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Genetic interactions between the Drosophila tumor suppressor gene ept and the stat92E transcription factor.

Gilbert MM, Beam CK, Robinson BS, Moberg KH - PLoS ONE (2009)

Bottom Line: We find that this phenotype is very sensitive to the genetic dose of stat92E, the transcriptional effector of the Jak-Stat signaling pathway, and that this pathway undergoes strong activation in ept mutant cells.Genetic evidence indicates that stat92E contributes to cell cycle deregulation and excess cell size phenotypes that are observed among ept mutant cells.These findings identify ept as a cell-autonomous inhibitor of the Jak-Stat pathway and suggest that excess Jak-Stat signaling makes a significant contribution to proliferative and tissue architectural phenotypes that occur in ept mutant tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, United States of America.

ABSTRACT

Background: Tumor Susceptibility Gene-101 (TSG101) promotes the endocytic degradation of transmembrane proteins and is implicated as a mutational target in cancer, yet the effect of TSG101 loss on cell proliferation in vertebrates is uncertain. By contrast, Drosophila epithelial tissues lacking the TSG101 ortholog erupted (ept) develop as enlarged undifferentiated tumors, indicating that the gene can have anti-growth properties in a simple metazoan. A full understanding of pathways deregulated by loss of Drosophila ept will aid in understanding potential links between mammalian TSG101 and growth control.

Principal findings: We have taken a genetic approach to the identification of pathways required for excess growth of Drosophila eye-antennal imaginal discs lacking ept. We find that this phenotype is very sensitive to the genetic dose of stat92E, the transcriptional effector of the Jak-Stat signaling pathway, and that this pathway undergoes strong activation in ept mutant cells. Genetic evidence indicates that stat92E contributes to cell cycle deregulation and excess cell size phenotypes that are observed among ept mutant cells. In addition, autonomous Stat92E hyper-activation is associated with altered tissue architecture in ept tumors and an effect on expression of the apical polarity determinant crumbs.

Conclusions: These findings identify ept as a cell-autonomous inhibitor of the Jak-Stat pathway and suggest that excess Jak-Stat signaling makes a significant contribution to proliferative and tissue architectural phenotypes that occur in ept mutant tissues.

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Related in: MedlinePlus

Dome localization in ept mutant tissue.(A-A″) A clone of ept2 mutant eye disc cells marked by the absence of GFP (green) stained for Dome (blue) and the endocytic marker Hrs (red) shows extensive accumulation of Dome in Hrs-positive structures (yellow arrowhead denotes example of magenta overlap). (B-B″) A confocal image of a section of an ept2 eye-antennal tumor expressing domeΔCYT (using the eyFLP;Actin>CD2>Gal4, UAS-GFP system) and stained for anti-pYStat92E (blue); GFP (green) marks cells that express the domeΔCYT transgene. The anti-pY-Stat92E epitope is strongly reduced in cells that express domeΔCYT but not in the patch of GFP-negative, ept2 mutant cells that do not express domeΔCYT.
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pone-0007083-g006: Dome localization in ept mutant tissue.(A-A″) A clone of ept2 mutant eye disc cells marked by the absence of GFP (green) stained for Dome (blue) and the endocytic marker Hrs (red) shows extensive accumulation of Dome in Hrs-positive structures (yellow arrowhead denotes example of magenta overlap). (B-B″) A confocal image of a section of an ept2 eye-antennal tumor expressing domeΔCYT (using the eyFLP;Actin>CD2>Gal4, UAS-GFP system) and stained for anti-pYStat92E (blue); GFP (green) marks cells that express the domeΔCYT transgene. The anti-pY-Stat92E epitope is strongly reduced in cells that express domeΔCYT but not in the patch of GFP-negative, ept2 mutant cells that do not express domeΔCYT.

Mentions: The autonomous effect of ept loss of pY-Stat92E suggests that this phenotype is not only an indication of Jak-Stat activation, but may also reveal a requirement for ept in controlling an intracellular step in the Jak-Stat cascade. Consequently, we sought to test whether ept loss effects trafficking of the transmembrane receptor Dome. Dome traffics through the late endosome [34], and loss of the hrs gene, which acts at the step immediately preceding ept [reviewed in 42], blocks Dome trafficking and activation even in the presence of Upd [43]. These observations have led to the proposal that movement of Dome into and through the endosomal system is a significant regulatory step in Jak-Stat signaling [43]. Since loss of ept leads to accumulation of certain apical trans-membrane proteins in the late endosome [5], we tested whether ept loss might also affect levels or localization of Dome. An antibody specific to the Dome protein [34] detects much higher levels of Dome in ept cells than in surrounding normal cells (Fig. 6A-A″). This Dome appears as puncta that partially co-localize with the endosomal protein Hrs (see arrowheads in Fig. 6). Loss of ept may also have more mild non-autonomous effects on Dome (see cells to the right of the clone in Fig. 5A″). Since Hrs-dependent movement of Dome into the late-endosome has been proposed to be required for activation of Stat92E [43], we next tested whether the accumulation of the pY-Stat92E epitope in ept mutant cells was dependent on Dome activity. The Actin>CD2>Gal4 “flip-out” chromosome [44] was used in combination with a UAS-GFP transgene and a transgene carrying a dominant-negative form of dome that lacks the C-terminal tail (UAS-domeΔCYT) [45] to produce GFP-positive/domeΔCYT-expressing clones in the background of an ept tumor. ept mutant cells that express the domeΔCYT allele (GFP-positive area in Fig. 6B-B″) do not display excess anti-pY-Stat92E staining, whereas those that do not express domeΔCYT (GFP-negative area in Fig. 6B-B″) retain high levels of the pY-Stat92E epitope. From these data, we conclude that ept loss alters Dome localization and levels in eye imaginal disc cells and this correlates with Dome-dependent accumulation of the pY-Stat92E epitope. This data agrees with a proposed model in which Dome must access the Hrs-positive late endosome in order to activate signaling [43]. The trapping of Dome in this ‘activation’ compartment in ept mutants may therefore contribute to high-level activation of the Jak-Stat pathway observed in these cells.


Genetic interactions between the Drosophila tumor suppressor gene ept and the stat92E transcription factor.

Gilbert MM, Beam CK, Robinson BS, Moberg KH - PLoS ONE (2009)

Dome localization in ept mutant tissue.(A-A″) A clone of ept2 mutant eye disc cells marked by the absence of GFP (green) stained for Dome (blue) and the endocytic marker Hrs (red) shows extensive accumulation of Dome in Hrs-positive structures (yellow arrowhead denotes example of magenta overlap). (B-B″) A confocal image of a section of an ept2 eye-antennal tumor expressing domeΔCYT (using the eyFLP;Actin>CD2>Gal4, UAS-GFP system) and stained for anti-pYStat92E (blue); GFP (green) marks cells that express the domeΔCYT transgene. The anti-pY-Stat92E epitope is strongly reduced in cells that express domeΔCYT but not in the patch of GFP-negative, ept2 mutant cells that do not express domeΔCYT.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747001&req=5

pone-0007083-g006: Dome localization in ept mutant tissue.(A-A″) A clone of ept2 mutant eye disc cells marked by the absence of GFP (green) stained for Dome (blue) and the endocytic marker Hrs (red) shows extensive accumulation of Dome in Hrs-positive structures (yellow arrowhead denotes example of magenta overlap). (B-B″) A confocal image of a section of an ept2 eye-antennal tumor expressing domeΔCYT (using the eyFLP;Actin>CD2>Gal4, UAS-GFP system) and stained for anti-pYStat92E (blue); GFP (green) marks cells that express the domeΔCYT transgene. The anti-pY-Stat92E epitope is strongly reduced in cells that express domeΔCYT but not in the patch of GFP-negative, ept2 mutant cells that do not express domeΔCYT.
Mentions: The autonomous effect of ept loss of pY-Stat92E suggests that this phenotype is not only an indication of Jak-Stat activation, but may also reveal a requirement for ept in controlling an intracellular step in the Jak-Stat cascade. Consequently, we sought to test whether ept loss effects trafficking of the transmembrane receptor Dome. Dome traffics through the late endosome [34], and loss of the hrs gene, which acts at the step immediately preceding ept [reviewed in 42], blocks Dome trafficking and activation even in the presence of Upd [43]. These observations have led to the proposal that movement of Dome into and through the endosomal system is a significant regulatory step in Jak-Stat signaling [43]. Since loss of ept leads to accumulation of certain apical trans-membrane proteins in the late endosome [5], we tested whether ept loss might also affect levels or localization of Dome. An antibody specific to the Dome protein [34] detects much higher levels of Dome in ept cells than in surrounding normal cells (Fig. 6A-A″). This Dome appears as puncta that partially co-localize with the endosomal protein Hrs (see arrowheads in Fig. 6). Loss of ept may also have more mild non-autonomous effects on Dome (see cells to the right of the clone in Fig. 5A″). Since Hrs-dependent movement of Dome into the late-endosome has been proposed to be required for activation of Stat92E [43], we next tested whether the accumulation of the pY-Stat92E epitope in ept mutant cells was dependent on Dome activity. The Actin>CD2>Gal4 “flip-out” chromosome [44] was used in combination with a UAS-GFP transgene and a transgene carrying a dominant-negative form of dome that lacks the C-terminal tail (UAS-domeΔCYT) [45] to produce GFP-positive/domeΔCYT-expressing clones in the background of an ept tumor. ept mutant cells that express the domeΔCYT allele (GFP-positive area in Fig. 6B-B″) do not display excess anti-pY-Stat92E staining, whereas those that do not express domeΔCYT (GFP-negative area in Fig. 6B-B″) retain high levels of the pY-Stat92E epitope. From these data, we conclude that ept loss alters Dome localization and levels in eye imaginal disc cells and this correlates with Dome-dependent accumulation of the pY-Stat92E epitope. This data agrees with a proposed model in which Dome must access the Hrs-positive late endosome in order to activate signaling [43]. The trapping of Dome in this ‘activation’ compartment in ept mutants may therefore contribute to high-level activation of the Jak-Stat pathway observed in these cells.

Bottom Line: We find that this phenotype is very sensitive to the genetic dose of stat92E, the transcriptional effector of the Jak-Stat signaling pathway, and that this pathway undergoes strong activation in ept mutant cells.Genetic evidence indicates that stat92E contributes to cell cycle deregulation and excess cell size phenotypes that are observed among ept mutant cells.These findings identify ept as a cell-autonomous inhibitor of the Jak-Stat pathway and suggest that excess Jak-Stat signaling makes a significant contribution to proliferative and tissue architectural phenotypes that occur in ept mutant tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, United States of America.

ABSTRACT

Background: Tumor Susceptibility Gene-101 (TSG101) promotes the endocytic degradation of transmembrane proteins and is implicated as a mutational target in cancer, yet the effect of TSG101 loss on cell proliferation in vertebrates is uncertain. By contrast, Drosophila epithelial tissues lacking the TSG101 ortholog erupted (ept) develop as enlarged undifferentiated tumors, indicating that the gene can have anti-growth properties in a simple metazoan. A full understanding of pathways deregulated by loss of Drosophila ept will aid in understanding potential links between mammalian TSG101 and growth control.

Principal findings: We have taken a genetic approach to the identification of pathways required for excess growth of Drosophila eye-antennal imaginal discs lacking ept. We find that this phenotype is very sensitive to the genetic dose of stat92E, the transcriptional effector of the Jak-Stat signaling pathway, and that this pathway undergoes strong activation in ept mutant cells. Genetic evidence indicates that stat92E contributes to cell cycle deregulation and excess cell size phenotypes that are observed among ept mutant cells. In addition, autonomous Stat92E hyper-activation is associated with altered tissue architecture in ept tumors and an effect on expression of the apical polarity determinant crumbs.

Conclusions: These findings identify ept as a cell-autonomous inhibitor of the Jak-Stat pathway and suggest that excess Jak-Stat signaling makes a significant contribution to proliferative and tissue architectural phenotypes that occur in ept mutant tissues.

Show MeSH
Related in: MedlinePlus