InteractiveFly: GeneBrief

kibra ortholog: Biological Overview | References

Gene name - kibra ortholog

Synonyms - CG33967

Cytological map position- 88D1-88D1

Function - signaling

Keywords - regulation the Hippo kinase cascade via direct binding to Hippo and Salvador, tumor suppressor

Symbol - kibra

FlyBase ID: FBgn0262127

Genetic map position - 3R:10,524,252..10,549,978 [-]

Classification - WW domain and C-terminal C2 kibra domain protein

Cellular location - cytoplasmic

NCBI link: EntrezGene
kibra orthologs: Biolitmine
Recent literature
Jin, A., Neufeld, T. P. and Choe, J. (2015). Kibra and aPKC regulate starvation-induced autophagy in Drosophila.Biochem Biophys Res Commun 468: 1-7. PubMed ID: 26551466
Autophagy is a bulk degradation system that functions in response to cellular stresses such as metabolic stress, endoplasmic reticulum stress, oxidative stress, and developmental processes. During autophagy, cytoplasmic components are captured in double-membrane vesicles called autophagosomes. The autophagosome fuses with the lysosome, producing a vacuole known as an autolysosome. The cellular components are degraded by lysosomal proteases and recycled. Autophagy is important for maintaining cellular homeostasis, and the process is evolutionarily conserved. Kibra is an upstream regulator of the hippo signaling pathway, which controls organ size by affecting cell growth, proliferation, and apoptosis. Kibra is mainly localized in the apical membrane domain of epithelial cells and acts as a scaffold protein. This study found that Kibra is required for autophagy to function properly. The absence of Kibra caused defects in the formation of autophagic vesicles and autophagic degradation. It was also found that the well-known cell polarity protein aPKC interacts with Kibra, and its activity affects autophagy upstream of Kibra. Constitutively active aPKC decreased autophagic vesicle formation and autophagic degradation. The interaction between aPKC and Kibra was confirmed in S2 cells and Drosophila larva. Taken together, these data suggest that Kibra and aPKC are essential for regulating starvation-induced autophagy.

Williams, A.A., White, R., Siniard, A., Corneveaux, J., Huentelman, M. and Duch, C. (2016). MECP2 impairs neuronal structure by regulating KIBRA. Neurobiol Dis [Epub ahead of print]. PubMed ID: 27015692
MECP2 is a chromatin-associated protein that binds methylated CpGs and can both activate and repress transcription. Using a Drosophila model of MECP2 gain-of-function, this study identified memory associated Kibra as a target of MECP2 in regulating dendritic growth. It was found that expression of human MECP2 increases kibra expression in Drosophila, and targeted RNAi knockdown of kibra in identified neurons fully rescues dendritic defects as induced by MECP2 gain-of-function. Validation in mouse confirmed that Kibra is similarly regulated by Mecp2 in a mammalian system. It was found that Mecp2 gain-of-function in cultured mouse cortical neurons causes dendritic impairments and increases Kibra levels. Accordingly, Mecp2 loss-of-function in vivo leads to decreased Kibra levels in hippocampus, cortex, and cerebellum. Together, these results functionally link two neuronal genes of high interest in human health and disease and highlight the translational utility of the Drosophila model for understanding MECP2 function.

Ma, X., Guo, X., Richardson, H. E., Xu, T. and Xue, L. (2018). POSH regulates Hippo signaling through ubiquitin-mediated expanded degradation. Proc Natl Acad Sci U S A 115(9): 2150-2155. PubMed ID: 29440430
The Hippo signaling pathway is a master regulator of organ growth, tissue homeostasis, and tumorigenesis. The activity of the Hippo pathway is controlled by various upstream components, including Expanded (Ex), but the precise molecular mechanism of how Ex is regulated remains poorly understood. This study identified Plenty of SH3s (POSH), an E3 ubiquitin ligase, as a key component of Hippo signaling in Drosophila. POSH overexpression synergizes with loss of Kibra to induce overgrowth and up-regulation of Hippo pathway target genes. Furthermore, knockdown of POSH impedes dextran sulfate sodium-induced Yorkie-dependent intestinal stem cell renewal, suggesting a physiological role of POSH in modulating Hippo signaling. Mechanistically, POSH binds to the C-terminal of Ex and is essential for the Crumbs-induced ubiquitination and degradation of Ex. These findings establish POSH as a crucial regulator that integrates the signal from the cell surface to negatively regulate Ex-mediated Hippo activation in Drosophila.
Bonello, T. T., Cai, D., Fletcher, G. C., Wiengartner, K., Pengilly, V., Lange, K. S., Liu, Z., Lippincott-Schwartz, J., Kavran, J. M. and Thompson, B. J. (2023). Phase separation of Hippo signalling complexes. Embo j 42(6): e112863. PubMed ID: 36807601
The Hippo pathway was originally discovered to control tissue growth in Drosophila and includes the Hippo kinase (Hpo; MST1/2 in mammals), scaffold protein Salvador (Sav; SAV1 in mammals) and the Warts kinase (Wts; LATS1/2 in mammals). The Hpo kinase is activated by binding to Crumbs-Expanded (Crb-Ex) and/or Merlin-Kibra (Mer-Kib) proteins at the apical domain of epithelial cells. This study shows that activation of Hpo also involves the formation of supramolecular complexes with properties of a biomolecular condensate, including concentration dependence and sensitivity to starvation, macromolecular crowding, or 1,6-hexanediol treatment. Overexpressing Ex or Kib induces formation of micron-scale Hpo condensates in the cytoplasm, rather than at the apical membrane. Several Hippo pathway components contain unstructured low-complexity domains and purified Hpo-Sav complexes undergo phase separation in vitro. Formation of Hpo condensates is conserved in human cells. It is proposed that apical Hpo kinase activation occurs in phase separated "signalosomes" induced by clustering of upstream pathway components.
Tokamov, S. A., Nouri, N., Rich, A., Buiter, S., Glotzer, M., Fehon, R. G. (2023). Apical polarity and actomyosin dynamics control Kibra subcellular localization and function in Drosophila Hippo signaling. Dev Cell, 58(19): 1864-1879 PubMed ID: 37729921>
The Hippo pathway is an evolutionarily conserved regulator of tissue growth that integrates inputs from both polarity and actomyosin networks. An upstream activator of the Hippo pathway, Kibra, localizes at the junctional and medial regions of the apical cortex in epithelial cells, and medial accumulation promotes Kibra activity. This study demonstrates that cortical Kibra distribution is controlled by a tug-of-war between apical polarity and actomyosin dynamics. This study show sthat while the apical polarity network, in part via atypical protein kinase C (aPKC), tethers Kibra at the junctional cortex to silence its activity, medial actomyosin flows promote Kibra-mediated Hippo complex formation at the medial cortex, thereby activating the Hippo pathway. This study provides a mechanistic understanding of the relationship between the Hippo pathway, polarity, and actomyosin cytoskeleton, and it offers novel insights into how fundamental features of epithelial tissue architecture can serve as inputs into signaling cascades that control tissue growth, patterning, and morphogenesis.
Tokamov, S. A., Buiter, S., Ullyot, A., Scepanovic, G., Williams, A. M., Fernandez-Gonzalez, R., Horne-Badovinac, S., Fehon, R. G. (2024). Cortical tension promotes Kibra degradation via Par-1. Mol Biol Cell, 35(1):ar2 PubMed ID: 37903240
The Hippo pathway is an evolutionarily conserved regulator of tissue growth. Multiple Hippo signaling components are regulated via proteolytic degradation. However, how these degradation mechanisms are themselves modulated remains unexplored. Kibra is a key upstream pathway activator that promotes its own ubiquitin-mediated degradation upon assembling a Hippo signaling complex. This study demonstrates that Hippo complex-dependent Kibra degradation is modulated by cortical tension. Using classical genetic, osmotic, and pharmacological manipulations of myosin activity and cortical tension, it was shown that increasing cortical tension leads to Kibra degradation, whereas decreasing cortical tension increases Kibra abundance. Our study also implicates Par-1 in regulating Kib abundance downstream of cortical tension. Par-1 promotes ubiquitin-mediated Kib degradation in a Hippo complex-dependent manner and is required for tension-induced Kib degradation. Collectively, these results reveal a previously unknown molecular mechanism by which cortical tension affects Hippo signaling and provide novel insights into the role of mechanical forces in growth control.


The Hippo signaling pathway regulates organ size and tissue homeostasis from Drosophila to mammals. Central to this pathway is a kinase cascade wherein Hippo (Hpo), in complex with Salvador (Sav), phosphorylates and activates Warts (Wts), which in turn phosphorylates and inactivates the Yorkie (Yki) oncoprotein, known as the YAP coactivator in mammalian cells. The FERM domain proteins Merlin (Mer) and Expanded (Ex) are upstream components that regulate Hpo activity through unknown mechanisms. This study identified Kibra as another upstream component of the Hippo signaling pathway. This study shows that Kibra functions together with Mer and Ex in a protein complex localized to the apical domain of epithelial cells, and that this protein complex regulates the Hippo kinase cascade via direct binding to Hpo and Sav. These results shed light on the mechanism of Ex and Mer function and implicate Kibra as a potential tumor suppressor with relevance to neurofibromatosis (Yu, 2010).

In multicellular organisms, cell growth, proliferation, and death must be coordinated in order to attain proper organ size during development and to maintain tissue homeostasis in adult life. Recent studies in Drosophila have led to the discovery of the Hippo signaling pathway as a key mechanism that controls organ size by impinging on cell growth, proliferation, and apoptosis. Central to the Hippo pathway is a kinase cascade comprised of four tumor suppressors, including the Ste20-like kinase Hippo (Hpo) and its regulatory protein Salvador (Sav), the NDR family kinase Warts (Wts) and its regulatory protein Mats. The Hpo-Sav complex phosphorylates and activates the Wts-Mats complex, which in turn phosphorylates and inactivates the oncoprotein Yki, which normally functions as a coactivator for the TEAD/TEF family transcription factor Scalloped (Sd). Recent studies have also implicated the atypical cadherin Fat (Ft) as well as the membrane-associated FERM-domain proteins Expanded (Ex) and Merlin (Mer) as upstream components of the Hippo pathway. How these proteins are biochemically linked to the Hippo kinase cascade remains largely unknown, although Ex can at least partially regulate the Hippo pathway by directly binding and sequestering Yki in the cytoplasm (Badouel, 2009). Ft differs from Ex, Mer, and core components of the Hippo kinase cascade in that, besides tissue growth, Ft also regulates planar cell polarity (PCP), for which it interacts with another cadherin Dachsous (Ds). Most recently, it was shown that a gradient of Ds activity in imaginal discs can modulate Hippo-mediated growth regulation, thus potentially linking PCP to the Hippo kinase cascade, although the biochemical mechanism of this linkage remains to be determined (Yu, 2010).

The physiological function of the Hippo pathway is best understood in Drosophila imaginal discs, where inactivation of the Hippo pathway tumor suppressors, or overexpression of the Yki oncoprotein, results in tissue overgrowth characterized by excessive cell proliferation, diminished apoptosis, and increased transcription of Hippo pathway target genes such as the cell death inhibitor diap1 and the microRNA bantam, as well as ex and mer as part of a negative feedback regulatory loop. Recent studies further implicated the Hippo pathway as a conserved mechanism of organ size control and tissue homeostasis in mammals. Thus, the mammalian homologs of Hpo (Mst1/2), Sav (WW45), Wts (Lats1/2), and Yki (YAP) constitute an analogous kinase cascade, and transgenic overexpression of YAP or inactivation of Mst1/2 led to massive organomegaly and rapid progression to tumorigenesis. Furthermore, NF2, the mammalian homolog of mer, is a well-established tumor suppressor gene whose mutations lead to neurofibromatosis (Yu, 2010 and references therein).

Besides its prominent role in controlling imaginal disc growth, the Hippo pathway is required during Drosophila oogenesis for the proper maturation of posterior follicle cells (PFCs). In the absence of Hippo signaling, the PFCs fail to undergo a Notch-mediated mitotic cycle-endocycle switch and accumulate in extra layers of follicular epithelium. The PFC maturation defects, in turn, lead to a disruption of the anterior-posterior (AP) polarity of the underlying oocyte, which manifests itself as mislocalization of the oocyte nucleus and AP axis determinants such as the RNA-binding protein Staufen (Stau). Interestingly, the oocyte polarity defect is observed in mutants for components of the Hippo kinase cascade as well as ex and mer, but not ft, suggesting that the canonical Hippo pathway may integrate different signals in different developmental contexts (Yu, 2010).

This study identifies Kibra as an upstream component of the Hippo pathway. Loss of kibra leads to oogenesis defects, imaginal disc overgrowth, and aberrant gene expression characteristic of defective Hippo signaling. Kibra functions together with Mer and Ex in an apical protein complex, which, through direct binding to the Hpo-Sav complex, regulates the Hippo kinase cascade and thus Yki phosphorylation. These findings uncover an important missing link in the Hippo signaling pathway and shed light on the molecular mechanism of the Ex and Mer tumor suppressor proteins (Yu, 2010).

In a genetic screen for oocyte polarity mutants based on FRT/FLP-induced mitotic clones in follicle cells, four lethal P element insertion lines on chromosome 3R were identified that caused mislocalization of Stau-GFP and Stau to the center of the oocyte when the PFCs were made homozygous mutant for the P element insertions. This polarity defect was observed with variable penetrance depending on the specific P element line analyzed, likely due to their hypomorphic nature. These lethal lines (264/09, 1156/7, f06952, and EP3494) fail to complement each other and all carry a P element insertion near the 5' UTR or within the first intron of CG33967. CG33967 encodes a 1288 amino acid protein that shares 39% identity with KIBRA, a cytoplasmic protein named after its predominant expression in kidney and brain in humans (Kremerskothen, 2003). Both CG33967 and KIBRA contain two N-terminal WW domains and one C-terminal C2 domain. CG33967 is referred to as kibra to distinguish it from its human ortholog KIBRA (Yu, 2010).

This study identifies Kibra as a tumor suppressor and an essential component of the Hippo pathway. A model is proposed in which Kibra functions together with Mer and Ex in an apical protein complex to transduce growth-regulatory signals to the Hpo-Sav complex, which, through the canonical Hippo kinase cascade, controls Yki phosphorylation and target gene transcription. Of note, the findings do not exclude the possibility that Kibra, Ex, or Mer may interact with additional Hippo pathway components besides Hpo-Sav, especially given the recent report that Ex can directly bind Yki (Badouel, 2009). How Kibra, Ex, and Mer function together to integrate upstream signals remains to be determined. One possibility is that these proteins function redundantly in receiving signals from the same upstream regulator(s). Alternatively, each protein may be regulated by distinct upstream regulator(s) (Yu, 2010).

A commonly used assay for Hippo signaling in Drosophila S2 cells involves examining mobility shifts of the Wts protein on SDS-PAGE. Given its large size and that not all protein phosphorylation causes discernable mobility shift on SDS-PAGE, this assay is less sensitive in detecting Wts phosphorylation than the phospho-specific antibody used in the present study. Indeed, overexpression of Ex in S2 cells has no effect on Wts mobility, yet this study demonstrates that Ex induces robust Wts phosphorylation at its hydrophobic motif. The fact that this hydrophobic motif is a well-established direct phosphorylation site by Hpo homologs in mammalian cells (Chan, 2005; Praskova, 2008) further suggests that Ex, as well as Mer and Kibra, regulates Wts through the canonical Hippo kinase cascade. Indeed, it was found that Ex-induced Wts phosphorylation is Hpo dependent. These results are not incompatible with recent report that Ex can also regulate the Hippo pathway in a kinase-independent manner (Badouel, 2009). Using a well-established assay for Yki transcriptional activity, this study found that while Ex, Mer plus Kibra, or Hpo could all suppress the activity of a Yki-Gal4 fusion protein, only Ex was able to suppress the activity of a Yki-Gal4 fusion protein in which all the possible Wts-phosphorylation sites are mutated. These observations are consistent with the view that Ex can regulate the Hippo pathway through both Wts-dependent and -independent mechanisms (Yu, 2010).

A comparison of the loss-of-function phenotypes of mer, ex, and kibra in egg chambers and imaginal discs reveals tissue-specific differences in the relative contribution of each gene to Hippo pathway regulation. For example, loss of ex alone, but not mer or kibra, is sufficient to cause robust diap1 upregulation in imaginal discs, suggesting that ex has a more essential role in diap1 transcriptional regulation. However, the converse is true in the ovary, where loss of mer or kibra results in stronger oocyte polarity and Notch signaling defects than loss of ex. In fact, the severity of mer or kibra mutant phenotypes in oogenesis are comparable to those of core components of the Hippo pathway such as hpo and sav, even though the former display much milder overgrowth than the latter in imaginal discs. Perhaps the most extreme case of tissue-specific requirement is provided by the ft tumor suppressor gene, which is required for Hippo pathway regulation in the imaginal discs but dispensable in developing egg chambers. While the underlying molecular basis remains to be determined, such tissue-specific requirements suggest that the core Hippo kinase cascade may function as a signal integrator of multiple inputs in a dynamic and versatile manner, and that additional cell surface receptors besides Ft may signal to the Hippo pathway (Yu, 2010).

Considerable efforts have been directed at identifying the key signaling pathways regulated by the NF2/Merlin tumor suppressor protein. These investigations have led to the identification of a number of effector mechanisms downstream of NF2/Merlin, such as growth control pathways mediated by Ras, Rac, STAT, or PI3K, contact inhibition mediated by cell surface receptors or adherens junctions, and endocytosis/degradation of various membrane proteins. The recent identification of Mer as an upstream regulator of Hpo in Drosophila provides yet another plausible mechanism through which Mer functions as a tumor suppressor protein. The identification of Kibra as a regulator of the Hippo pathway further strengthens the case for a functional link between NF2/Mer and the Hippo pathway. The observation that NF2/Mer and KIBRA can synergistically stimulate Lats1/2 phosphorylation in mammalian cells not only supports an NF2/Mer-Hippo connection, but further implicates KIBRA as a potential tumor suppressor in humans with relevance to neurofibromatosis (Yu, 2010).

The identification of Kibra as an upstream regulator of the Hippo pathway has implications for understanding memory-related functions of the human KIBRA gene (Papassotiropoulos, 2006). Besides its well-established roles in growth control, the Hippo pathway is also required for differentiation and morphogenesis of certain postmitotic neurons in Drosophila (Mikeladze-Dvali, 2005; Emoto, 2006). It is speculated that modulation of the Hippo pathway may influence the growth or differentiation of memory-related neuronal structures, a hypothesis that can be directly tested by genetic manipulation of Hippo signaling activity in animal models (Yu, 2010).

Kibra is a regulator of the Salvador/Warts/Hippo signaling network

The Salvador (Sav)/Warts (Wts)/Hippo (Hpo) (SWH) network controls tissue growth by inhibiting cell proliferation and promoting apoptosis. The core of the pathway consists of a MST and LATS family kinase cascade that ultimately phosphorylates and inactivates the YAP/Yorkie (Yki) transcription coactivator. The FERM domain proteins Merlin (Mer) and Expanded (Ex) represent one mode of upstream regulation controlling pathway activity. This study identified Kibra as a member of the SWH network. Kibra, which colocalizes and associates with Mer and Ex, also promotes the Mer/Ex association. Furthermore, the Kibra/Mer association is conserved in human cells. Finally, Kibra complexes with Wts and kibra depletion in tissue culture cells induces a marked reduction in Yki phosphorylation without affecting the Yki/Wts interaction. It is suggested that Kibra is part of an apical scaffold that promotes SWH pathway activity (Genevet, 2010).

An in vivo screen was performed in the fly wing in order to identify genes implicated in growth control. Transgenic flies bearing RNA interference (RNAi) constructs generated by the Vienna Drosophila RNAi Centre (VDRC) were crossed to the hedgehog-GAL4 (hh-GAL4) driver, leading to target gene silencing in the posterior compartment of the wing. A collection was screened of 12,000 lines targeting genes conserved between Drosophila and mammals. Expressing an RNAi line directed against kibra induced overgrowth of the posterior wing compartment compared to control flies. This phenotype was also observed upon wts depletion. Driving the same kibra RNAi line in the eye also led to increased organ size, similarly to a wts RNAi line. Adult eye sections revealed that kibra knockdown retinas present an excess of interommatidial cells (IOCs). The IOCs, the last population of cells to differentiate in the eye primordium, give rise to the secondary and tertiary pigment cells that optically isolate the ommatidia in the compound eye from each other. Extra IOCs are produced during normal development but are then eliminated by apoptosis at the pupal stage to give rise to the adult lattice. The presence of extra IOCs is a hallmark of SWH network loss of function, which reduces retinal apoptosis, as seen in wts RNAi adult eye sections. Thus, depletion of kibra elicits a similar phenotype to SWH network mutants, suggesting a potential role for Kibra in Hpo signaling (Genevet, 2010).

To study kibra loss of function, the kibraΔ32 allele loss of function allele was generated by imprecise excision of the EP747 transposon. This deletion allele, which removes the translation initiation site, is homozygous lethal and may be a null allele for kibra. kibraΔ32 FLP/FRT mutant clones in 40 hr after-puparium-formation (APF) retinas present extra IOCs, similarly to what was observed in adult eyes with kibra knockdown. Duplication of bristles or missing bristles can also be observed. Apoptotic indexes were determined during the retinal apoptosis wave (28 hr APF) in pupal retinas containing kibra mutant clones stained with an anti-active Caspase-3 antibody. kibra mutant tissue presents a reduced apoptotic index compared with wild-type (WT) areas in the same retinas. Thus, extra IOCs persist in kibra mutant clones as a result of decreased developmental apoptosis (Genevet, 2010).

The proliferation rate of kibra mutant cells was assessed in imaginal discs, the larval precursors to the adult appendages. By using the FLP/FRT system under the control of the heat-shock promoter, kibra mutant cells and their WT sister clones were generated through single recombination events from heterozygous mother cells. After several rounds of divisions, the sizes of mutant clones (no GFP) and WT twin spots (two copies of GFP) were compared, allowing estimation of the relative proliferation rates of mutant versus WT cells. The total kibra clone area is 1.57-fold larger than the control twin spot area, compared to a ratio of 0.98 when both clones and twin spots are WT, indicating that kibraΔ32 mutant cells grow 1.6 times faster than WT cells (Genevet, 2010).

In addition to cell cycle rates, the timing of cell cycle exit can readily be measured in the eye disc, where cell divisions follow a spatially determined pattern. During the third larval instar, the morphogenetic furrow, a wave of differentiation, sweeps the eye disc from posterior to anterior. Anterior to the furrow cells still proliferate asynchronously, while in the furrow cells synchronize in G1. Immediately posterior to the furrow, cells enter a final round of synchronous S phases, the second mitotic wave (SMW). Posterior to the SMW, most cells permanently exit the cell cycle. Thus, in WT discs, no S phases can be observed posterior to the SMW. As expected, hpo mutant cells fail to exit from the cell cycle in a timely manner and present ectopic EdU-positive staining posterior to the SMW. kibra mutant cells exhibit a less pronounced but similar phenotype. Thus, kibra mutant tissues have a proliferative advantage and an apoptosis defect, consistent with an involvement in the SWH network. The overgrowth defect appears more subtle than that of core pathway members such as wts and is more akin to upstream regulators (e.g., ex and mer) (Genevet, 2010).

Several transcriptional targets of the SWH network have been identified, such as the Drosophila Inhibitor of Apoptosis 1 (DIAP1) gene, the cell cycle regulator cycE, the miRNA bantam, as well as ex. In kibra mutant wing or eye discs, no strong change in DIAP1, CycE, or ex-lacZ reporter levels could be detected. Since overgrowth of kibra mutant cells in the wing is subtle compared to wts mutants, it is possible that Kibra plays a relatively minor role in SWH signaling in the wing. Accordingly, using an anti-Kibra antibody, it was noted that Kibra staining in the wing disc is weak and consists of a punctate apical staining which can clearly be observed when kibra is overexpressed in a stripe of cells. Thus, the extent to which Kibra is required may vary in different tissues (Genevet, 2010).

Ovarian posterior follicle cells (PFCs) are particularly sensitive to SWH loss of function, leading to a study the kibraΔ32 phenotype in the ovary. First, it was noted that Kibra protein levels are higher in follicle cells than in the wing discs. Kibra staining is mainly apical and is severely reduced in kibraΔ32 clones. Similarly to hpo or wts loss of function, kibra loss of function in the PFCs induces an upregulation of the ex-lacZ reporter. hpo or wts mutant PFCs also show a misregulation of the Notch (N) pathway and ectopic cell divisions. The N target Hindsight (Hnt) is normally repressed in all follicle cells up to stage 6 and switched on from stage 7 to stage 10B. Cut, which is repressed by Hnt, presents an opposite pattern of expression. In kibra mutant PFCs from stage 7-10B egg chambers, Hnt expression is lost, while Cut is ectopically expressed. This indicates that N signaling is downregulated in kibra mutant PFCs. Loss of kibra also leads to perturbation of epithelial integrity, as mutant PFCs show an accumulation of the apical polarity protein aPKC and the N receptor as well as multilayering of the follicular epithelium. Ectopic mitotic divisions are also observed in PFCs clones after stage 6, as detected by phospho-histone H3 (PH3) staining. Together, these phenotypes are identical to those observed in hpo or wts loss of function, suggesting that Kibra is indeed a member of the SWH network (Genevet, 2010).

To further explore the role of Kibra in the SWH network, genetic interaction and epistasis experiments were performed. Overexpressing kibra in the eye under the GMR (Glass Multimer Reporter) promoter elicits the formation of a small rough eye with frequent ommatidial fusions. This phenotype can be partially rescued by removing one copy of the hpo gene. In contrast, overexpressing kibra could not rescue the hpo-like overgrowth phenotype induced by yki overexpression, suggesting that Kibra may be an upstream regulator of the pathway (Genevet, 2010).

To conduct epistasis experiments between kibra and yki, the MARCM system was used to generate clones of mutant cells while simultaneously overexpressing or depleting other pathway components. MARCM clones expressing yki RNAi generated with eyFLP lead to the formation of a normal eye, because yki-depleted cells are eliminated by apoptosis and replaced by WT cells. As expected, eyFLP kibra MARCM clones cause eye overgrowth. This overgrowth is rescued by yki depletion in the mutant cells, indicating that the kibra overgrowth phenotype is yki dependent. Furthermore, overexpressing kibra in the eye under the GMR promoter induces apoptosis in third instar eye discs, which is suppressed by loss of hpo. Together, these epistasis experiments are consistent with Kibra being a member of the SWH network acting upstream of Yki and Hpo (Genevet, 2010).

Genetic interactions between kibra, mer, and ex, upstream members of the SWH network, were then investigated. Expressing a kibra, an ex, or a mer RNAi line in the eye under the GMR promoter induces eye overgrowth. Combined depletion of either Ex/Kibra or Mer/Kibra shows stronger phenotypes than individual depletion of these proteins. The MARCM technique was used to evaluate epistatic relationships between those three genes. hsFLP MARCM clones of various genotypes were generated and scored according to the severity of the wing overgrowth phenotypes, with type 0 representing normal wings and type 4 the strongest overgrowth. Overexpressing ex or mer in kibra mutant clones significantly rescues the overgrowth of kibra mutant clones. Reciprocally, kibra overexpression was also able to suppress the ex overgrowth phenotype. Thus, a strict epistatic relationship between kibra, ex, and mer could not be determined, consistent with a model whereby kibra, ex, and mer cooperate to control SWH pathway activity (Genevet, 2010).

As well as being an upstream regulator of the SWH network, ex is also one of its transcriptional targets, as are other upstream regulators (e.g., mer, four-jointed, dachsous). Since epistasis experiments place Kibra at the level of Mer and Ex, it was of interest to test whether this is also the case for kibra. Kibra levels were highly upregulated in mer;ex or hpo clones, showing an apical localization. The same is true in hpo clones in follicle cells. Similarly, hpo-depleted cultured Drosophila S2R+ cells have increased Kibra levels. To determine whether kibra is a transcriptional SWH network target, quantitative RT-PCR experiments were performed on yki-overexpressing and control wing imaginal discs. As expected, ex mRNA levels were increased in yki-expressing discs compared to control discs. Interestingly, kibra mRNA levels were also upregulated in yki-expressing discs, confirming that kibra is a Yki transcriptional target and suggesting the existence of a possible negative feedback loop regulating Kibra expression (Genevet, 2010).

Because hpo clones present increased levels of Kibra as well as Mer and Ex, these constitute a good system to evaluate the colocalization of those proteins. Indeed, Kibra colocalizes with Mer in the wing disc. As expected, Mer and Ex also colocalize. Thus, Kibra, Mer, and Ex colocalize apically in imaginal disc cells, but are dispensable for each other's apical sorting, because Kibra is still apical in mer;ex clones and Mer/Ex are normally localized in kibra clones (Genevet, 2010).

Because Kibra colocalizes with Mer/Ex, a possible association between those proteins was examined by conducting coimmunoprecipitation (co-IP) assays in S2R+ cells. Kibra was found to co-IP with Ex and Mer, but not with Hpo or with the negative regulator of Hpo, dRASSF (Polesello, 2006). Kibra possesses two WW domains, which are predicted to mediate protein-protein interactions by binding to PPXY motifs. Furthermore, the first WW domain of human KIBRA was shown to recognize the consensus motif RXPPXY in vitro (Kremerskothen, 2003). In flies, Mer does not contain any PPXY sites, while Ex has two PPXY sites (P786PPY and P1203PPY) and an RXPPXY site (R842DPPPY). The association between Kibra and Ex was investigated by mutating amino acids that are known to be required for WW domains and PPXY sites to interact. A Kibra protein mutant for its first WW domain (P85A) could no longer co-IP WT Ex. Reciprocally, WT Kibra could not co-IP an Ex protein deficient for its RXPPXY site (P845A). Thus, Kibra associates with Ex through its first WW domain and the Ex RXPPXY motif (Genevet, 2010).

Because Kibra complexes with Ex and a Yki/Ex interaction has recently been described (Badouel, 2009), attempts were made to determine whether Kibra can affect Yki activity. S2R+ cells were treated with RNAi against several SWH pathway components, and Yki phophorylation on Ser168 was monitored by western blotting. The phosphorylation of Yki by Wts at Ser168 leads to Yki inactivation and sequestration in the cytoplasm, where it has been reported to bind Ex, Wts, Hpo, and 14.3.3. lacZ RNAi-treated cells show a high basal level of phospho-Yki (P-Yki). As expected, Yki phosphorylation is abolished when Wts is depleted, and mildly reduced when the Wts cofactor Mats is depleted. In wts treated RNAi cells, a Yki downward shift can also be observed using a pan-Yki antibody. ex RNAi treatment has only a mild effect on P-Yki levels. Interestingly, kibra depletion leads to a marked reduction in P-Yki. When depleted in conjunction with ex, the P-Yki signal becomes even further reduced (Genevet, 2010).

This suggests that Kibra and Ex are required for Wts activity on Yki, which prompted an investigation of whether Kibra could associate with Wts. Co-IP assays reveal that Kibra interacts with Wts. Wts does not seem to compete with Ex for Kibra association, because it could still complex with a form of Kibra mutant for its first WW domain. Because Kibra associates with Wts and Ex interacts with Yki, whether Wts requires Kibra/Ex to bind Yki was investigated. Endogenous IPs between Yki and Wts were performed in S2 cells treated with various dsRNAs. In these conditions, the effect of kibra and ex depletion on Yki phosphorylation can also be observed. In control cells, Wts binding to Yki is detected after immunoprecipitating Yki. This endogenous interaction is unaffected by the individual or combined depletion of ex and kibra. These results suggest that Ex and Kibra are required to activate the SWH pathway by nucleating an active Hpo/Wts kinase cassette, rather than promoting the Wts/Yki interaction (Genevet, 2010).

These data identify Kibra as a regulator of the SWH network that associates with Ex and Mer, with which it is colocalized apically and transcriptionally coregulated. Given that the apical surface of epithelial cells is instrumental in both cell-cell signaling and tissue morphogenesis, it is speculated that Kibra may cooperate with Ex and Mer to transduce an extracellular signal, or relay information about epithelial architecture, via the SWH network, to control tissue growth and morphogenesis (Genevet, 2010).

Recent data have suggested that an apical scaffold machinery containing Hpo, Wts, and Ex recruits Yki to the apical membrane, facilitating its inhibitory phosphorylation by Wts (Badouel, 2009; Oh, 2009). Since Kibra associates with Ex and is also apically localized, it is hypothesized that Kibra is also part of this scaffold and participates in nucleating an active Hpo/Wts complex and recruiting Yki for inactivation. This view is supported by the finding that Kibra complexes with Wts and that combined depletion of Kibra and Ex leads to a strong decrease in Yki phosphorylation, but does not disrupt the Wts/Yki interaction. The data also suggest that the importance of Kibra may be tissue-specific since robust phenotypes were observed in ovaries and hemocyte-derived S2R+ cells, but weaker effects in imaginal discs. Thus, considering the relative levels of expression of Ex, Mer, and Kibra may be important in determining pathway activation. Finally, since mammalian KIBRA complexes with the NF2/MER tumor suppressor, these findings raise the possibility that human KIBRA may contribute to tumor suppression in human neurofibromas and potentially other tumors (Genevet, 2010).

The WW domain protein Kibra acts upstream of Hippo in Drosophila

The conserved Hippo kinase pathway plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis. Whereas the function of the core kinase cascade, consisting of the serine/threonine kinases Hippo and Warts, in phosphorylating and thereby inactivating the transcriptional coactivator Yorkie is well established, much less is known about the upstream events that regulate Hippo signaling activity. The FERM domain proteins Expanded and Merlin appear to represent two different signaling branches that feed into the Hippo pathway. Signaling by the atypical cadherin Fat may act via Expanded, but how Merlin is regulated has remained elusive. This study shows that the WW domain protein Kibra is a Hippo signaling component upstream of Hippo and Merlin. Kibra acts synergistically with Expanded, and it physically interacts with Merlin. Thus, Kibra predominantly acts in the Merlin branch upstream of the core kinase cascade to regulate Hippo signaling (Baumgartner, 2010).

Overexpression of Drosophila Kibra in the developing eye has been shown to decrease the size of the adult organ (Tseng, 2002). Four different loss-of-function alleles of Kibra were generated to define its function in growth control. Deletion of the first exon (harboring the translational start site) by imprecise excision of a P element resulted in the alleles Kibra1 and Kibra2. Kibra3, a mutation in the initiating ATG, was generated by means of an EMS reversion mutagenesis of the EP-mediated Kibra overexpression phenotype. Finally, the entire Kibra locus was removed by the hybrid element insertion (HEI) technique. All alleles were lethal when homozygous and failed to complement each other but were complemented by the precise P element excision used as a control throughout this study. All mutants displayed the same growth phenotypes, and homozygous mutant animals died as first-instar larvae. It is concluded that all Kibra alleles are genetically null (Baumgartner, 2010).

Kibra mutant heads were enlarged in comparison to controls. Similarly, wings containing posterior compartments largely mutant for Kibra were larger than control wings. The presence of a UAS-Kibra overexpression construct, without any Gal4 driver, rescued the lethality of Kibra homozygous mutant flies as well as the size defects of Kibra mutant organs, proving that the growth alterations are caused by the loss of Kibra function. Thus, Kibra is a general regulator of growth that is required to restrict organ size (Baumgartner, 2010).

To determine the cause of the Kibra mutant overgrowth phenotypes, a clonal analysis in wing imaginal discs was performed. Clones of Kibra mutant cells were larger than their corresponding wild-type sister clones. The number of cells per clone was increased in Kibra mutant clones compared to wild-type clones but not to the same extent as the clone size. However, FACS analysis revealed that cell size was unchanged in Kibra mutant cells, suggesting a change in cellular architecture in cells devoid of Kibra function. It is concluded that Kibra mutant clones in the wing imaginal disc were enlarged because Kibra mutant cells exhibit a proliferative advantage over wild-type cells (Baumgartner, 2010).

Tangential sections were analyzed of mosaic compound eyes consisting of Kibra mutant cells surrounded by heterozygous cells. The mutant ommatidia were normally structured and the different cell types properly differentiated, but the interommatidial regions were enlarged compared to the control. The increased distance between mutant ommatidia was due to more cells, because clones of Kibra mutant cells in the pupal retina displayed an increase in the number of interommatidial cells. Supernumerary interommatidial cells are a hallmark of inactivation of the Hippo pathway. Whereas a complete loss of Hippo signaling causes a pronounced excess of interommatidial cells, a mild extra interommatidial cell phenotype is observed in mutants that reduce but do not abrogate Hippo signaling, such as ex or Mer (Baumgartner, 2010).

A reduction in Hippo signaling activity results in extra interommatidial cells because the developmental apoptosis in pupal retinae is largely eliminated. Conversely, overexpression of hpo or ex induces apoptosis in third instar eye discs. Overexpression of Kibra in clones in the wing imaginal disc reduced clone size. Kibra-overexpressing clones contained fewer cells than control clones. To investigate whether overexpression of Kibra induces apoptosis, Kibra overexpression clones were generated in the third instar eye disc by using the Gene-Switch system. Indeed, the Kibra-overexpressing clones located anterior to the morphogenetic furrow (MF) showed an increase in programmed cell death as judged by staining for cleaved Caspase-3 and TUNEL staining, suggesting that overexpression of Kibra induces inappropriate apoptosis of proliferating cells. Consistently, co-overexpression of Diap1, a direct Yorkie transcriptional target, partially rescued the small eye phenotype associated with Kibra overexpression. Co-overexpression of CycE, another target of the Hippo pathway, also resulted in a partial rescue of the small eye. The size of Kibra-overexpressing eyes was further restored by concomitant overexpression of Diap1 and CycE. These results suggest that the effects elicited by Kibra overexpression are at least partly due to a reduction in the expression of the Hippo pathway target genes Diap1 and CycE (Baumgartner, 2010).

The striking similarities of the Kibra, ex, and Mer phenotypes prompted a genetic test of whether Kibra restricts tissue size via Hippo signaling. Interaction studies were started at the level of the transcriptional coactivator yki, which induces target genes promoting cell proliferation and cell survival and is inactivated by Hippo signaling. Three lines of evidence suggest that Kibra acts via inactivation of Yki. First, the coexpression of Kibra and yki during eye development suppressed the eye size reduction caused by Kibra and resulted in the same overgrowth phenotype as observed in eyes overexpressing yki alone. Second, the growth advantage of Kibra mutant cells was completely abolished by the concomitant loss of yki function. Third, a pupal lethal hypomorphic combination of Kibra alleles was rescued to viability by removal of a single copy of yki (Baumgartner, 2010).

To determine whether (and at which level) Kibra acts in the Hippo pathway to inactivate Yki, a series of epistasis tests were performed. It was found that the loss-of-function phenotypes of hpo, sav, and wts were epistatic to the Kibra overexpression phenotype, indicating that Kibra acts upstream of Hpo (Baumgartner, 2010).

Next, interaction with the upstream components Ex and Mer was tested. Overexpression of ex in a Kibra mutant background resulted in an intermediate phenotype. Vice versa, overexpression of Kibra also yielded an additive effect in an ex mutant head. Conversely, Kibra overexpression failed to reduce organ size in a Mer mutant head, indicating that Kibra requires Mer to exert its function. The eyFlp/FRT recombination system (without cell lethal) was used to generate mosaic animals with heads largely homozygous for ex and Mer mutations, as well as ex Kibra and Mer Kibra double mutations, respectively. Both ex and Mer mosaic heads showed only mild overgrowth. Strikingly, pupae with mosaic heads doubly mutant for ex and Kibra did not eclose, and normal head structures were displaced by overgrown tissue. In contrast, flies with Mer Kibra mosaic heads were viable. However, Mer Kibra double mutant clones showed stronger overgrowth than Mer clones. Reducing ex function during eye development by the expression of a hairpin RNAi construct did not alter the wild-type eye size but resulted in a severe enhancement of the Kibra loss-of-function phenotype, and the resulting eyes resembled those of hpo mutants. Reducing Mer function caused subtle overgrowth but enhanced the Kibra mutant phenotype much less (Baumgartner, 2010).

Whereas single mutants for ex and Mer cause a mild overgrowth phenotype, ex Mer double mutants display strong synergistic effects, suggesting that the two FERM domain proteins act in separate branches to activate Hippo signaling. These findings suggest that Kibra acts primarily upstream of Mer. However, since Mer Kibra double mutant clones show stronger overgrowth than Mer mutant clones and a reduction of Mer function enhances the Kibra loss-of-function phenotype, Kibra also contributes to Mer-independent regulation of Yki activity (Baumgartner, 2010).

To confirm that Kibra acts via Hippo signaling, whether Kibra mutant clones upregulated the expression of a Diap1 enhancer element (diap1-GFP4.3) that had been published to be a minimal Hippo responsive element (HRE; Zhang, 2008) was tested. A pronounced upregulation of diap1-GFP4.3 was evident in clones of hpo mutant cells posterior and, to a weaker extent, anterior to the MF in eye imaginal discs. Cells lacking Kibra function also upregulated diap1-GFP4.3 expression, although to a lesser degree and with restriction to the differentiating tissue posterior to the MF. Clones of ex mutant cells, in resemblance to hpo clones, upregulated diap1-GFP4.3 strongly behind and somewhat weaker before the MF, whereas Mer mutant cells, like Kibra mutant cells, upregulated diap1-GFP4.3 expression weakly and solely posterior to the MF. Thus, the loss of Kibra results in an upregulation of a Hippo signaling reporter gene. The similar response of diap1-GFP4.3 to loss of Kibra or Mer suggests that Kibra and Mer act in the same way on Hippo signaling to regulate the HRE (Baumgartner, 2010).

This study provides genetic and biochemical evidence that the WW domain protein Kibra is a Hippo signaling component. Several lines of evidence indicate that Kibra acts predominantly in the Mer branch. First, the mild overgrowth phenotype caused by loss of Kibra function is akin to the Mer phenotype. Second, genetic epistasis experiments place Kibra upstream of Mer. Third, the effects of Kibra and Mer loss-of-function on a reporter for Hippo signaling activity are very similar. Fourth, Kibra and Mer synergise with ex in a similar fashion. Fifth, Kibra physically interacts with Mer. However, since the genetic analysis of Kibra also revealed a synergism with Mer, Kibra also acts on Yki activity in a Mer-independent manner (Baumgartner, 2010).

FERM domain proteins, such as Mer, have been suggested to connect membrane proteins with the underlying cortical cytoskeleton in order to integrate signals from the membrane and initiate intracellular signaling cascades. Thus, it is conceivable that Mer, together with as yet unknown proteins, assembles downstream cytoplasmic components of the Hippo pathway at the membrane and that controlled assembly and stabilization of such multiprotein complexes regulates the activity of the Hippo kinase cascade. In such a scenario, adaptor proteins providing multiple protein-protein interaction domains are of special interest (Baumgartner, 2010).

The WW domain protein Kibra binds Mer and could enable signaling events at the membrane/cytoskeleton interface that activate the Hpo kinase cascade. Since a truncated Kibra protein lacking the WW domains interacts more fiercely with Mer, it is likely that the physical association of Kibra and Mer is modulated by binding of other factors to the WW domains of Kibra (Baumgartner, 2010).

Interestingly, the effects caused by the concomitant loss of ex and Kibra functions are more severe than those elicited by mutated Hippo signaling core components. In addition to massively overgrowing, clones of ex Kibra double mutant cells round up, a behavior that was never observed in clones of hpo mutant cells. Furthermore, the diap1-GFP4.3 reporter indicates higher Yki activity in proliferating ex Kibra mutant eye imaginal disc cells as compared to hpo mutant cells. It thus appears that Yki activity is unleashed in cells lacking both ex and Kibra functions. Since Ex has been shown to directly bind Yki, it is tempting to speculate that Kibra participates in a distinct (Mer-independent) mechanism to prevent nuclear Yki localization (Baumgartner, 2010).

Drosophila Pez acts in Hippo signaling to restrict intestinal stem cell proliferation

The conserved Hippo signaling pathway acts in growth control and is fundamental to animal development and oncogenesis. Hippo signaling has also been implicated in adult midgut homeostasis in Drosophila. Regulated divisions of intestinal stem cells (ISCs), giving rise to an ISC and an enteroblast (EB) that differentiates into an enterocyte (EC) or an enteroendocrine (EE) cell, enable rapid tissue turnover in response to intestinal stress. The damage-related increase in ISC proliferation requires deactivation of the Hippo pathway and consequential activation of the transcriptional coactivator Yorkie (Yki) in both ECs and ISCs. This study identified Pez, an evolutionarily conserved FERM domain protein containing a protein tyrosine phosphatase (PTP) domain, as a novel binding partner of the upstream Hippo signaling component Kibra. Pez function (but not its PTP domain) is essential for Hippo pathway activity specifically in the fly midgut epithelium. Thus, Pez displays a tissue-specific requirement and functions as a negative upstream regulator of Yki in the regulation of ISC proliferation (Poernbacher, 2012).

The WW domain protein Kibra has recently been shown to function as a tumor suppressor in the Hippo pathwa. Because Kibra is an adaptor molecule, attempts were made to identify physical binding partners of Kibra to further explore upstream Hippo signaling. Affinity purification-mass spectrometry (AP-MS) analysis with Kibra as bait identified Pez as a novel interaction partner of Kibra in Drosophila cultured cells. The same result was recently obtained in a large-scale proteomic study of Drosophila cultured cells. The binding between Pez and Kibra was confirmed by reciprocal coimmunoprecipitation (co-IP) experiments with epitope-tagged proteins. Furthermore, a yeast two-hybrid (Y2H) experiment revealed that the Kibra-Pez interaction is robust and direct (Poernbacher, 2012).

To address a possible function of Pez in the Hippo pathway, two loss-of-function alleles of Pez that were generated by different methods. Pez1 is an EMS-induced allele resulting in an early premature translational stop codon. Pez2 was generated by imprecise excision of the P element P{GawB}NP4748, removing most of the Pez coding sequence. Homozygotes for either Pez allele as well as heteroallelic Pez1/Pez2 flies are viable but smaller than controls. Combinations of the Pez alleles with the deficiency Df(2L)ED384 uncovering the Pez locus are also viable and cause a similar reduction in body size as the homozygous or heteroallelic combinations. One copy of a GFP-tagged Pez genomic rescue construct (gPez) restores normal body size. Therefore, both Pez1 and Pez2 are likely to represent strong or null alleles. For further experiments, heteroallelic Pez1/Pez2 flies were used as Pez mutant flies (Poernbacher, 2012).

In addition to their reduced body size, Pez mutant flies exhibit a developmental delay of 2 days and decreased fertility, all hallmarks of starvation. Pez mutant larvae are small and have decreased triglyceride (TAG) stores and increased expression of the starvation marker genes lipase-3 and 4E-BP. Clones of Pez mutant cells in larval fat bodies did not affect lipid droplets, thus excluding a fat body-autonomous requirement for Pez in lipid metabolism. Surprisingly, overexpression of Drosophila Pez in the developing eye or wing decreased the size of the adult organs, indicating that Pez restricts growth rather than promoting it. It is proposed that the starvation-like phenotype of Pez mutants is due to indirect effects on metabolism arising from a failure in nutrient utilization. Clones of Pez mutant cells in wing imaginal discs did not show growth defects in comparison to their corresponding wild-type sister clones. However, Pez mutant flies exhibit hyperplasia and extensive multilayering of the adult midgut epithelium. One copy of gPez restores normal tissue architecture. The structure of the larval midgut epithelium, as well as that of the other larval and adult epithelia, is not disturbed in Pez mutants. Thus, Pez specifically functions to restrict growth of the adult midgut epithelium (Poernbacher, 2012).

The Pez protein contains two conserved structural elements: an amino-terminal FERM domain (band 4.1-ezrin-radixin- moesin family of adhesion molecules) and a carboxyterminal protein tyrosine phosphatase (PTP) domain. A truncated version of the protein lacking the FERM domain (DFERM-Pez) or a phosphatase-dead protein (PezPD) still rescued the Pez mutant gut phenotype when overexpressed in ECs. However, overexpression of DFERM-Pez in the developing wing failed to decrease wing size, whereas overexpression of PezPD or of a truncated protein lacking the PTP domain (DPTP-Pez) caused a similar phenotype as overexpression of wild-type Pez, suggesting that the FERM domain is required for the growth-regulatory function of endogenous Pez but becomes dispensable when DFERM-Pez is overexpressed in ECs. In contrast, the potential phosphatase activity of Pez is clearly not needed for its function in growth control (Poernbacher, 2012).

Two other FERM domain proteins, Merlin (Mer) and Expanded (Ex), act in upstream Hippo signaling to control organ size in Drosophila. Together with the WW domain protein Kibra, Ex and Mer constitute the KEM complex that assembles at the apical junction of epithelial cells and regulates the core Hippo pathway kinase cassette (Baumgartner, 2010; Genevet, 2010; Yu, 2010). Overexpression of Kibra, Ex, or Mer in ECs of Pez mutant flies significantly suppressed the Pez gut phenotypes. Thus, Pez is not an essential mediator of Hippo signaling downstream of the KEM complex. Mer and Ex did not detectably coimmunoprecipitate with Pez in Drosophila S2 cells. However, Kibra and Pez coimmunoprecipitated and colocalized in S2 cells. This was dependent on the first WW domain of Kibra, whereas the FERM and PTP domains of Pez as well as two potential ligands of WW domains, a PPPY motif and a PPSGY motif, in the central linker region of Pez were dispensable. A fragment encompassing a proline-rich stretch of Pez (amino acids 368-627; PezPro) was sufficient for the binding to Kibra, whereas the remaining linker region (amino acids 622-967; PezLink) did not bind Kibra. Importantly, knockdown of Kibra via Myo1A-Gal4 caused mild overgrowth of the adult midgut epithelium, and overexpressed Kibra recruited gPez-GFP from the cell cortex of ECs into cytoplasmic punctae. The subcellular localizations of overexpressed Kibra, Ex, or Mer were not affected when Pez was absent (Poernbacher, 2012).

It is concluded that Pez and Kibra function together in a protein complex to regulate Hippo signaling in adult midgut ECs. The results establish that the Drosophila Pez protein acts as a component of upstream Hippo signaling, restricts transcriptional activity of Yki in epithelial cells of the adult midgut, and plays a crucial role in the control of ISC proliferation. Importantly, the involvement of Hippo signaling in intestinal regeneration is conserved in the mammalian system ] (Poernbacher, 2012).

The two mammalian homologs of Drosophila Pez are the widely expressed, cytosolic nonreceptor tyrosine phosphatases PTPD1/PTPN21 and PTPD2/PTP36/PTPN14/Pez. All three proteins share a similar domain structure including the well-conserved terminal FERM and PTP domains. The central region shows extensive sequence divergence but it contains several shorter regions of conservation that may function as adaptors in signal transduction. PTPD1 is a component of a cortical scaffold complex nucleated by focal adhesion kinase (FAK) and thus regulates a proliferative signaling pathway through a scaffolding function. PTPD2 has been implicated in the regulation of cell adhesion, as an inducer of TGF-β signaling, and in lymphatic development of mammals and choanal development of humans. Interestingly, PTPD2 is a potential tumor suppressor, based on sporadic mutations in breast cancer cells and colorectal cancer cells. It is tempting to speculate that mammalian PTPD2 shares the function of its fly homolog as a component of Hippo signaling that restrains the oncogenic potential of gut regeneration (Poernbacher, 2012).

Functions of Kibra orthologs in other species

Evolutionary and molecular facts link the WWC protein family to Hippo signaling

The scaffolding protein KIBRA (also called WWC1) is involved in the regulation of important intracellular transport processes and the establishment of cell polarity. Furthermore, KIBRA/WWC1 is an upstream regulator of the Hippo signaling pathway that controls cell proliferation and organ size in animals. KIBRA/WWC1 represents only one member of the WWC protein family which also includes the highly similar proteins WWC2 and WWC3. Whereas the function of KIBRA/WWC1 was studied intensively in cells and animal models, the importance of WWC2 and WWC3 was not yet elucidated. This study describes evolutionary, molecular and functional aspects of the WWC family. The WWC genes arose in the ancestor of bilateral animals (clades such as insects and vertebrates) from a single founder gene most similar to the present KIBRA/WWC1-like sequence of Drosophila. This situation was still maintained until the common ancestor of lancelet and vertebrates. In fish, a progenitor-like sequence of mammalian KIBRA/WWC1 and WWC2 is expressed together with WWC3. Finally, in all tetrapodes, the three family members KIBRA/WWC1, WWC2 and WWC3, are found except for a large genomic deletion including WWC3 in Mus musculus. At the molecular level, the highly conserved WWC proteins share a similar primary structure, the ability to form homo- and heterodimers and the interaction with a common set of binding proteins. Furthermore, all WWC proteins negatively regulate cell proliferation and organ growth due to a suppression of the transcriptional activity of YAP, the major effector of the Hippo pathway (Wennmann, 2014).

Selective erasure of distinct forms of long-term synaptic plasticity underlying different forms of memory in the same postsynaptic neuron

Generalization of fear responses to non-threatening stimuli is a feature of anxiety disorders. It has been challenging to target maladaptive generalized memories without affecting adaptive memories. Synapse-specific long-term plasticity underlying memory involves the targeting of plasticity-related proteins (PRPs) to activated synapses. If distinct tags and PRPs are used for different forms of plasticity, one could selectively remove distinct forms of memory. Using a stimulation paradigm in which associative long-term facilitation (LTF) occurs at one input and non-associative LTF at another input to the same postsynaptic neuron in an Aplysia sensorimotor preparation, this study found that each form of LTF is reversed by inhibiting distinct isoforms of protein kinase M (PKM), putative PRPs, in the postsynaptic neuron. A dominant-negative (dn) atypical PKM selectively reversed associative LTF, while a dn classical PKM selectively reversed non-associative LTF. Although both PKMs are formed from calpain-mediated cleavage of protein kinase C (PKC; see Drosophila PKC) isoforms, each form of LTF is sensitive to a distinct dn calpain expressed in the postsynaptic neuron. Associative LTF is blocked by dn classical calpain, whereas non-associative LTF is blocked by dn small optic lobe (SOL) calpain. Interfering with a putative synaptic tag, the adaptor protein KIBRA (see Drosophila Kibra), which protects the atypical PKM from degradation, selectively erases associative LTF. Thus, the activity of distinct PRPs and tags in a postsynaptic neuron contribute to the maintenance of different forms of synaptic plasticity at separate inputs, allowing for selective reversal of synaptic plasticity and providing a cellular basis for developing therapeutic strategies for selectively reversing maladaptive memories (Hu, 2017).

The Hippo pathway regulator KIBRA promotes podocyte injury by inhibiting YAP signaling and disrupting actin cytoskeletal dynamics
(KIdney BRAin protein (KIBRA); see Drosophila Kibra), an upstream regulator of the Hippo signaling pathway encoded by the Wwc1 gene, shares the pro-injury properties of its putative binding partner dendrin and antagonizes the pro-survival signaling of the downstream Hippo pathway effector YAP (Yes-associated protein; see Drosophila Yorkie) in Drosophila and MCF10A cells. YAP has been identified an essential component of the glomerular filtration barrier that promotes podocyte survival by inhibiting dendrin pro-apoptotic function. This study tested the hypothesis that KIBRA promotes podocyte injury. Increased expression of KIBRA and phosphorylated YAP (P-YAP) protein in glomeruli of patients with biopsy-proven focal segmental glomerulosclerosis (FSGS). KIBRA/WWc1 overexpression in murine podocytes promoted LATS kinase phosphorylation, leading to subsequent YAP S127 phosphorylation, YAP cytoplasmic sequestration, and reduction in YAP target gene expression. Functionally, KIBRA overexpression induced significant morphological changes in podocytes including disruption of the actin cytoskeletal architecture and reduction of focal adhesion size and number, all of which were rescued by subsequent YAP overexpression. Conversely, constitutive KIBRA knockout mice displayed reduced P-YAP and increased YAP expression at baseline. These mice were protected from acute podocyte foot process effacement following protamine sulfate perfusion. KIBRA knockdown podocytes were also protected against protamine-induced injury. These findings suggest an important role for KIBRA in the pathogenesis of podocyte injury and the progression of proteinuric kidney disease (Meliambro, 2017).


Search PubMed for articles about Drosophila Kibra

Badouel, C., et al. (2009) The FERM-domain protein Expanded regulates Hippo pathway activity via direct interactions with the transcriptional activator Yorkie. Dev. Cell 16: 411-420. PubMed ID: 19289086

Baumgartner, R., Poernbacher, I., Buser, N., Hafen, E. and Stocker, H. (2010). The WW domain protein Kibra acts upstream of Hippo in Drosophila. Dev. Cell 18(2): 309-16. PubMed ID: 20159600

Chan, E. H., et al. (2005). The Ste20-like kinase Mst2 activates the human large tumor suppressor kinase Lats1. Oncogene 24: 2076-2086. PubMed ID: 15688006

Emoto, K., Parrish, J. Z., Jan, L. Y. and Jan, Y. N. (2006). The tumour suppressor Hippo acts with the NDR kinases in dendritic tiling and maintenance. Nature 443: 210-213. PubMed ID: 16906135

Genevet, A., et al. (2010). Kibra is a regulator of the Salvador/Warts/Hippo signaling network. Dev. Cell 18(2): 300-8. PubMed ID: 20159599

Hu, J., Ferguson, L., Adler, K., Farah, C. A., Hastings, M. H., Sossin, W. S. and Schacher, S. (2017). Selective erasure of distinct forms of long-term synaptic plasticity underlying different forms of memory in the same postsynaptic neuron. Curr Biol 27(13): 1888-1899 e1884. PubMed ID: 28648820

Kremerskothen, J., et al. (2003). Characterization of KIBRA, a novel WW domain-containing protein. Biochem. Biophys. Res. Commun. 300: 862-867. PubMed ID: 12559952

Mikeladze-Dvali, T., et al. (2005). The growth regulators warts/lats and melted interact in a bistable loop to specify opposite fates in Drosophila R8 photoreceptors. Cell 122: 775-787. PubMed ID: 16143107

Meliambro, K., Wong, J. S., Ray, J., Calizo, R. C., Towne, S., Cole, B., El Salem, F., Gordon, R. E., Kaufman, L., He, J. C., Azeloglu, E. U. and Campbell, K. N. (2017). The Hippo pathway regulator KIBRA promotes podocyte injury by inhibiting YAP signaling and disrupting actin cytoskeletal dynamics. J Biol Chem 292(51):21137-21148. PubMed ID: 28982981

Oh, H., Reddy, B. V. and Irvine, K. D. (2009). Phosphorylation-independent repression of Yorkie in Fat-Hippo signaling. Dev. Biol. 335: 188-197. PubMed ID: 19733165

Papassotiropoulos, A., et al. (2006). Common Kibra alleles are associated with human memory performance. Science 314: 475-478. PubMed ID: 17053149

Poernbacher, I., Baumgartner, R., Marada, S. K., Edwards, K. and Stocker, H. (2012). Drosophila Pez acts in Hippo signaling to restrict intestinal stem cell proliferation. Curr. Biol. 22(5): 389-96. PubMed ID: 22305752

Polesello, C., et al. (2006). The Drosophila RASSF homolog antagonizes the Hippo pathway. Curr. Biol. 16: 2459-2465. PubMed ID: 17174922

Praskova, M., Xia, F. and Avruch, J. (2008). MOBKL1A/MOBKL1B phosphorylation by MST1 and MST2 inhibits cell proliferation. Curr. Biol. 18: 311-321. PubMed ID: 18328708

Tseng, A. S. and Hariharan, I. K. (2002). An overexpression screen in Drosophila for genes that restrict growth or cell-cycle progression in the developing eye. Genetics 162: 229-243. PubMed ID: 12242236

Wennmann, D. O., Schmitz, J., Wehr, M. C., Krahn, M. P., Koschmal, N., Gromnitza, S., Schulze, U., Weide, T., Chekuri, A., Skryabin, B. V., Gerke, V., Pavenstadt, H., Duning, K. and Kremerskothen, J. (2014). Evolutionary and molecular facts link the WWC protein family to Hippo signaling. Mol Biol Evol [Epub ahead of print]. PubMed ID: 24682284

Yu, J., et al. (2010). Kibra functions as a tumor suppressor protein that regulates Hippo signaling in conjunction with Merlin and Expanded. Dev. Cell 18(2): 288-99. PubMed ID: 20159598

Zhang, L., et al. (2008). The TEAD/TEF family of transcription factor Scalloped mediates Hippo signaling in organ size control. Dev. Cell 14: 377-387. PubMed ID: 18258485

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date revised: 10 June 2024

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