|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 6 February 2008
doi: 10.1242/dev.015255
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Howard Hughes Medical Institute, Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers The State University of New Jersey, Piscataway, NJ 08854, USA.
* Author for correspondence (e-mail: irvine{at}waksman.rutgers.edu)
Accepted 7 January 2008
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Fat, Growth, Hippo, Transcription, Drosophila
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Saucedo and Edgar, 2007).
These pathways act through Warts (Wts), a conserved Ser/Thr kinase, via two
distinct mechanisms. The Fat signaling pathway influences the levels of Wts
through a post-transcriptional process that requires the unconventional myosin
Dachs (Cho et al., 2006). Thus,
in fat mutant cells, levels of Wts are reduced. The Hippo pathway
influences the activity of Wts without affecting its levels, through the
kinase Hippo (Hpo), which phosphorylates Wts, and the co-factors Salvador
(Sav) and Mob as Tumor Suppressor (Mats), which bind to Hpo and Wts (reviewed
in Pan, 2007;
Saucedo and Edgar, 2007). The
tumor suppressors Expanded (Ex) and Merlin (Mer) act upstream of Hpo
(Hamaratoglu et al., 2006).
There is also crosstalk between these pathways, as Fat influences the levels
of Ex at the subapical membrane (Bennett
and Harvey, 2006; Feng and
Irvine, 2007; Silva et al.,
2006; Willecke et al.,
2006).
Activated Wts phosphorylates, and thereby inhibits, a non-DNA binding
transcriptional co-activator protein, Yorkie (Yki)
(Huang et al., 2005). Yki
promotes growth and inhibits apoptosis by enhancing the transcription of
downstream genes, including Cyclin E, Cyclin B, DIAP1 and
bantam (reviewed in Pan,
2007; Saucedo and Edgar,
2007). Mutation of yki inhibits growth and cell survival,
whereas overexpression of yki promotes overgrowth, presumably because
this overcomes negative regulatory mechanisms that normally limit its
activity. Genetically, yki acts downstream of wts, and
Wts-dependent phosphorylation can inhibit the transcriptional co-activator
activity of Yki, as monitored by experiments in which a Yki:Gal4 fusion
protein promotes transcription of a reporter gene in cultured
Drosophila cells (Huang et al.,
2005).
Most of the genes in the Fat and Hippo pathways are conserved in humans
(reviewed in Pan, 2007;
Saucedo and Edgar, 2007).
Loss-of-function mutations in several mammalian homologs of
Drosophila tumor suppressors have also been linked to cancers,
supporting their conserved action as tumor suppressors. The mammalian homolog
of Yki is YES-associated protein (YAP). YAP can rescue the lethality
associated with Hpo pathway hyperactivation in Drosophila, implying
functional conservation between Yki and YAP
(Huang et al., 2005). The
observation that overexpression of Yki promotes overgrowth would suggest that
YAP might act as an oncogene in mammals, and recent studies have borne out
this out expectation (Overholtzer et al.,
2006; Zender et al.,
2006).
The identification of Yorkie as the most downstream transcriptional regulator of the Fat and Hippo pathways provided an opportunity to elucidate molecular and cellular mechanisms through which tumor suppressors in these pathways affect growth control. Here, we show that Wts phosphorylates Yki in vivo at multiple sites and regulates its nuclear localization. Phosphorylation and nuclear localization of Yki are also modulated by upstream tumor suppressors of the Fat and Hippo pathways. We also show that phosphorylation of Yki mediates binding to 14-3-3 proteins, acting via a conserved Ser that also contributes to normal regulation of Yki activity in vivo. Taken together, our studies indicate that Yki is regulated by Warts-mediated phosphorylation at multiple sites to influence its subcellular localization.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Ectopic expression clones were created by Flip-out using yw hs-FLP[122]; act> y+>gal4 (AyGal4) or yw hs-FLP [122]; act> y+>gal4 UAS-GFP (AyGal4-GFP), UAS-yki, UAS-yki:GFP, UAS-ykiS168A:GFP, UAS-hpo and UAS-wts. Ectopic expression of UAS-yki, UAS-yki:GFP and UAS-yki-S168A:GFP was also induced with GMR-gal4, sd-gal4, dpp-gal4, ptc-Gal4, act-Gal4, tub-Gal4 ey-Gal4 and ci-gal4.
For rescue experiments in the wing imaginal discs, hs-FLP; ykiB5 FRT42D/CyO;Ci-gal4/TM6B and hs-FLP; arm-lacZ FRT42D; UAS-yki:GFP/TM6b were used. Among more than ten insertions each, we have retained four UAS-yki:GFP insertions, 4-4-Y and 4-12-1 (2nd chromosome), 4-3-Y and 4-9-Y (3rd chromosome), and four UAS-yki-S168A:GFP insertions, 10-2-1 and 10-12-1 (2nd chromosome) and 10-7-Y and 10-9-Y (3rd chromosome).
For reduction of ex or fat levels by RNAi, we used UAS-IR
constructs 9396 (fat) and 22994 (ex), together with
UAS-dicer2 (Vienna Drosophila RNAi Center)
(Dietzl et al., 2007).
Yorkie antibody preparation
Full-length Yki was cloned into pGEX-3X (Amersham Biosciences) at a
SmaI site. GST:Yki was in BL21(DE3) E. coli (Invitrogen) by
induction with 1 mM IPTG. Insoluble inclusion bodies contained the majority of
GST-Yki, and were used to immunize rabbits (Cocalico Biologicals).
Histology and imaging
Imaginal discs were fixed and stained as described previously
(Cho and Irvine, 2004), using
as primary antibodies rabbit anti-Yki (1:400), anti-En (Developmental Studies
Hybridoma Bank) and goat anti-β-gal (1:1000, Biogenesis). Fluorescent
stains were captured on a Leica TCS SP5 confocal microscope. For some panels,
maximum projection through multiple horizontal sections was performed to allow
visualization of staining in different focal planes.
Generation of transgenic flies
The S168A mutation was introduced into Yki by primer-mediated site-directed
mutagenesis. Yki and Yki-S168A with C terminal HA-tags
(Huang et al., 2005) were
amplified by PCR and cloned into pEGFP-N1 (Clontech) to construct
yki:GFP and yki-S168A:GFP; these were then cloned into pUAST
for P-element-mediated transformation (Duke University Model System
Genomics).
Protein blotting
Homozygous mutant wing discs were obtained from wandering third instar
larvae using exe1/CyO-GFP, ftGrv/L14,
exe1 ftGrv/CyO-GFP and wtsP2
FRT82B/TM6b stocks; Oregon-R was used as wild type. Larvae were collected
and dissected in PBS [10 mM sodium phosphate, 2.7 mM KCl, 137 mM NaCl (pH
7.4)], and wing discs were collected and lysed in CIP buffer [100 mM NaCl, 50
mM Tris-HCl (pH 7.9), 10 mM MgCl2, 1 mM DTT], 1 mM PMSF, 0.1% NP40,
protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail
(Calbiochem). For CIP treatment, phosphatase inhibitor cocktail was omitted,
and lysate was incubated at 37°C for 30 minutes in 20 units CIP (NEB) per
100 µl reaction. Lysates were cleared by centrifugation and subjected to
SDS-PAGE. To detect phosphorylated Yorkie in SDS-PAGE, we used Phos-tag
AAL-107 (FMS Laboratory) according to the manufacturer's instructions. Western
blotting was performed using rabbit anti-Yki (1:4000), mouse anti-Actin
(1:5000, Calbiochem), rat anti-HA:HRP (1:2000, Roche), mouse
anti-
-Tubulin (1:5000, Sigma) and mouse anti-V5:HRP (1:5000,
Invitrogen).
Plasmid constructs
For immunoprecipitation assays, 14-3-3
and 14-3-3
were
amplified by PCR using total cDNA of S2 cells as a template and then cloned
into pAc5.1/V5-HisA (Invitrogen). For assays of phosphorylation-dependent
mobility shifts, Yki:V5, Yki-S168A:V5, Yki-N:V5 and Yki-N-S168A:V5 were
constructed in pAc5.1/V5-HisA vector (Invitrogen). Flag-tagged Hpo, Myc-tagged
Wts and HA-tagged Sav expressing constructs were from J. Jiang
(Jia et al., 2003).
pUAST-Yki:FLAG(3x) and pUAST-Yki:S168A:FLAG(3x) were constructed by cloning
Yki and Yki-S168A into pUAST-FLAG(3x) (Y. Mao).
S2 cell assays and co-immunoprecipitation
S2 cells were cultured with Schneider's Drosophila medium
(Invitrogen) and 10% FBS (Sigma). Transfections were performed with Cellfectin
(Invitrogen) according to manufacturer's protocol. Co-immunoprecipitation
assays were performed as described previously
(Chen et al., 2003).
To make yki double-stranded RNA, the first 500 bp of yki
coding region was amplified by PCR and subcloned into pCR2.1-TOPO vector
(Invitrogen) and then both forward and reverse clones of yki were
amplified using M13 primers for use as templates for T7 polymerase in vitro
transcription. Generation of double strand RNA and RNAi in S2 cells was then
performed as described (Clemens et al.,
2000).
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
), we
investigated the subcellular localization of Yki in Drosophila. In
one approach, we created a yki:GFP fusion gene, and
expressed it under UAS-Gal4 control. Confirmation that this fusion protein is
functional was provided by the observation that expression of
yki:GFP under arm-Gal4 control was sufficient to
rescue the lethality of a yki null allele, ykiB5.
To examine Yki:GFP function specifically within the wing disc, we generated
ykiB5 mutant clones in animals in which Yki:GFP was
expressed in anterior cells under ci-Gal4 control. In posterior
cells, ykiB5 clones were rare and small
(Fig. 1A). By contrast, in
anterior cells, ykiB5 clones were readily recovered,
indicating that Yki:GFP rescues the growth and viability of
ykiB5 cells. Despite its presumed function as
transcriptional co-activator, when the localization of Yki:GFP was examined in
wing imaginal discs, it is predominantly cytoplasmic, although low levels
appear in the nucleus (Fig.
1B).
In a second approach to investigating the subcellular localization of Yki,
we generated antibodies against Yki. Confirmation of the specificity of
anti-Yki staining was provided by examining clones of cells mutant for
ykiB5, which encodes a protein null allele
(Huang et al., 2005). To
compensate for the poor growth and viability of yki clones, we
employed the Minute technique, which gives clones a growth advantage
over neighboring cells. This allowed recovery of yki mutant clones.
The absence of staining in these clones confirmed that our antisera recognizes
Yki (Fig. 1D,E). The wild-type
localization profile of Yki is similar to that of Yki:GFP; Yki is
predominantly cytoplasmic, although there are low levels in the nucleus. In
vertical sections through the disc epithelium
(Fig. 1E), Yki can be detected
throughout the apical to basal aspect of these cells, but appears excluded
from nuclei.
Yki is phosphorylated by Warts in vivo
Prior studies of Yki phosphorylation have all relied on experiments in
cultured cells. To investigate whether the phosphorylation status of Yki is
detectably influenced in vivo by Wts or other upstream tumor suppressors, we
examined endogenous Yki in lysates of wing imaginal discs by western blotting.
Because yki mutations are lethal, the specificity of anti-Yki sera on
western blots was characterized by RNAi treatment of cultured cells; Yki is
the predominant band detected (Fig.
2C). Western blotting of wing imaginal discs revealed a prominent
smeared band of the expected mobility (
50 kDa)
(Fig. 2). In a wts
mutant that permits survival of animals until the third larval instar
(wtsP2) the Yki band appears sharper, consistent with the
possibility that Yki phosphorylation is reduced, but the difference in
mobility is subtle (Fig. 2A).
Thus, to further evaluate the in vivo phosphorylation status of Yki, we
employed a new technique for separating phosphorylated isoforms, Phos-tag gels
(Kinoshita et al., 2006). The
Phos-tag is a phosphate binding compound that, when incorporated into
polyacrylamide gels, can result in an exaggerated mobility shift for
phosphorylated proteins, dependent upon the degree of phosphorylation
(Kinoshita et al., 2006). When
wing disc lysates are run on Phos-tag gels, several prominent Yki bands are
detected, implying that several distinct phophophorylated isoforms are present
in wild-type tissue (Fig. 2B).
Most of these isoforms are absent in wts mutants
(Fig. 2B), implying that the
bulk of Yki phosphorylation in vivo is Wts dependent. Confirmation that the
mobility shifts observed are a consequence of phosphorylation was provided by
treatment of lysates with calf intestinal alkaline phosphatase (CIP). CIP
treatment of lysates resulted in sharper bands, with slightly faster mobility,
on conventional gels, and collapsed the bands observed on Phos-tag gels into a
single major band, resulting in a mobility profile similar to that observed in
wtsP2 (Fig.
2). Thus, endogenous Yki is phosphorylated in vivo at multiple
sites in a Wts-dependent process.
|
|
). Ex acts
within the Hippo pathway, and regulates the activity of Wts
(Hamaratoglu et al., 2006).
Yki mobility in lysates of ex mutants was similar to that in
fat mutants: phosphorylated Yki isoforms appear reduced but not
eliminated, while unphosphorylated Yki appears increased
(Fig. 2B). Although it has been
reported that ex fat double mutants have the same phenotype as
fat or ex single mutants
(Bennett and Harvey, 2006;
Silva et al., 2006), a more
recent study revealed that they have actually act in parallel and have
additive phenotypes (Feng and Irvine,
2007). Consistent with this, we observed an additive effect on Yki
dephosphorylation in lysates from ex fat double mutants
(Fig. 2B).
|
We next investigated the influence of tumor suppressors that act upstream
of wts. Increased nuclear Yki was clearly observed within clones of
cells mutant for the Wts kinase hpo
(Fig. 3F), and the Wts
co-factor mats (Fig.
3G). Clones of cells doubly mutant for two more upstream
regulators of the Hippo pathway, ex and Mer, also exhibited
Yki staining throughout the cytoplasm and nucleus
(Fig. 3B), comparable with that
observed in wts. Thus, the action of these tumor suppressors within
the Hippo pathway can be directly visualized in vivo by their effect on Yki
localization. ex single mutant clones did not detectably increase
nuclear Yki (Fig. 3D),
presumably owing to the partial redundancy of ex with Mer.
fat mutants have effects on growth and Yki phosphorylation comparable
with ex mutants, and in most cases fat mutant clones also
failed to exhibit a noticeable increase in the levels of nuclear Yki
(Fig. 3H), although we did
identify two exceptional clones (out of over 100 examined) in which an effect
on Yki localization could be clearly observed
(Fig. 3C). ex fat
double mutants exhibit more severe phenotypes and effects on Yki
dephosphorylation than single mutants (Fig.
2B), reflective of their parallel action in the Fat and Hippo
pathways (Cho et al., 2006;
Feng and Irvine, 2007). ex
fat double mutant clones also consistently exhibited nuclear Yki staining
(Fig. 3E). This observation
suggests that both fat and ex can modulate the subcellular
localization of Yki, but only in double mutant clones is the effect strong
enough to be clearly detected. To further investigate the influence of
ex and fat, we examined Yki staining in wing discs in which
their levels were reduced in anterior cells throughout development by RNAi.
When double-stranded RNAs corresponding to fat or ex were
expressed under ci-Gal4 control, together with the RNAi enhancer
dicer2 (Dietzl et al.,
2007), a modest but consistent increase in nuclear Yki was
observed (Fig. 3I,J),
confirming that these genes can each individually influence Yki localization.
Expression of dicer2 alone had no effect on Yki (not shown). As the
ex and fat mutations we employed are thought to be null
alleles, we surmise that the apparently stronger RNAi phenotype results from
the perdurance of gene products within mutant clones. Altogether, our
observations establish a correlation among the overgrowth phenotypes of Fat
and Hippo pathway mutants, their effects on Yki phosphorylation, and their
effects on Yki nuclear localization.
|
). To
investigate whether the influence of Wts on Yki nuclear localization might
involve modulating binding to 14-3-3 proteins, we performed
co-immunoprecipitation assays on proteins expressed in cultured
Drosophila S2 cells. Epitope-tagged forms of Yki and the two
Drosophila 14-3-3 proteins ( and ), were transfected into
cells in the presence or absence of Wts and the Wts activators Hpo and Sav. As
the ability of Akt to modulate association of YAP with 14-3-3 proteins has
been mapped to a specific, conserved Ser residue
(Basu et al., 2003),
corresponding to Ser168 of Drosophila Yki, we also expressed a mutant
form of Yki in which this was changed to Ala (Yki-S168A:FLAG). In the absence
of exogenous kinases, weak association between 14-3-3 proteins and Yki could
be detected (Fig. 4A). This
interaction presumably depends on phosphorylation of Ser168 by endogenous
kinases, as it was eliminated by the S168A mutation. Notably, the interaction
between 14-3-3 and Yki was enhanced by expression and activation of Wts
(Fig. 4A). This enhanced
interaction was also abolished by the S168A mutation
(Fig. 4A). These observations
imply that Wts-mediated phosphorylation of Ser168 promotes binding of Yki to
14-3-3 proteins.
To compare the effects of Wts on 14-3-3 binding with its effects on Yki
phosphorylation, we examined Yki mobility on western blots of cultured cell
lysates. On conventional gels, activated Wts effected a substantial shift in
Yki mobility, implying that Yki is heavily phosphorylated when co-transfected
with activated Wts (Fig. 4B).
This shift was not detectably influenced by the S168A mutation, implying that
Ser168 is just one of multiple Wts-dependent phosphorylation sites on Yki. A
shift was also observed on Phos-tag gels for both wild type and S168A mutant
Yki (Fig. 4B). These
experiments also illustrated one limitation of the Phos-tag gels, as there was
often a decrease in the amount of Yki detected, which, as overall levels of
Yki were not decreased on conventional gels, presumably resulted from the
relatively poor transfer efficiency of heavily phosphorylated proteins out of
Phos-tag gels (Kinoshita et al.,
2006). Interestingly, on Phos-Tag gels, S168A mutant Yki also
exhibited a distinct, modestly shifted isoform that was not observed in
experiments with wild-type Yki. This was observed both in cultured cells
(Fig. 4B), and in wing disc
lysates (Fig. 4C). This
suggests that mutation of Ser168 can impair further phosphorylation of Yki.
However, as interpretation of this result was potentially complicated by a
reduced ability to detect heavily phosphorylated isoforms of Yki on Phos-tag
gels, we also examined the mobility of smaller fragments of Yki.
An N-terminal fragment of Yki (Yki-N:V5), comprising the first 240 amino acids, was shifted into several bands on Phos-tag gels by co-expression of activated Wts (Fig. 4D). Conversely, the same fragment with the S168A mutation was shifted into only three bands. These results confirm that Ser168 can be a site of Warts-mediated phosphorylation. As multiple phosphorylated isoforms appear to be affected by Ser168 mutation, they also suggest that phosphorylation of Yki at Ser168 might promote phosphorylation of Yki at additional sites, in other words, that it might act as a priming site. The influence of S168A mutation on Wts phosphorylation does not appear to be due to an effect on Wts-Yki binding, as physical interaction between them, as assayed by co-immunoprecipitation from S2 cells, was not detectably affected (not shown).
|
). Expression of UAS-yki under GMR-Gal4
control resulted in overgrown eyes (Fig.
5B), whereas expression of UAS-yki:GFP did not cause any
obvious eye overgrowth (Fig.
5C), even though UAS-yki:GFP was active in rescue
experiments (Fig. 1A). However,
examination of Yki levels by western blotting and by confocal microscopy
(Fig. 5E and data not shown)
revealed that UAS-yki directs higher expression levels of Yki than
any of our UAS-yki:GFP transgene insertions. Thus, Yki:GFP might be
less active simply because its expression levels are lower. In contrast to the mild effects of Yki:GFP expression under GMR-Gal4 control, expression of Yki-S168A:GFP under GMR-Gal4 control generated substantial eye overgrowths (Fig. 5D). In this case, transgene insertions that generated strong overgrowths were associated with Yki-S168A:GFP levels at or below those of wild-type Yki:GFP insertions that did not generate detectable effects on growth (Fig. 5E-G; data not shown). Thus, the stronger phenotypes of Yki-S168A:GFP in comparison with Yki:GFP imply that the S168A mutation hyperactivates Yki, presumably by making it resistant to an inhibitory mechanism that acts via Ser168 phosphorylation. Examination of Yki:GFP-expressing clones in imaginal discs further supported this conclusion. Clones expressing Yki:GFP exhibited irregular shapes and normal sizes (Fig. 5H, Fig. 6A). By contrast, clones expressing Yki-S168A:GFP were larger, and were also rounder (Fig. 5I, Fig. 6B), which is characteristic of mutations in Hippo and Fat pathway tumor suppressors. Mutation of Fat or Hippo pathway tumor suppressors is associated with the induction of downstream target genes, including thread (th, Diap1) and ex. Expression of th-lacZ (Fig. 5H,I) and ex-lacZ reporters (not shown) was elevated in association with Yki-S168A:GFP-expressing clones, but was not detectably affected by Yki:GFP-expressing clones. Altogether, these results indicate that S168A mutant Yki is hyperactivated.
|
S168A mutant Yki exhibits reduced sensitivity to Wts in vivo
To further investigate how Ser168 influences the regulation of Yki by Wts
in vivo, we examined the consequences of Wts activation on Yki-S168A activity
in imaginal discs. Overexpression of Hpo activates Wts, which inhibits Yki and
thereby promotes apoptosis. Thus, Hpo-expressing clones are not normally
recovered in imaginal discs (Fig.
6C; data not shown). When Yki:GFP was co-expressed with Hpo,
clones were tiny and rarely detected (Fig.
6D), indicating that expression of Yki:GFP was not sufficient to
block the effects of Hpo expression. However, when Yki-S168A:GFP was
co-expressed with Hpo, clones were larger and more numerous
(Fig. 6E). Thus, the S168A
mutation reduces the sensitivity of cells to the pro-apoptotic effects of Hpo
overexpression. To achieve even higher levels of Wts activation, we
co-expressed Wts together with Hpo. In this case, the size and frequency of
clones was greatly reduced even for Yki-S168A:GFP-expressing clones, and in
most cases no clones were recovered (Fig.
6F). Thus, Yki-S168A:GFP can still be affected by high level Wts
activation in vivo. However, at lower Wts activation levels (i.e. expression
of Hpo alone) mutation of Ser168 confers partial resistance to Wts
activation.
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
The observation that nuclear localization of Yki is regulated by Wts
defines a cellular mechanism by which Wts-dependent phosphorylation inhibits
Yki activity. In general there is a correlation between the phosphorylation of
Yki and its localization; i.e. genotypes that most strongly reduce Yki
phosphorylation (e.g. wts, ex fat) most strongly affect Yki
localization, while genotypes with weaker effects on phosphorylation (e.g.
fat, ex) have weaker effects on Yki localization. Although it is
formally possible that inhibition of nuclear localization is not the only
mechanism by which phosphorylation inhibits Yki activity, we think it more
likely that an increase in nuclear Yki accounts for all biological effects,
but that in some cases (e.g. ex or fat mutant clones), the
increase was below our sensitivity of detection. In favor of this, we note the
additive effects of ex and fat mutants on Yki localization,
which parallels their additive effects on Yki phosphorylation
(Fig. 2B), and their additive
effects on growth and gene expression (Feng
and Irvine, 2007). This would also be consistent with the
observation that Yki-S168A:GFP is hyperactivated, but its subcellular
localization is only slightly shifted. Additionally, yki is essential
for normal growth and viability, presumably via its influence on downstream
gene expression, yet endogenous Yki is barely detectable in the nucleus. In
this respect, Yki could be comparable with transcriptional co-activators of
other signaling pathways, such as the Notch intracellular domain, which can
have substantial biological activity even under conditions where its levels in
the nucleus are below detection using conventional antibody staining
techniques (Struhl and Adachi,
1998). As even very low levels of endogenous Yki have significant
biological effects, then even a slight increase might be sufficient to
substantially influence downstream gene expression and growth.
Several transcription factors have previously been reported to be regulated
by phosphorylation-dependent effects on nuclear localization, and a molecular
mechanism for this involving binding to 14-3-3 proteins has been described
(reviewed by Mackintosh,
2004). 14-3-3 binding also appears to contribute to the regulation
of Yki localization, as phosphorylation of Yki promotes binding to 14-3-3
proteins in cultured cells. Moreover, this binding is dependent upon Ser168,
which conforms to a consensus site for 14-3-3 binding, and which influences
Yki activity and localization in vivo. However, our results suggest that there
may be other cytoplasmic tethers that contribute to the retention of
phosphorylated Yki in the cytoplasm. Analysis on Phos-tag gels established
that Yki is phosphorylated on multiple sites. When Ser168 is mutated, binding
to 14-3-3 proteins was eliminated, consistent with the observation that this
is the only Ser within Yki that conforms to a 14-3-3 binding site consensus
sequence. However, the bulk of Yki-S168A:GFP was retained in the cytoplasm. By
contrast, when wts was mutant, Yki was equally distributed between
cytoplasm and nucleus. Additionally, the observation that the growth and
survival of cells expressing Yki-S168A:GFP could be blocked when Wts and Hpo
were co-expressed implies that nuclear localization of this mutant form of Yki
can still be blocked by Wts-mediated phosphorylation at other sites. Moreover,
the ability of Wts to promote phosphorylation of Yki was reduced but not
eliminated by mutation of Ser168. Altogether, these results suggest that
Ser168 can account only for part of the regulation of Yki by Wts, which, if it
is the only 14-3-3 interaction site, implies the existence of other
cytoplasmic tethers.
While this manuscript was under review, the observation that Yki and YAP
localization can be influenced by Warts-mediated phosphorylation was
independently reported (Dong et al.,
2007; Zhao et al.,
2007). Our results overlap with these studies in describing the
influence of Yki phosphorylation on Yki localization, and the binding to
14-3-3 proteins mediated by phosphorylation of Yki Ser168 (or its mammalian
equivalent, YAP Ser127). Our observations extend these published findings by
reporting, for the first time, the phosphorylation status of Yki in vivo. This
has allowed us to characterize the effects of upstream regulators under
endogenous expression conditions (as opposed to the extreme levels of
expression and phosphorylation associated with co-transfection experiments in
cultured cells). Importantly, our results imply that even subtle changes in
phosphorylation and nuclear localization, such as are associated with
fat mutants, can have dramatic effects on downstream gene expression
and growth. Our results also clearly indicate that multiple sites can
contribute to Yki phosphorylation and localization, in contrast to the
inference that Ser168 of Yki is the only relevant site
(Dong et al., 2007) but in
agreement with the inference that there are multiple Lats phosphorylation
sites on YAP (Zhao et al.,
2007).
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Basu, S., Totty, N. F., Irwin, M. S., Sudol, M. and Downward,
J. (2003). Akt phosphorylates the Yes-associated protein,
YAP, to induce interaction with 14-3-3 and attenuation of p73-mediated
apoptosis. Mol. Cell 11,11
-23.[CrossRef][Medline]
Bennett, F. C. and Harvey, K. F. (2006). Fat
cadherin modulates organ size in Drosophila via the Salvador/Warts/Hippo
signaling pathway. Curr. Biol.
16,2101
-2110.[CrossRef][Medline]
Chen, H. K., Fernandez-Funez, P., Acevedo, S. F., Lam, Y. C.,
Kaytor, M. D., Fernandez, M. H., Aitken, A., Skoulakis, E. M., Orr, H. T.,
Botas, J. et al. (2003). Interaction of Akt-phosphorylated
ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type
1. Cell 113,457
-468.[CrossRef][Medline]
Cho, E. and Irvine, K. D. (2004). Action of
fat, four-jointed, dachsous and dachs in distal-to-proximal wing signaling.
Development 131,4489
-4500.
Cho, E., Feng, Y., Rauskolb, C., Maitra, S., Fehon, R. and
Irvine, K. D. (2006). Delineation of a Fat tumor suppressor
pathway. Nat. Genet. 38,1142
-1150.[CrossRef][Medline]
Clemens, J. C., Worby, C. A., Simonson-Leff, N., Muda, M.,
Maehama, T., Hemmings, B. A. and Dixon, J. E. (2000). Use of
double-stranded RNA interference in Drosophila cell lines to dissect signal
transduction pathways. Proc. Natl. Acad. Sci. USA
97,6499
-6503.
Dietzl, G., Chen, D., Schnorrer, F., Su, K. C., Barinova, Y.,
Fellner, M., Gasser, B., Kinsey, K., Oppel, S., Scheiblauer, S. et al.
(2007). A genome-wide transgenic RNAi library for conditional
gene inactivation in Drosophila. Nature
448,151
-156.[CrossRef][Medline]
Dong, J., Feldmann, G., Huang, J., Wu, S., Zhang, N., Comerford,
S. A., Gayyed, M. F., Anders, R. A., Maitra, A. and Pan, D.
(2007). Elucidation of a universal size-control mechanism in
Drosophila and mammals. Cell
130,1120
-1133.[CrossRef][Medline]
Feng, Y. and Irvine, K. D. (2007). Fat and
Expanded act in parallel to regulate growth through Warts. Proc.
Natl. Acad. Sci. USA 104,20362
-20367.
Hamaratoglu, F., Willecke, M., Kango-Singh, M., Nolo, R., Hyun,
E., Tao, C., Jafar-Nejad, H. and Halder, G. (2006). The
tumour-suppressor genes NF2/Merlin and Expanded act through Hippo signalling
to regulate cell proliferation and apoptosis. Nat. Cell
Biol. 8,27
-36.[CrossRef][Medline]
Huang, J., Wu, S., Barrera, J., Matthews, K. and Pan, D.
(2005). The Hippo signaling pathway coordinately regulates cell
proliferation and apoptosis by inactivating Yorkie, the Drosophila Homolog of
YAP. Cell 122,421
-434.[CrossRef][Medline]
Jia, J., Zhang, W., Wang, B., Trinko, R. and Jiang, J.
(2003). The Drosophila Ste20 family kinase dMST functions as a
tumor suppressor by restricting cell proliferation and promoting apoptosis.
Genes Dev. 17,2514
-2519.
Kinoshita, E., Kinoshita-Kikuta, E., Takiyama, K. and Koike,
T. (2006). Phosphate-binding tag, a new tool to visualize
phosphorylated proteins. Mol. Cell. Proteomics
5, 749-757.
Mackintosh, C. (2004). Dynamic interactions
between 14-3-3 proteins and phosphoproteins regulate diverse cellular
processes. Biochem. J.
381,329
-342.[CrossRef][Medline]
Overholtzer, M., Zhang, J., Smolen, G. A., Muir, B., Li, W.,
Sgroi, D. C., Deng, C. X., Brugge, J. S. and Haber, D. A.
(2006). Transforming properties of YAP, a candidate oncogene on
the chromosome 11q22 amplicon. Proc. Natl. Acad. Sci.
USA 103,12405
-12410.
Pan, D. (2007). Hippo signaling in organ size
control. Genes Dev. 21,886
-897.
Saucedo, L. J. and Edgar, B. A. (2007). Filling
out the Hippo pathway. Nat. Rev. Mol. Cell Biol.
8, 613-621.[CrossRef][Medline]
Silva, E., Tsatskis, Y., Gardano, L., Tapon, N. and McNeill,
H. (2006). The tumor-suppressor gene fat controls tissue
growth upstream of expanded in the Hippo signaling pathway. Curr.
Biol. 16,2081
-2089.[CrossRef][Medline]
Struhl, G. and Adachi, A. (1998). Nuclear
access and action of notch in vivo. Cell
93,649
-660.[CrossRef][Medline]
Willecke, M., Hamaratoglu, F., Kango-Singh, M., Udan, R., Chen,
C. L., Tao, C., Zhang, X. and Halder, G. (2006). The fat
cadherin acts through the Hippo tumor-suppressor pathway to regulate tissue
size. Curr. Biol. 16,2090
-2100.[CrossRef][Medline]
Zender, L., Spector, M. S., Xue, W., Flemming, P., Cordon-Cardo,
C., Silke, J., Fan, S. T., Luk, J. M., Wigler, M., Hannon, G. J. et al.
(2006). Identification and validation of oncogenes in liver
cancer using an integrative oncogenomic approach. Cell
125,1253
-1267.[CrossRef][Medline]
Zhao, B., Wei, X., Li, W., Udan, R. S., Yang, Q., Kim, J., Xie,
J., Ikenoue, T., Yu, J., Li, L. et al. (2007). Inactivation
of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition
and tissue growth control. Genes Dev.
21,2747
-2761.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. Mao, B. Kucuk, and K. D. Irvine Drosophila lowfat, a novel modulator of Fat signaling Development, October 1, 2009; 136(19): 3223 - 3233. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. W. Peng, M. Slattery, and R. S. Mann Transcription factor choice in the Hippo signaling pathway: homothorax and yorkie regulation of the microRNA bantam in the progenitor domain of the Drosophila eye imaginal disc Genes & Dev., October 1, 2009; 23(19): 2307 - 2319. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Zhang, C. C. Milton, P. O. Humbert, and K. F. Harvey Transcriptional Output of the Salvador/Warts/Hippo Pathway Is Controlled in Distinct Fashions in Drosophila melanogaster and Mammalian Cell Lines Cancer Res., August 1, 2009; 69(15): 6033 - 6041. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Hamaratoglu, K. Gajewski, L. Sansores-Garcia, C. Morrison, C. Tao, and G. Halder The Hippo tumor-suppressor pathway regulates apical-domain size in parallel to tissue growth J. Cell Sci., July 15, 2009; 122(14): 2351 - 2359. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Zhao, J. Kim, X. Ye, Z.-C. Lai, and K.-L. Guan Both TEAD-Binding and WW Domains Are Required for the Growth Stimulation and Oncogenic Transformation Activity of Yes-Associated Protein Cancer Res., February 1, 2009; 69(3): 1089 - 1098. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. V. V. G. Reddy and K. D. Irvine The Fat and Warts signaling pathways: new insights into their regulation, mechanism and conservation Development, September 1, 2008; 135(17): 2827 - 2838. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. O. Ishikawa, H. Takeuchi, R. S. Haltiwanger, and K. D. Irvine Four-jointed Is a Golgi Kinase That Phosphorylates a Subset of Cadherin Domains Science, July 18, 2008; 321(5887): 401 - 404. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. A. Baena-Lopez, I. Rodriguez, and A. Baonza The tumor suppressor genes dachsous and fat modulate different signalling pathways by regulating dally and dally-like PNAS, July 15, 2008; 105(28): 9645 - 9650. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
