0
Laymen’s summary
In multicellular organisms control over organ size and shape is of utter importance.
However, the regulation of control is rather complex. An involved signalling route is the
Hippo pathway. In this review, the core cascade and downstream target of the Hippo
pathway will be discussed. The first components of the pathway, Warts, Salvador, Hippo
and Mats, were discovered in a search for tumour suppressors in the fruitfly (Drosophila
melanogaster). The downstream target is the transcription co-activator Yorkie (YKI),
which targets many genes implicated in proliferation and apoptosis. These processes
are involved in growth control. Deregulation can, therefore, result in severe effects, such
as tumourigenesis. Furthermore, the upstream regulators of the Hippo pathway will be
disclosed. Various factors are shown to regulate the Hippo pathway core kinases and/or
Yorkie. The vastness of the upstream regulators is required for tissue, timing and
organism specificity.
In order for the Hippo pathway to affect the overall tissue, communication between
cells is necessary. Therefore, cell-cell interactions are required to tightly control and
modulate Hippo activity between neighbouring cells. Cross talk between the Hippo
pathway and other mechanisms is necessary to coordinate the complex development
and homeostasis of tissue size, shape and patterning.
Taken together, the Hippo pathway is important for processes, such as proliferation and
apoptosis. Even though, the regulation of the pathway is understood more and more,
research is required to elucidate the signalling route even further. Since deregulation of
the pathway can result in tumour formations, better understanding of the pathway
could help better understand tumour development and progression. Moreover, insights
in the signalling route might help develop therapeutic targets for cancer and other
diseases.
1
Contents
ABSTRACT
3
INTRODUCTION
4
CHAPTER 1. THE DISCOVERY OF THE CORE COMPONENTS OF THE HIPPO PATHWAY
6
CHAPTER 2. DOWNSTREAM TARGET OF THE CORE CASCADE OF THE HIPPO PATHWAY
10
CHAPTER 3. REGULATORS OF THE HIPPO PATHWAY CORE COMPONENTS
15
CHAPTER 4. CELL-CELL INTERACTIONS INFLUENCE THE REGULATION OF THE HIPPO
PATHWAY
22
DISCUSSION
28
APPENDIX
33
2
Abstract
In multicellular organisms control over tissue/organ size, shape and patterning is of
utter importance. However, the regulation of control is rather complex. An involved
signalling route is the Hippo pathway. In this review, the core cascade and downstream
target of the Hippo pathway will be discussed. The first components of the pathway,
Warts, Salvador, Hippo and Mats, were discovered in a search for tumour suppressors
in Drosophila melanogaster. The downstream target is the transcription co-activator
Yorkie (YKI), which targets many genes implicated in proliferation and apoptosis. These
processes are involved in growth control. Deregulation can, therefore, result in severe
effects, such as tumourigenesis. Other processes influenced by Yki targeting are cellcycle exit, differentiation, polarization, migration and metabolism. Furthermore, the
upstream regulators of the Hippo pathway will be disclosed. Various factors are shown
to regulate the Hippo pathway core kinases and/or Yorkie. The vastness of the
upstream regulators is required for tissue, timing and organism specificity.
In order for the Hippo pathway to orchestrate effects in the overall tissue,
communication between cells is necessary. Therefore, cell-cell interactions are required
to tightly control and modulate Hippo activity between neighbouring cells. Cross talk
between the Hippo pathway and other mechanisms is necessary to coordinate the
complex development and homeostasis of tissue size, shape and patterning.
Taken together, the Hippo pathway is important for processes, such as proliferation and
apoptosis. Even though, the regulation of the pathway is understood more and more,
research is required to elucidate the signalling route even further. Since deregulation of
the pathway can result in tumour formations, better understanding of the pathway
could help better understand tumour development and progression. Moreover, insights
in the signalling route might help develop therapeutic targets for cancer and other
diseases.
3
Introduction
Tissue and organ size control is an important and complicated process in multicellular
organisms. On a single cell level, size can be controlled by cellular growth. Mass
accumulation is mostly regulated by synthesis of proteins and macromolecules.
Proliferation can decrease cell size in that the mass is distributed over two cells1.
However, size control is more intricate in tissue respect. Here, division does not
negatively affect overall size, since division does not affect the overall size of the tissue.
Rather, proliferation positively affects tissue size, because an increase in cell number
allows a larger number of cells to also increase in size, eventually leading to tissue
growth2. Tissue size, is therefore, controlled in part by proliferation and is negatively
regulated by processes such as apoptosis1, 3-7. Cells have their own growth properties,
which thus need to be coordinated in order to elicit size, shape and patterning on a
whole tissue scale. In other words, communication between cells in a tissue is of
importance for cells to regulate tissue size2. The effects of proliferation and apoptosis,
therefore, need not be programmed (intrinsic), but dynamic; adapting to changing
environmental signals. There is a need for growth allowing conditions, such as sufficient
space, nutrient availability and an absence of stress factors. For communication,
however, signal transduction, via cell-cell interactions in the tissue, is required2.
Furthermore, a mechanism/integrator regulating growth in response to these various
signal inputs is required.
One such mechanism identified in the recent years is the Hippo pathway. The
Hippo pathway has been shown capable of transducing outside signals to affect gene
expression and consequently influence tissue size, shape and patterning. The first
components of the Hippo pathway were discovered in a screen for tumour suppressors
in Drosophila3, 5-9. The identification of components in such a screen did not come as a
surprise, since regulators involved in growth control, could well affect tumour
development and progression. In fact, the Hippo pathway was seen deregulated in
various cancer cell lines and hyperproliferative growths in vivo1, 3, 5, 6, 10-15. These studies
show that the deregulation of the Hippo pathway often leads to hyperproliferation and
might even result in tumourgenesis. However, the role of the Hippo pathway seems to
encompass more than the modulation of proliferation and apoptosis alone. Studies have
identified its involvement in differentiation, cell-cycle exit and migration as well14, 16-18.
As explained in the ‘Hallmarks of Cancer19’, development and progression of
cancer is dependent on deregulation of several processes. For tumour development an
important property is the capability of inducing and sustaining growth. Furthermore,
programs, which negatively regulate cell proliferation, such as apoptosis, need to be
circumvented. In tumour progression processes, such as the deregulation of polarity
and stimulation of migration/invasion, are also involved. In this review, the known
characteristics of the function of the Hippo pathway will be discussed to help
understand its frequent involvement in tumour development and progression.
Here, the identification of the Hippo pathway core components, Hpo, Wts, Sav
and Mats, will be discussed (chapter 1). Furthermore, regulation and function of the
most known downstream regulator, Yki/Yap, a transcription co-activator targeting
many genes, will be disclosed (chapter 2). The extensive upstream regulators of both
the Hippo pathway core components and Yki/Yap, will be reviewed as well (chapter 3).
Lastly, the ‘communication’ aspect, consisting of input signals and transducers involved
in Hippo signalling, will be considered (chapter 4). Taken together, the extent and
complexity of the pathway will become clear. As mentioned previously, intrinsic
4
regulation of cell growth control does not impact on tissue scale, therefore, better
understanding of the regulation on tissue scale will become more clear when focussing
on all interacting aspects. Regulation on tissue scale requires ‘communication’ between
neighbouring cells in which many factors and mechanisms/pathways are involved. The
importance of the mechanism is further emphasized by the conservation of many of the
core components (Table 1, appendix). The complexity of the mechanism seems related
to differing functions in timing, tissues and organisms.
Overall, the pathway is complex due to the various regulatory factors and targets, cross
talk with other mechanisms and feedback. There is no one way to describe the function
and mechanism of action. Even though, understanding of the pathway has increased
enormously, much remains elusive. The Hippo pathway is known to be important in
tissue/organ development and homeostasis of size, shape and patterning. Deregulation
of the pathway can result in severe defects, such as cancer. However, it is also
implicated in other diseases, such as obesity, immunity and organ deformations 14, 15, 2022. Further research is required to elucidate the mechanism of tissue/organ
development and homeostasis control. Better understanding of the Hippo pathway
might help better understand the process underlying tumour development and
progression. Consequently, it could help in the development of therapeutic targets for
cancer and other diseases.
5
Chapter 1. The discovery of the core components of the Hippo
pathway
Even though, the pathway is often called the Hippo pathway, the first discovered
component of the pathway was in fact not Hippo9.
In the mid nineties new screens in the model organism Drosophila melanogaster
were developed to facilitate the discovery of new tumour suppressors. Many tumour
suppressors in this organism were previously found using homozygous mutants.
However, several mutations in tumour suppressors are embryonic lethal, making other
more sensitive methods to study and discover them necessary. One of the newer
methods emerging at these times was the use of mosaic organisms. This method proved
an efficient system in that it is analogous to that of tumour growth in humans. In
patients usually only minor groups of cells are mutated and cancerous amongst a
majority of wildtype cells. In mosaic organisms a comparable situation is present of
mutated groups of cells amongst wildtype cells. Furthermore, the wildtype and mutated
groups of cells can easily be compared to detect phenotypic differences, such as
proliferation rate and morphology, in screens. The genetic mosaic in Drosophila could
be induced using different arising techniques. The use of the FLP/FRT recombination
system, a site-specific mitotic recombination system adapted from yeast, gave rise to
the discovery of the first component of the Hippo pathway1, 2. The mosaic screen for
highly proliferative phenotypes performed by ‘Xu T., et al., 19959’ led to the
identification of the tumour suppressor Warts (Wts/Large tumour suppressor/Lats).
The gene encodes a serine-threonine kinase. Sequence comparisons showed that it has
a sequence similarity to three proteins involved in the regulation of cell-cycle control,
cell growth and proliferation in the budding yeast Neurospora. This similarity further
emphasised the possible tumour suppressor function. Later on, the human homologues
Lats1 and Lats2 were found, indicating a high degree of conservation in function and
sequence8, 23.
Since the initial use of FLP/FRT recombination in Drosophila by ‘Xu T., et al.,
19959’ in mosaic screens, several other tumour suppressors were discovered. It was not
until seven year later, though, that another component of the Hippo pathway was
discovered1, 6. The mosaic screens performed in Drosophila became more refined by
focusing on proliferation differences in the compound eye. Subtle changes in
proliferation and morphology were easily detected when comparing the size and
arrangement of specifically shaped ommatidia. The development of the complex
compound eye is tightly regulated by both cell proliferation and apoptosis1, 5. This focus
on both processes led to an important discovery. A mosaic screen by ‘Tapon N., et al.,
20021’ gave rise to the identification of, Salvador (Sav/Shar-pei), a novel tumour
suppressor involved in both processes. To this point relatively few tumour suppressors,
active in both proliferation and apoptosis, were known.
Sav mutants showed both elevated levels in cyclin E (CycE) and Drosophila inhibitor of
apoptosis 1 (DIAP1). The prior protein is required for the modulation of cell-cycle exit.
The down-regulation of cycE has been shown to induce cell-cycle exit and limit
proliferation subsequently24. Reversely, the increase of protein levels resulted in a
higher degree of proliferation. The latter protein, DIAP1, is involved in apoptosis1, 3. This
protein is capable of inhibiting apoptosis by binding to caspases. An up-regulation will
thus result in holding off apoptosis25. The tumour suppressor function of Sav was
stressed even more by further research. Sav proved to be highly conserved from C.
6
elegans to humans. Moreover, the human orthologue was mutated in three cancer cell
lines1, 5, 6.
The fact that Sav is a component of the Hippo pathway was discovered when
looking at a molecular level. The protein contained a C-terminal coiled-coil region and a
N-terminal WW domain. Proteins containing WW domains are usually capable of
protein-protein interactions. The use of double mutants and GST pull-down assays
revealed the fact that Sav is capable of interacting with Wts1, 5. Further experimentation
proved this interaction and a scaffolding function for Sav was proposed1, 5, 26.
Short after the discovery of Sav, five individual papers mentioned the discovery
of another component of the pathway: Hippo (Hpo) itself3, 4, 26-28. Again, the tumour
suppressor, which encodes a serine-threonine kinase part of the Sterile 20 family, was
discovered in mosaic screens. In these screens mutations that resulted in
overproliferation, an increase in cell growth and impaired apoptosis were selected.
Figure 1. shows the overgrowth phenotype in hpo loss of function mutants3. These
results are similar with the above mentioned wts and sav loss of function mutant
phenotypes (data not shown). Since, both cycE and diap1 were up-regulated in hpo
mutants as well, there was an indication that there could be a connection with Wts and
Sav3, 4, 26-28. Several interaction and binding assays proved that there was in fact a
physical connection. Yeast two-hybrid experiments using both Hpo and Sav as bait
showed both proteins could interact3, 4. Furthermore, binding assays showed that it was
in fact the C-terminal coiled-coil region of Sav that was capable of binding the Cterminal region of Hpo. Hpo, then, stabilises Sav by phosphorylation 3, 4, 26-29. Binding
assays showed that there was a physical interaction between Hpo and Wts as well.
However, it is not the C-terminal, but the N-terminal region of Hpo that is necessary for
the interaction with Wts3, 27. The catalytically active N-terminal region of Hpo is able to
phosphorylate and activate Wts3. Interaction of Hpo with Sav facilitates this
phosphorylation. Furthermore, genetic epistasis showed Hpo is active downstream of
Sav, but upstream of Wts in the pathway11.
In contrast to the other components of the pathway, Hpo is able to affect the
process of apoptosis directly. Hpo is able to directly phosphorylate DIAP1 in order to
induce its subsequent degradation4, 28. Furthermore, Hpo can induce the expression of
the pro-aptotic protein Head involuted defect (hid), which can bind and inhibit DIAP11, 4.
7
Figure 1. Overgrowth effects in hpo loss of function clones in Drosophila melanogaster. (A-F)
Scanning electron micrographs (SEM) of wildtype (A) and hpo mutant clone containing (B-F)
Drosophila tissue. (A-B) Show the fly head. (C-D) Show a side view of the compound eye. (D)
Shows a less severe hpo mutant. (E) Shows the fly notum. Dashed lines outline the hpo mutant
clones. (F) High magnification of the epidermal cells. Dashed lines show the border between
wiltype (left) and mutant (right) clones. (G) Drosophila wing tissue containing hpo mutant
clones. Mutant clones are outlined by red dashed lines. (H) Drosophila leg portion containing
hpo mutant clones. Mutant clones are outlined by red dashed lines. (I) Section of adult
Drosophila eye tissue containing wildtype (upper right) and hpo mutant clones (lower left).
Boundary between clones is marked by red dashed lines. Wildtype clones contain pigment. Note
the increased spacing in mutant clones between mutant photoreceptor clusters, due to an
increase in interommatidial cells (data not shown). Results from Wu, S., et al., 20033 (check to
see used genotypes produced by FLP/FRT recombinase).
So far, three of the core components of the Hippo pathway were known. The
interaction of the scaffolding protein Sav with the kinase Hpo is needed to facilitate the
phosphorylation of the kinase Wts. Nonetheless, it was still unknown how the catalytic
activity of Wts was required for the modulation of proliferation and apoptosis. Other
mosaic screens in the compound eyes of Drosophila were performed in hopes of finding
a phosphorylation target of Wts. The screen performed by ‘Lai Z.C., et al., 20057’ led to
the identification of the tumour suppressor Mats. Mats encodes a non-catalytical
protein, part of the Mob superfamily7, 30, 31. However, subsequent binding assays
showed the protein was not a Wts target for phosphorylation. In fact, Mats interacts
synergistically, as an activating subunit, with Wts. The protein is functionally and
genetically conserved in mammals. Furthermore, its tumour suppressor function is
emphasised by the fact that the gene is mutated in mammalian cancer cells7, 30, 31. More
resent studies on the mammalian orthologues Mob1A and Mob1B showed that Mst1
8
and Mst2 (Hpo orthologues) phosphorylate Mob in order to enhance its ability to bind
Lats1 (Wts orthologue) 31.
Similar to the previously discovered components of the Hippo pathway, Hpo
itself is evolutionary conserved with human orthologues Mst1 and Mst2. Even the
phosphorylation of Wts homologues Lats1 and Lats2 by Mst2 through the catalytic Cterminal conserved29. The high degree of physical and functional conservation of many
of the pathway components indicate a tissue size control mechanism which might be
universal in all organisms3, 26-29.
All in all, the use of mosaic screens to identify new tumour suppressors led to the
discovery of the four core components of the Hippo pathway (Figure 2). Hpo binds and
phosphorylates the scaffolding protein Sav, which is consequently stabilised. The
interaction between Hpo and Sav is necessary to facilitate the interaction between Hpo
and Mats and Wts. Hpo is able to phosphorylate Mats in order to enhance the ability of
the latter to bind Wts. The binding of Wts to Mats is needed for Hpo to phosphorylate
and activate Wts. Eventually; the activation of the pathway will result in the modulation
of proliferation and apoptosis. To this point, the direct downstream targets of these core
components remain unknown, though. In the next chapter, the discovery of the direct
target of Wts will be discussed.
Figure 2. Schematic overview of the
Hippo pathway core kinases in
Drosophila. Hpo interaction with the
scaffolding protein Sav is required for
Hpo phosphorylation activity. The
interaction between Sav and Hpo is
stabilized by Sav phosphorylation. Hpo
phosphorylates
the
downstream
targets Mats and Wts. Mats functions
as a scaffolding protein required for
the activity of Wts. Phosphyralation by
Hpo stabilizes the complex and
activates Wts. Wts negatively regulates
proliferation and levels of CycE and
DIAP1 via a downstream target. Hpo
can directly modulate DIAP1 levels.
Human Hippo pathway regulation is
fairly similar, apart from Cyclin E
transcription. Conserved mammalian
genes are shown in Table 1, appendix.
9
Chapter 2. Downstream target of the core cascade of the Hippo
Pathway
Previous studies have shown that the Hippo signalling pathway is involved in the
regulation of tissue size; tightly regulating proliferation as well as apoptosis. Several
genes, cycE and diap1, implicated in proliferation and apoptosis are involved, albeit
indirectly. The exact manner by which the tumour suppressor core components of the
pathway elicited the size control remained unclear.
It was not until a Yeast Two-Hybrid experiment was performed, mentioned in
‘Huang J., et al., 200510’ (Figure 3), that a target for the core components was discovered.
In this experiment Wts was used as bait resulting in the detection of an interacting
protein. The Drosophila protein, later named Yorkie (Yki), showed a 31% identity with
is human orthologue Yes-associated protein (YAP), which is a transcription co-activator.
The functional conservation is emphasised by the fact that Yap can rescue the
phenotype the yki loss of function mutants10. Further in vitro binding assays and in vivo
kinase assays showed that Yki could indeed associate with Wts and was phosphorylated
upon its association10, 15. This interaction required the WW domains in the C-terminal
region of Yki and the non-catalytical N-terminal region of Wts10. In vivo, Yki
overexpressing clones showed overgrowth phenotypes similar to that of loss of function
hpo, wts or sav mutants. Furthermore, overexpression of Yki led to an increase in
expression of DIAP1 and CycE, showing Yki activation is involved in increased
transcription of diap1 and cycE. Epistatic analysis led to the inclination that Yki
functioned downstream of the Hippo signalling cascade; Yki being in fact inhibited
through the phosphorylation by Wts10, 15.
Figure
3.
Yeast
Two-Hybrid
experiment showing Yki is capable of
binding Wts. Sos-Wts is used as bait
for library screening. Sos is an empty
bait containing Sos only. Myr is used
as
control
prey,
containing
myrosylation signal only. 18-418,
186-418 and 229-418 are three
independent Yki clones isolated from
the screen. The number represents
the starting and ending position of
the Yki polypeptides in the clones.
25°C is used as control temperature,
whereas 37°C shows the results of
the Y 2-H screen. Results from 43
Elucidation of the function of Yki was made possible by looking at its subcellular
localisation, using epitope tagged proteins and antibody staining11, 15, 33. Yki localises to
both the cytoplasm and nucleus in absence of the Hippo pathway core components. Coexpression of these components localises Yki less to the nucleus and more to the
cytoplasm. As a transcription co-activator this re-localisation is inhibiting its function,
since its transcription co-activating function can only be performed inside the nucleus.
10
There are other transcription regulators known to shuttle to the cytoplasm.
Many of these proteins are shown to interact with the cytoplasmic localisation protein,
14-3-315. Prior to the discovery of the involvement of Yki in the Hippo pathway, its
human orthologue Yap was found accumulated in the cytoplasm as a complex with 143-334. The interaction between Yap and 14-3-3 suggested an interaction between Yki
and 14-3-3 was possible as well. Furthermore, Taz, another human orthologue of Yki,
was known to be able to bind 14-3-3 upon phosphorylation of a single serine residue of
a certain motif34. This motif is conserved at S127 in Yap and at S168 in Yki. In vivo
experiments show the phosphorylation of these residues was found necessary to induce
cytoplasmic Yap/Yki localisation and sequestration. Mutating either S127 or S168 to a
non-phosphorylatable variant phenocopies both Yki overexpression and hpo, wts or sav
loss of function mutants15, 32, 33. Therefore, binding to 14-3-3 keeps Yki in the cytoplasm.
The phosphorylation of the S168 residue and subsequent sequestration of Yki by
14-3-3 is not sufficient to explain all the effects of the Hippo pathway on Yki though15, 32,
33, 35, 36. Other mechanisms are involved in the inhibition of Yki. Experiments, using nonphosphorylable mutants encompassing more than the S168 region (Yki: V53SA) showed
more dramatic overgrowth effects, indicating the importance of other regions33, 36.
Within this larger region reside the two other speculated phosphorylation sites S111
and S250. Experiments, using Phos-tag gels, phenotype screens and subcellular
localisation assays, showed that these sites can indeed be phosphorylated by Wts,
resulting in inhibition and cytoplasmic localisation of Yki36. However, the inhibition via
phosphorylation of these sites is independent from Yki binding to 14-3-337. Moreover,
Yki:V53SA alone showed more overgrowth than with co-expression of components of
the Hippo pathway, indicating the presence of an inhibition mechanism which is not
dependent on phosphorylation. This indication is strengthened by the reduced nuclear
localisation when Hpo and/or Wts were co-expressed38. Another tumour suppressor,
Expanded (Ex), which will be looked at more in depth in the next chapter, showed a
similar Yki inhibitory function independent of phosphorylation. Affinity
chromatography coupled with mass spectrometry showed that Yki is in fact a direct
binding partner of Ex35. The direct binding of Ex, Hpo and Wts seems dependent on the
WW domain of Yki and the PPXY motifs present in Ex, Hpo and Wts35, 38.
All in all, many phosphorylation dependent and independent processes are
involved in the inhibition of Yki. The greatest inhibiting influence on Yki is via its
phosphorylation at the S168, though. The alteration is necessary to facilitate Yki binding
to 14-3-3, which shuttles and sequesters Yki from the nucleus to the cytoplasm35, 36, 38.
Furthermore, the importance of the phosphorylation dependent inhibition of Yki
emphasises the necessity of regulation via the Hippo pathway. Next, the function of Yki
in relation to proliferation and apoptosis regulation will be explained.
As mentioned previously, the overexpression of Yki resulted in an overgrowth
phenotype. The cause of this phenotype was the transcriptional up-regulation of several
genes with proliferative and anti-apoptotic functions. Among these genes in Drosophila
cycE and diap1, but also dMyc have been shown to be a Yki target1, 3, 10, 15, 24, 25, 39.
Furthermore, bantam miRNA is discovered as a Hpo pathway transcription target. The
bantam miRNA targets both proliferation and apoptosis, phenocopying Yki
overexpression40, 41. In mammals, several similar targets, including some diap1 and
dMyc homologues, are known. Other targeted genes are proliferation-involved genes,
encoding proteins such as Ki67, sox4, H19 and AFP, and the anti-apoptotic gene
encoding MCL1. However, the up-regulation of Cyclin E1 or Cyclin E2 has not yet been
discovered. In Drosophila, the CycE up-regulation is tissue specific, showing elevated
11
levels in the eye imaginal discs and around the morphogenetic furrow only15. The
numerous transcriptional targets, which are not present in all tissues or not always
conserved, suggest a more tissue specific targeting role for Yki. Nevertheless, knowing
Yki as a transcription co-activator, a direct binding partner was still necessary, yet
remained elusive.
Several transcription factors were speculated to associate with Yap, among
these were p73, Runx2, ErbB4 and members of the TEAD/TEF family10, 34. ‘Vassilev, A.,
et al., 200134’ showed an interaction between human orthologue Yap and the four
members of the TEAD/TEF family was possible, before the connection with Yap and the
Hippo pathway was made. Subsequent studies focussing on the direct binding partner
of Yap found that transcription factors of the TEAD/TEF family were indeed the most
important activating binding partners of Yap32, 42. Furthermore, the Drosophila
orthologue Scalloped (Sd) was found to be able to bind Yki in several in vitro and in vivo
experiments as well13, 43, 44. The C-terminal region of Sd can associate with a conserved
N-terminal domain of Yki. The binding of Sd to Yki is independent of phosphorylation38.
Upon binding, the Sd-Yki complex localises to the nucleus where transcription of target
genes is enhanced13, 34, 43, 44. Interestingly, the association of Sd and Yki is a more
complex interaction than it seems. The down-regulation of Sd causes an increase in
bantam miRNA expression instead of the expected decrease13. However, mutations of sd
phenocopy loss of function mutations in yki, indicating a similar function13, 43, 44. The
different effects on the transcription of the target gene bantam miRNA, indicate there
might be a more complex mechanism involved. Dosage experiments mentioned in ‘Wu,
S., et al., 200843’ further emphasise the delicate interdependence between Yki and Sd.
Although, Sd and the TEAD/TEF orthologues might be the most important
transcription factor, small phenotypic differences comparing sd and yki mutants
indicate it is possibly not the only transcription factor involved13, 43, 44. There are other
transcription factors known to interact with Yki/Yap. Both the TALE-homeodomain
protein Homethorax (Hth) and the zinc finger transcription factor Teashirt (Tsh) have
been shown to interact with Yki and up-regulate transcription41. However, both
transcription factors are active only in the eye imaginal discs, further emphasising the
possible spatiotemporal mechanism underlying Yki function.
Research performed over the years underscore the importance of the Hippo
pathway targets in development, tissue size homeostasis and its apparent oncogenic
predisposition. The suggestion of Yki/Yap being oncogenic is not surprising. The first
indication is the strong overgrowth phenotypes visible in several mutants1-3. Secondly,
Yki target proteins are often up-regulated in many cancer cell lines and tumours, for
example Myc and IAP family members10, 15, 32. Thirdly, in vivo experiments show short
induction of overexpression of Yap in mice result in hepatomegaly (Figure 4). Moreover,
longer induction of overexpression results in tumour growth15. However, the discovery
of down-regulated Yap in cancer patients indicates that it may not be an oncoprotein.
This controversy, together with the above-mentioned questions surrounding the actual
mechanism, stresses the complexity of the transcriptional regulation facilitated by
Yki/Yap.
12
Figure 4. Effects of Yap overexpression in vivo in mouse livers. Mouse strains were created
crossing ApoE/rtTA mice (reverse tetracycline transactivator (rtTA) under control of the liver
specific ApoE promotor) with Yap mice (human Yap cDNA driven by minimal CMV promotor
and tetracycline response element). The cross resulted in a ApoE/rtTA-Yap strain, in which Yap
was expressed upon treatment with the tetracyclin containing drug Doxycyclin (Dox).
Treatment consisted of a 0.2 mg/ml Dox dosage starting at 3 weeks of age. (A-B) Show
hepatomegaly after 1 week and 4 of Yap overexpression (right) compared to wildtype livers
(left). (C-D) Shows prolonged overexpression of Yap, (eight weeks (C) and 3 months (D), leading
to tumourigenesis. Arrow heads in (C) point out nodules scattered throughout the liver. Results
from Dong J., et al., 200715.
Taken together, the tumour suppressive Hippo components are the main regulator of
the activity of Yki by inhibiting its function. If not inhibited, Yki can bind possibly
several different transcription factors as a transcription co-activator. The most known
and supposedly the most important interacting transcription factor is Sd. By binding a
transcription factor, Yki can regulate the transcription of several genes involved in
proliferative and anti-apoptotic processes. A schematic overview of Yki regulation and
signalling is depicted in Figure 5. Having elucidated the downstream mechanism and
thus the effect of the Hippo pathway further, the question of the cause of its
(in)activation still remains. Hence, the upstream regulation of the Hippo pathway will
be discussed in the following chapter.
13
Figure 5. Schematic overview of Yki regulation and signalling in Drosophila. Yki can be
phosphorylated directly by Wts. The phosphorylation enables binding to 14-3-3 resulting in
cytosolic sequestration. Hpo is also shown to inhibit Yki, however, the specific mechanism
remains unknown. Ex is shown to directly bind and inhibit Yki in a phosphorylation
independent manner. If active, Yki is able to bind transcription factors such as Tsh, Hth and Sd.
Of these transcription factors, Sd is the best understood and possibly the most important.
Binding of Yki activates transcription of several genes involved in proliferation and apoptosis.
Human Yki regulation and signalling is fairly similar, apart from Cyclin E transcription.
Conserved mammalian genes are shown in Table 1, appendix.
14
Chapter 3. Regulators of the Hippo pathway core components
The results of many studies over the years have led to the unfolding of a complex and
highly regulated network of proteins being involved in the Hippo pathway. A recent
study, ‘Kwon Y., et al., 201345’, developed a protein-protein interaction network (PPIN)
of the Hippo pathway. The network was created using mass spectrometry, using some
of the most described and important existing pathway components as bait. The
resulting network comprised 153 different proteins and 204 interactions, revealing the
extensiveness of the Hippo pathway. Since, the Hippo pathway is this extensive, only the
most known and some of the most recent interacting proteins will be discussed in this
chapter.
Perhaps the most known proteins regulating the Hippo pathway in Drosophila
are Merlin (Mer) and Expanded (Ex). Merlin was already a known tumour suppressor in
mammals. The human orthologue of Mer is also known as the neurofibromatosis type-2
(NF2) gene, which gives rise to a familial cancer syndrome in the central nervous
system when mutated. Mer is part of the protein 4.1 superfamily of adaptor proteins
with a FERM domain. A related protein containing a FERM domain is Ex 12. Binding
assays and in vivo localisation shows both proteins can interact and localise to
adherence junctions independently of one another12.
Mosaic screens showed double mutant mer and ex clones have a tissue
overgrowth phenotype, indicating a role in growth control. The overgrowth phenotype,
caused by defects in proliferation and apoptosis, is similar to that of the previously
mentioned hpo, sav and wts mutations (Figure 6) 12. Furthermore, an up-regulation of
CycE and DIAP1 levels was also found in the mutants. These results indicate a possible
link between Mer and Ex and the Hippo pathway12. The overexpression of either Ex
and/or Hpo in different mutant backgrounds showed that the proteins indeed act in the
same pathway. Ex overexpression caused undergrowth phenotypes, which could be
rescued by hpo loss of function. However, Hpo overexpression could not be suppressed
by loss of either mer or ex. These results show that even though they act in the same
pathway, Mer and Ex act upstream of Hpo. Later on, Mer and Ex were found to act
upstream of all the Hpo core kinases and Yki as well. A Yki reporter gene assay shows
that Mer and Ex can repress the activity of Yki in a similar way as Hpo, Sav and Wts10, 12.
Since many components of the Hippo pathway are conserved in vertebrates both
genetically as well as functionally, it is likely that Mer and Ex orthologues act upstream
of the Hippo core kinases in vertebrates12.
Accordingly, the regulation of the Hippo core kinases can involve both Mer and
Ex. This is not surprising, since both proteins have been shown to interact.
Furthermore, the co-expression of both proteins can enhance the phosphorylation of
Wts in cultured cells, while expression of one of the proteins alone has a smaller effect.
Therefore, Mer and Ex act synergistically to induce the Hippo pathway, possibly by
acting parallel to each other12, 46.
15
Figure 6. Mer and Ex regulate tissue growth in Drosophila mid-pupal retina. The retinae were
stained with anti-Discs large (Dlg) antibodies that localize to apicolateral junctions and visualise
cell outlines. (A) Shows wildtype retina. (B) Shows mer mutant clone retina. (C) Shows ex
mutant clone retina. (D) Shows hpo mutant clone retina. (E) Shows retina with wildtype (red
and green staining) and mer;ex double mutant (absence of red and green staining) clones. (F)
Shows retina with wildtype (red and green staining) and TSC1 (Tuberous Sclerosis Complex 1)
mutant clones (absence of red and green staining). TSC1 is a growth control gene affecting
growth via a Hippo pathway independent mechanism. Interommatidial cell number was
affected in both mer and ex single mutants. However, only the mer;ex double mutant
phenocopies the hpo mutant severity. Phenotypical differences in the TSC1 mutant clone
indicate the involvement of Mer or Ex in a distinct growth control mechanism. Results from
‘Hamaratoglu F., et al., 200612.
The differences between the proteins are further emphasised when looking at Ex in
particular. As mentioned in chapter 2, Ex can function as a Yki binding protein,
indicating a dual role of the protein when it comes to influencing the Hippo pathway 35.
Ex is able to abrogate the function of Yki, by relocalising it from the nucleus to the
cytoplasm. This inhibition of Yki takes place downstream of Wts and in concert with the
inhibition via 14-3-335. These results, together with the above-mentioned results, imply
that Ex can either function as an upstream regulator of the Hippo core kinases or
function as a direct binding partner and inhibitor of Yki12, 35.
Looking more in depth at hpo, sav and wts mutant cells, elevated levels of Mer
and Ex were visible. This elevation of protein levels was due to derepressed
transcription. Apparently, the Hippo pathway contains a feedback mechanism to keep
the signalling in a steady state. The activity of the Hippo pathway is tightly controlled in
a steady state, which can be influenced either positively or negatively. Overactivation of
the Hippo pathway leads to inhibition of Yki and thus decreased transcription of targets
including Ex and Mer. When the Hippo pathway is repressed, Yki activity is not inhibited
resulting in transcription of targets including the upstream regulators of Hippo
pathway12.
16
The feedback mechanism notwithstanding, it can be concluded that Ex and Mer
are required for Hpo regulation. Nonetheless, immunoprecipitation experiments
showed no direct interaction of Mer or Ex with Hpo or Wts. There was a link missing
between the Hippo core kinases and the upstream regulators Mer and Ex 12 and this link
was discovered a few years later. RNAi and mosaic screens picked up on a protein,
Kibra, showing an overgrowth phenotype. The overgrowth was caused by
overproliferation and inhibition of apoptosis, indicating a possible involvement in the
Hippo pathway. The phenotypes show less overgrowth compared to mutations in the
Hippo pathway core kinases. Rather, the phenotypes are similar to mutations in either
mer or ex, implying a similar function 46-48. Immunoprecipitation showed Kibra could
interact with both Mer and Ex, increasing the interaction between Mer and Ex 47, 48. This
result suggests a scaffolding function for Kibra. Additionally, results from RNAi screens
and mosaic screens confirm that Kibra functions upstream of the Hippo pathway core
kinases46, 47. In fact, co-expression of all three proteins, Kibra, Ex and Mer, increases the
activation of the Hippo pathway, indicating again a synergistic effect 48. Furthermore,
kibra is a Yki transcription target just as ex and mer, indicating its role in feedback of the
Hippo pathway as well47 . However, a double mutant of kibra and ex shows an
overgrowth phenotype which is even more severe than hpo, sav or wts mutants. In these
double mutants Yki transcription is up-regulated more than in Hippo pathway core
kinase mutants, suggesting that overgrowth via Yki activation can be inhibited
dependent or independent of the Hippo pathway. As mentioned above, this could be
facilitated by Ex, which can bind Yki directly. Moreover, these double mutant results
indicate Kibra might possible be involved in Hippo pathway independent inhibition of
Yki as well46.
Kibra is shown to co-localise with Mer and Ex. This co-localisation is near the
apical membrane, indicating a possible function of transducing signals or information
from the extracellular environment6, 7. All three proteins can localise independently of
one another. More importantly, this localisation seems necessary for the membrane
localisation of the Hippo kinase complex. The loss of Kibra, Ex and Mer results in a
decrease of membrane associated Hpo. Furthermore, it is this localisation of the Hippo
pathway core kinases, which is suggested to be necessary for its activation48.
Apparently, the three proteins function as an apical scaffolding mechanism, necessary
for the localisation of the Hippo pathway core kinases. This localisation could be
necessary in order to stimulate the relay of external signals to internal regulation of the
Hippo pathway48.
Other than the interaction of Kibra with Mer and Ex, yeast 2-hybrid experiments
show Kibra can directly bind Sav, via the WW domain in Kibra. Co-immunoprecipitation
shows that Kibra is able to interact with Hpo in a Sav dependent manner. Continuation
of yeast 2-hybrid experiments showed Mer is capable of interacting with Sav and Ex
with Hpo, contradictory to results mentioned in ‘Hamaratoglu F., et al., 200612’. Further
co-immunoprecipitation confirmed these results. Apparently, many of the upstream
regulators can interact with each other and/or interact with downstream Hippo
pathway core kinases as well48. All together, the specific mechanism remains unclear,
yet the formation of interactions seems required for the enhancement and stimulation
of the Hippo pathway.
Interestingly, the interactions and importance of different components seems
tissue specific. Experiments performed in the eye imaginal discs showed Ex as a more
important upstream regulator than Mer and/or Kibra46-48. On the other hand, Mer and
Kibra seem more important in the ovaries47, 48. The Hippo pathway core kinases seem to
17
act as a signal integrator receiving information from multiple inputs depending on the
tissue type and possibly other factors. However, in order to enhance the view on this
tissue specificity understanding of the (trans)membrane factors involved in the Hippo
pathway is necessary. These factors and the non-cell autonomous implications will be
discussed in the next chapter.
The above mentioned interactions and involvedness in the Hippo pathway of
Mer, Ex and Kibra are all researched in Drosophila. All three proteins are evolutionary
conserved. Although, the functional conservation has not fully been explored, a
conservation of mechanism is expected. For instance, the synergistic phosphorylation of
Lats 1 and 2 in vitro by co-expression of mammalian Kibra and Mer is comparable to
that of the phosphorylation of Wts in Drosophila48. However, the tumour suppressive
function of Kibra shown in Drosophila might not be fully conserved in mammals. For
example, the knockdown of Kibra in a human cancer cell line causes impaired
proliferation and migration instead of the expected increase of proliferation and
migration. This suggests Kibra has an oncogenic rather than a tumour suppressive
function. Note that in this case, instead of the Hippo pathway being involved; the MEKERK-RSK cascade seems involved49. Taken together, the regulation of the Hippo
pathway in mammals might be more complex than in Drosophila. Furthermore, the
regulatory function of Ex, Mer and Kibra should be elucidated further in both mammals
and Drosophila.
In the past years, other factors involved in the regulation of the Hippo pathway core
kinases have been discovered as well. Among them are RASSF, STRIPAK and AKT,
negatively regulating Hpo. Mutants of the Drosophila orthologue, drassf (Ras associated
factor), are viable and show growth defects. The mutants are about 15% lighter than
wildtype due to a reduced cell number. Co-immunoprecipitation shows the protein is
able to interact with Hpo via the SARAH (Sav, RASSF, Hpo) domain26, 50, 51. Apparently,
there is a binding competition between dRASSF and Sav for Hpo, suggesting an
oncogenic function for dRASSF. However, dRASSF might still contain a tumour
suppressive function as well. Especially when looking at the fact that a RAS1 loss of
function results in reduced proliferation and increased apoptosis, which can be
substantially rescued by drassf mutation. Therefore, dRASSF seems to have both an
oncogenic function via the Hippo pathway as a tumour suppressive function via RAS1 50.
In mammals these functions seem to be split over two proteins. The mammalian
dRASSF orthologue RASSF1 encodes two proteins, RASSF1A and RASSF1C. The prior
protein is shown to interact with and activate Mst 1 both in vitro and in vivo51. The
latter protein was shown to have oncogenic properties. Deletion of RASSF1A in mice,
while RASSF1C remains intact, causes an increase in spontaneous tumour formation52.
Even though, dRASSF showed to be a negative regulator of Hpo, a phosphatase
able to directly inactivate Hpo remained unknown. A genome wide RNAi screen
identified the Drosophila Striatin-interacting Phosphatase and Kinase (dSTRIPAK) PP2A
complex as a possible phosphatase53. Affinity purification coupled to mass spectrometry
and co-immunoprecipitation indicated dSTRIPAK could indeed associate with
Hpo/RASSF complexes and thereby dephosphorylating Hpo. The striatin family member
Cka was necessary for the interaction with Hpo and was at least in part dependent on
dRASSF. The association of Hpo with dRASSF prevents Hpo from binding Sav. dSTRIPAK
is recruited, subsequently, to dephosphorylate Hpo. The STRIPAK mechanism seems
conserved, since orthologues are able to control the phosphorylation levels of Mst 1 and
253.
18
Another negative regulator of the Hippo pathway is Akt, also known as Protein
Kinase B (PKB). Akt is involved in cell survival via evasion of apoptosis. The evasion of
apoptosis is elicited via different routes, though. A newly discovered route is the Hippo
pathway. Upon cleavage, activated Mst can activate c-jun N-terminal Kinase (JNK)
mediated apoptosis. Akt can inhibit the cleavage of Mst in vivo and thus inhibit the
activation of apoptosis20, 21. Moreover, Hpo is able to inhibit Akt as well. Overexpression
of Yki shows an up-regulation of akt mRNA levels, showing Akt is a target-gene of Yki20.
This is conserved in a human epithelial breast cancer cell line where knock down of
Lats1 results in an up-regulation of Akt dependent on Yap function13. It is interesting to
note that Akt signalling is implicated in metabolism. Hence, the cross talk between Akt
and the Hippo pathway could be necessary to link the availability of nutrients to growth
control13.
The link between nutrient sensing and the Hippo pathway is further emphasised
by another genome wide RNAi screen. This screen led to the discovery of Salt inducible
kinases 2 and 3 (Sik 2 and Sik 3) as negative regulators of Hippo signalling 22. The coexpression of Sik 2 and 3 led to overgrowth phenotypes and up-regulation of ex, similar
to when yki is up-regulated. Co-immunoprecipitation revealed the Drosophila Sik’s can
interact with and phosphorylate Sav. Upon phosphorylation Sav’s ability to induce Hpo
activity is inhibited. The phosophylation site on Sav is not conserved in mammals.
However, Sik2 does promote Yap dependent transcription in human cells, indicating it
might still be functionally conserved in some other way22.
Other than Hpo being regulated, Wts is modulated by several factors as well. Coimmunoprecipitation shows Wts can interact with the large myosin protein Dachs 54.
This protein was previously identified as a downstream component of the Fat signalling
route, which will be discussed in the next chapter. Epistasis experiments show Dachs
functions parallel to Hpo, inhibiting Wts upon interaction. Dachs might function as a
scaffolding protein necessary for the assembly of a degradation complex ensuing the
binding of Wts. Furthermore, Dachs localises apically in presence of Fat 54. An
interacting partner of Dachs, required for the inhibition of Wts, was discovered during a
genetic screen. The LIM domain containing protein, Zyxin (Zyx), is shown to interact
with Dachs. Loss of Zyx reduces Yki activity and thus tissue overgrowth. Evidently, Zyx
positively regulates co-activator Yki, by disabling its inhibitor55. A model is proposed
where the stability of Wts is regulated via the localisation of Dachs. When Dachs is
localised apically, it is able to interact with Zyx to stimulate its interaction with Wts,
thereby inducing its degradation. Interestingly, in mammals, Zyx in implicated in linking
effects of mechanical strain to cell behaviour, since it can modulate the cytoskeletal
organisation of actin bundles. This provides a possible link for mechanical strain and
growth control via gene regulation55.
Apparently, external factors such as nutrient availability and mechanical strain are
involved in the Hippo pathway. Another factor involved is the apical basolateral cell
polarity. Many of the proteins involved have actually been shown to localise apically47,
48. The importance of this polarity is emphasised by the fact that in many neoplastic
tumours polarity genes are mutated. Disruption of apical basal cell polarity can lead to
uncontrolled cell proliferation of epithelial cells, resulting in an epithelial to
mesenchymal transition (EMT). This transition often underlies the transition to
cancer56. Among these polarity genes are the conserved tumour suppressors Lethal
giant larvae (Lgl), Scribbled (Scr) and Disc large (Dlg) 56-58. Lgl opposes the apical
19
localised aPKC complex, consisting of the atypical Protein Kinase C, par-3 and par-6.
Normally, aPKC and Lgl are in a sort of balanced state, inhibiting one another. aPKC is
able to interact with and phosphorylate Lgl, excluding it from the cell cortex. In lgl
mutants, the polarity and epithelial integrity is often disrupted, increasing proliferation
and neoplastic tissue overgrowth. Furthermore, DIAP1 and CycE levels are upregulated. These observations indicate a link with the Hippo pathway.
Indeed, depletion of Lgl activates Yki. Localisation assays show that Hpo and
dRASSF are mislocalised away from the apical cortex in lgl mutants. aPKC activation
mislocalises Hpo and dRASSF in a comparable way as seen in lgl mutants57, 58. Together,
these results imply that Lgl is required for the localisation of Hpo and dRASSF and that
it can be affected by phosphorylation by aPKC. As mentioned previously, the cortical
localisation is required for Hippo pathway activation. The balance, between Lgl and
aPKC, can be affected by Scr and Dlg, as a loss of polarity is responsible for a balance
shift57, 58. Unexpectedly, in lgl mutant clones, the boundary cells are eliminated via JNK
mediated apoptosis. The up-regulation of JNK signalling indicates that the Hippo
pathway is involved58-60. Since, lack of lgl would cause the Hippo pathway to be
mislocalised and deregulated, another non-cell autonomous mechanism is involved58.
Another factor involved in promoting basolateral cell polarity in follicular
epithelium is the Sterile 20 family member, Tao, is also known for controlling
microtubule dynamics61, 62. However, it has been implicated in modulating the activity
of Hpo as well. Co-immunoprecipitation shows it can interact with and phosphorylate
Hpo. The protein is conserved since its mammalian orthologue, TAOK3, affects the Mst
kinases in a similar way62. Tao forms no complex with the other known upstream
regulators Mer or Ex, though. This is not surprising since Tao can control cytoskeletal
organisation and polarity via microtubule dynamics, indicating a parallel function. Again
there seems to be a link between polarity and growth control61, 62.
So far, the involvedness of JNK signalling has been mentioned various instances.
Nevertheless, there is some controversy surrounding the function of JNK signalling in
the Hippo pathway. A recent study described in ‘Sun G., and Irvine K.D., 201359’, shows
its involvedness in regenerative growth. Regenerative growth requires the activation
JNK signalling. This signalling route is activated under stress, for example by wounding,
irradiation or oxidation. As a reaction to stress it can induce apoptosis and proliferation.
Notwithstanding the controversy, both can be regulated via the Hippo pathway. JNK can
be activated by Hpo to mediate apoptosis and activate Yki. In the case of regenerative
growth, Yki gene transcription is up-regulated resulting in an increase in proliferation
and a decrease in apoptosis. Epistasis experiments show JNK acts upstream of Wts to
inhibit it from phosphorylating Yki. This inhibition requires the activity of dJub, a
Drosophila orthologue of Ajuba LIM domain proteins59, 63. In Drosophila, loss of dJub
phonocopies Yki deficiency. Co-immunoprecipitation shows dJub is able to interact with
Sav and Wts, which is conserved as mammalian Ajuba proteins can interact with Lats
and ww45. The interaction of dJub inhibits the phosophorylation of Yki 63. In vitro, the
mammalian orthologue of JNK is shown to stimulate the binding of Ajuba proteins to
Lats1 via phosphorylation. The binding of the Ajuba proteins prevent Lats1 to be
phosphorylated by Mst59. Therefore, there is a connection between JNK and the Hippo
pathway in the regulation of apoptosis and proliferation. Understanding of the exact
mechanism of action requires further examination, though.
20
Taken together, there are many different regulators of the Hippo pathway.
Furthermore, these regulators sometimes suggest connections to other mechanisms,
such as polarity and metabolism. A schematic overview of the regulation is depicted in
Figure 7. In order to look at Hippo signalling on a broader scale, the connections
between cells in a tissue should be discussed. Therefore, cell-cell interactions
influencing the Hippo pathway will be the main focus in the next chapter.
Figure 7. Schematic overview of the upstream regulation of the Hippo pathway in Drosophila.
Ex, Mer and Kibra positively regulate Hippo signalling upstream of Hpo and Sav. The precise
mechanism remains elusive. Tao-1 phosphorylates and activates Hpo. dRASSF and dStripak
negatively regulate Hippo signalling by dephosphorylating Hpo. The balance in polarity caused
by mutual antagonism of aPKC and Lgl positively regulates the Hippo pathway. JNK signalling is
required for the inhibition of Wts via the phosphorylation of dJub. There is a direct link between
Hpo and JNK as well, however, surrounding this link lies a lot of controversy. Metabolism
associated proteins like Akt and Sik-1/-2 negatively regulate the Hippo pathway via inhibition
of Hpo and Sav respectively. Mechanical stress can influence Hippo signalling via a protein
complex consisitng of Dachs, Zyx and App. The binding of Dachs to Wts inhibits its function
possibly by facilitating the degradation of Wts. Note that some of the upstream Hippo pathway
regulators are among the Yki targeted genes indicating a feedback mechanism. Many of the
factors are conserved in mammals (Table 1, appendix), however, there are differences in
regulation. For example, mammals have various RASSF homologues. RASSF1A is shown to
positively regulate Mst1 (Hpo orthologue), whereas, RASSF1C is suggested to negatively
regulate the Hippo pathway.
21
Chapter 4. Cell-cell interactions influence the regulation of the
Hippo pathway
The development and maintenance of tissues and organs is a tightly regulated and
complex process. Many mechanisms are required to control size, shape and patterning.
In order to coordinate this, communication between neighbouring cells and tissues is
necessary. Therefore, a connection between the external environment and the internal
signalling routes, to tightly control the regulation, is necessary. In the previous chapters
a few inputs of the Hippo pathway were disclosed. Here, factors capable of transducing
different input signals to regulate the Hippo pathway will be discussed.
The transmembrane protein, Fat, mentioned briefly in chapter 3, is a factor capable of
transducing external information. The gene was first described in a search for
hyperplastic tumour suppressors in Drosophila64. Fat and Fat-like proteins are
protocadherins that are part of the cadherin super family. Its distinctive extracellular
domain, consisting of a large amount of cadherin repeats, sets these proteins apart from
conventional cadherins. The proteins have a wide range of functions, i.e. functioning in
cell-cell interactions, regulation of planar cell polarity (PCP), migration and growth
control65, 66. Not surprisingly, its influence on the latter function, links the protein to
Hippo signalling.
Fat mutants in Drosophila phenocopy hpo mutants, thereby showing overgrowth
and up-regulated diap1 and cycE. Several epistasis experiments placed it upstream of
the Hippo pathway core kinases54, 67. Furthermore, co-immunoprecipitation indicated
Fat negatively affects the phosphorylation of Wts. The link between Fat and Dachs,
Zyxin and App was already explained in chapter 3. Fat inhibits the phosphorylation of
Wts via inhibition of these proteins, thereby positively regulating the Hippo pathway54.
The use of several cleaved fragments of Fat revealed that the intracellular domain of Fat
is required for its influence on the Hippo pathway67. Actually, expression of the
intracellular domain of Fat can rescue the fat mutant overgrowth phenotype, indicating
Fat can compete for Dachs binding54. These results suggest Fat, with its extra- and
intracellular domain, can function as a signal transducing receptor in the Hippo
pathway. However, the fat mutant phenotypes are not as severe as complete loss of
Hippo signalling, indicating it is not the only upstream regulator54, 67. The epistasis
experiments performed showed Fat acts upstream of Ex as well, while functioning in
parallel with Mer54, 67, 68. Fat revealed to be required for the localisation of Ex68.
Upstream regulators of Fat and Fat-like cadherins were already revealed in a
study focussing on planar cell polarity. Fat is able to interact with another
protocadherin Dachsous (Ds). Furthermore, the Golgi protein Four-jointed (Fj) is
involved69. Fat activity is negatively regulated upon Ds interaction. In fact, Ds regulates
the phosphorylation of Fat via Disc overgrown/Casein Kinase 1δ/ε (DCO). Coimmunoprecipitation of several Fat deletion mutants show DCO is able to interact with
the intracellular domain of Fat and phosphorylate several sites70. Fj negatively regulates
Ds activity. In vitro and in cell culture, Fj was shown to be able to phosphorylate Fat and
Ds, inhibiting the binding of the two protocadherins71. Additionally, the protein lowfat
(Lft) is able to bind the intracellular domains of both Ds and Fat. Lft expression is
responsible for elevated levels of Fat and Ds. Immunostaining shows Lft affects the
protein levels, not the mRNA levels of Fat and Ds. These results suggest Lft regulates Fat
22
and Ds levels posttranscriptionally72. A schematic overview of Fat signalling in relation
to Hippo signalling is depicted in Figure 10.
An intriguing result arose when looking deeper into the regulation of Fat
signalling. Both overexpression and depletion of Ds caused a down-regulation of Yki
target genes73, 74. Figure 8 shows ds gain of function during Drosophila wing
development causes growth defects. The down-regulation of the genes indicated
induction of the Hippo pathway. However, it was controversial to see both a gain and a
loss of function of ds causing similar effects. These results suggested it was not the
absolute amount of the protein modulating the Hippo pathway. Moreover, when looking
at mutant clones a clear pattern was visible. The Hippo pathway seemed repressed in
the boundary of mutant clones rather than inside the mass. Apparently, the position of a
cell on/near a clone boundary influenced the Hippo pathway. Subsequent experiments
showed it was in fact the differences in Ds levels between neighbouring cells causing the
effect. The same observations could be seen in fj mutant clones. Fj and Ds are expressed
in a gradient71, 73, 74. At boundaries cells can interact with cells with different amount of
Fj and/or Ds activity. Cells on both sides of the boundary can induce Yki target
transcription in gradients up to several cells away from the boundary73, 74.
Figure 8. Uniform expression of Ds and Fj inhibit growth in Drosophila adult wings. A tub-Gal4
transgene was used for ubiquitous expression. (A) Shows wildtype wing size, no UAS transgene
was used. (B) Shows wings size when Ds is uniformely expressed. (C) Shows wing size when Fj
is uniformely expressed. (D) Shows wing size when both Ds and Fj are uniformly expressed. Ds
and Fj single mutants show an intermediate phenotype when compared to the more severe
double mutant. The double mutant phenocopies Dachs depletion mutants, emphasising the link
to Fat signalling. Results from Rogulja D., et al., 200873.
The mechanism where Hippo signalling is controlled by differential levels of factors of
juxtapositioning cells was called boundary signalling/the boundary effect (Figure 9).
The existence of boundary signalling facilitates a link between PCP and growth control.
PCP can cause gradient differences of proteins in a tissue. Differences of protein activity
can subsequently modulate Fat and Hippo signalling. In fact, a known morphogen
distributed in a gradient along the anterior-posterior axis in Drosophila wing
development is involved in the expression gradient formation of Fj and Ds73. Another
interesting implication of the boundary effect is the function of Ds. The extracellular
domain of Ds can interact with Fat and modulate Fat signalling. For the generation of
23
the boundary signal, though, the expression of only the intracellular domain was
sufficient. The intracellular domain was insufficient to render the cell capable of
responding to the boundary. In order to generate the boundary effect and respond to
the boundary signal both the intracellular and extracellular domain of Ds are required.
The data suggest that, like Fat, Ds acts as a transducing receptor74. Even though, the
specific mechanism of boundary signalling remains unknown, it is required for the
modulation of the Hippo pathway and linking it to PCP.
Figure 9. Schematic overview of the boundary effect.
Cells in adjacent tissues have differing levels of Ds
and Fj. At borders of tissue types, cells with the
differing protein levels are juxtapositioned causing
inhibition of the Hippo pathway. Note, that the
boundary effect is explained here using proteins
taking part in Fat signalling. The boundary effect
was shown in Crb signalling as well.
The involvement of Fat signalling in Hippo pathway modulation seems conserved in the
vertebrate system. Genetically, the proteins are conserved. Furthermore, a homolog of
Fat (Fat1) has similar results in Yap target regulation in Zebrafish (Danio rerio) 75.
Interestingly, the homolog of Scr can interact with Fat1 and is required for the Fat1
regulation of Hippo signalling. Since, Scr is a polarity regulating protein, the link
between polarity and Hippo signalling is emphasised in Fat1 signalling. The cross talk
between different signalling cascades seems required to properly define pronephros
size and structure in Zebrafish75.
Another transmembrane protein involved in Hippo signalling is Crumbs (Crb). Crb is
known to be involved in the regulation of apical basal cell polarity57, 76, 77. The protein
localises at the apical cortex by self-recruitment, upon interaction between the
extracellular domain of two Crb proteins in neighbouring cells78. The apical polarisation
is antagonised by the polarity regulating proteins Dlg and Lgl, mentioned in chapter 3 77,
78. Mutations in crb phenocopy hpo mutants, with up-regulated Yki target transcription
and induced proliferation. These results suggested Crb might function as an upstream
regulator of the Hippo pathway76, 79. Epistasis experiments showed the transmembrane
protein indeed functioned upstream of the Hippo pathway. Furthermore, Ex localisation
seemed affected in crb mutants. Instead of localising apically, Ex distributed more
diffusely upon crb mutation57, 76, 77. Co-immunoprecipitation coupled to cleavage
experiments showed the intracellular domain of Crb was required for binding to and
apical localisation of Ex76, 77. The domain required for Ex localisation differs from the
polarisation regulation domain, though, indicating Crb can regulate growth and
polarisation simultaneously77. Moreover, the apical recruitment of Ex, and Kibra as well,
have been shown to stabilise Crb at the apical membrane in Drosophila follicle cells78.
Normally, the antagonistic effects of Lgl induce endocytosis of Crb and subsequent loss
of apical membrane identity. Recruitment of the Crb complex and the upstream Hippo
signalling factors prevent this from happening78.
Interestingly, follow up experimentation showed a loss of function mutation of
crb causing the same phenotype as a gain of function mutation76, 79. These seemingly
24
contradictory results could be explained by the previously mentioned boundary effect73,
74, 79. Apoptosis seemed to be induced especially at clonal boundaries of mosaic animals.
At these boundaries the levels of transmembrane Crb differed. Apparently, if Crb levels
are similar, either high or low, in neighbouring cells, the Hippo pathway is downregulated and proliferation is induced. However, if Crb levels in adjacent cells differ, the
Hippo pathway is induced and apoptosis is induced. These results suggest a non-cell
autonomous function for Crb in tissue morphology. A cell can transduce a signal to
induce apoptosis via the extracellular domain of Crb in a neighbouring cell79. This signal
transduction could be used as a size sensing mechanism. As organs grow, mechanical
forces could alter the geometric structure of an organ. This structure change can also
lead to changes in Crb interactions. The competition between neighbouring cells and the
added boundary effect will induce apoptosis and reduce organ size subsequently.
Apoptosis is most often induced in both cells at the boundary. However, when boundary
signalling and mutations in Hippo signalling are coupled, super competitive clones can
consequently arise. Boundary signalling would induce apoptosis via crb in both
wildtype and Hippo signalling mutant clones. Though, the Hippo pathway mutation
induces Yki target transcription, such as diap1, making apoptosis evasion possible79.
Therefore, instead of the induction of apoptosis in neighbouring cells at the boundary,
the hpo mutant cells will be able to survive.
In conclusion, Crb is an important transmembrane protein able to integrate
signals from neighbouring cells to modulate the Hippo pathway accordingly.
An additional transmembrane receptor involved in Hippo signalling was found in the
human glioblastoma multiforme (BGM) cancer cell line. CD44 encodes a cell surface
hyaluronan receptor. Depletion of the protein leads to sustained phosphorylation and
activation of Mst1/2 and Lats1/2, resulting in inactivation of Yap. A reduced expression
of cIAP1/2, human orthologues of diap1, was also observed14. High expression of the
receptor gave the opposite effect, indicating the negative regulation function on the
Hippo pathway in GBM cells. Overgrowth visible upon expression of CD44 was due to
evasion of apoptosis mediated by the Hippo pathway via JNK signalling14. The
connection of the CD44 receptor to the Hippo pathway did not come as a surprise since
a link has been suggested previously. The cytoplasmic domain of CD44 has been shown
to interact with Merlin. The binding of Merlin can inhibit the hyaluronan-CD44
interaction and thus prevent CD44 activity80. These results suggest CD44 is a negative
regulator of the Hippo pathway.
Recent studies have proposed a role for cytoskeleton integrity in modulating the Hippo
pathway. Immunofluorescence assays show Mst can co-localise with Filamentous actin
(F-actin) structures in mammalian cell lines 60. Furthermore, induction of extra F-actin
polymerisation was shown to induce an overgrowth phenotype in Drosophila imaginal
discs81. F-actin polymerisation was stimulated by depletion of capping proteins and
expression an F-actin formation triggering protein (Diaphanous). Besides the
overgrowth phenotype, F-actin formation leads to several Hippo pathway typical
phenotypes, e.g. activation of Yki, up-regulation of diap1 and ex. These results show that
changes in F-actin can modulate the Hippo pathway81.
Evidence for a link between the Hippo pathway and the actin-cytoskeleton was
then further emphasised in mammalian cell lines. Studies suggested the proteins Ajuba
and Angiomotin (Amot) as possible links between the actin-cytoskeleton, F-actin and
the Hippo pathway82, 83.
25
All in all, a connection between the actin-cytoskeleton and the Hippo pathway is
clear. A possible functional explanation for this connection lies in the external to
internal information transduction aspect. In Hela cells, the loss of cell-cell interactions
led to the induction of F-actin polymerisation84. Apparently, the loss of information
from neighbouring cells can be the cause for actin-cytoskeletal changes. As was
mentioned previously, the F-actin changes leads to the disruption of the Hippo pathway
and induction of Yki target genes necessary for proliferation and migration. This
mechanism can be implicated in development. However, there is likely a role in wound
healing as well. Cell interactions are often lost during wound formation. Stimulation of
processes, such as proliferation and migration, can thus promote the healing process84.
Taken together, these results indicate cell-cell interactions are required to transduce
signals for the regulation of the Hippo pathway. Furthermore, the dual role of many
factors in both polarity (PCP) and growth control is likely not a coincidence. Cross talk
between both processes is likely necessary to achieve proper tissue and organ size,
shape and patterning. The different intracellular signals involved in Hippo signalling
are depicted in Figure 10.
Figure 10. Schematic overview of intracellular interactions regulating the Hippo pathway. The
apically localised transmembrane protein Crb can interact with Crb proteins from neighbouring
cells. This interaction is required for its function in the localisation and consequent activation of
Ex. Crb localisation and activity is affected by cell polarisation. The transmembrane protein Fat
is localised apically as well. Binding with the transmembrane protein Ds of a neighbouring cell
facilitates the phosphorylation of the intracellular domain of Fat by DCO. The intracellular
domain of Fat is required for the inhibition of Dachs, an intracellular protein known to inhibit
Hippo signalling. Protein levels of Fat and Ds are positively regulated by Lft. Both Crb signalling
and Fat signalling, via Ds and Fj are regulated via the boundary effect, where differing protein
levels in adjacent cells are required for Hippo pathway modulation. Another cell-cell interaction
modulator of the Hippo pathway acts through adherens junctions (AJ). dJub negatively regulates
Hippo signalling via these junctions. In humans dJub connects the adherens junctions via αcatenin to the actin-cytoskeleton via F-actin. This interaction connects the actin-cytokeleton to
the Hippo pathway. However, this mechanism is not shown in Drosophila and is therefore
26
annotated by the asterisks. The fact that F-actin polymerization is able to modulate Hippo
signalling in Drosophila indicates there might a similar mechanism, though. Most of the other
regulators are conserved in mammals (Table 1, appendix). An additional transmembrane
regulator in humans is CD44. This protein is shown to negatively regulate Hippo signalling in
the glioblastoma multiforme cancer cell line.
27
Discussion
Mechanisms involved in cell growth regulate normal development and homeostasis of
shape, size and patterning of tissues/organs in multicellular organisms31, 85. One of the
first mechanisms discovered involved in cell growth, by affecting both proliferation and
apoptosis, is the Hippo pathway1, 3, 6, 8. Deregulation of this mechanism leads to severe
effects involved in the development and progression of cancer. In the previous chapters
many components of the Hippo pathway were shown deregulated in cancer cell lines1, 5,
6, 10, 12-15, 32. Furthermore, prolonged deregulation of the main effector, Yki, resulted in
tumour formations in the liver of mice in vivo15. The severe impact of deregulated
components on the balanced mechanism underscores the importance of tight regulation
of the mechanism.
In this review, the identification and the function of components of the Hippo
pathway were reviewed. The core kinases, Hpo, Sav, Mats and Wts, of the pathway were
first discovered in Drosophila melanogaster (chapter 1). Since the kinases negatively
regulate growth they have been identified as tumour suppressors1, 3, 5, 6, 8, 26, 31. The
Hippo pathway core kinases modulate the downstream activity of the oncogene yki via
phosphorylation and subsequent inactivation of the encoding protein. Yki functions as a
transcription co-activator, regulating the transcription of several target genes involved
in proliferation and apoptosis evasion (chapter 2) 10. More recently, the function of the
Hippo pathway has been ascribed a broader function than growth control alone. Several
of the downstream targets have been implicated in processes such as cell cycle exit,
differentiation and migration as well1, 4, 26, 86-88. In any case, Hippo signalling has been
identified as an important mechanism for tissue/organ development and homeostasis.
The importance of the core kinases and Yki is emphasized by the evolutionary
conservation (Table 1, appendix) 2, 89, 90. Figure 11, shows these core components are
conserved in Drosophila and humans. A recent publication, ‘Hilman D., and Gat U.,
201390’, shows that conservation of the core kinases can even be deduced to organisms
such as bakers yeast (S. cerevisae). The conservation of the key regulator, Yki, seems
less apparent, though. For instance, whether the transcription co-activator is conserved
in C. elegans, is still debated. Recent findings suggest the newly identified gene yap-1 as
a homolog91, 92. Yap-1 partially shares the basal characteristics, sequence similarity in
the N-terminal TEAD binding domain and WW domain, of mammalian Yap.
Furthermore, knockdown of the C. elegans homologs of Wts and 14-3-3 show nuclear
accumulation of Yap-191. These results suggest that Yap-1 is negatively regulated by
Hippo pathway components, similarly to the regulation of Yki/Yap in other organisms.
However, a regulative connection between the C. elegans homologs of Hpo and Yap-1
remains absent. It could be the connection is hard to show. However, it could also be
that the Hippo pathway in C. elegans is conserved without Hpo itself taking part in the
signalling route. This does not have to come as a surprise, since C. elegans is known as
an organism with a fixed cell number and high invariance in development93. The already
existing tight regulation of cell size, shape and patterning might thus make full activity
of a Hippo pathway redundant. Bioinformatics might help to elucidate the evolutionary
conservation of the Hippo pathway further.
28
Figure 11. Evolutionary conservation of the Hippo pathway core components. The schematic
overview shows the genetic and functional conservation of the core components of the Hippo
pathway. The most important downstream target is conserved as well. Note the duplication of
Mst1/2 (Hpo), Lats1/2(Wts), Mob1A/B(Mats) and Yap/Taz (Yki).
While the conservation of the core components of the Hippo pathway seems relatively
clear, the conservation of the upstream regulators is less apparent. The upstream
regulation of Hippo signalling is rather extensive (chapter 3) 45. Many factors are
involved, amongst which Ex, Mer and Kibra are probably the best known12, 46-48. The
extensiveness of the regulation makes many of the components at least partially
redundant between many tissues and organisms. An example is the tissue and timing
dependent requirement for Ex and Mer in Drosophila, where Mer is more required in
posterior follicle cells in the oocyte and Ex more in eye imaginal discs during larval
development47, 48. Furthermore, there seem to be differences in the conservation of
many factors between organisms89, 90, 92. For instance, several factors, within the core
pathway as well, have more than one homolog in more complex organisms89, 90. Figure
11 shows situations where Drosophila has only one copy of a certain gene and humans
have more. Duplication followed by diversification is a process commonly observed
when looking at the evolution of a mechanism. The more complex an organism becomes
the more complex signalling routes are required. The rise in complexity can be due to an
increase in timing and/or tissue specificity, but also due to increased cross talk between
mechanisms and/or tissues. The integrator model mentioned in ‘Yu J., et al., 201048’ is in
concordance with this model of evolution. The core kinases may act as signal
integrators and are well conserved and ubiquitously required in most organisms. The
upstream signals come from different regulators, which are less conserved and required
in a timing or tissue specific manner. However, note that some of the Hippo pathway
factors have been shown to modulate cell growth independently from the core kinases
as well12, 47, 48. Ex is probably the most explanatory example, since it appears to
negatively regulate Yki activity independently of the core kinases12. Furthermore, other
signalling routes, such as Wnt and GPCR, have been shown able to regulate Yki/Yap
29
activity94. Therefore, even though the core of the Hippo pathway is important and
conserved, it is only a small piece of the puzzle.
Aside from the internal regulators, many external signals are integrated to
modulate Hippo signalling. Growth and other processes can be regulated in one cell on
its own. However, for proper development and homeostasis of size, shape and
patterning of tissues, various inputs from the exterior are required (chapter 4). Many
transmembrane proteins are involved in receiving external signals and transducing
them interiorly to the Hippo pathway. Among these proteins are Fat and Crb66, 67, 76, 77, 95,
96. Ligand binding to the extracellular domain of either proteins results in Hippo
pathway activation. This is a mechanism through which overall tissue size and shape
can be controlled. When the shape of a tissue/organ increases the density of cells
increases as well. The increase in density or ‘crowding’ is thought to facilitate cell-cell
interactions. Subsequent to the increase in interactions, the Hippo pathway is activated
to prevent further proliferation and/or induce apoptosis79. These specific reactions
counter the ‘crowding’ effect and overall size is maintained/reduced. In other words,
contact inhibition takes place to modulate tissue/organ size. Vice versa, when tissue size
is smaller the lack of interactions causes reduced Hippo signalling activity, resulting
thus in overall tissue growth. However, in both Fat and Crb signalling the boundary
effect plays a role as well. Differing protein levels in adjacent tissues are required for
proper Hippo signalling modulation73, 74.
Not surprisingly, polarization is required for proper functioning of these
transmembrane proteins57, 74, 76-79, 82. When active, the transmembrane proteins are
mostly localized at the apical membrane, where many of the Hippo pathway
components localize as well. Proper localization of many of the Hippo pathway
components is actually dependent on proper localization of upstream regulators56-58, 77,
82, 97. The loss of polarity might thus indirectly affect the Hippo pathway by
mislocalization of several apically required factors. Furthermore, the above mentioned
boundary effect requires polarization. Note that, at least in the case of Fat signalling,
gradient formation of the ligand is required. The formation of this gradient is dependent
on a morphogen gradient affecting the patterning of Drosophila wing during
development73. This example illustrates how not only polarity within a cell but also
patterning within a tissue is required for proper Hippo regulation. A clue that polarity
requirement in Hippo signalling might be more intricate arises when looking at
migration. Tumour progression is often accompanied by invasion. In order for invasion
to take place certain polarity needs to be lost16. Or, in other words, contact inhibition
between neighbouring cells needs to be lost. Experiments done in human ovarian
cancer cell lines showed the loss of contact inhibition was promoted by overexpression
of Yap2. Furthermore, the overexpression induces migration subsequently98. In
Drosophila border cell migration regulation of the Hippo pathway was shown to be
involved as well16. Apparently, Wts is able to phosphorylate and inhibit ENA, an actin
regulator required for migration speed. Within the cluster of cells, cell-cell interactions
are affluent and Hippo signalling is activated. Therefore, cells inside the cluster will not
readily migrate because of ENA inhibition. Cells on the border of the cluster, though,
lack many of the cell-cell interactions. The lack of Hippo signalling makes the cells in the
outer rim capable of migration. The migration of the border cells is thought to ‘pull’ the
rest of the cluster ‘along’16. Both these studies suggest the interactions between cells in
a tissue are required for proper Hippo regulation. Cell-cell interactions are regulated in
part by polarisation, making it thus an important Hippo pathway modulator. Once
polarity is lost cells are no longer bound to their position and migration/invasion is
30
facilitated. The regulation of migration of course can be important in processes such as
wound healing. However, as mentioned previously, when deregulated it is an important
factor involved in tumour progression.
When addressing the topic of tissue organisation, the influence of mechanical
stress and the involvement of the actin-cytoskeleton need to be mentioned. Cell-cell
interactions are not only elicited by transmembrane proteins, such as Fat and Crb,
connections can be made via adherens junctions also81, 84. The actin-cytoskeleton of
neighbouring cells is connected via these adherens junctions. Mechanical stress is
known to influence the (de)polymerization of the actin-cytoskeleton in cells55, 81, 84. The
stress signals can be transduced via the adherens junctions. Transduction of these
signals makes it thus possible to facilitate cytoskeletal changes throughout a tissue.
Moreover, F-actin changes can modulate the Hippo pathway81. There is a lot of cross
talk visible between the cytoskeleton conformation and the Hippo pathway55, 83, 84, 99, 100.
However, how one mechanism/process regulates the other precisely and vice versa is
still relatively unknown. Taken together, the connections between cells are again
required for the organisation, size and shape of tissues via the Hippo pathway.
Another factor, which might be involved in the modulation of the Hippo pathway,
is nutrient availability. If the Hippo pathway is indeed involved in integrating external
signals for proper tissue development and homeostasis, this does not come as a
surprise. Controlling growth to situations where nutrients are available seems
advantageous. Indeed, proteins such as Akt and Sik, which are involved in metabolism,
have connections to the Hippo pathway13, 22. Furthermore, a recent study shows Hpo is
able to modulate fat storage through adipocyte proliferation in Drosophila101.
The diverse and extensive regulation via several input signals and upstream
regulators are not the only factors making the Hippo pathway a complex mechanism.
The Hippo signalling pathway does not just act as an on/off switch. There are several
feedback loops involved to coordinate a strict balance50. As mentioned in chapter 3, Yki
activation targets the transcription of several upstream regulators of the Hippo
pathway, such as Ex, Mer and Kibra12, 46-48. Here, transcription negatively regulates its
own activity. Hippo activity also affects the abovementioned processes such as polarity
and cytoskeletal conformation, thus, indirectly affecting the Hippo pathway itself as
well84, 94. Note that the changes can also affect neighbouring cells; the feedback loops
have more than just a cell autonomous effect.
Taken together, the Hippo pathway is a complex and highly regulated mechanism
involved in growth control. A schematic overview of the various input signals and
downstream effects is illustrated in Figure 12. Regulation of the pathway differs
between tissues and organisms. Furthermore, there is feedback and cross talk with
many other mechanisms. Conservation of many of the core components shows the
mechanism is of great importance. The importance is further emphasised by the severe
tumorigenic effects caused when the signalling route is deregulated. Therefore,
extensive research to further elucidate the different mechanisms and involved
regulators to fully understand its function in relation to tissue development and
homeostasis is essential.
31
Figure 12. Schematic overview of the various input signals and downstream effects of the Hippo
pathway. In tissues/organs various signals can affect the Hippo pathway core kinases (Hippo
core) to facilitate downstream regulation. Polarity, dynamics of the actin-cytoskeleton, cell-cell
interactions and metabolism are among these input signals. However, there might be other
signals, which have not been discovered. The downstream target of the Hippo core is the
transcription co-activator Yki. Here, the co-activated transcription factor is indicated as Tcf. Yki
can be regulated directly by some regulators of the Hippo core and possibly other factors as
well. Targets of Yki-transcription factor signalling are genes involved in processes such as
proliferation, apoptosis, cell-cycle exit, differentiation, polarity migration, metabolism and
Hippo pathway regulators. The input signals and downstream effects can be tissue, timing
and/or organism specific.
32
Appendix
Components of the Hippo pathway in Drosophila and mice (mammals). Conserved protein
functions are mentioned in the third column. From Halder G., and Johnson R.L., 2006 2.
*The mammalian Mst1 and Mst 2 kinases require cleavage prior to activation. Drosophila does
require similar cleavage3, 4, 26-28.
IMammalian Yap and Taz contain a PDZ binding motif, required for nuclear localization and proapoptotic signalling, which is not present in Drosophila Yki. Yap and Taz are phosphorylated by
Lats and a different kinase as well. Phosphorylation mediates the ubiquitination of Yap and Taz
and label the proteins for subsequent degradation.
±Drosophila does not have a CD44 homolog.
§There is no direct homolog of Dachs known in vertebrates.
¶The sequence of the mammalian Ex homologs diverge from Drosophila Ex12.
33
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