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Pez: Biological Overview | References

BIOLOGICAL OVERVIEW

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 pathway. 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).

Suppressor of Deltex mediates Pez degradation and modulates Drosophila midgut homeostasis

Pez functions as an upstream negative regulator of Yorkie (Yki) to regulate intestinal stem cell (ISC) proliferation and is essential for the activity of the Hippo pathway specifically in the Drosophila midgut epithelium. This study reports that Suppressor of Deltex (Su(dx)) acts as a negative regulator of Pez. Su(dx) was shown to target Pez for degradation both in vitro and in vivo. Overexpression of Su(dx) induced proliferation in the fly midgut epithelium, which could be rescued by overexpressed Pez. The study also demonstrated that the interaction between Su(dx) and Pez, bridged by WW domains and PY/PPxY motifs, is required for Su(dx)-mediated Pez degradation. Furthermore, Kibra, a binding partner of Pez, was shown to stabilize Pez via WW-PY/PPxY interaction. Moreover, PTPN14, a Pez mammalian homolog, is degraded by overexpressed Su(dx) or Su(dx) homologue WWP1 in mammalian cells. These results reveal a previously unrecognized mechanism of Pez degradation in maintaining the homeostasis of Drosophila midgut (Wang, 2015).

The protein tyrosine phosphatase Pez is the Drosophila homologue of non-receptor type protein tyrosine phosphatase 14 (PTPN14), a regulator of the TGF-β pathway (Smith, 1995; Wyatt, 2007; Wyatt, 2008). PTPN14 overexpression activates TGF-β signalling and causes epithelial-mesenchymal transition (EMT) (Wyatt, 2007). Its overexpression is also correlated with lymphatic function, choanal development, angiogenesis and hereditary haemorrhagic telangiectasia (Au, 2010; Benzinou, 2012). Recent studies have revealed that PTPN14 negatively regulates the oncogenic function of Yes-associated protein (YAP) through retaining YAP in the cytoplasm and sustaining the phosphorylation state of YAP (Huang, 2013, Liu, 2013; Wang, 2012; Michaloglou, 2013). YAP is the transcription co-activator downstream of Hippo signalling to mediate the expression of various genes to promote growth, and its upregulation was found in a variety of human tumours and cancers (Wang, 2015).

Drosophila midgut, where the intestinal stem cells (ISCs) are under tight control to maintain homeostasis, has been developed as an excellent model to study adult stem cells in recent years. The Hippo signalling pathway has been shown to play an essential role in the regulation of ISC proliferation. Pez has been identified as a negative upstream regulator of Yorkie (Yki), the Drosophila homologue of YAP, and is required for the activity of the Hippo pathway in the regulation of ISC proliferation (Poernbacher, 2012). However, how the stability and function of Pez are regulated remains unclear (Wang, 2015).

Suppressor of Deltex (Su(dx)) is a member of the NEDD4 (neural precursor cell-expressed developmentally downregulated gene 4) family E3 ubiquitin ligase (Cornell, 1999). There are three typical NEDD4 family members in Drosophila, dSmurf, Su(dx) and NEDD4. Each of them contains an N-terminal phospholipid binding C2 domain, four WW domains and a C-terminal HECT-type ligase domain. Su(dx) was first reported as a negative regulator of the Notch signaling pathway (Fostier, 1998). It downregulates the expression of Notch target genes through promoting Notch endosomal sorting (Hori, 2004; Wilkin, 2004; Wang, 2015 and references therein).

This study shows that Su(dx) targets Pez for degradation both in vitro and in vivo. Su(dx) overexpression induces cell proliferation in Drosophila midgut by downregulating Pez protein levels. It was also demonstrated that Su(dx) directly interacts with Pez via its WW domains and Pez's PY/PPPY motifs. This interaction subsequently promotes Pez ubiquitylation. Furthermore, Kibra, a WW domain containing Pez binding partner, was found to stabilize Pez on interaction. Moreover, evidence is provided that overexpression of Su(dx) or its homologue WWP1 is able to degrade PTPN14 in mammalian culture cells, indicating a possibility that a conserved mechanism of Pez degradation may play an essential role in maintaining tissue homeostasis (Wang, 2015).

This study reports the identification of Su(dx) as an E3 ligase of Pez. Su(dx) was first identified as an E3 ligase regulating the Notch signalling pathway. But the direct substrate of Su(dx) was unclear. The present study identified Pez as a Su(dx) substrate. Furthermore, whether Pez regulates the Notch signalling pathway was examined. Pez knockdown induced notched wings and a decrease of Cut in wing discs that is very similar to what have caused by Su(dx) overexpression, indicating a dysfunction of the Notch pathway. However, another typical marker of the Notch pathway, wingless, was not affected by the absence of Pez. It is possible that Pez is not a canonical regulator of the Notch pathway and Su(dx) might have other substrates under this circumstance (Wang, 2015).

According to the observations, Su(dx) overexpression in ECs only induced midgut epithelial proliferation to some extent, and it did not fully mimic the loss of pez induced phenotypes. It is speculated that the difference was largely due to the incomplete degradation efficiency of Pez by Su(dx) overexpression in ECs (Wang, 2015).

PTPN14 has been reported as an inhibitor of YAP1 in mammalian cells. It can suppress the activity of YAP1 through retaining YAP1 in the cytoplasm and sustaining the phosphorylation state of YAP1 (Wang, 2012). However, in the current experiments, Pez overexpression slightly upregulates Yki phosphorylation level without obvious Yki localization change in S2 cells. It is speculated that the mechanism of YAP regulation by PTPN14 may not be conserved in Drosophila (Wang, 2015).

Furthermore, this work presents evidence that Kibra, a WW domain containing Pez partner, stabilizes Pez, providing an interesting model that WW–PY/PPxY interaction play a role in the regulation of protein stabilization. In addition, it was found that other WW-containing proteins, such as Sav, were unable to stabilize Pez. On the basis of these observations, the regulation of Pez stabilization by Su(dx) and Kibra is speculated to be a specific event (Wang, 2015).

It was also found that PTPN14, the human homologue of Pez, can be degraded by overexpressed Su(dx) and its human homologue WWP1. However, in the following experiments, it was found that WWC1, the human homologue of Kibra, did not stabilize PTPN14. These data suggest that, although the similar regulation of Pez/PTPN14 by degradation exists in Drosophila and mammalian cells, the detailed mechanism may vary (Wang, 2015).

It has been reported that PTPN14 sporadic mutations were found in breast cancer cells and colorectal cancer cells, indicating a potential tumour suppressor function of PTPN14. Moreover, amplification and overexpression of WWP1 has been found in breast and prostate cancers. Therefore, the current study may provide new insights into cancer development. Further characterization of the relationship of Su(dx)-Pez in mice and examination of their correlation in clinical cancers may provide potential targeting therapy for cancer treatments (Wang, 2015).

Comparative analysis of the Band 4.1/ezrin-related protein tyrosine phosphatase Pez from two Drosophila species: implications for structure and function

The FERM-PTPs are a group of proteins that have FERM (Band 4.1, ezrin, radixin, moesin homology) domains at or near their N-termini, and PTP (protein tyrosine phosphatase) domains at their C-termini. Their central regions contain either PSD-95, Dlg, ZO-1 homology domains or putative Src homology 3 domain binding sites. The known FERM-PTPs fall into three distinct classes, which are named BAS, MEG, and PEZ, after representative human PTPs. This study analyzed Pez, a novel gene encoding the single PEZ-class protein present in Drosophila. Pez cDNAs were sequenced from the distantly related flies Drosophila melanogaster and Drosophila silvestris and were found to be highly conserved except in the central region, which contains at least 21 insertions and deletions. Comparison of fly and human Pez reveals several short conserved motifs in the central region that are likely protein binding sites and/or phosphorylation sites. Novel invertebrate members of the BAS and MEG classes were identified using genome data, and an alignment was generated of vertebrate and invertebrate FERM domains of each class. 'Specialized' residues were identified that are conserved only within a given class of PTPs. These residues highlight surface regions that may bind class-specific ligands; for PEZ, these residues cluster on and near FERM subdomain F1. Finally, the PTP domain of fly Pez was modeled based on known PTP tertiary structures, and it is concluded that Pez is likely a functional phosphatase despite some unusual features of the active site cleft sequences. Biochemical confirmation of this hypothesis and genetic analysis of Pez are currently underway (Edwards, 2001).

Search PubMed for articles about Drosophila Pez

Au, A. C., Hernandez, P. A., Lieber, E., Nadroo, A. M., Shen, Y. M., Kelley, K. A., Gelb, B. D. and Diaz, G. A. (2010). Protein tyrosine phosphatase PTPN14 is a regulator of lymphatic function and choanal development in humans. Am J Hum Genet 87: 436-444. PubMed ID: 20826270

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 Citation: 20159600

Benzinou, M., et al. (2012). Mouse and human strategies identify PTPN14 as a modifier of angiogenesis and hereditary haemorrhagic telangiectasia. Nat Commun 3: 616. PubMed ID: 22233626

Cornell, M., Evans, D. A., Mann, R., Fostier, M., Flasza, M., Monthatong, M., Artavanis-Tsakonas, S. and Baron, M. (1999). The Drosophila melanogaster Suppressor of deltex gene, a regulator of the Notch receptor signaling pathway, is an E3 class ubiquitin ligase. Genetics 152: 567-576. PubMed ID: 10353900

Edwards, K., Davis, T., Marcey, D., Kurihara, J. and Yamamoto, D. (2001). Comparative analysis of the Band 4.1/ezrin-related protein tyrosine phosphatase Pez from two Drosophila species: implications for structure and function. Gene 275: 195-205. PubMed ID: 11587846

Fostier, M., Evans, D. A., Artavanis-Tsakonas, S. and Baron, M. (1998). Genetic characterization of the Drosophila melanogaster Suppressor of deltex gene: A regulator of notch signaling. Genetics 150: 1477-1485. PubMed ID: 9832525

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

Hori, K., Fostier, M., Ito, M., Fuwa, T. J., Go, M. J., Okano, H., Baron, M. and Matsuno, K. (2004). Drosophila deltex mediates suppressor of Hairless-independent and late-endosomal activation of Notch signaling. Development 131: 5527-5537. PubMed ID: 15496440

Huang, J. M., Nagatomo, I., Suzuki, E., Mizuno, T., Kumagai, T., Berezov, A., Zhang, H., Karlan, B., Greene, M. I. and Wang, Q. (2013). YAP modifies cancer cell sensitivity to EGFR and survivin inhibitors and is negatively regulated by the non-receptor type protein tyrosine phosphatase 14. Oncogene 32: 2220-2229. PubMed ID: 22689061

Liu, X., Yang, N., Figel, S. A., Wilson, K. E., Morrison, C. D., Gelman, I. H. and Zhang, J. (2013). PTPN14 interacts with and negatively regulates the oncogenic function of YAP. Oncogene 32: 1266-1273. PubMed ID: 22525271

Michaloglou, C., Lehmann, W., Martin, T., Delaunay, C., Hueber, A., Barys, L., Niu, H., Billy, E., Wartmann, M., Ito, M., Wilson, C. J., Digan, M. E., Bauer, A., Voshol, H., Christofori, G., Sellers, W. R., Hofmann, F. and Schmelzle, T. (2013). The tyrosine phosphatase PTPN14 is a negative regulator of YAP activity. PLoS One 8: e61916. PubMed ID: 23613971

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 Citation: 22305752

Smith, A. L., Mitchell, P. J., Shipley, J., Gusterson, B. A., Rogers, M. V. and Crompton, M. R. (1995). Pez: a novel human cDNA encoding protein tyrosine phosphatase- and ezrin-like domains. Biochem Biophys Res Commun 209: 959-965. PubMed ID: 7733990

Wyatt, L., Wadham, C., Crocker, L. A., Lardelli, M. and Khew-Goodall, Y. (2007). The protein tyrosine phosphatase Pez regulates TGFbeta, epithelial-mesenchymal transition, and organ development. J Cell Biol 178: 1223-1235. PubMed ID: 17893246

Wyatt, L. and Khew-Goodall, Y. (2008). PTP-Pez: a novel regulator of TGFbeta signaling. Cell Cycle 7: 2290-2295. PubMed ID: 18677119

Wang, C., Zhang, W., Yin, M.X., Hu, L., Li, P., Xu, J., Huang, H., Wang, S., Lu, Y., Wu, W., Ho, M.S., Li, L., Zhao, Y. and Zhang, L. (2015). Suppressor of Deltex mediates Pez degradation and modulates Drosophila midgut homeostasis. Nat Commun 6: 6607. PubMed ID: 25814387

Wang, W., Huang, J., Wang, X., Yuan, J., Li, X., Feng, L., Park, J. I. and Chen, J. (2012). PTPN14 is required for the density-dependent control of YAP1. Genes Dev 26: 1959-1971. PubMed ID: 22948661

Wilkin, M. B., Carbery, A. M., Fostier, M., Aslam, H., Mazaleyrat, S. L., Higgs, J., Myat, A., Evans, D. A., Cornell, M. and Baron, M. (2004). Regulation of notch endosomal sorting and signaling by Drosophila Nedd4 family proteins. Curr Biol 14: 2237-2244. PubMed ID: 15620650

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 Citation: 20159598

date revised: 22 February 2022

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