InteractiveFly: GeneBrief

myopic: Biological Overview | References


Gene name - myopic

Synonyms -

Cytological map position - 70F4-70F5

Function - signaling protein

Keywords - Hippo/Warts pathway, regulation of receptor endocytosis, integrin trafficking, controls Yorkie endosomal association and protein levels, tumor suppressor Egfr pathway, Toll pathway - immune reponse, wing, eye

Symbol - mop

FlyBase ID: FBgn0036448

Genetic map position - chr3L:14762441-14769229

Classification - Protein-interacting, N-terminal, Bro1-like domain of mammalian His-Domain type N23 protein tyrosine phosphatase and related domains

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Pradhan-Sundd, T. and Verheyen, E. M. (2015). The Myopic-Ubpy-Hrs nexus enables endosomal recycling of Frizzled. Mol Biol Cell [Epub ahead of print]. PubMed ID: 26224310
Summary:
Endosomal trafficking of signaling proteins plays an essential role in cellular homeostasis. The seven-pass transmembrane protein, Frizzled (Fz) is a critical component of the Wnt signaling. Although Wnt signaling is proposed to be regulated by endosomal trafficking of Fz, the molecular events that enable this regulation are not completely understood. This study shows that the endosomal protein Myopic (Mop) regulates Fz trafficking in the Drosophila wing disk by inhibiting the ubiquitination and degradation of Hrs. Deletion of Mop or Hrs results in endosomal accumulation of Fz and therefore reduced Wnt signaling. The in situ Proximity Ligation Assay revealed a strong association between Mop and Hrs in the Drosophila wing disk. Overexpression of Hrs rescues the trafficking defect caused by mop knockdown. Mop aids in the maintenance of Ubpy which deubiquitinates (and thus stabilizes) Hrs. In the absence of the ubiquitin ligase Cbl, Mop is dispensable. These findings support a previously unknown role for Mop in endosomal trafficking of Fz in Wnt-receiving cells.

Loncle, N., Agromayor, M., Martin-Serrano, J. and Williams, D. W. (2015). An ESCRT module is required for neuron pruning. Sci Rep 5: 8461. PubMed ID: 25676218
Summary:
Neural circuits are refined by both functional and structural changes. Structural remodeling by large-scale pruning occurs where relatively long neuronal branches are cut away from their parent neuron and removed by local degeneration. Until now, the molecular mechanisms executing such branch severing events have remained poorly understood. This study reveal a role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery during neuronal remodeling. The data show that a specific ESCRT pruning module, including members of the ESCRT-I and ESCRT-III complexes, but not ESCRT-0 or ESCRT-II, are required for the neurite scission event during pruning. Furthermore it was shown that this ESCRT module requires a direct, in vivo, interaction between Shrub/CHMP4B and the accessory protein Myopic/HD-PTP.
BIOLOGICAL OVERVIEW

Protein tyrosine phosphatases (PTPs) are a group of tightly regulated enzymes that coordinate with protein tyrosine kinases to control protein phosphorylation during various cellular processes. Using genetic analysis in Drosophila non-transmembrane PTPs, one role was identified that Myopic (Mop), the Drosophila homolog of the human His domain phosphotyrosine phosphatase (HDPTP), plays in cell adhesion. Depletion of Mop results in aberrant integrin distribution and border cell dissociation during Drosophila oogenesis. Interestingly, Mop phosphatase activity is not required for its role in maintaining border cell cluster integrity. Rab4 GTPase was further identified as a Mop interactor in a yeast two-hybrid screen. Expression of the Rab4 dominant-negative mutant leads to border cell dissociation and suppression of Mop-induced wing-blade adhesion defects, suggesting a critical role of Rab4 in Mop-mediated signaling. In mammals, it has been shown that Rab4-dependent recycling of integrins is necessary for cell adhesion and migration. This study found that human HDPTP regulates the spatial distribution of Rab4 and integrin trafficking. Depletion of HDPTP resulted in actin reorganization and increased cell motility. Together, these findings suggest an evolutionarily conserved function of HDPTP-Rab4 in the regulation of endocytic trafficking, cell adhesion and migration (Chen, 2012).

Cell adhesion and cell migration are essential for the development and coordinated function of multicellular organisms. Aberrant regulation of these processes often results in the progression of many diseases, including cancer invasion and metastasis. Accumulating evidence has indicated that dynamic and reversible protein tyrosine phosphorylation is essential for the regulation of cell migration and cell adhesion. While many studies have been devoted to the role of protein tyrosine kinases in these processes, the function of protein tyrosine phosphatases (PTPs) in cell adhesion and migration remains unclear (Chen, 2012).

The dynamic change of integrin-mediated focal adhesions plays a critical role in cell adhesion and migration. Many focal adhesion regulators such as focal adhesion kinase (FAK), Src, p130Cas, and paxillin are tyrosine phosphorylated. The tyrosine phosphorylation of these proteins affects focal adhesion dynamics. Phosphorylation of tyrosine 397 in FAK promotes its association with Src, and the activated FAK-Src complex subsequently regulates focal adhesion dynamics by signaling downstream targets. Several PTPs have been implicated in integrin signaling, cell adhesion and motility. One study has shown that SHP-2 phosphatase influences FAK activity. SHP-2 also promotes Src kinase activation by inhibiting Csk. Depletion of PTP-PEST has been found to lead to the hyperphosphorylation of p130Cas, FAK and paxillin, and a marked increase in focal adhesions. Moreover, PTP1B and PTPα, have also been found to regulate Src phosphorylation and integrin-mediated adhesion (Chen, 2012).

In Drosophila, a total of sixteen putative classical PTPs have been identified. Compared to mammalian PTPs, Drosophila PTP family members are relatively simple, most containing only one gene corresponding to each subtype (except for DPTP10D and DPTP4E, which share similar domain structures). Therefore, Drosophila can serve as an excellent model system for the study of the physiological and developmental function of PTPs. While much research has been devoted to the function of receptor PTPs, the role of non-transmembrane PTPs (NT-PTPs) in Drosophila development remains unknown. One of the most well studied Drosophila NT-PTPs is Corkscrew (Csw). Csw is the ortholog of human SHP-2 which has two SH2 domains at the N-terminus and a PTP domain at the C-terminus. Csw functions as a downstream effecter of Sevenless PTK and is essential for the development of the R7 photoreceptor. Phenotypic analysis showed that Csw can also act downstream of many receptor tyrosine kinases, such as the Drosophila epidermal growth factor receptor (DER) and the fibroblast growth factor (Breathless). PTP-ER has been shown to function as a negative regulator downstream of Ras1 and to be involved in RAS1/MAPK-mediated R7 photoreceptor differentiation. PTP61F, the Drosophila ortholog of human PTP1B and TCPTP, has been reported to interact with Dock, an adapter protein required for axon guidance. PTP61F has recent been shown to coordinate with dAbl in regulating actin cytoskeleton organization via reversible tyrosine phosphorylation of Abi and Kette ). Moreover, dPtpmeg, a FERM and PDZ domain-containing NT-PTP, is reported to be involved in the formation of neuronal circuits in the Drosophila brain, though its molecular function in this process is not known (Chen, 2012).

To explore the functional role of Drosophila NT-PTPs in cell adhesion and migration, genetic analyses was performed to identify NT-PTPs that could modulate border cell migration during oogenesis. This study found that Myopic (Mop), the Drosophila homolog of the human His domain phosphotyrosine phosphatase (HDPTP), plays an important role in maintaining border cell cluster integrity. Depletion of Mop altered the normal distribution of integrin receptor. While Mop has recently been reported to regulate EGFR and Toll receptor signaling (Miura, 2008; Huang, 2010), its molecular mechanism has remained elusive. This study found that Mop interacts with Rab4 GTPase in controlling integrin distribution and cell adhesion. It was further demonstrated that human HDPTP is essential for the intracellular positioning of Rab4, integrin trafficking, and cell migration. These findings provide some insight into the mechanisms underlying HDPTP in the regulation of cell adhesion and migration (Chen, 2012).

Accumulating evidence has indicated that vesicular trafficking regulates the distribution of plasma membrane content as well as the localization of cytoskeletal proteins during cell adhesion and migration. Drosophila border cells migrate as a cluster of strongly adherent cells during the development of the egg chamber. During this process, JNK signaling and endocytosis-mediated spatial distribution of receptor tyrosine kinases play a critical role, though mechanisms involved in this process have remained elusive. This identified Mop, the Drosophila homolog of human HDPTP, as a regulator of integrin trafficking. Mop is essential for proper integrin localization and for maintaining border cell integrity during oogenesis. It was further demonstrated that Mop and HDPTP interacts with Rab4 GTPase in both Drosophila and mammals. Rab4 has been shown to regulate integrin recycling and cell migration (Roberts, 2001; White, 2007). The current findings indicate that Mop/HDPTP-mediated endocytic trafficking plays an essential role in integrin-mediated cell adhesion and migration. Mop has been predicted as a nontransmembrane-PTP (Andersen, 2005). However, amino acid sequence analysis revealed that Mop displays several differences from conserved PTP motifs within the phosphatase domain. For example, the catalytic essential aspartic acid (D) within motif 8 (WPDXGXP) is replaced by a lysine residue (K). Although the active site cysteine (C) in the catalytic motif 9 (VHCSAGXGR[T/S]G) could be found, the overall signature motif of Mop was much more divergent compared to other PTPs. Moreover, no Mop tyrosine phosphatase activity could be detected using in vitro phosphatase assays. These results suggest that Drosophila Mop may not be enzymatic active. Alternatively, Mop may exhibit weak phosphatase activity which can not be detected using either pNPP or in gel phosphatase assay. A recent study by Lin (2011) indicated that human PTPN23/HDPTP exhibits relatively low activity that is comparable with the specific activity of PTP1B D181E mutant. This study also found that expression of Mop-C/S mutant, in which the catalytic cysteine in the active site is replaced by serine, or Mop phosphatase domain deletion mutant rescued the Mop-RNAi induced border cell dissociation defects as effectively as the wild-type Mop, indicating that the putative tyrosine phosphatase activity is not essential for maintaining border cell cluster integrity (Chen, 2012).

In addition to having a C-terminus phosphatase domain, Mop has a sequence similar to that of yeast Bro1 at the N-terminus. The Bro1 domain consists of a folded core of about 370 residues and has been found in many proteins, including Bro1, Alix, and Rim20, known to regulate endosome trafficking (Kim, 2005). The Bro1 domain has been shown to bind with multivesicular body components (ESCRT-III proteins) such as yeast Snf7 and mammalian CHMP4B for targeting Bro1 domain-containing proteins to endosomes (Ichioka, 2007). Interestingly, mutational analysis revealed that CHMP4B binding is not required for HDPTP function, suggesting that the Bro1 domain may have other functions (Doyotte, 2008). This study identified Rab4 as an interactor with Mop and HDPTP through the Bro1 domain. The Rab GTPases cycle between the GTP-bound active and the GDP-bound inactive forms and are necessary for efficient membrane vesicle transport between different subcellular compartments (Stenmark, 2009). Among them, Rab4 and Rab11 have been implicated in controlling membrane trafficking through the endocytic recycling pathways (Grant, 2009). Several lines of evidence suggest that Rab4 is involved in Mop-mediated integrin distribution. First, it was found that expression of dominant-negative Rab4S22N or depletion of Rab4 in migrating border cells resulted in integrin redistribution and border cell dissociation. Second, genetic analysis revealed that Rab4S22N and Rab4- RNAi suppressed Mop-induced wing blistering phenotypes. Third, in mammalian cells, Rab4 regulated integrin recycling from early endosomes and was required for cell adhesion and spreading. This study found that downregulation of HDPTP disrupted integrin distribution, focal adhesion formation, actin organization, and enhanced cancer cell molility. Moreover, since Mop interacts with Rab4 in a nulcleotide-independent manner, Mop may act as an adaptor rather than an effector of Rab4 during endosomal trafficking processes. In mammals, Rab4 has been shown to regulate fast integrin recycling and persistent cell migration. This study also noticed that dominant-negative Rab4 caused integrin redistribution to the perinuclear region. However, it was found that expression of HDPTP could not rescue the dominant-negative Rab4-induced integrin redistribution. This is consistent with the genetic data that Rab4 acts downstream of Mop. It is proposed that Mop/HDPTP may act as an adaptor to keep Rab4 in proper endosomal domains. Depletion of HDPTP resulted in Rab4 mislocalization, changes in the integrin dynamics and increases in cell migration. Indeed, it was found that misexpression of βPS-integrin resulted in a marked increase in border cell cluster dissociation and βPS-integrin knockdown suppressed MopRNAi-induced border cell dissociation phenotype. Mop/HDPTP is likely to function via its association with Rab4 on early endosomes to regulate integrin sorting and recycling (Chen, 2012).

Integrins activate multiple signaling pathways involved in regulating cell proliferation, survival and migration. One major signaling event stimulated by integrins is mediated by the FAK and Src tyrosine kinases. FAK and Src are crucial regulators of cell adhesion and motility which they regulate by controlling the formation and turnover of focal adhesions. Several reports have shown interactions between HDPTP and FAK-Src complex. HDPTP has been proposed to act as a molecular bridge between FAK and Src in regulating endothelial and bladder carcinoma cell motility (Mariotti, 2009b; Mariotti, 2009a). Moreover, Lin identified PTPN23/HDPTP as a negative regulator of cell invasion in mammary epithelial cells (Lin, 2011). In those studies, FAK and Src were found to be substrates of HDPTP, suggesting that loss of HDPTP may increase FAK-Src activity to promote cell motility. Intriguingly, in Drosophila, this study showed that Mop regulates the border cell association in a PTP domain-independent manner. Recent studies in Drosophila and mammalian cells also found that the phosphatase activity of Mop and HDPTP is not required for its biological function in receptor signaling and tumor suppression (Miura, 2008; Gingras, 2009; Huang, 2010). The current findings on Mop/HDPTPRab4 interaction and the regulation of integrin recycling provide new insights into the role of Mop/HDPTP in the regulation of cell adhesion and migration, and they are not mutually exclusive from previous findings (Chen, 2012).

A screen for conditional growth suppressor genes identifies the Drosophila homolog of HD-PTP as a regulator of the oncoprotein Yorkie

Mammalian cancers depend on 'multiple hits,' some of which promote growth and some of which block apoptosis. A screened was performed for mutations that require a synergistic block in apoptosis to promote tissue overgrowth and myopic (mop), the Drosophila homolog of the candidate tumor-suppressor and endosomal regulator His-domain protein tyrosine phosphatase (HD-PTP), was identified. Myopic was found to regulate the Salvador/Warts/Hippo (SWH) tumor suppressor pathway: Myopic PPxY motifs bind conserved residues in the WW domains of the transcriptional coactivator Yorkie, and Myopic colocalizes with Yorkie at endosomes. Myopic controls Yorkie endosomal association and protein levels, ultimately influencing expression of some Yorkie target genes. However, the antiapoptotic gene diap1 is not affected, which may explain the conditional nature of the myopic growth phenotype. These data establish Myopic as a Yorkie regulator and implicate Myopic-dependent association of Yorkie with endosomal compartments as a regulatory step in nuclear outputs of the SWH pathway (Gilbert, 2011).

This study describes a screening strategy to identify mutations in Drosophila that require a synergistic block in cell death in order to promote tissue overgrowth. Using this approach, the endosomal protein Myopic, which is the Drosophila homolog of the candidate mammalian tumor suppressor HD-PTP, was identified as a regulator of the SWH growth inhibitory pathway. Through multiple approaches, this study demonstrates that Mop regulates Yki activity via a mechanism involving direct binding and modulation of Yki endosomal association (Gilbert, 2011).

This study defines a pool of cytoplasmic Yki that binds Mop and colocalizes with it on endosomes. Data from discs and cultured cells indicate Mop controls endosomal association of this pool of Yki and that a positive correlation exists between Yki colocalization with EEA1-positive early endosomes, and Yki levels and activity. A growing body of genetic and molecular data support a role for endosomes as key signaling centers for signal transduction pathways that influence the nuclear translocation of latent cytoplasmic transcription factors. For example, the activated c-Met receptor associates with the STAT3 transcription factor on EEA1-positive endosomes prior to STAT3 nuclear accumulation, and c-Met delivery to a perinuclear endosomal compartment is necessary to sustain nuclear STAT3. The enrichment of Yki on EEA1 endosomes and activation of a subset of Yki nuclear targets in mop mutant cells suggests that Yki, perhaps in association with receptor complexes, may take a similar route to the nucleus. Intriguingly, microtubule-regulated perinuclear transport of Merlin (Mer) controls nucleocytoplasmic shuttling of Yki). The direct link between Mer transport and Yki shuttling is not clear. However, as Mer can control internalization of transmembrane receptors, perinuclear transport of Mer might in turn modulate endosomal internalization and transit of Yki:receptor complexes en route to the nucleus (Gilbert, 2011).

Genetic data show that exogenous Mop is sufficient to restrict ectopic expression of the Yki-target ex but not diap1 and that loss of endogenous Mop upregulates a set of Yki targets that do not include diap1. Mop thus appears to define a regulatory step in determining outputs of the SWH pathway, perhaps as part of the endosomal sorting process. Trafficking of transmembrane proteins down alternate endosomal routes contributes to the activation of different nuclear programs in the Notch, Jak/STAT and Akt pathways. Similarly, association of Yki-containing complexes with different endosomal compartments may shift Yki nuclear output, perhaps by bringing Yki into contact with post-translational modifiers or binding partners that affect its ability to activate its suite of target promoters. Further studies will be required to establish whether loss of Mop indeed alters Yki post-translational modification or the assembly of Yki transcriptional complexes (Gilbert, 2011).

In the context of SWH signaling, the differential effect of mop loss on ex and diap1 expression place Mop within the growth-regulatory arm of the SWH network. Differential effects on the growth and apoptotic outputs of the SWH pathway is also a feature of mutations in ex and mer, which preferentially drive Yki-dependent clonal growth or anti-apoptotic signals respectively and whose combined mutant phenotypes are more severe than those of single mutants. The synergy between ex and mop alleles on IOC number extends this model and supports the hypothesis that Ex is downstream of wts in growth control but upstream of wts in apoptotic control (Gilbert, 2011).

mop mutant cells undergo high rates of caspase-dependent apoptosis in developing eye and wing imaginal discs (this study and Miura, 2008). It is probable that this apoptosis is not caused by an effect on diap1 expression but rather a requirement for Mop in additional pro-survival mechanisms. Knockdown of vertebrate HD-PTP/PTPN23 elevates levels of tyrosine phosphorylated focal adhesion kinase (FAK), which is implicated in cell migration and integrin-mediated survival signals. Mop facilitates trafficking of the EGFR receptor into late endosomal compartments and promotes Ras/MAPK signaling downstream of EGFR in the developing retina (Miura, 2008). Because the Ras/MAPK module is required to restrain cell death pathways, reduced EGFR-dependent signaling seems likely to contribute to a subset of the apoptotic phenotype of mop mutant cells (Gilbert, 2011).

The Mop:Yki interaction involves a WW:PPxY interaction mechanism shared by the SWH proteins Ex, Wts, and Hpo that can bind Yki directly and regulate its activity independent of S168 phosphorylation status. Mop represses growth driven by the YkiS3A mutant, indicating that its repressive mechanism is not dependent on Wts kinase activity. As Mop controls the distribution of Yki across endosomal compartments, the paired Bro1 and PPxY domains in Mop could function as a bridge between Yki-containing SWH signaling complexes in the cytoplasm and complexes on the outer membrane of endosomes such as ESCRTs. These complexes could be fairly static or they could assemble and disassemble in response to specific signals. The fact that Mop, Ex, Hpo and Wts share a WW:PPxY binding mechanism suggests these proteins might compete for Yki binding in the cytosol, or that Mop acts as an endocytic scaffolding factor in a 'hand-off' mechanism from the upstream components Ex, Hpo, and Wts. Indeed understanding the dynamics and composition of the Mop:Yki complex is a significant question going forward. Intriguingly loss of the Lgl kinase, which regulates cell polarity and membrane compartmentalization, elevates Yki activity by mislocalizing Hpo and the SWH component RASSF in the cytoplasm of disc cells, suggesting that Hpo and RASSF proteins participate in dynamic and localized interactions in the cytoplasm that are important for their Yki-regulatory function (Gilbert, 2011).

The human HD-PTP/PTPN23 gene resides in a region of the genome (3p21.3) associated with loss-of-heterozygosity (LOH) in greater than 90% of small cell (SCLC) and non-small cell (NSCLC) lung cancers. Yap protein is predominantly nuclear in a subset of primary NSCLC samples, promotes cell proliferation and invasion in NSCLC cell lines, and its expression correlates with poor prognosis in NSCLC patients. Thus mutations that deregulate Yap levels and activity are predicted to promote the inappropriate growth and invasiveness of lung epithelial cells. The mechanism of growth suppression by HD-PTP is not known, but its ability to suppress colony formation of human renal cancer cells is independent of catalytic PTPase activity in much the same way regulation of Yki by Mop does not require PTPase activity. Although HD-PTP lacks a canonical PPxY motif, genetic data indicate that Mop retains the ability to inhibit Yap activity in the Drosophila eye. The extent to which HD-PTP binds Yap or Taz has yet to be examined, but if the relationship between the orthologous Drosophila proteins is conserved in vertebrates, this link to Yki/Yap may contribute to growth regulatory roles of vertebrate HD-PTP proteins in development and disease (Gilbert, 2011).

Endocytic pathway is required for Drosophila Toll innate immune signaling

The Toll signaling pathway is required for the innate immune response against fungi and Gram-positive bacteria in Drosophila. This study shows that the endosomal proteins Myopic (Mop) and Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) are required for the activation of the Toll signaling pathway. This requirement is observed in cultured cells and in flies, and epistasis experiments show that the Mop protein functions upstream of the MyD88 adaptor and the Pelle kinase. Mop and Hrs, which are critical components of the ESCRT-0 endocytosis complex, colocalize with the Toll receptor in endosomes. It is concluded that endocytosis is required for the activation of the Toll signaling pathway (Huang, 2010).

The Toll signaling pathway is essential for the Drosophila innate immune response to infections by fungi or Gram-positive bacteria. Most of the Toll signaling components were identified through genetic screens for mutants defective in embryonic dorsal-ventral patterning. This study describes Mop, a putative protein tyrosine phosphatase, as a regulator of the Toll pathway. Mop is an endosomal protein that colocalizes with Hrs, a subunit of the ESCRT-0 complex. Knocking down mop by RNAi inhibits Toll pathway activation both in vitro and in vivo. Epistasis studies show that Mop functions upstream of MyD88 and Pelle, at the same level as the Toll receptor. This study shows that Hrs is required for signal-dependent Cactus degradation, and Hrs is present in a complex together with Mop and the Toll receptor. The findings strongly suggest endocytosis plays an essential role in Drosophila Toll signaling (Huang, 2010).

Mop contains two conserved domains, an N-terminal Bro1 domain and a C-terminal PTP domain. Although Mop ortholog has not been identified in the yeast genome, the Bro1 domain itself is evolutionarily conserved from yeast to humans. The yeast Bro1 protein is a component of the ESCRT machinery and is localized to endosomes through the interaction with Snf7, an ESCRT-III subunit. Bro1 recruits the Doa4 deubiquitinating enzyme to endosomes and also functions as a cofactor to activate Doa4, which removes the ubiquitin moiety of ubiquitinated membrane proteins before the cargos invaginate into MVB vesicles. The presence of the Bro1 domain in Mop suggests that Mop is an endosomal protein, which is supported by the data. However, mutant Mop protein lacking the entire Bro1 domain (Mop deltaBro1) still localizes to endosomes. The C-terminal region of the yeast Bro1 protein, outside the Bro1 domain, also contributes to its endosomal location. It is likely that the Mop protein is targeted to endosomes through the nonconserved sequences between two domains and/or the Bro1 domain. This hypothesis is consistent with the finding that HD-PTP, the human Mop homolog, is endosomal in HeLa cells but is distributed throughout the cytoplasm in Drosophila S2 cells. Although Mop deltaBro1 localizes to endosomes, it cannot complement the function of wild-type Mop in Toll pathway activation. The Bro1 domain may target Mop to the specific endosomal domain or recruit other proteins involved in endocytosis or signaling (Huang, 2010).

This study also found that Mop proteins bearing a mutation in the putative phosphatase catalytic motif or missing the phosphatase domain can substitute for the endogenous Mop protein, indicating that the putative phosphatase activity is not required for Toll signaling. Immunoprecipitation experiments show that Hrs, Mop, and Toll are present in the same complex. Mop may act as an adaptor to interact with different proteins to facilitate endocytosis. During the preparation of this work, Mop was reported as an endosomal protein required for EGFR signaling during photoreceptor differentiation in Drosophila eye imaginal disk (Miura, 2008). Genetic evidence suggests that activated Drosophila EGFR is ubiquitylated and sorted through the endocytosis machinery for lysosomal degradation by a mechanism similar to the mammalian EGFR. A mop allele carrying a point mutation in the putative phosphatase catalytic motif functions as well as the wild-type allele in EGFR signaling of eye discs. These observations show that the putative phosphatase activity of Mop is not required for Toll or EGFR signaling. A recent paper demonstrated that the Human Mop homolog, HD-PTP, does not possess enzymatic activity (Huang, 2010).

The Toll signaling pathway has been characterized extensively during Drosophila embryonic development. The Toll protein has been shown to be present at the plasma membrane in the syncytial blastoderm. The majority of MyD88 and Tube also localize to the plasma membrane. However, a significant fraction of these could be detected as punctate structures in syncytial embryos. Mop and Hrs are required for Spätzle-dependent Cactus degradation, and both are essential endosomal proteins. These observations suggest that endocytosis of the Toll receptor is necessary for normal Toll signaling. Expression of chimeric Tube or Pelle proteins fused to the N-terminal 90 amino acid residues of Src activates Toll signaling without ligand binding in Drosophila embryos. The N-terminal region of Src contains a bipartite targeting sequence including the myristylation signal, and the Src protein is known to shuttle between the plasma membrane and endosomes. It is possible that the signal is initiated from endosomes under those experimental conditions (Huang, 2010).

Endocytosis is a dynamic process that regulates various signaling pathways in both a positive and negative manner. Mutations in the Drosophila tumor suppressor gene lethal giant discs (lgd) result in endosomal defects and overactivation of the Notch signaling pathway. In addition to the Toll signaling, endocytic pathway is required for EGFR activation and Wingless signaling. Mammalian TLR4 induces TRAM-TRIF-dependent IRF3 activation from endosomes after initiating the TIRAP-MyD88-dependent NFkappaB signaling at the plasma membrane (Kagan, 2008). The findings that Mop and Hrs are required for Toll signaling suggest that endocytosis has an evolutionarily conserved role in Drosophila Toll and mammalian TLR4 signaling. However, it is interesting to note that endocytosis is required for IRF3, but not NF-kappaB signaling in mammalian cells, but is required for NF-kappaB signaling in Drosophila (Huang, 2010).

Myopic acts in the endocytic pathway to enhance signaling by the Drosophila EGF receptor

Endocytosis of activated receptors can control signaling levels by exposing the receptors to novel downstream molecules or by instigating their degradation. Epidermal growth factor receptor (EGFR) signaling has crucial roles in development and is misregulated in many cancers. Myopic, the Drosophila homolog of the Bro1-domain tyrosine phosphatase HD-PTP, promotes EGFR signaling in vivo and in cultured cells. myopic is not required in the presence of activated Ras or in the absence of the ubiquitin ligase Cbl, indicating that it acts on internalized EGFR, and its overexpression enhances the activity of an activated form of EGFR. Myopic is localized to intracellular vesicles adjacent to Rab5-containing early endosomes, and its absence results in the enlargement of endosomal compartments. Loss of Myopic prevents cleavage of the EGFR cytoplasmic domain, a process controlled by the endocytic regulators Cbl and Sprouty. It is suggested that Myopic promotes EGFR signaling by mediating its progression through the endocytic pathway (Miura, 2008).

The Epidermal growth factor receptor (EGFR) is required for cell differentiation and proliferation in numerous developmental systems, and activation of the human EGFR homologs, ERBB1-4, is implicated in many cancers. EGFR signaling events are terminated following removal of the receptor from the cell membrane by endocytosis. Ubiquitylation of EGFR by Cbl, an E3 ubiquitin ligase, initiates its internalization into clathrin-coated vesicles and its transit through early and late endosomes, which differ by the exchange of Rab7 for Rab5. EGFR can either return to the cell surface in Rab11-containing recycling endosomes, or reach the lysosome for degradation. Delivery of receptors to the lysosome requires sorting from the limiting membrane of late endosomes into the internal vesicles of multivesicular bodies (MVBs), a process mediated by four endosomal sorting complexes required for transport (ESCRT-0, I, II and III). Ubiquitylated receptors are bound by Hepatocyte growth factor regulated tyrosine kinase substrate (Hrs) in ESCRT-0, Tumor susceptibility gene 101 (Tsg101; also known as Erupted) in ESCRT-I and Vacuolar protein sorting 36 (Vps36) in ESCRT-II, and their deubiquitylation and internalization are coordinated by ESCRT-III (Miura, 2008).

Genetic or pharmacological blocks of endocytosis prevent degradation of EGFR and other receptors. In Drosophila, Hrs mutations block MVB invagination, trapping receptor tyrosine kinases (RTKs) and other receptors on the outer membrane of the MVB, and sometimes leading to enhanced signaling. Mutations in the ESCRT complex subunits Tsg101 (ESCRT-I) and Vps25 (ESCRT-II) cause overproliferation owing to the accumulation of mitogenic receptors such as Notch and Thickveins. In mammalian cells, loss of Hrs (also known as Hgs) or Tsg101 results in increased EGFR signaling. However, other studies have demonstrated a positive role for endocytosis in receptor signaling. Mutations affecting the Drosophila trafficking protein Lethal giant discs dramatically increase Notch signaling only in the presence of Hrs, indicating that signaling is maximized at a specific point in the endocytic process. Wingless (Wg) signaling is enhanced by internalization into endosomes, where it colocalizes with downstream signaling molecules. In mammalian cells, EGFR encounters the scaffolding proteins Mek1 partner (Mp1) and p14, which are required for maximal phosphorylation of the downstream component mitogen-activated protein kinase (MAPK), only on endosomes (Miura, 2008).

The characterization of the novel Drosophila gene myopic (mop) is described here. Loss of mop affects EGFR-dependent processes in eye and embryonic development, and reduces MAPK phosphorylation by activated EGFR in cultured cells. Mop acts upstream of Ras activation to promote the function of activated, internalized EGFR. Mop is homologous to human HD-PTP (PTPN23 - Human Gene Nomenclature Database) (Toyooka, 2000), which contains a Bro1 domain that is able to bind the ESCRT-III complex component SNF7 (CHMP4B - Human Gene Nomenclature Database) (Ichioka, 2007; Kim, 2005) and a tyrosine phosphatase domain. Mop is present on intracellular vesicles, and cells lacking mop have enlarged endosomes and reduced cleavage of the EGFR cytoplasmic domain. It is proposed that Mop potentiates EGFR signaling by enhancing its progression through endocytosis. Consistent with this hypothesis, it was found that components of the ESCRT-0 and ESCRT-I complexes are also required for EGFR signaling in Drosophila cells (Miura, 2008).

This study shows that Mop is necessary for EGFR signaling in vivo and in cultured cells. Mop is located on endosomes and affects endosome size, promotes cleavage of EGFR and lysosomal entry of its ligand, and is not required in the absence of the Cbl or Sty proteins that regulate endocytic trafficking of EGFR. These data suggest that Mop enhances EGFR signaling by facilitating its progression through the endocytic pathway. Consistent with this model, Hrs and ESCRT-I subunits also have a positive effect on EGFR signaling (Miura, 2008).

The Bro1 domain of yeast Bro1 is sufficient for localization to late endosomes through its binding to the ESCRT-III subunit Snf7 (Kim, 2005), and this domain is present in many proteins involved in endocytosis. Bro1 itself is required for transmembrane proteins to reach the vacuole for degradation; it promotes protein deubiquitylation by recruiting and activating Doa4, a ubiquitin thiolesterase. Since mutations in the E3 ubiquitin ligase gene Cbl can rescue mop mutant clones, recruiting deubiquitylating enzymes might be one of the functions of Mop. The vertebrate Bro1-domain protein Alix (also known as AIP1 and Pdcd6ip) inhibits EGFR endocytosis by blocking the ubiquitylation of EGFR by Cbl, and by preventing the binding of Ruk (Sh3kbp1), which recruits endophilins, to the EGFR-Cbl complex. However, CG12876, not Mop, is the Drosophila ortholog of Alix (Miura, 2008).

A closer vertebrate homolog of Mop, which has both Bro1 and tyrosine phosphatase domains, has been named HD-PTP in human (Toyooka, 2000) and PTP-TD14 in rat. HD-PTP shares with Alix the ability to bind Snf7 and Tsg101, but does not bind to Ruk (Ichioka, 2007). PTP-TD14 was found to suppress cell transformation by Ha-Ras, and required phosphatase activity for this function (Cao, 1998). The activity of Mop described in this study appears distinct in that Mop acts upstream of Ras activation, and no requirement was demonstrated for the catalytic cysteine in its predicted phosphatase domain. If Mop does act as a phosphatase, Hrs would be a candidate substrate because tyrosine phosphorylation of Hrs by internalized receptors promotes its degradation, and Hrs levels appear reduced in mop mutant clones (Miura, 2008).

Endocytosis has been proposed to play several different roles in receptor signaling. Most commonly, endocytosis followed by receptor degradation terminates signaling. However, endocytosis can also prolong the duration of signaling or influence its subcellular location. Receptors may also signal through different downstream pathways localized to specialized endosomal compartments (Miura, 2008).

Genetic studies in Drosophila have emphasized the importance of endocytic trafficking for receptor silencing. Mutations in Hrs, Vps25 or Tsg101 result in the accumulation of multiple receptors on the perimeter membrane of the MVB, leading to enhanced signaling. Depletion of Hrs or Tsg101 in mammalian cells also results in increased EGFR signaling, although the two molecules have distinct effects on MVB morphology. By contrast, this study found that mop and Hrs mutants exhibit diminished EGFR signaling in vivo, and depletion of mop, Tsg101 or Vps28 reduces EGFR signaling in S2 cells. Progression through the endocytic pathway may thus be required for maximal EGFR signaling, at least in some contexts (Miura, 2008).

Several possible mechanisms could explain such a requirement for endocytic progression. MAPK phosphorylation may be enhanced in the presence of signaling components present on late endosomes. Cleavage of the EGFR cytoplasmic domain, which requires Mop activity, might enhance EGFR signaling. The cleaved intracellular domain of ErbB4 has been shown to enter the nucleus and regulate gene expression (Sardi, 2006), suggesting the possibility that Mop affects a nuclear function of EGFR in addition to promoting MAPK phosphorylation. Alternatively, the reduction in EGFR signaling in mop mutants could be due to a failure to recycle the receptor to the cell surface. Mutations in the yeast Vps class C genes, which are required for trafficking to late endosomes, also prevent the recycling of cargo proteins. Recycling is essential for EGFR-induced proliferation of mammalian cells, and may promote the localized RTK signaling that drives directional cell migration (Miura, 2008).

Despite the reduction in EGFR signaling in mop mutants, signaling by other receptors such as Notch, Smoothened and Torso is unaffected. This phenotypic specificity could be due to a dedicated function of Mop in the EGFR pathway, or to high sensitivity of EGFR signaling to a general process that requires Mop. Although the Mop-related protein Alix has been found in a complex with EGFR (Schmidt, 2004), no physical interaction of Mop with EGFR could be detected. The function of mop is not limited to promoting EGFR signaling; it also promotes trafficking of Wg and expression of the Wg target gene sens. In addition, mop is required for normal cellularization of the embryo, and its cellularization phenotype is not rescued by removal of Cbl (data not shown) (Miura, 2008). Additional studies will be required to determine whether all endosomes, or only a specific subclass, are affected by mop. Interestingly, EGF treatment of mammalian cells induces EGFR trafficking through a specialized class of MVBs. Although significant colocalization of activated EGFR with Mop was not seen, EGFR may transiently pass through Mop-containing endosomes before accumulating in another compartment. The wing disc appears less sensitive than the eye disc to the effect of mop on EGFR signaling. This might be due to differences in the endogenous levels of Cbl or other mediators of EGFR internalization, or in the strength or duration of signaling necessary to activate target genes, or to the use of a different ligand with distinct effects on receptor trafficking (Miura, 2008).

Taken together, these results identify a positive role for progress through the endocytic pathway and for the novel molecule Mop in EGFR signaling in Drosophila. The importance of upregulation of the trafficking proteins Rab11a, Rab5a and Tsg101 for EGFR signaling in hepatomas and breast cancers highlights the potential value of specific effectors of EGFR endocytosis as targets for anti-cancer therapies (Miura, 2008).


REFERENCES

Search PubMed for articles about Drosophila Myopic

Andersen, J. N., Del Vecchio, R. L., Kannan, N., Gergel, J., Neuwald, A. F. and Tonks, N. K. (2005). Computational analysis of protein tyrosine phosphatases: practical guide to bioinformatics and data resources. Methods 35: 90-114. PubMed ID:15588990

Cao, L., Zhang, L., Ruiz-Lozano, P., Yang, Q., Chien, K. R., Graham, R. M. and Zhou, M. (1998). A novel putative protein-tyrosine phosphatase contains a BRO1-like domain and suppresses Ha-ras-mediated transformation. J Biol Chem 273: 21077-21083. PubMed ID: 9694860

Chen, D. Y., Li, M. Y., Wu, S. Y., Lin, Y. L., Tsai, S. P., Lai, P. L., Lin, Y. T., Kuo, J. C., Meng, T. C. and Chen, G. C. (2012). The Bro1-domain-containing protein Myopic/HDPTP coordinates with Rab4 to regulate cell adhesion and migration. J Cell Sci 125: 4841-4852. PubMed ID:22825871

Doyotte, A., Mironov, A., McKenzie, E. and Woodman, P. (2008). The Bro1-related protein HD-PTP/PTPN23 is required for endosomal cargo sorting and multivesicular body morphogenesis. Proc Natl Acad Sci U S A 105: 6308-6313. PubMed ID:18434552

Gilbert, M. M., Tipping, M., Veraksa, A. and Moberg, K. H. (2011). A screen for conditional growth suppressor genes identifies the Drosophila homolog of HD-PTP as a regulator of the oncoprotein Yorkie. Dev Cell 20: 700-712. PubMed ID:21571226

Gingras, M. C., Zhang, Y. L., Kharitidi, D., Barr, A. J., Knapp, S., Tremblay, M. L. and Pause, A. (2009). HD-PTP is a catalytically inactive tyrosine phosphatase due to a conserved divergence in its phosphatase domain. PLoS One 4: e5105. PubMed ID:19340315

Grant, B. D. and Donaldson, J. G. (2009). Pathways and mechanisms of endocytic recycling. Nat Rev Mol Cell Biol 10: 597-608. PubMed ID:19696797

Huang, H. R., (2010). Endocytic pathway is required for Drosophila Toll innate immune signaling. Proc. Natl. Acad. Sci. 107(18): 8322-7. PubMed ID: 20404143

Ichioka, F., Takaya, E., Suzuki, H., Kajigaya, S., Buchman, V. L., Shibata, H. and Maki, M. (2007). HD-PTP and Alix share some membrane-traffic related proteins that interact with their Bro1 domains or proline-rich regions. Arch. Biochem. Biophys. 457: 142-149. PubMed ID: 17174262

Kagan, J. C., Su, T., Horng, T., Chow, A., Akira, S. and Medzhitov, R. (2008). TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-beta. Nat Immunol 9: 361-368. PubMed ID: 18297073

Kim, J., Sitaraman, S., Hierro, A., Beach, B. M., Odorizzi, G. and Hurley, J. H. (2005). Structural basis for endosomal targeting by the Bro1 domain. Dev. Cell 8: 937-947. PubMed ID: 15935782

Lin, G., Aranda, V., Muthuswamy, S. K. and Tonks, N. K. (2011). Identification of PTPN23 as a novel regulator of cell invasion in mammary epithelial cells from a loss-of-function screen of the 'PTP-ome'. Genes Dev 25: 1412-1425. PubMed ID: 21724833

Mariotti, M., Castiglioni, S. and Maier, J. A. (2009a). Inhibition of T24 human bladder carcinoma cell migration by RNA interference suppressing the expression of HD-PTP. Cancer Lett 273: 155-163. PubMed ID:18835089

Mariotti, M., Castiglioni, S., Garcia-Manteiga, J. M., Beguinot, L. and Maier, J. A. (2009b). HD-PTP inhibits endothelial migration through its interaction with Src. Int J Biochem Cell Biol 41: 687-693. PubMed ID:18762272

Miura, G. I., Roignant, J. Y., Wassef, M. and Treisman, J. E. (2008). Myopic acts in the endocytic pathway to enhance signaling by the Drosophila EGF receptor. Development 135(11): 1913-22. PubMed ID: 18434417

Roberts, M., Barry, S., Woods, A., van der Sluijs, P. and Norman, J. (2001). PDGF-regulated rab4-dependent recycling of alphavbeta3 integrin from early endosomes is necessary for cell adhesion and spreading. Curr Biol 11: 1392-1402. PubMed ID:11566097

Sardi, S. P., Murtie, J., Koirala, S., Patten, B. A. and Corfas, G. (2006). Presenilin-dependent ErbB4 nuclear signaling regulates the timing of astrogenesis in the developing brain. Cell 127: 185-197. PubMed ID: 17018285

Schmidt, M. H., Hoeller, D., Yu, J., Furnari, F. B., Cavenee, W. K., Dikic, I. and Bogler, O. (2004). Alix/AIP1 antagonizes epidermal growth factor receptor downregulation by the Cbl-SETA/CIN85 complex. Mol. Cell. Biol. 24: 8981-8993. PubMed ID: 15456872

Stenmark, H. (2009). Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 10: 513-525. PubMed ID:19603039

Toyooka, S., Ouchida, M., Jitsumori, Y., Tsukuda, K., Sakai, A., Nakamura, A., Shimizu, N. and Shimizu, K. (2000). HD-PTP: A novel protein tyrosine phosphatase gene on human chromosome 3p21.3. Biochem. Biophys. Res. Commun. 278: 671-678. PubMed ID: 11095967

White, D. P., Caswell, P. T. and Norman, J. C. (2007). alpha v beta3 and alpha5beta1 integrin recycling pathways dictate downstream Rho kinase signaling to regulate persistent cell migration. J Cell Biol 177: 515-525. PubMed ID:17485491


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date revised: 5 February 2013

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