting Yki for inactivation. This view
is supported by our finding that Kibra complexes with Wts and
that combined depletion of Kibra and Ex leads to a strong
decrease in Yki phosphorylation, but does not disrupt the Wts/
Yki interaction. Our data also suggest that the importance of
Kibra may be tissue-specific since we observe robust pheno-
types in ovaries and hemocyte-derived S2R+ cells, but weaker
effects in imaginal discs. Thus, considering the relative levels
of expression of Ex, Mer, and Kibra may be important in deter-
mining pathway activation. Finally, since mammalian KIBRA
complexes with the NF2/MER tumor suppressor, our findings
raise the possibility that human KIBRA may contribute to tumor
suppression in human neurofibromas and potentially other
tumors.
EXPERIMENTAL PROCEDURES
For Drosophila genotypes, primer sequences, and further experimental
details, see Supplemental Experimental Procedures.
kibra Mutant
The P element of EP747 (Bloomington stock center) was mobilized using
standard genetic techniques, and excisions were screened by PCR (see
Supplemental Experimental Procedures for details).
Immunostainings
Immunostainings and confocal image acquisitions were performed as in
Genevet et al. (2009). Mouse b-galactosidase (Promega), rabbit anti-Cleaved
Caspase-3 (Asp175) (Cell Signaling Technology), rabbit anti-aPKC (Santa
Cruz), and mouse NICD (C17.9C6, Development Studies Hybridoma Bank)
antibodies were used at 1/500. Mouse anti-Arm, anti-Cut, and anti-Hnt (N2
7A1, 2B10 and 1G9, DSHB) were used at 1/10 and 1/20. The EdU staining
was performed as described in the Click-iT EdU Alexa Fluor Imaging Kit (Invi-
trogen). Guinea pig anti-Mer, a gift from R. Fehon, was used at 1/7500, and
rabbit anti-Ex, a gift from A. Laughon, at 1/400. Rabbit anti-Kibra antibody
(Kib18, 1/100) was generated by Eurogentec SA (Seraing, Belgium) against
a peptide corresponding to the last 15 amino acids of Kibra.
Developmental Cell
Hippo Signaling and Kibra
306 Developmental Cell 18, 300–308, February 16, 2010 ª2010 Elsevier Inc.
Page 7
Quantification of Apoptotic Indexes in Mutant Clones
versus WT Tissue
Apoptotic indexes were quantified on 6 retinas as described in Colombani
et al. (2006).
Standard Growth Conditions and Size Measurements
The experiment was performed as described in Genevet et al. (2009). Ten wing
discs for each genotype were analyzed.
Fly Eye Scanning Electron Microscopy
Scanning microscopy of adult flies was performed as described in Polesello
et al. (2006).
RNAi Treatment and Coimmunoprecipitation of Proteins
For RNAi treatment, S2 or S2R+ cells were treated with dsRNAs as indicated
for 4 days. For coimmunoprecipitations, S2R+ or S2 cells transfected with the
indicated plasmids were used. See Supplemental Experimental Procedures
for details.
Quantitative RT-PCR
MS1096 > > (control) and MS1096 > > yki wing discs were dissected in PBS
and snap-frozen in liquid nitrogen. RNA isolation and subsequent qRT-PCR
reactions were performed as described in Genevet et al. (2009). See Supple-
mental Experimental Procedures for primer sequences.
Statistical Analysis
All error bars displayed represent standard deviations. All statistical analyses
performed (except epistasis analysis) were assessed by Mann-Whitney
nonparametric tests using the website http://elegans.swmed.edu/leon/
stats/utest.html.
The epistasis analysis was made by pairwise comparison after correction
for the batch effect on 42 to 310 flies of each genotype, divided in 4 to 6
cohorts. The approach used was a three-way log-linear model, against a
null-hypothesis of no interaction between phenotype and population. The p
value indicates whether the pair of populations differ in their phenotype
profiles: p value(kibra
/
; kibra
/
+ UAS ex) = 2.69 3 10
63
, p value(kibra
/
;
kibra
/
+UAS mer) = 6.07 3 10
35
, p value(ex
/
;ex
/
+ UAS kibra) = 1.76 3
10
20
.
SUPPLEMENTAL INFORMATION
Supplemental Information includes four figures, Supplemental Experimental
Procedures, and Supplemental References and can be found with this article
online at doi:10.1016/j.devcel.2009.12.011.
ACKNOWLEDGMENTS
We thank D. Pan, R. Fehon, G. Halder, A. Laughon, K. Irvine, J. Kremer-
skothen, G. Struhl, T. Igaki, H. McNeill, the Bloomington and VDRC stock
centres, and the DGRC for fly stocks and reagents. We are very grateful to
G. Dietzl, B. Dickson, and S. Cohen for their support in carrying out the RNAi
screen. We are grateful to K. Blight, A. Weston, and L. Collinson from the
LRI Electron Microscopy Facility and to P. Jordan from the Light Microscopy
Facility for technical help, to G. Kelly for help with statistics, to F. Josue
´
for
help with apoptotic index analysis, as well as to T. Gilbank, S. Murray, and
S. Maloney from the Fly Facility for technical support. We thank S. Cohen
and C. Polesello for discussions and comments on the manuscript. We thank
H. Stocker and E. Hafen for discussing data prior to publication. M.C.W. is sup-
ported by an EMBO long-term fellowship. The Tapon and Thompson laborato-
ries are supported by Cancer Research UK.
Received: May 27, 2009
Revised: October 20, 2009
Accepted: December 24, 2009
Published: February 15, 2010
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  • Source
    • "promoter (Xu and Rubin, 1993; Lee and Luo, 1999). Flies of the following genotypes were generated: yw hsFLP UAS-nucGFPmyc; FRT42D hpo 42-47 / FRT42D tubGal80; tubGal4/+ (Wu et al., 2003); yw hsFLP UAS-nucGFPmyc; FRT42D hpo JM1 /FRT42D tubGal80; tubGal4/+ (Jia et al., 2003); yw hsFLP, tubGal4, UAS-nucGFPmyc; FRT82B wts X1 /FRT82B tubGal80 (Xu et al., 1995); yw hsFLP, tubGal4, UAS-nucGFPmyc; FRT82B kib 32 /FRT82B tubGal80 (Genevet et al., 2010); yw hsFLP; ex AP50 FRT40A/FRT40A ubi-GFP (Hamaratoglu et al., 2006); yw hsFLP; ex AP50 FRT40A/FRT40A ubi-GFP; FRT82B kib 32 / FRT82B ubi-GFP; yw hsFLP, tubGal4, UAS-nucGFPmyc; FRT82B cpb M143 / FRT82B tubGal80 (Fernández et al., 2011 ); yw hsFLP, tubGal4, UASnucGFPmyc ; UAS.cpb/+; FRT82B wts X1 /FRT82B tubGal80; yw hsFLP UASnucGFPmyc ; FRT42D hpo 42-47 , ena 210 /FRT42D tubGal80; tubGal4/+. The slbo-lacZ enhancer trap line was obtained from the Bloomington Stock Center (Bloomington, IN) and the upd-lacZ enhancer trap line has been described previously (Shaw et al., 2010). "
    Full-text · Dataset · May 2016
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    • "Overexpression of all three human WWC proteins showed similar (although weaker) effects as DmKibra: a rough eye phenotype as well as a reduction of the posterior wing size compartment (fig. 6; Baumgartner et al. 2010; Genevet et al. 2010; Yu et al. 2010). These observations indicate that the ability of the WWC proteins to modulate the Hippo pathway, to inhibit cell proliferation , and to regulate tissue growth is evolutionarily conserved from fly to men. "
    Full-text · Dataset · May 2016
  • Source
    • "Negative feedback mechanism in a signaling pathway maintains homeostasis of output effects. The presence of negative feedback in the Hippo pathway has been suggested in early Drosophila studies, showing that Expanded, Merlin, and Kibra are targets of Yorkie [21, 22]. In addition, our group previously showed that Hippo pathway component proteins are increased in the Sav1 knock-out mouse model [23]. "
    [Show abstract] [Hide abstract] ABSTRACT: The Hippo pathway represses YAP oncoprotein activity through phosphorylation by LATS kinases. Although variety of upstream components has been found to participate in the Hippo pathway, the existence and function of negative feedback has remained uncertain. We found that activated YAP, together with TEAD transcription factors, directly induces transcription of LATS2, but not LATS1, to form a negative feedback loop. We also observed increased mRNA levels of Hippo upstream components upon YAP activation. To reveal the physiological role of this negative feedback regulation, we deleted Lats2 or Lats1 in the liver-specific Sav1-knockout mouse model which develops a YAP-induced tumor. Additional deletion of Lats2 severely enhanced YAP-induced tumorigenic phenotypes in a liver specific Sav1 knock-out mouse model while additional deletion of Lats1 mildly affected the phenotype. Only Sav1 and Lats2 double knock-down cells formed larger colonies in soft agar assay, thereby recapitulating accelerated tumorigenesis seen in vivo. Importantly, this negative feedback is evolutionarily conserved, as Drosophila Yorkie (YAP ortholog) induces transcription of Warts (LATS2 ortholog) with Scalloped (TEAD ortholog). Collectively, we demonstrated the existence and function of an evolutionarily conserved negative feedback mechanism in the Hippo pathway, as well as the functional difference between LATS1 and LATS2 in regulation of YAP.
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