EGF receptor : Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

Gene name - EGF receptor

Synonyms - DER, Ellipse, torpedo

Cytological map position - 57F1

Function - transmembrane signaling

Keywords - Epidermal growth factor pathway

Symbol - Egfr

FlyBase ID:FBgn0003731

Genetic map position - 2-[97]

Classification - receptor tyrosine kinase

Cellular location - surface TM protein



NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Seong, K.H., Tsuda, M., Tsuda-Sakurai, K. and Aigaki, T. (2015). The plant homeodomain finger protein MESR4 is essential for embryonic development in Drosophila. Genesis [Epub ahead of print]. PubMed ID: 26467775
Summary:
Misexpression Suppressor of Ras 4 (MESR4), a PHD finger protein with nine zinc-finger motifs has been implicated in various biological processes including the regulation of fat storage and innate immunity in Drosophila. However, the role of MESR4 in the context of development remains unclear. This study shows that MESR4 is a nuclear protein essential for embryonic development. Immunostaining of polytene chromosomes using anti-MESR4 antibody revealed that MESR4 binds to numerous bands along the chromosome arms. The most intense signal was detected at the 39E-F region, which is known to contain the histone gene cluster. P-element insertions in the MESR4 locus are homozygous lethal during embryogenesis with defects in ventral ectoderm formation and head encapsulation. In the mutant embryos, expression of Fasciclin 3 (Fas3), an EGFR signal target gene is greatly reduced, and the level of EGFR signal-dependent double phosphorylated ERK (dp-ERK) remains low. However, in the context of wing vein formation, genetic interaction experiments suggest that MESR4 is involved in the EGFR signaling as a negative regulator. These results suggest that MESR4 is a novel chromatin-binding protein required for proper expression of genes including those regulated by the EGFR signaling pathway during development.

Jin, Y., Ha, N., Fores, M., Xiang, J., Glasser, C., Maldera, J., Jimenez, G. and Edgar, B. A. (2015). EGFR/Ras signaling controls Drosophila intestinal stem cell proliferation via Capicua-regulated genes. PLoS Genet 11: e1005634. PubMed ID: 26683696
Summary:
Epithelial renewal in the Drosophila intestine is orchestrated by Intestinal Stem Cells (ISCs). Following damage or stress the intestinal epithelium produces ligands that activate the epidermal growth factor receptor (EGFR) in ISCs. This promotes their growth and division and, thereby, epithelial regeneration. This study demonstrates that the HMG-box transcriptional repressor, Capicua (Cic), mediates these functions of EGFR signaling. Depleting Cic in ISCs activated them for division, whereas overexpressed Cic inhibited ISC proliferation and midgut regeneration. Epistasis tests showed that Cic acted as an essential downstream effector of EGFR/Ras signaling, and immunofluorescence showed that Cic's nuclear localization was regulated by EGFR signaling. ISC-specific mRNA expression profiling and DNA binding mapping using DamID indicated that Cic represses cell proliferation via direct targets including string (Cdc25), Cyclin E, and the ETS domain transcription factors Ets21C and Pointed (pnt). pnt was required for ISC over-proliferation following Cic depletion, and ectopic pnt restored ISC proliferation even in the presence of overexpressed dominant-active Cic. These studies identify Cic, Pnt, and Ets21C as critical downstream effectors of EGFR signaling in Drosophila ISCs.

Eichenlaub, T., Cohen, S.M. and Herranz, H. (2016). Cell competition drives the formation of metastatic tumors in a Drosophila model of epithelial tumor formation. Curr Biol [Epub ahead of print]. PubMed ID: 26853367
Summary:
Cell competition is a homeostatic process in which proliferating cells compete for survival. Elimination of otherwise normal healthy cells through competition is important during development and has recently been shown to contribute to maintaining tissue health during organismal aging. The mechanisms that allow for ongoing cell competition during adult life could, in principle, contribute to tumorigenesis. However, direct evidence supporting this hypothesis has been lacking. This study provides evidence that cell competition drives tumor formation in a Drosophila model of epithelial cancer. Cells expressing EGFR together with the conserved microRNA miR-8 acquire the properties of supercompetitors. Neoplastic transformation and metastasis depend on the ability of these cells to induce apoptosis and engulf nearby cells. miR-8 expression causes genome instability by downregulating expression of the Septin family protein Peanut. Cytokinesis failure due to downregulation of Peanut is required for tumorigenesis. The study provides evidence that the cellular mechanisms that drive cell competition during normal tissue growth can be co-opted to drive tumor formation and metastasis. Analogous mechanisms for cytokinesis failure may lead to polyploid intermediates in tumorigenesis in mammalian cancer models.

Chen, L.P., Wang, P., Sun, Y.J. and Wu, Y.J. (2016). Direct interaction of avermectin with epidermal growth factor receptor mediates the penetration resistance in Drosophila larvae. Open Biol 6. PubMed ID: 27249340
Summary:
With the widespread use of avermectins (AVMs) for managing parasitic and agricultural pests, the resistance of worms and insects to AVMs has emerged as a serious threat to human health and agriculture worldwide. The reduced penetration of AVMs is one of the main reasons for the development of the resistance to the chemicals. However, the detailed molecular mechanisms remain elusive. This study used the larvae of Drosophila melanogaster as a model system to explore the molecular mechanisms underlying the development of penetration resistance to AVMs. It was shown that the chitin layer is thickened and the efflux transporter P-glycoprotein (P-gp) is overexpressed in the AVM-resistant larvae epidermis. The activation of the transcription factor Relish by the over-activated Epidermal growth factor receptor (EGFR)/AKT/ERK pathway was found to induce the overexpression of the chitin synthases DmeCHS1/2 and P-gp in the resistant larvae. Interestingly, AVM was found to directly interact with EGFR and lead to the activation of the EGFR/AKT/ERK pathway, which activates the transcription factor Relish and induces the overexpression of DmeCHS1/2 and P-gp. These findings provide new insights into the molecular mechanisms underlying the development of penetration resistance to drugs. 

Fregoso Lomas, M., De Vito, S., Boisclair Lachance, J.F., Houde, J. and Nilson, L.A. (2016). Determination of EGFR signaling output by opposing gradients of BMP and JAK/STAT activity. Curr Biol [Epub ahead of print]. PubMed ID: 27593379
Summary:
A relatively small number of signaling pathways drive a wide range of developmental decisions, but how this versatility in signaling outcome is generated is not clear. In the Drosophila follicular epithelium, localized epidermal growth factor receptor (EGFR) activation induces distinct cell fates depending on its location. Posterior follicle cells respond to EGFR activity by expressing the T-box transcription factors Midline and H15, while anterior cells respond by expressing the homeodomain transcription factor Mirror. This study shows that the choice between these alternative outputs of EGFR signaling is regulated by antiparallel gradients of JAK/STAT and BMP pathway activity and that mutual repression between Midline/H15 and Mirror generates a bistable switch that toggles between alternative EGFR signaling outcomes. JAK/STAT and BMP pathway input is integrated through their joint and opposing regulation of both sides of this switch. By converting this positional information into a binary decision between EGFR signaling outcomes, this regulatory network ultimately allows the same ligand-receptor pair to establish both the anterior-posterior (AP) and dorsal-ventral (DV) axes of the issue.

Jussen, D., von Hilchen, J. and Urbach, R. (2016). Genetic regulation and function of epidermal growth factor receptor signalling in patterning of the embryonic Drosophila brain. Open Biol 6(12). PubMed ID: 27974623
Summary:
The specification of distinct neural cell types in central nervous system development crucially depends on positional cues conferred to neural stem cells in the neuroectoderm. This study investigated the regulation and function of the epidermal growth factor receptor (EGFR) signalling pathway in early development of the Drosophila brain. Localized EGFR signalling in the brain neuroectoderm was found to rely on a neuromere-specific deployment of activating (Spitz, Vein) and inhibiting (Argos) ligands. Activated EGFR controls the spatially restricted expression of all dorsoventral (DV) patterning genes in a gene- and neuromere-specific manner. Further, a novel role of DV genes-ventral nervous system defective (vnd), intermediate neuroblast defective (ind), Nkx6-in regulating the expression of vein and argos, which feed back on EGFR, indicating that EGFR signalling stands not strictly atop the DV patterning genes. Within this network of genetic interactions, Vnd acts as a positive EGFR feedback regulator. Further, it was shown that EGFR signalling becomes dependent on single-minded-expressing midline cells in the posterior brain (tritocerebrum), but remains midline-independent in the anterior brain (deuto- and protocerebrum). Finally, it was demonstrated that activated EGFR controls the proper formation of brain neuroblasts by regulating the number, survival and proneural gene expression of neuroectodermal progenitor cells. These data demonstrate that EGFR signalling is crucially important for patterning and early neurogenesis of the brain.
Chabu, C., Li, D. M. and Xu, T. (2017). EGFR/ARF6 regulation of Hh signalling stimulates oncogenic Ras tumour overgrowth. Nat Commun 8: 14688. PubMed ID: 28281543
Summary:
Multiple signalling events interact in cancer cells. Oncogenic Ras cooperates with Egfr, which cannot be explained by the canonical signalling paradigm. In turn, Egfr cooperates with Hedgehog signalling. How oncogenic Ras elicits and integrates Egfr and Hedgehog signals to drive overgrowth remains unclear. Using a Drosophila tumour model, this study shows that Egfr cooperates with oncogenic Ras via Arf6, which functions as a novel regulator of Hh signalling. Oncogenic Ras induces the expression of Egfr ligands. Egfr then signals through Arf6, which regulates Hh transport to promote Hh signalling. Blocking any step of this signalling cascade inhibits Hh signalling and correspondingly suppresses the growth of both, fly and human cancer cells harbouring oncogenic Ras mutations. These findings highlight a non-canonical Egfr signalling mechanism, centered on Arf6 as a novel regulator of Hh signalling. This explains both, the puzzling requirement of Egfr in oncogenic Ras-mediated overgrowth and the cooperation between Egfr and Hedgehog.
Xiang, J., Bandura, J., Zhang, P., Jin, Y., Reuter, H. and Edgar, B. A. (2017). EGFR-dependent TOR-independent endocycles support Drosophila gut epithelial regeneration. Nat Commun 8: 15125. PubMed ID: 28485389
Summary:
Following gut epithelial damage, epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) signalling triggers Drosophila intestinal stem cells to produce enteroblasts (EBs) and enterocytes (ECs) that regenerate the gut. As EBs differentiate into ECs, they become postmitotic, but undergo extensive growth and DNA endoreplication. This study reports that EGFR/RAS/MAPK signalling is required and sufficient to drive damage-induced EB/EC growth. Endoreplication occurs exclusively in EBs and newborn ECs that inherit EGFR and active MAPK from fast-dividing progenitors. Mature ECs lack EGF receptors and are refractory to growth signalling. Genetic tests indicated that stress-dependent EGFR/MAPK promotes gut regeneration via a novel mechanism that operates independently of Insulin/Pi3K/TOR signalling, which is nevertheless required in nonstressed conditions. The E2f1 transcription factor is required for and sufficient to drive EC endoreplication, and Ras/Raf signalling upregulates E2f1 levels posttranscriptionally. This study illustrates how distinct signalling mechanisms direct stress-dependent versus homeostatic regeneration, and highlight the importance of postmitotic cell growth in gut epithelial repair.
Kim, S., Nahm, M., Kim, N., Kwon, Y., Kim, J., Choi, S., Choi, E. Y., Shim, J., Lee, C. and Lee, S. (2017). Graf regulates hematopoiesis through GEEC endocytosis of EGFR. Development 144(22): 4159-4172. PubMed ID: 28993397
Summary:
GTPase regulator associated with focal adhesion kinase 1 (GRAF1) is an essential component of the GPI-enriched endocytic compartment (GEEC) endocytosis pathway. Mutations in the human GRAF1 gene are associated with acute myeloid leukemia, but its normal role in myeloid cell development remains unclear. This study shows that Graf, the Drosophila ortholog of GRAF1, is expressed and specifically localizes to GEEC endocytic membranes in macrophage-like plasmatocytes. Loss of Graf impairs GEEC endocytosis, enhances EGFR signaling and induces a plasmatocyte overproliferation phenotype that requires the EGFR signaling cascade. Mechanistically, Graf-dependent GEEC endocytosis serves as a major route for EGFR internalization at high, but not low, doses of the predominant Drosophila EGFR ligand Spitz (Spi), and is indispensable for efficient EGFR degradation and signal attenuation. Finally, Graf interacts directly with EGFR in a receptor ubiquitylation-dependent manner, suggesting a mechanism by which Graf promotes GEEC endocytosis of EGFR at high Spi. Based on these findings, a model is proposed in which Graf functions to downregulate EGFR signaling by facilitating Spi-induced receptor internalization through GEEC endocytosis, thereby restraining plasmatocyte proliferation.
Crespo-Yanez, X., Aguilar-Gurrieri, C., Jacomin, A. C., Journet, A., Mortier, M., Taillebourg, E., Soleilhac, E., Weissenhorn, W. and Fauvarque, M. O. (2018). CHMP1B is a target of USP8/UBPY regulated by ubiquitin during endocytosis. PLoS Genet 14(6): e1007456. PubMed ID: 29933386
Summary:
Integration and down-regulation of cell growth and differentiation signals rely on plasma membrane receptors endocytosis and sorting towards either recycling vesicles or degradative lysosomes via multivesicular bodies (MVB). In this process, the endosomal sorting complex-III required for transport (ESCRT-III) controls membrane deformation and scission triggering intraluminal vesicle (ILV) formation at early endosomes. This study shows that the ESCRT-III member CHMP1B can be ubiquitinated within a flexible loop known to undergo conformational changes during polymerization. This study demonstrates further that CHMP1B is deubiquitinated by the ubiquitin specific protease USP8 (syn. UBPY) and found fully devoid of ubiquitin in a ~500 kDa large complex that also contains its known ESCRT-III partner IST1. Moreover, EGF stimulation induces the rapid and transient accumulation of ubiquitinated forms of CHMP1B on cell membranes. Accordingly, CHMP1B ubiquitination is necessary for CHMP1B function in both EGF receptor trafficking in human cells and wing development in Drosophila. From these observations, it is proposed that CHMP1B is dynamically regulated by ubiquitination in response to EGF and that USP8 triggers CHMP1B deubiquitination possibly favoring its subsequent assembly into a membrane-associated ESCRT-III polymer.
Courgeon, M., He, D., Liu, H. H., Legent, K. and Treisman, J. E. (2018). The Drosophila Epidermal Growth Factor Receptor does not act in the nucleus. J Cell Sci. PubMed ID: 30158176
Summary:
Mammalian members of the ErbB family, including the Epidermal Growth Factor Receptor (EGFR), can regulate transcription, DNA replication and repair through nuclear entry of either the full-length proteins or their cleaved cytoplasmic domains. In cancer cells, these nuclear functions contribute to tumor progression and drug resistance. This study examined whether the single Drosophila EGFR can also localize to the nucleus. A chimeric EGFR protein fused at its cytoplasmic C-terminus to DNA-binding and transcriptional activation domains strongly activated transcriptional reporters when overexpressed in cultured cells or in vivo. However, this activity was independent of cleavage and endocytosis. Without an exogenous activation domain, EGFR fused to a DNA-binding domain did not activate or repress transcription. Addition of the same DNA-binding and transcriptional activation domains to the endogenous Egfr locus by genome editing produced no detectable reporter expression in wild type or oncogenic contexts. These results show that when expressed at physiological levels, the cytoplasmic domain of the Drosophila EGFR does not have access to the nucleus. Nuclear EGFR functions are likely to have evolved after vertebrates and invertebrates diverged.
Eichenlaub, T., Villadsen, R., Freitas, F. C. P., Andrejeva, D., Aldana, B. I., Nguyen, H. T., Petersen, O. W., Gorodkin, J., Herranz, H. and Cohen, S. M. (2018). Warburg effect metabolism drives neoplasia in a Drosophila genetic model of epithelial cancer. Curr Biol. PubMed ID: 30293715
Summary:
Cancers develop in a complex mutational landscape. Genetic models of tumor formation have been used to explore how combinations of mutations cooperate to promote tumor formation in vivo. This study identified lactate dehydrogenase (LDH), a key enzyme in Warburg effect metabolism, as a cooperating factor that is both necessary and sufficient for epidermal growth factor receptor (EGFR)-driven epithelial neoplasia and metastasis in a Drosophila model. LDH is upregulated during the transition from hyperplasia to neoplasia, and neoplasia is prevented by LDH depletion. Elevated LDH is sufficient to drive this transition. Notably, genetic alterations that increase glucose flux, or a high-sugar diet, are also sufficient to promote EGFR-driven neoplasia, and this depends on LDH activity. This study provides evidence that increased LDHA expression promotes a transformed phenotype in a human primary breast cell culture model. Furthermore, analysis of publically available cancer data showed evidence of synergy between elevated EGFR and LDHA activity linked to poor clinical outcome in a number of human cancers. Altered metabolism has generally been assumed to be an enabling feature that accelerates cancer cell proliferation. These findings provide evidence that sugar metabolism may have a more profound role in driving neoplasia than previously appreciated.
BIOLOGICAL OVERVIEW

The Drosophila EGF receptor homolog, commonly called Torpedo or DER, has a complex biology. It is found in multiple sites and at varying times during development; in signaling processes that determine egg polarity, during early development to determine the identity of cells in the ventral ectoderm, during neurogenesis, in the development of the Malpighian tubules, and during larval stages in the development of the eye and wing.

Egfr interacts with three different ligands: Gurken, Spitz (the principle ligand), and Argos. Two accessory proteins modulate Egfr signaling: Rhomboid and Star. The signaling molecules downstream of Egfr include Shc ( the Drosophila homolog of a mammalian oncogene), DRK (an adaptor protein that docks onto Shc bound to Egfr and a homolog of mammalian GRB2), a guanine nucleotide exchange factor (SOS) activated by DRK, and downstream targets like Ras, Raf and Rolled (Map kinase)(Lai, 1995).

All these downstream signaling molecules are members of the RAS-RAF-MAPK pathway that amplifies and transmits receptor signals to various parts of the cell. Signals to the cytoskeleton result in changes in cell shape; signals to the nucleus result in gene activation.

The signaling pathway used by Egfr is also used by other receptor tyrosine kinases, including the Torso receptor and Sevenless, another Drosophila EFG receptor homolog. How does a single pathway used by a number of different receptors permit the wide variety of specific cell differentiation effects that are receptor-dependent? This question is currently being investigated in a number of developmental pathways, but as yet, no answer is available. The immune system, that most intensely studied cell interaction system, is not driven by a single ligand receptor interaction, but by numerous signaling events involving a number of receptors and a number of ligands. There is no reason to believe that Drosophila differentiation will be any less complex. Thus signals through the Egfr will be one of a number of signals integrated into differentiation decisions.

The complexity of receptor tyrosine kinase signaling is revealed by studies of eye and wing differentiation. Signaling in the eye involves three ligands and two receptors as well as Rhomboid and Star, the two accessory proteins of Egfr signaling. The Boss-Sevenless receptor-ligand pair is involved, as well as the Spitz-Egfr receptor-ligand pair. An additional ligand, Argos, functions either directly or indirectly to inhibit the Egfr signaling (Freeman, 1994).

Rhomboid's particiption in Egfr signaling in the wing currently presents a fascinating mystery. Rhomboid is expressed in a geometric pattern that establishes the future sites of wing vein differentiation. Rhomboid enhances Egfr signaling at the sites of future veins, but exactly how this enhancement occurs is currently unknown. It appears that Rhomboid sets the stage for Egfr signaling and the resultant induction of wing veins (Sturtevant, 1995).

A more detailed review of Gurken's role in signaling dorsoventral and anterior-posterior polarity in the developing oocyte will be found at the gurken site. Maternally transcribed Egfr is used in this signaling process. Egfr has two promoters and two first exons. The different developmental roles for each of the two transcripts have not yet been documented (Clifford, 1994).

Egfr and the determination of wing and leg cell fates

Wing and leg precursors of Drosophila are recruited from a common pool of ectodermal cells expressing the homeobox gene Dll. Induction by Dpp promotes this cell fate decision toward the wing and proximal leg. The receptor tyrosine kinase Egfr antagonizes the wing-promoting function of Dpp and allows recruitment of leg precursor cells from uncommitted ectodermal cells. By monitoring the spatial distribution of cells responding to Dpp and Egfr, it has been shown that nuclear transduction of the two signals peaks at different positions along the dorsoventral axis when the fates of wing and leg discs are specified and that the balance of the two signals assessed within the nucleus determines the number of cells recruited to the wing. Differential activation of the two signals and the cross talk between them critically affect this cell fate choice (Kubota, 2000).

In a screen for genes expressed in the embryonic limb primordia, rhomboid was found to be transiently expressed in the central part of Dll-expressing limb primordia in stage 11 embryos. rho transcription is the rate-limiting step of the activation of an EGFR ligand Spitz. As expected from the role of rho as a stimulator of Egfr, a transient expression of an activated, phosphorylated form of MAPK (dpMAPK) is detected in the nucleus of limb primordial cells surrounding the rho-expressing cells. The dpMAPK expression starts after the initiation of Dll transcription and diminishes before the separation of the wing and leg disc primordium. The dpMAPK expression is undetectable in null mutants of rho or Egfr. The peak of dpMAPK expression is located ventrally to the cells expressing dpp. The results suggest that rho-mediated stimulation of Egfr and MAPK occurs at the time of cell fate specification of wing and leg discs (Kubota, 2000).

The spatial distribution of cells responding to Dpp and its relationship to Egfr signals was studied. To this end, an antibody specific to phosphorylated C-terminal sequence of Mad was produced. The phosphorylated sequence corresponds to the site at which the type I BMP receptor phosphorylates SMad1. The antibody detects an antigen distributed in a pattern similar to, but broader than, that of DPP mRNA. This immunoreactivity is dependent on Dpp signaling, as it is absent in stage 11 mutants of thick veins encoding type I Dpp receptor and in dpp mutants. This indicates that other extant TGFbeta-related signaling molecules present in Drosophila embryos do not substitute for Dpp to induce this immunoreactivity. Conversely, ectopic expression of Dpp results in high accumulation of this immunoreactivity. These results suggest that the antibody detects a Dpp-specific signaling event, most likely the phosphorylation and nuclear transport of Mad. Hereafter, the immunoreactivity detected by this antibody is called pSSVS (Kubota, 2000).

pSSVS is found mainly localized in the nucleus and distributed in regions a few cells wider in diameter than those of dpp-expressing cells. These properties are consistent with the previous findings that Mad transduces the Dpp signal to the nucleus. Double labeling of pSSVS and DLL mRNA shows that pSSVS expression is higher in the dorsal region of Dll-expressing cells. Combined with the double-labeling results of dpMAPK and Dll or dpp, it is concluded that cells responding to Dpp and Egfr overlap, but the peak of the responses are shifted. Such differential distribution of the two signals results in an arrangement of cells responding to a different strength of Dpp and Egfr along the dorsoventral axis (Kubota, 2000).

To study the role of Egfr at the stage of wing and leg cell fate determination, specific marker gene expression was examined in Egfr signaling mutants. DLL mRNA is expressed in the entire limb primordium at stage 11 and becomes restricted to distal leg cells at stage 15. Esg protein expression was used to detect both wing and proximal leg cells. In rho mutants, the size of limb primordia at stage 11 is the same as the control, but the later development of leg discs is abnormal. The number of leg disc cells expressing Dll and/or Esg at stage 15 is reduced, and these cells no longer show the circular arrangement typical of leg disc precursors. Amorphic mutation of Egfr cause a ventral expansion of limb primordia as a result of a loss of the early function of Egfr, but the expression of leg markers is severely reduced at stage 15. A similar phenotype is observed in mutants lacking both maternal and paternal copies of Dsor1, which encodes a MAP kinase kinase. In all cases described above, Esg-expressing cells at the dorsal part of leg discs are most frequently lost, suggesting that the development of dorsoproximal leg cells is most sensitive to the loss of Egfr activity. In contrast, wing and leg disc development is normal in vein mutants, suggesting the putative ligand of Egfr encoded by this gene is dispensable. These results suggest that MAPK activation induced by Rho and Egfr is essential for normal leg development (Kubota, 2000).

The temporal requirement for Egfr was studied by the temperature-sensitive allele Egfrf1. When the temperature is increased to the restrictive temperature at 5 hours after egg laying (AEL) prior to the induction of the limb primordium, the expression of Dll is expanded to the ventral midline, as was also observed with the strong Egfr mutants. When the temperature is increased at 6 hour AEL, the initial Dll expression is not altered, but the leg disc development is severely affected. Only mild defect is found in leg discs when the temperature is increased at 7 hours AEL, suggesting that Egfr must function between 6 and 7 hours AEL to correctly specify the leg cell fate. This is the time when the transient activation of MAPK is observed. Furthermore, whether Egfr is required autonomously in limb primordial cells was examined by expressing a dominant-negative form of Egfr using Dll-Gal4. Expression of this driver starts in the limb primordium at stage 11 and persists in a subset of wing discs and in entire leg discs at stage 15 because of the persistence of Gal4 activity. Imaginal disc-specific inhibition of Egfr interfers with leg disc development, while leaving the wing disc intact. These results demonstrated that a transient activation of Egfr in stage 11 limb primordia is essential for the leg disc development (Kubota, 2000).

In contrast to the severe defects in leg discs, none of the mutations in Egfr signaling interfer with wing disc formation. In these mutants, wing primordia consistently express Esg and another wing disc marker Vestigial, and invaginate to form discs. However, an increase in the number of wing disc cells has been noted in Egfr signaling mutants. This effect was analyzed in rho mutants; unlike Egfr mutants, in rho mutants the number of limb primordial cells at stage 11 is the same as the control. The number of Esg-expressing wing disc cells in rho mutants is increased compared to the control, while the number of the proximal leg disc cells is severely reduced. It is concluded that Egfr signaling is required to limit wing disc cell differentiation in limb primordial cells that are not yet fully committed. It is inferred that a subset of prospective leg cells that do not receive a sufficient amount of Egfr signaling fail to differentiate as proximal leg and instead adopt a wing fate (Kubota, 2000).

The increase in the number of wing disc cells in rho mutants resembles the overexpression phenotype of Dpp and raises a possibility that Egfr might prevent wing disc development by negatively regulating Dpp signaling. Such a cross talk could occur at several levels including the following: (1) regulation of dpp transcription, (2) signal transduction from Dpp receptors to the nucleus, and (3) transcriptional regulation of downstream target genes. The analyses excluded the first two possibilities for two reasons. (1) The expression pattern of DPP mRNA is unaffected by the mutation of rho. A previous report showing an expansion of dpp expression in Egfr mutants probably reflects the global patterning role of Egfr in the earlier stage. (2) pSSVS expression around limb primordia does not change in rho mutants. Conversely, the expression pattern of dpMAPK is not changed by a null mutation of tkv. These results suggest that the differential distribution of cells responding to Dpp and Egfr is set up independently of each other's activity (Kubota, 2000).

Dpp and Egfr were found to antagonize each other after signal transduction into the nucleus. Hyperactivation of Egfr by an ectopic expression of an Egfr ligand Spitz causes a great accumulation of dpMAPK. As expected from the negative effect of Egfr on the wing development, this treatment completely eliminates wing disc formation and, in addition, causes a malformation of the leg disc. Since it was found that cells migrating out of the leg primordium express dpMAPK, it is unlikely that the failure in wing disc formation is due to the prevention of cell migration or to cell death. It has been suggested that hyperactivation of Egfr prevents limb primordial cells from adopting the wing cell fate. It is likely that those cells adopt the epidermal fate instead. Overexpression of Dpp causes an accumulation of pSSVS and an increase in the number of wing disc cells. Coexpression of Dpp with Spi partially restores the development of both wing and leg discs, suggesting that wing disc development overcomes the negative effect of Egfr if provided with a sufficient amount of Dpp. The restored wing primordia migrate with high levels of pSSVS and dpMAPK, further supporting the notion that Dpp and Egfr signals are transduced independently of one another (Kubota, 2000).

dad is an immediate transcriptional target gene of Dpp, the expression of which closely parallels that of pSSVS expression in embryos and is inducible by Dpp. dad expression is not affected in Egfr or rho mutants. Furthermore, elevated dad expression induced by Dpp is not affected by sSpi, suggesting that at least one of the immediate transcriptional responses to Dpp is unaffected by elevated Egfr signaling (Kubota, 2000).

The antagonism between Dpp and Egfr during wing disc development raises a question: what is the default state of the wing and leg primordia in the absence of the two signals? Double mutant phenotypes of Dpp and Egfr signaling were examined. tkv mutants lack wing discs and their leg discs are malformed. This phenotype reflects a disc cell autonomous requirement for Dpp signaling, because the phenotype is reproduced by the disc-specific inhibition of Dpp signaling by dad, which inhibits Mad. The phenotype of either tkv;rho or tkv;Egfr double mutants is a simple addition of each mutation, in which wing discs are lost completely and leg discs are severely reduced. Since Dll-expressing limb primordial cells are present in tkv;Egfr double mutants in stage 11, it has been concluded that these cells fail to differentiate as wing discs and their ability to differentiate as leg discs is also compromised. A few Esg-positive cells remain at the position of the leg, and it is speculated that this reflects the presence of a second leg-inducing signal. These results suggest that Dpp is absolutely required for wing disc development irrespective of the activity of Egfr (Kubota, 2000).

Egfr affects the choice of wing vs. leg developmental options differently; it promotes leg development while it inhibits wing development. These two activities of Egfr are the earliest of known events of leg specification, and occur prior to the establishment of proximodistal axis in the leg. In the absence of late functions of Dpp and Egfr, limb primordia are specified but fail to differentiate into wing disc and most of leg disc. Thus it is proposed that early limb primordium at stage 11 consists of cells not yet fully committed to either wing or leg disc fate, and the cells are exposed to different amounts of Dpp and Egfr signaling according to their dorsoventral location. Dpp recruits the cells to the wing disc fate. Egfr antagonizes the cellular response to the wing-inducing function of Dpp and allows the development of wing discs only in the dorsal region. Thus the dorsoventral difference in Dpp and Egfr signaling in the limb primordium provides key information to the separation and differentiation of the wing and leg discs. In contrast to the opposing roles of Dpp and Egfr on wing disc development, leg discs requires both signals. The effect of the loss of Egfr activity on leg disc development is not compensated for by a simultaneous loss of Dpp signaling, indicating that Egfr has an additional activity to promote leg development separately from its role to antagonize Dpp. Because dorsal and ventral limb primordial cells respond to Egfr differently, it is speculated that at least one additional dorsoventral factor influences leg disc formation at stage 11. This idea is consistent with the fact that residual proximal leg cells can still be induced in the almost complete absence of Egfr and Dpp activity. One candidate for the factor is Wg, which is expressed in the limb primordium (Kubota, 2000).

The nuclear transduction of the Dpp signal, as visualized by the distribution of pSSVS and expression of dad, is unaffected by Egfr. The results suggest that the antagonistic effect of Egfr on Dpp signaling occurs after transduction into the nucleus. Therefore, the mechanism of SMad inhibition by direct phosphorylation by MAP kinase does not play a major role in this case (Kubota, 2000).

The finding that Egfr is activated in the limb primordium and prevents wing disc formation suggests that Egfr is a key factor in the diversification of the wing and leg fate. It is proposed that the differential activation of Dpp and Egfr, and the dorsal cell migration brings a subset of limb primordial cells out of the range of Egfr signaling, and thereby allows Dpp to induce wing development. It follows that dorsally migrating cells acquire the wing cell identity only after the separation from leg-promoting signals. Consistent with this idea, expression of wing-specific markers Vg and Sna, start only after the separation of the two primordia. Mechanisms that promote the dorsal cell migration remain to be identified. Given that the basic genetic components for the induction of the wing and leg have been identified in the model organism Drosophila, it can now be asked how the genetic mechanism of wing and leg specification has evolved by comparing the expression and function of these genes in limb primordial cells of primitive insects (Kubota, 2000).

Egfr and midline glia maturation

A good example of the function of Egfr in regulating cell development is found by examining the role of Egfr in midline glia maturation. The midline glial cells are required for correct formation of the axonal pattern in the embryonic ventral nerve cord. Initially, six midline cells form an equivalence group with the capacity to develop as glial cells. By the end of embryonic development three to four cells are singled out as midline glial cells. Midline glia development occurs in two steps, both of which depend on activation of the Egfreceptor and subsequent Ras1/Raf-mediated signal transduction (See Drosophila Ras1). In the first step six midline cells in each segment, originating from the anterior-most three of a total of eight midline progenitor cells, are determined as the midline glia equivalence group. The process of generation of the midline glia equivalence group involves Notch function and segmentation genes. It might also depend on the function of single minded, the master regulatory gene of midline development. The single minded transcript accumulates in the midline glia and, depending on the context, can act either as a transcriptional activator or repressor. By the end of embryogenesis the final number of midline glial cells is about 3 to 4. Thus, the final number of cells has to be selected from the initially defined equivalence group in a second step (Scholz, 1997).

Egfr mutants show a reduced number of midline glial cells and argos mutants, which possibly exhibit an increased activation of Egfr in the midline, show an increased number of midline glial cells. Furthermore, expression of activated ras1 (or activated raf) in the midline results in the appearance of extra midline cells. This model suggests that activation of ras1 signaling in the entire midline glial equivalence group promotes survival of all cells in this cluster. Thus, in wild-type flies, about 2-3 cells in each group down-regulate Egfr signaling and are destined for cell death. Both Rhomboid and Argos control activation of the Egfr during midline glia development. It is thought that a graded activation of Egfr is brought about by the activity of Rhomboid, which is thought to promote EGF receptor signaling, possibly by cell autonomous activation of the Egfr ligand Spitz. Ectopic rhomboid leads to extra midline glial cells. Egfr activates PointedP2 through phosphorylation; Pointed in turn activates the transcription of argos. Argos negatively regulates Egfr signaling non-cell autonomously and competes with Spitz function. pointed mutants form extra glial cells. Yan antagonizes PointedP2A in midline glial cells, just as it does in the developing photoreceptor cells (Scholz, 1997).

Egfr and head midline development

Egfr signaling is required in a narrow medial domain of the head ectoderm (here called ‘head midline’) that includes the anlagen of the medial brain (including the dorsomedial and ventral medial domain of the brain, termed DMD and VMD respectively), the visual system (optic lobe, larval eye) and the stomatogastric nervous system (SNS). These head midline cells differ profoundly from their lateral neighbors in the way they develop. Three differences are noteworthy: (1) Like their counterparts in the mesectoderm, the head midline cells do not give rise to typical neuroblasts by delamination, but stay integrated in the surface ectoderm for an extended period of time. (2) The proneural gene l’sc, which transiently (for approximately 30 minutes) comes on in all parts of the procephalic neurectoderm while neuroblasts delaminate, is expressed continuously in the head midline cells for several hours. (3) Head midline cells, similar to ventral midline cells of the trunk, require the Egfr pathway. In embryos carrying loss-of-function mutations in Egfr, spi, rho, S and pnt, most of the optic lobe, larval eye, SNS and dorsomedial brain are absent. This phenotype arises by a failure of many neurectodermal cells to segregate (i.e., invaginate) from the ectoderm; in addition, around the time when segregation should take place, there is an increased amount of apoptotic cell death, accompanied by reaper expression, which removes many head midline cells. In embryos where Egfr signaling is activated ectopically by inducing rho, or by argos (aos) or yan loss-of-function, head midline structures are variably enlarged. A typical phenotype resulting from the overactivity of Egfr signaling is a ‘cyclops’ like malformation of the visual system, in which the primordia of the visual system stay fused in the dorsal midline. The early expression of cell fate markers, such as sine oculis in Spitz-group mutants, is unaltered (Dumstrei, 1998).

About the time of gastrulation, Egfr signaling is activated in both mesectoderm and the head midline. The ventromedial domain of Egfr activation, as monitored by rho, aos and phosphorylated ERK expression, continues from the ventral mesectodermal domain into the head ectoderm. The ventromedial stripe of aos expression bifurcates at the level of the stomodeal plate and continues dorsally. Approaching the dorsal midline, it turns posteriorly and continues all the way to the posterior boundary of the head neurectoderm. There is an anterior-median patch of aos expression corresponding to the anlage of the stomatogastric nervous system. At later stages (stage 10-12) expression of aos in head midline structures becomes more complex; however, a high level of expression persists in large parts of the optic lobe, SNS, VMD and DMD. The domain of rho expression and ERK phosphorylation matches closely the aos expression domain. pnt and yan, two transcription factors executing the signal passed to the nucleus by the Egfr signaling cascade, are also expressed in structures of the head midline The Egfr pathway is required in the head midline for proper cytodifferentiation and epithelial maintenance. Both Egfr and its ligand Spitz are ubiquitously expressed in the embryo (Dumstrei, 1998).

The Egfr loss-of-function phenotype in the head midline and the mesectoderm is quite compatible with the postulated function of Egfr signaling in the Drosophila compound eye and in various vertebrate systems where Egfr signaling also promotes differentiation and prevents cell death. In the head midline, no evidence for a transformation of cell fate is observed similar to the that takes place in the ventral neurectoderm of the Drosophila embryo. A transformation of fate would imply that the structures missing (e.g., optic lobe, dorsomedial brain) are replaced by other structures, which then would have to expand in size in order to occupy the space normally allotted to them, as well as the additional space normally taken up by the midline structures. However, there does not appear to be an expansion of the lateral neurectoderm, such as takes place in ventral neurectoderm: for example, the number and pattern of neuroblasts delaminating from the lateral head neurectoderm in Egfr or Spi mutant embryos is normal. Thus, it appears that the loss of head midline structures observed in Egfr-signaling-deficient embryos is effected mainly by cell death (Dumstrei, 1998).

The gain of function of EGFR signaling causes a complex phenotype. In yan loss-of-function mutants, a hyperplasia of both dorsomedial brain and stomatogastric nervous system is observed. In case of the latter, cells of the DMD clearly overproliferate; ultimately, these cells express neural markers and become incorporated into the brain. The optic lobe phenotype is more difficult to interpret. Thus, cells located medial to the optic lobes express fasII, resulting in a single optic lobe (‘cyclops’ phenotype) overgrown by the enlarged brain. One might interpret this phenotype as a cell fate transformation: dorsomedial cells, which would normally not express fasII (and would not become part of the optic lobe) turn on this gene if the output of EGFR signaling is increased. However, to clearly interpret this phenotype, one would have to know much better what normally happens to the dorsomedial cells located between the optic lobes. At the blastoderm stage, the anlagen of the optic lobe map to a dorsomedial position. Shortly thereafter, dorsomedial cells move laterally, generating a thin membrane of amnioserosa like cells in between them. A great deal of cell death takes place in this region, prompting one to speculate that cell death is important for the lateral shift of the optic lobe primordia. Supporting this idea it has been found that in embryos in which no cell death occurs due to a deficiency that removes the reaper complex of genes, the optic lobe primordia are frequently extended toward the midline, similar to what can be observed in yan or aos loss-of-function. It is therefore possible that increased activity of Egfr signaling, rather than inducing another fate in the dorsomedial cells, rescues dorsomedial cells from cell death; the expression of FasII might reflect the fate that these cells would normally show if they were permitted to live. Clearly, more experimental studies are required to grasp the effect of Egfr signaling in this region (Dumstrei, 1998).

In head midline structures, in particular the optic lobe and SNS, there may be a late phase of EGFR signaling (as assayed by the expression of aos and activated ERK) whose significance is not yet known. EGFR signaling could be involved in modifying the inhibitory feed-back loop between neurogenic and proneural genes that exists in other neurectoderm cells. In the head midline neurectoderm, regulation of proneural and neurogenic genes has to be different. Thus, instead of a short burst of proneural gene expression in proneural clusters that is resolved into expression in individual neuroblasts, proneural genes are expressed for a long period of time; at the same time, the expression is never restricted to single neuroblasts. Since genes of the E(spl) complex are expressed in the same cells that express l’sc, the inhibitory loop between E(spl)-C and proneural genes must be interrupted at some level. It is possible that Egfr signaling is causing the interruption of this inhibitory loop. Based on genetic studies of Notch and Egfr signaling in the compound eye, it has been speculated that one of the consequences of Egfr activation (which ultimately is required for all ommatidial cell types to differentiate) is to inhibit N signaling, since constitutively active N inhibits ommatidial cell differentiation by preventing response to differentiative signals. However, the same effect could be achieved if Egfr signaling, similar to what is proposed here for the midline neurectoderm, interrupts the inhibition of proneural genes by E(spl). Although this would not prevent N signaling, it would cancel the effect of N signaling on downregulating proneural genes and thereby keep cells in a state of competency to respond to signals (Dumstrei, 1998).

Coordinated sequential action of EGFR and Notch signaling pathways regulates proneural wave progression in the Drosophila optic lobe.

During neurogenesis in the medulla of the Drosophila optic lobe, neuroepithelial cells are programmed to differentiate into neuroblasts at the medial edge of the developing optic lobe. The wave of differentiation progresses synchronously in a row of cells from medial to the lateral regions of the optic lobe, sweeping across the entire neuroepithelial sheet; it is preceded by the transient expression of the proneural gene lethal of scute [l(1)sc] and is thus called the proneural wave. This study found that the epidermal growth factor receptor (EGFR) signaling pathway promotes proneural wave progression. EGFR signaling is activated in neuroepithelial cells and induces l(1)sc expression. EGFR activation is regulated by transient expression of Rhomboid (Rho), which is required for the maturation of the EGF ligand Spitz. Rho expression is also regulated by the EGFR signal. The transient and spatially restricted expression of Rho generates sequential activation of EGFR signaling and assures the directional progression of the differentiation wave. This study also provides new insights into the role of Notch signaling. Expression of the Notch ligand Delta is induced by EGFR, and Notch signaling prolongs the proneural state. Notch signaling activity is downregulated by its own feedback mechanism that permits cells at proneural states to subsequently develop into neuroblasts. Thus, coordinated sequential action of the EGFR and Notch signaling pathways causes the proneural wave to progress and induce neuroblast formation in a precisely ordered manner (Yasugi, 2010).

Loss of EGFR function in progenitor cells caused failure of L(1)sc expression and differentiation into neuroblasts (see A model of progression of the proneural wave). In addition, elevated EGFR signaling resulted in faster proneural wave progression and induced earlier neuroblast differentiation. The activation of the EGFR signal is regulated by a transient expression of Rho, which cleaves membrane-associated Spi to generate secreted active Spi. This study also demonstrated that Rho expression itself depends on EGFR function, and thus the sequential induction of the EGFR signal progresses the proneural wave. Clones of cells mutant for pnt were not recovered unless Minute was employed, suggesting that the EGFR pathway is required for the proliferation of neuroepithelial cells. However, the progression of the proneural wave is not regulated by the proliferation rate per se (Yasugi, 2010).

The function of the Notch signaling pathway in neurogenesis is known as the lateral inhibition. A revision of this notion has recently been proposed for mouse neurogenesis, in which levels of the Notch signal oscillate in neural progenitor cells during early stages of embryogenesis, and thus no cell maintains a constant level of the signal. The oscillation depends mainly on a short lifetime and negative-feedback regulation of the Notch effecter protein Hes1, a homolog of Drosophila E(spl). This prevents precocious neuronal fate determination. The biggest difficulty in analysis of Notch signaling is the random distribution of different stages of cells in the developing ventricular zone, which is thus called a salt-and-pepper pattern. In medulla neurogenesis, however, cell differentiation is well organized spatiotemporally and the developmental process of medulla neurons can be viewed as a medial-lateral array of progressively aged cells across the optic lobe. Such features allowed the functions of Notch to be precisely analyzed. Cells are classified into four types according to their developmental stages: neuroepithelial cells expressing PatJ, neuronal progenitor I expressing a low level of Dpn, neuronal progenitor II expressing L(1)sc and neuroblasts expressing high levels of Dpn. The Notch signal is activated in neuronal progenitor I and II. The EGFR signal turns on in the neuronal progenitor II stage and progresses the stage by activating L(1)sc expression. Cells become neuroblasts when the Notch and EGFR signals are shut off. Cells stay as neuronal progenitor I when Notch signal alone is activated, whereas cells stay as neuronal progenitor II when the Notch signal is activated in conjunction with the EGFR signal. Although the Notch signal is once activated, it must be turned off to let cells differentiate into neuroblasts. In neuronal progenitor II, E(spl)-C expression is induced by Notch signaling, and the increased E(spl)-C next downregulates Dl expression and subsequent activation of the Notch signal (Yasugi, 2010).

What does Notch do in medulla neurogenesis? It is infered that the Notch signal sustains cell fates, whereas the EGFR signal progresses the transitions of cell fate. This was well documented when a constitutively active form of each signal component was induced. EGFR, or its downstream Ras, induces expression of L(1)sc but does not fix its state, even though the constitutively active form is employed. At the same time, a constitutively active Notch sustains cell fates in a cell-autonomous manner. Constitutively active N receptors, by contrast, autonomously determine cell fates depending on the context: cells become neuronal progenitor I in the absence of EGFR and neuronal progenitor II in the presence of EGFR. The precocious neurogenesis caused by the impairment of Notch signaling suggests that Notch keeps cells in the progenitor state for a certain length of time in order to allow neuroepithelial cells to grow into a sufficient population. In the prospective spinal cord of chick embryo (Hammerle, 2007), the development from neural stem cells to neurons progresses rostrocaudally, during which the transition from proliferating progenitors to neurogenic progenitors is regulated by Notch signaling (Yasugi, 2010).

Although Notch plays a pivotal role in determining cell fate between neural and non-neural cells, the function may be context dependent and can be classified into three categories. (1) Classical lateral inhibition is seen in CNS formation in embryogenesis and SOP formation in Drosophila. Cells that once expressed a higher level of the Notch ligand maintain their cell states and become neuroblasts. (2) Oscillatory activations are found in early development of the mouse brain (Shimojo, 2008). Progenitor cells are not destined to either cell types. (3) An association with the proneural wave found in Drosophila medulla neurogenesis as is described in this study. The Notch signal is transiently activated only once and then shuts off in a synchronized manner. The notable difference in the outcome is the ratio of neural to non-neural cells; a small number of cells from the entire population become neuroblasts or neural stem cells in the former cases (1 and 2), whereas most of the cells become neuroblasts in the latter case (3). The differences between (1) and (2) can be ascribed at least in part to the duration of development. Hes1 expression has been shown to oscillate within a period of 2 hours in the mouse, whereas in Drosophila embryogenesis, selection of neuroblasts from neuroectodermal cells takes place within a few hours. Thus, even if Drosophila E(spl) has a half-life time equivalent to Hes1, the selection process during embryogenesis probably finishes within a cycle of the oscillation. The process of medulla neuroblast formation continues for more than 1 day, but Notch signaling is activated for a much shorter period in any given cell. This raises the possibility that E(spl)/Hes1 may have a similarly short half-life but outcome would depend on the developmental context (Yasugi, 2010).

The functions of EGFR and Notch described in this study resemble their roles in SOP formation of adult chordotonal organ development; the EGFR signal provides an inductive cue, whereas the Notch signal prevents premature SOP formation. In addition, restricted expression of rho and activation of the EGFR signal assure reiterative SOP commitment. Several neuroblasts are also sequentially differentiated from epidermal cells in adult chordotonal organs (Yasugi, 2010).

Unpaired, a ligand of the JAK/STAT pathway is expressed in lateral neuroepithelial cells and shapes an activity gradient that is higher in lateral and lower in the medial neuroepithelium. The JAK/STAT signal acts as a negative regulator of the progression of the proneural wave (Yasugi, 2008). This report has shown that activation of both EGFR and Notch signaling pathways depends on the activity of the JAK/STAT signal. The JAK/STAT signal probably acts upstream of EGFR and Notch signals in a non-autonomous fashion. These three signals coordinate and precisely regulate the formation of neuroblasts (Yasugi, 2010).

Protein tyrosine phosphatase PTPN3 inhibits lung cancer cell proliferation and migration by promoting EGFR endocytic degradation

Epidermal growth factor receptor (EGFR) regulates multiple signaling cascades essential for cell proliferation, growth and differentiation. Using a genetic approach, this study found that Drosophila FERM and PDZ domain-containing protein tyrosine phosphatase, dPtpmeg, negatively regulates border cell migration and inhibits the EGFR/Ras/mitogen-activated protein kinase signaling pathway during wing morphogenesis. EGFR pathway substrate 15 (Eps15) was further identified as a target of dPtpmeg and its human homolog PTPN3. Eps15 is a scaffolding adaptor protein known to be involved in EGFR endocytosis and trafficking. Interestingly, PTPN3-mediated tyrosine dephosphorylation of Eps15 promotes EGFR for lipid raft-mediated endocytosis and lysosomal degradation. PTPN3 and the Eps15 tyrosine phosphorylation-deficient mutant suppress non-small-cell lung cancer cell growth and migration in vitro and reduce lung tumor xenograft growth in vivo. Moreover, depletion of PTPN3 impairs the degradation of EGFR and enhances proliferation and tumorigenicity of lung cancer cells. Taken together, these results indicate that PTPN3 may act as a tumor suppressor in lung cancer through its modulation of EGFR signaling (Li, 2014).

Reversible tyrosine protein phosphorylation by protein tyrosine kinases and protein tyrosine phosphatases (PTPs) acts as a molecular switch that regulates a variety of biological processes. The receptor tyrosine kinase epidermal growth factor receptor (EGFR), the best characterized member of the ErbB family receptors, acts as a critical regulator of numerous cellular processes, including growth, proliferation and differentiation. Upon activation by its growth factor ligands, EGFR undergoes dimerization and activation, leading to tyrosine phosphorylation of the intracellular region of the receptor as well as many cytoplasmic substrates. The activated EGFR is then internalized by clathrin-mediated endocytosis and sorted into the endosomal compartments, through which it is either recycled back to the plasma membrane or transported to the lysosome for degradation. Because overexpression or constitutive activation of EGFR has been implicated in the pathogenesis and progression of a variety of human malignancies, it is therefore crucial to understand how EGFR signaling is regulated. Several PTPs have been implicated in the regulation of EGFR signaling. Among them, PTPrk, DEP-1 (PTPRJ), PTP1B (PTPN1), SHP-1 (PTPN6), TCPTP (PTPN2), PTPN9 and PTPN12 have been shown to downregulate EGFR signaling by dephosphorylating EGFR. The receptor-type PTP DEP-1 dephosphorylates EGFR on the cell surface and inhibits its internalization. On the other hand, the endoplasmic reticulum-localized PTP1B has been reported to regulate EGFR signaling from endosomes. PTP1B promotes the sequestration of EGFR onto internal vesicles of multivesicular bodies. The ESCRT (endosomal sorting complex required for transport) complexes are known to play an important role in sorting EGFR to multivesicular bodies. Recently, it has been shown that the ESCRT accessory protein HD-PTP/PTPN23 coordinates with the ubiquitin-specific peptidase UBPY to drive EGFR sorting to the multivesicular bodies. A better understanding of the role of PTPs in regulating EGFR signaling will help to provide insights into the molecular mechanisms behind EGFR-mediated tumorigenesis (Li, 2014).

PTPN3 (PTPH1) and the closely-related PTPN4 (PTPMEG) are non-transmembrane PTPs that contain an N-terminal FERM (Band 4.1, Ezrin, Radixin, Moesin homology) domain followed by a single PDZ (PSD95, Dlg, ZO1) domain and the C-terminal PTP domain. They have been implicated in the regulation of cell growth and proliferation. However, their role in receptor protein tyrosine kinase signaling is not clear. The dPtpmeg is the Drosophila homolog of mammalian PTPN3 and PTPN4. Phenotypic analyses have revealed that dptpmeg mutants exhibit aberrant mushroom body axon projection patterns in the brain. Besides its role in regulating neuronal wiring, the molecular function of dPtpmeg has remained largely unknown. This study has identified EGFR pathway substrate 15 (Eps15) as a substrate of dPtpmeg and PTPN3. Eps15 is known to be an endocytic adaptor involved in the regulation of EGFR trafficking. PTPN3 dephosphorylated Eps15 and promoted EGFR for lipid raft-mediated endocytosis and lysosomal degradation. The ectopic expression of PTPN3 or Eps15-Y850F mutant in the non-small-cell lung cancer (NSCLC) cells inhibited cell proliferation, migration and tumor growth. These findings uncover a novel role for PTPN3 in the regulation of EGFR endocytic trafficking, degradation and signaling (Li, 2014).

The EGFR belongs to the ErbB family of protein tyrosine kinases and is a major regulator for both normal development and cancer progression. PTPs, which include receptor-like PTPs and nonreceptor PTPs, are a group of tightly regulated enzymes thought to regulate tyrosine phosphorylation by antagonizing the action protein tyrosine kinases. This study provides the first evidence that PTPN3 inhibits the EGFR signaling by targeting the receptor for lysosomal degradation. In Drosophila, dPtpmeg antagonizes receptor tyrosine kinase activity and plays a role in controlling border cell migration during oogenesis. Moreover, dPtpmeg negatively regulates the EGFR/Ras/MAPK pathway during wing morphogenesis. Substrate-trapping and biochemical analysis further identified Eps15 as a substrate for dPTPmeg and PTPN3. The results demonstrate that PTPN3 dephosphorylates Eps15 and promotes EGFR for degradation in lung cancer cells (Li, 2014).

Eps15 is a multidomain adaptor protein that plays an important role in regulating endocytic trafficking. In mammalian cells, Eps15 can be phosphorylated by EGFR at tyrosine residue 850 upon EGF stimulation. It has been shown that ectopic expression of Eps15-Y850F mutant impairs the internalization of EGFR. On the contrary, using immunofluorescence and flow cytometry, this study shows that ectopic expression of PTPN3 and Eps15-Y850F does not affect the internalization of EGFR. Since a dramatic reduction of EGFR levels was found in cells expressing PTPN3 and Eps15-Y850F, one explanation for the results being contradictory to previous findings might be because of the difference in detection sensitivity. In addition to its role in endocytic trafficking, Eps15 was reported to localize at the trans-Golgi network and regulate vesicle trafficking during the secretory process. However, immunofluorescence analysis of the intracellular distribution of endogenous PTPN3 or exogenously expressed HA-tagged PTPN3 indicated no significant localization at the trans-Golgi network, and this might suggest that PTPN3 is not involved in Eps15-mediated protein sorting and vesicle trafficking at the trans-Golgi network (Li, 2014).

The ligand-activated EGFR and the transforming growth factor-β receptor have been reported to be endocytosed through a clathrin-dependent as well as a clathrin-independent pathway. Segregation of these cell surface receptors through distinct endocytic pathways is known to regulate downstream signal duration and receptor trafficking, although it is unclear how it does this. Several lines of evidence indicate that PTPN3 and Eps15-Y850F accelerate downregulation of EGFR via a clathrin-independent but lipid raft-dependent pathway. First, EGF-488 trafficking assay revealed that EGF-488 was largely colocalized with lipid raft-associated protein caveolin-1 but not with clathrin in cells expressing PTPN3 or Eps15-Y850F. Second, analysis of EGFR profile by sucrose gradient fractionation showed that EGFR was concentrated in clathrin-enriched non-lipid fractions in control cells. However, overexpression of PTPN3 and Eps15-Y850F led to a redistribution of EGFR to caveolin-1-enriched lipid raft fractions. Third, disruption of lipid rafts with MβCD and filipin suppressed PTPN3- or Eps15-Y850F-induced EGFR degradation. How does PTPN3-mediated tyrosine dephosphorylation of Eps15 regulate EGFR for lipid raft-dependent endocytosis and lysosomal degradation? Accumulating evidence has shown that EGFR ubiquitination is not essential for its internalization, but appears to play an important role in endosomal sorting and lysosomal targeting of the receptor. One possibility is that tyrosine dephosphorylation of Eps15 by PTPN3 may affect the ubiquitination status of EGFR, accelerating EGFR for lysosomal degradation. Recently, an endosomally localized Eps15 isoform (Eps15b) has been identified that interacts with the Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) complex to mediate EGFR degradation. This study found that like Eps15, Eps15b is also a substrate of PTPN3, indicating a dual role for PTPN3 in the regulation of endocytic trafficking and endosomal sorting of EGFR (Li, 2014).

Accumulating evidence has indicated that PTPs can function as tumor suppressors or oncogenes depending on the substrate involved and the cellular context. It has been reported that PTPN3 expresses in gastric cancer cells and may play a role in gastric cancer progression and differentiation. PTPN3 has been found to coordinate with p38γ MAPK to promote Ras oncogenesis in colon cancer, and it has been found to stimulate breast cancer growth by inducing and stabilizing the protein expression of vitamin D receptor. Interestingly, recent studies have also indicated that PTPN3 plays a role in tumor suppression. Mutational analysis of the tyrosine phosphatome found PTPN3 along with five other PTPs (PTPRF, PTPRG, PTPRT, PTPN13 and PTPN14) are mutated in colorectal cancer. Moreover, the transcriptome of two NSCLC cell lines were analyzed, and one allele of PTPN3 was found to be mutated in the NSCLC cell line H2228. It was further shown that ectopic expression of PTPN3 inhibits the growth of NSCLC cells, although the molecular mechanisms underlying the growth inhibition remain unknown. The current data support a model in which PTPN3-mediated tyrosine dephosphorylation of Eps15 leads to EGFR degradation and tumor suppression in NSCLC cells. This study demonstrated that PTPN3 and Eps15-Y850F overexpression reduced EGFR protein levels and impeded the proliferation and migration of NSCLC cells. Moreover, PTPN3 and Eps15-Y850F significantly suppressed NSCLC tumor growth in a subcutaneous xenograft model. Conversely, depletion of PTPN3 enhanced EGFR stabilization and promoted NSCLC tumorigenicity both in vitro and in vivo. The findings are consistent with the idea that PTPN3 acts as a tumor suppressor in NSCLC (Li, 2014).

In conclusion, this study has identified Eps15 as an evolutionarily conserved dPtpmeg/PTPN3 substrate that regulates EGFR signaling. The finding that PTPN3-mediated tyrosine dephosphorylation of Eps15 modulates EGFR-dependent cancer progression may help contribute to the development of a targeting intervention in NSCLC (Li, 2014).


GENE STRUCTURE

The structure of the cDNAs indicates the use of alternative 5' exons. Thus the gene encodes two proteins differing at their N-terminus. The splicing alternatives show similar distribution during development. Most of the coding region of Egfr is located within four exons separated by three small introns (Schejter, 1986).

The alternative 3' exons, termed Type 1 and Type 2 exons, are separated from the downstream common sequences by 45 kb. Each alternative exon codes for a signal sequence. The Type 1 exon codes for 101 amino acids while the Type 2 exon codes for 52 amino acids (Clifford, 1994).

Genomic DNA length - 60 kb


PROTEIN STRUCTURE

Amino Acids - 1458 and 1409

Structural Domains

cDNA clones of Egfr were isolated and sequenced. The deduced amino acid sequence shows a similar degree of homology to the human epidermal growth factor receptor and to the rat and human neu proteins; the most striking difference is the addition of a third cysteine-rich extracellular domain in Egfr. In the extracellular region the homology between Egfr and HER (human EGF receptor) is 37% (Schejter, 1986 and Price, 1989). The intracellular region contains one kinase domain (Clifford, 1994).


EGF receptor: Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References
date revised: 30 September 2000 

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