Cell-cell signaling coordinates proliferation of metazoan tissues during development, and its alteration can induce malignant transformation. Endocytosis regulates signaling by controlling the levels and activity of transmembrane receptors, both prior to and following ligand engagement. Vps25, a component of the endosomal sorting complex required for transport (ESCRT) machinery that regulates endocytic sorting of signaling receptors, has been identified as an unconventional type of Drosophila tumor suppressor. vps25 mutant cells in the eye disc undergo autonomous neoplastic-like transformation, but they also stimulate nonautonomous cell proliferation. Endocytic trafficking defects in vps25 cells cause endosomal accumulation of the signaling receptor Notch and enhanced Notch signaling. Increased Notch activity leads to ectopic production of the mitogenic JAK-STAT pathway ligand Unpaired, which is secreted from mutant cells to induce overproliferation of the surrounding epithelium. The data show that defects in endocytic sorting can both transform cells and, through heterotypic signaling, alter the behavior of neighboring wild-type tissue (Vaccari, 2005).

Clones mutant for vps25 in the leg disc mimic those expressing an activated form of the Dpp receptor (Thickveins, Tkv). Activated Tkv signaling represses expression of Wingless, allowing Hedgehog to activate expression of the Dpp ligand in ventral cells and generate ventral leg outgrowths. vps25 mutant clones located on the ventral side of the leg imaginal disc similarly upregulate expression of the Dpp ligand, observed with a dpp-lacZ transgene. In contrast, the dpp-lacZ transgene is not activated in vps25 mutant clones in wing discs, where the antagonistic Dpp-Wg regulatory loop does not operate and dpp-lacZ is not induced by Dpp signaling. However, mutant cells in wing discs still mimic overexpressed Tkv, revealed by upregulation of the Dpp target gene, omb-lacZ. These results indicate that Dpp receptor (Tkv) signaling is upregulated in vps25 mutant cells and is responsible for generating ectopic DV organizer activity and consequent ventral leg outgrowths (Thompson, 2005).

Metazoan animals must coordinate the proliferation of tissues to permit normal development of individual organs and the organism as a whole. Coordination of tissue growth occurs through signal transduction networks that regulate the underlying cell cycle machinery. These signal transduction networks rely on surface receptors to communicate with neighboring cells. Proper regulation of this network is essential, since aberrant cell-cell communication arising from alterations in receptor number or function can lead to uncoordinated proliferation and the formation of benign and malignant tumors (Vaccari, 2005).

While the importance of spatiotemporally controlled ligand and receptor synthesis in controlling cell-cell signaling has long been known, a recently appreciated mode of signal regulation involves endocytosis. Endocytosis of ligand-bound, activated receptors can turn off signaling by removing the receptor from the cell surface, or it can promote signaling by bringing an activated receptor in proximity to internally localized signal transduction components. In addition to these mechanisms, endocytosis can control the steady-state level of nonactivated receptors on the cell surface, which will determine the cell’s sensitivity to ligand (Vaccari, 2005).

While the stimuli for internalization vary among receptors, ubiquitination of endocytosed receptors is an emerging common theme. Ubiquitination may play a role in promoting receptor internalization per se, as well as the eventual fate of the internalized receptor. Internalized receptors generally traffic along one of two itineraries: either they are returned to the cell surface via a recycling endosome, or they are transported to the lysosome for degradation. Ubiquitination often promotes the latter route. Many ubiquitinated receptors reach a prelysosomal endocytic compartment called the multivesicular body (MVB). Inward budding of the limiting membrane then carries these receptors into internal vesicles of the MVB lumen, which permits the entire receptor to be degraded upon subsequent fusion with the lysosome (Vaccari, 2005).

The cellular machinery that sorts ubiquitinated proteins for lysosomal degradation has been identified through yeast genetic screening. Mutations in a group of genes called 'class E vps' (vesicular protein sorting) genes give rise to an expanded endosomal compartment that accumulates ubiquitinated transmembrane proteins destined for degradation. Eleven of the identified class E vps proteins form four complexes, termed Hrs/Stam and ESCRT (endosomal sorting complex required for transport) -I, -II, and -III. Extensive biochemical and cell biological experiments (Babst, 2005) have led to a model in which Hrs (see Drosophila Hrs) binds ubiquitinated cargo in the early endosome and then recruits the ESCRT-I complex from the cytoplasm (See Drosophila Tumor suppressor protein 101 as an example of an ESCRT-I protein). Endosomal recruitment brings ESCRT-I in proximity to and activates ESCRT-II, which receives the ubiquitinated cargo. ESCRT-II then induces the assembly of ESCRT-III, to which ESCRT-II passes its cargo in kind. The net result of this process is to concentrate lysosome-destined cargo into a membrane subdomain that will invaginate inward, isolating transmembrane receptors from the general cellular cytoplasm. Since receptor signaling can occur from endosomal environments as well as from the cell surface, sorting by ESCRT proteins may modulate the amplitude and kinetics of various signal transduction pathways (Vaccari, 2005).

This study shows that the ESCRT-II complex member Vps25 acts as an unconventional type of Drosophila tumor suppressor. In addition to cell-autonomous loss of cell polarity and proliferation control, mutations in vps25 cause ectopic mitogenic signaling. In mutant tissue, Notch is trapped in an endosomal compartment, and inappropriate Notch activation results. This ectopic Notch activity induces production of the secreted signal Unpaired (Upd), which stimulates excess cell division in neighboring wild-type cells. A mutation in a single gene thus causes both persistent proliferation of mutant cells and also induction of overproliferation in surrounding tissue (Vaccari, 2005).

A model is presented for tissue transformation in vps25 mosaic epithelia. In wild-type epithelial cells, Notch is endocytosed and degraded via MVB sorting in endosomes. In vps25 mutant cells, Notch is endocytosed but fails to be degraded due to impaired MVB sorting; thus, it accumulates in enlarged endosomes. vps25 mutant cells also fail to polarize, to exit the cell cycle and to differentiate; they are later eliminated by apoptosis. Due to ectopic Notch activation, vps25 mutant cells produce and secrete Upd. Via the JAK-STAT pathway, the ectopic Upd promotes extra growth of the neighboring wild-type epithelium. This heterotypic signaling process echoes aspects of the tumor-host interactions observed during malignant transformation of mammalian tissues (Vaccari, 2005).

Class E vps proteins have been studied in cultured vertebrate cells (Babst, 2005), but the early lethality of mutant mice and cell cycle arrest seen in tissue-specific inactivation have hampered functional analyses in mammals. As in yeast and mammals, loss of ESCRT-II function in flies causes accumulation of ubiquitinated proteins in an enlarged endosomal structure, indicating that the cell biological role of ESCRT-II is conserved across phylogeny. Trapping of Notch in an early endosomal compartment in hrs mutants, and in an Hrs-positive compartment in vps25 mutants, is also consistent with the ordering of class E protein functions in yeast. ESCRT-II physically interacts with both ESCRT-I and ESCRT-III, and identical phenotypes on endosomal organization and sorting are seen in yeast mutant for any ESCRT complex member. Thus, the phenotype described here probably represents that of general ESCRT absence in flies (Vaccari, 2005).

It was surprising to find strong differences between the Drosophila mutant phenotypes of vps25 and hrs. In yeast, deletion of the hrs homolog vps27 causes an endosomal and cargo accumulation phenotype that is indistinguishable from deletion of ESCRT complex members. However, the immunoreactivity to Notch, ubiquitin, and the early endosomal marker Avalanche (Avl), which is largely regular in hrs cells (Jekely, 2003), is highly irregular in vps25 cells. Loss of vps25, which acts downstream of hrs in the endosomal sorting pathway in yeast (Babst, 2002b) thus causes a more severe disruption in endosomal organization than hrs. It remains possible that other Drosophila gene products can partially substitute for Hrs or that the N-terminally truncated protein produced by the single hrs allele (Lloyd, 2002) retains some function. The distinct endosomal phenotypes of hrs and vps25 are significant given their different affects on signaling pathways and dramatically disparate functions in controlling both autonomous and nonautonomous proliferation (Vaccari, 2005).

The autonomous overproliferation of vps25 mutant cells strongly resembles that of tissues mutant for other Drosophila neoplastic tumor suppressor genes, including the endocytic syntaxin-encoding avl (Bilder, 2004; Lu, 2005). In addition to the immense increase of cell numbers seen in mutant eye discs, vps25 cells resemble avl cells in misdistribution of polarized proteins and cellular junctions, and both genes are required for the endocytic trafficking and degradation of Notch (Lu, 2005). However, their effects on Notch localization and Notch pathway activity are quite different. In vps25 cells, Notch is trapped internally in the early endosome, and high levels of Notch activity are seen. In avl tissue, where Notch is trapped at the cell surface, ectopic pathway activation is not seen despite the elevated pools of Notch protein. Similarly, ectopic Notch pathway activation is not seen in cells mutant for other characterized neoplastic tumor suppressor genes (D. Bilder, unpublished data cited in Vaccari, 2005). While a requirement for Notch in the cell-autonomous overproliferation of vps25 mutant tissue has not been directly tested, the avl phenotype suggests that altered activities of other mistrafficked membrane proteins, perhaps including the apical membrane determinant Crumbs (Lu, 2005), are responsible for the persistent proliferation of mutant cells (Vaccari, 2005).

By contrast, the nonautonomous tissue growth induced by vps25 (but not avl) cells is spurred by Notch-mediated production of the extracellular ligand Upd. Overexpression of Upd, either alone or in response to ectopic Notch expression, can cause hyperproliferation of cells anterior to the morphogenetic furrow resembling the phenotype of vps25 mosaic clones. Moreover, Upd overexpression phenotypes, like vps25 mutant phenotypes, are suppressed by STAT92E heterozygosity. Importantly, Notch also acts through Upd to regulate eye disc tissue growth during wild-type development. Thus, activation of Upd by Notch in vps25 mutant eye cells is an appropriate cellular response in an inappropriate developmental context that leads to tumorous tissue growth (Vaccari, 2005).

In mammalian immune cells, ectopic Notch pathway activity can lead to lymphomas, while, during normal development, Notch activation induces production of interleukin-4 and related molecules, which, like Upd, signal through the JAK-STAT pathway. Intriguingly, a recent report describes a critical role for Interleukin-8 expression in neovascularization of Ras-transformed human cells. While a number of cytokines are among the secreted factors that can be produced in tumors, there are no examples in which mammalian Upd orthologs specifically have been shown to modify the tumor environment of Notch-induced malignancies, for instance by promoting angiogenesis or recruiting stroma. Nevertheless, it is speculated that heterotypic signaling by Upd-like factors might alter the proliferation rates of untransformed cells relative to tumor cells, ultimately favoring tumor expansion. Further investigations will be required to establish whether the vps25 mutant phenotype represents a novel inductive mechanism relevant to mammalian tumor-host interactions (Vaccari, 2005).

Notch and its ligands are distributed widely throughout development, yet Notch activity is highly localized to specific times and places. Many posttranslational mechanisms are involved in restricting Notch activity, including ligand presentation, modification of the receptor by sugars, and proteolytic processing to create active receptor forms. Each of these processes both provides a potential point of regulation and ensures that inappropriate activation does not occur, which is critical due to the potent effects of Notch-mediated signals. The vps25 phenotype described here highlights another mechanism that prevents inappropriate activation: endocytic sorting of receptor that is being cleared from the cell surface. In wild-type cells, Notch is continuously internalized and degraded in the lysosome to maintain steady-state levels of surface expression and therefore receptor availability. For this process to accurately control signaling, the cell must ensure that the unliganded Notch is not activated during internalization. The results indicate that prevention of Notch activation requires MVB sorting, and they suggest that rapid transit through the endosomal environment is required to prevent this inappropriate activation (Vaccari, 2005).

How could the endosomal accumulation of Notch in vps25 cells lead to ectopic Notch signaling? The terminal proteolytic cleavage in Notch activation is mediated by the membrane bound γ-secretase activity provided by Presenilin and its associated proteins. The site of γ-secretase activity is controversial, with some evidence pointing toward the cell surface and other evidence pointing to an endosome. Interestingly, ubiquitination of Notch has recently been linked to both its internalization and its activation. A partially processed form of mammalian Notch requires ubiquitination for efficient γ-secretase cleavage and activation, while several Drosophila ubiquitin ligases seem to influence an endosomal sorting decision specifying degradation rather than activation of unliganded Notch. In this regard, it is notable that although hrs and vps25 mutant cells both contain elevated levels of ubiquitinated proteins, only vps25 mutants show ectopic Notch signaling. The differences in Notch signaling activity could in theory arise from differences in the amount of Notch trapped in the different endocytic mutants. However, very high amounts of Notch are present in avl mutant cells, which do not show ectopic Notch signaling (Lu, 2005). Therefore, the possibility is favored that ectopic Notch activity may be due to the locus of endocytic trapping, which differs between avl, hrs, and vps25 mutant cells. Possible mechanisms for inappropriate activation include coaccumulation of Notch and its ligands, prolonged exposure to γ-secretase, or eventual dissociation of the heterodimer in the endosomal environment. These possibilities are not mutually exclusive, and the altered organization of the vps25 endosome in addition to the absence of flux through the compartment is likely to contribute to inappropriate activation of Notch signaling. Future studies will discriminate among these possible mechanisms of ectopic Notch activation (Vaccari, 2005).

While this discusssion has concentrated on the Notch pathway, it is clear that many molecules are trapped in vps25 endosomes and that vps25 mutations are phenotypically pleiotropic due to alterations in a number of signaling pathways. For instance, STAT92E suppression of vps25 phenotypes is less complete than STAT92E suppression of overproliferation mediated by Upd alone, suggesting that additional factors contribute to vps25-induced tissue overgrowth. One candidate that merits exploration is the MAPK signaling cascade, since vps25 mutants enhance gain-of-function alleles of EGFR (Elp) and MAPK (Sem). The latter evidence is consistent with the persistent MAPK signaling described in several class E mutant tissues, including hrs in flies and TSG101 in mammals (Vaccari, 2005).

Thus, the complexity of the vps25 mutant phenotype emphasizes that endosomal sorting is a point of contact between diverse signaling pathways, and a likely regulatory nexus for normal development and for pathology. Since human tumors benefit from the coordinated disruption of multiple signaling pathways, subversion of endosomal sorting may be one susceptible route toward malignant transformation. Increasing evidence implicates defects in trafficking of specific receptors in the ontogeny of mammalian tumors. Moreover, the ESCRT-I complex member TSG101 was originally isolated for a tumor suppressive function in cultured cells, although such a role in vivo has not been established. The accessibility of Drosophila tissues, along with the availability of mutations that block specific steps of endocytic traffic, will help to elucidate how endocytosis affects metazoan signaling and the consequent effects on cell proliferation during development as well as tumorigenesis (Vaccari, 2005).

GENE STRUCTURE

cDNA clone length - 682 bp

Bases in 5' UTR - 75

Exons - 2

Bases in 3' UTR - 82

PROTEIN STRUCTURE

Amino Acids - 174

Structural Domains

See Pfam listing for ESCRT-II

EVOLUTIONARY HOMOLOGS

ESCRT-II in yeast

Sorting of ubiquitinated endosomal membrane proteins into the MVB pathway is executed by the class E Vps protein complexes ESCRT-I, -II, and -III, and the AAA-type ATPase Vps4. This study characterizes ESCRT-II, a soluble approximately 155 kDa protein complex formed by the class E Vps proteins Vps22, Vps25, and Vps36. This protein complex transiently associates with the endosomal membrane and thereby initiates the formation of ESCRT-III, a membrane-associated protein complex that functions immediately downstream of ESCRT-II during sorting of MVB cargo. ESCRT-II in turn functions downstream of ESCRT-I, a protein complex that binds to ubiquitinated endosomal cargo. It is proposed that the ESCRT complexes perform a coordinated cascade of events to select and sort MVB cargoes for delivery to the lumen of the vacuole/lysosome (Babst, 2002b).

The multivesicular-body (MVB) pathway delivers transmembrane proteins and lipids to the lumen of the endosome. The multivesicular-body sorting pathway has crucial roles in growth-factor-receptor downregulation, developmental signalling, regulation of the immune response and the budding of certain enveloped viruses such as human immunodeficiency virus. Ubiquitination is a signal for sorting into the MVB pathway, which also requires the functions of three protein complexes, termed ESCRT-I, -II and -III (endosomal sorting complex required for transport). This study reports the crystal structure of the core of the yeast ESCRT-II complex, which contains one molecule of the Vps protein Vps22, the carboxy-terminal domain of Vps36 and two molecules of Vps25, and has the shape of a capital letter 'Y'. The amino-terminal coiled coil of Vps22 and the flexible linker leading to the ubiquitin-binding NZF domain of Vps36 both protrude from the tip of one branch of the 'Y'. Vps22 and Vps36 form nearly equivalent interactions with the two Vps25 molecules at the centre of the 'Y'. The structure suggests how ubiquitinated cargo could be passed between ESCRT components of the MVB pathway through the sequential transfer of ubiquitinated cargo from one complex to the next (Hierro, 2004).

ESCRT-I, -II, and -III protein complexes are sequentially recruited to endosomal membranes, where they orchestrate protein sorting and MVB biogenesis. In addition, they play a critical role in retrovirus budding. Structural understanding of ESCRT interaction networks is largely lacking. The 3.6 Å structure of the yeast ESCRT-II core presented in this study reveals a trilobal complex containing two copies of Vps25, one copy of Vps22, and the C-terminal region of Vps36. Unexpectedly, the entire ESCRT-II core consists of eight repeats of a common building block, a 'winged helix' domain. Two PPXY-motifs from Vps25 are involved in contacts with Vps22 and Vps36, and their mutation leads to ESCRT-II disruption. Purified ESCRT-II binds directly to the Vps20 component of ESCRT-III. Surprisingly, this binding does not require the protruding N-terminal coiled-coil of Vps22. Vps25 is the chief subunit responsible for Vps20 recruitment. This interaction dramatically increases binding of both components to lipid vesicles in vitro (Teo, 2004).

Down-regulation of plasma membrane receptors via the endocytic pathway involves their monoubiquitylation, transport to endosomal membranes and eventual sorting into multi vesicular bodies (MVB) destined for lysosomal degradation. Successive assemblies of endosomal sorting complexes required for transport (ESCRT-I, -II and III) largely mediate sorting of plasma membrane receptors at endosomal membranes, the formation of multivesicular bodies and their release into the endosomal lumen. In addition, the human ESCRT-II has been shown to form a complex with RNA polymerase II elongation factor ELL in order to exert transcriptional control activity. The crystal structure of Vps25 is reported at 3.1 Å resolution. Vps25 crystallizes in a dimeric form and each monomer is composed of two winged helix domains arranged in tandem. Structural comparisons detect no conformational changes between unliganded Vps25 and Vps25 within the ESCRT-II complex composed of two Vps25 copies and one copy each of Vps22 and Vps36. This structural analysis present a framework for studying Vps25 interactions with ESCRT-I and ESCRT-III partners. Winged helix domain containing proteins have been implicated in nucleic acid binding and it remains to be determined whether Vps25 has a similar activity which might play a role in the proposed transcriptional control exerted by Vps25 and/or the whole ESCRT-II complex (Wernimont, 2004).

Mammalian ESCRT-II complex

CHMP6 (charged multivesicular body protein 6) is a human orthologue of yeast Vps (vacuolar protein sorting) 20, a component of ESCRT-III. Various CHMP6 orthologues in organisms ranging from yeast to humans contain the N-myristoylation consensus sequence at each N-terminus. Metabolic labelling of HEK-293 (human embryonic kidney) cells showed the incorporation of [3H]myristate into CHMP6 fused C-terminally to GFP (green fluorescent protein) (CHMP6-GFP). Interactions of CHMP6 with another ESCRT-III component CHMP4b/Shax [Snf7 (sucrose non-fermenting 7) homologue associated with Alix] 1, one of three paralogues of human Vps32/Snf7, and with EAP20 (ELL-associated protein 20), a human counterpart of yeast Vps25 and component of ESCRT-II, were observed by co-immunoprecipitation of epitope-tagged proteins expressed in HEK-293 cells. The in vitro pull-down assays using their recombinant proteins purified from Escherichia coli demonstrated direct physical interactions which were mediated by the N-terminal basic half of CHMP6. Overexpressed CHMP6-GFP in HeLa cells exhibited a punctate distribution throughout the cytoplasm especially in the perinuclear area, as revealed by fluorescence microscopic analysis. Accumulation of LBPA (lysobisphosphatidic acid), a major phospholipid in internal vesicles of an MVB (multivesicular body), was observed in the CHMP6-GFP-localizing area. FLAG-tagged EAP20 distributed diffusely, but exhibited a punctate distribution on co-expression with CHMP6-GFP. Overexpression of CHMP6-GFP caused reduction of transferrin receptors on the plasma membrane surface, but caused their accumulation in the cytoplasm. Ubiquitinated proteins and endocytosed EGF continuously accumulated in CHMP6-GFP-expressing cells. These results suggest that CHMP6 acts as an acceptor for ESCRT-II on endosomal membranes and regulates cargo sorting (Yorikawa, 2005).

date revised: 5 April 2006

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