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The Drosophila RASSF homolog antagonizes the hippo pathway.

Polesello C, Huelsmann S, Brown NH, Tapon N - Curr. Biol. (2006)

Bottom Line: Wts phosphorylates Yki and thus inhibits its activity and reduces expression of Yki target genes such as the caspase inhibitor DIAP1 and the micro RNA bantam.In this study, we show that the Drosophila RASSF ortholog (dRASSF) restricts Hpo activity by competing with Sav for binding to Hpo.In addition, we observe that dRASSF also possesses a tumor-suppressor function.

View Article: PubMed Central - PubMed

Affiliation: Apoptosis and Proliferation Control Laboratory, Cancer Research UK, London Institute, 44 Lincoln's Inn Fields, London, United Kingdom.

ABSTRACT
Correct organ size is determined by the balance between cell death and proliferation. Perturbation of this delicate balance leads to cancer formation . Hippo (Hpo), the Drosophila ortholog of MST1 and MST2 (Mammalian Sterile 20-like 1 and 2) is a key regulator of a signaling pathway that controls both cell death and proliferation . This pathway is so far composed of two Band 4.1 proteins, Expanded (Ex) and Merlin (Mer), two serine/threonine kinases, Hpo and Warts (Wts), the scaffold proteins Salvador (Sav) and Mats, and the transcriptional coactivator Yorkie (Yki). It has been proposed that Ex and Mer act upstream of Hpo, which in turn phosphorylates and activates Wts. Wts phosphorylates Yki and thus inhibits its activity and reduces expression of Yki target genes such as the caspase inhibitor DIAP1 and the micro RNA bantam. However, the mechanisms leading to Hpo activation are still poorly understood. In mammalian cells, members of the Ras association family (RASSF) of tumor suppressors have been shown to bind to MST1 and modulate its activity . In this study, we show that the Drosophila RASSF ortholog (dRASSF) restricts Hpo activity by competing with Sav for binding to Hpo. In addition, we observe that dRASSF also possesses a tumor-suppressor function.

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dRASSF Antagonizes Hpo Activity(A) Hpo/Sav and Hpo/dRASSF are two distinct complexes. Hpo-Myc, Sav-HA, and GFP-dRASSF were cotransfected in Kc cells. Anti-Myc, anti-HA, anti-dRASSF, or control (anti-Flag) immunoprecipitates were blotted for GFP-dRASSF, Sav-HA, Hpo-Myc, and phospho-MST1. Hpo interacts with Sav and dRASSF (lane 2). Sav and dRASSF interact only with Hpo and not with each other (lanes 3 and 4). The Hpo fraction bound to Sav is highly phosphorylated (lane 3), and that bound to dRASSF is not (lane 4).(B) dRASSF inhibits Hpo phosphorylation. Kc cells were treated for 4 days with eGFP, dRASSF, or Hpo dsRNAi. The addition of STS 3 hr prior to lysis induced Hpo phosphorylation (lane 1). Western blots were probed with dRASSF, Hpo34, and phospho-MST1 antibodies. In the presence of dRASSF dsRNAi, Hpo phosphorylation increased (lane 2). As expected, the Hpo band disappeared upon Hpo dsRNAi treatment (lane 3).(C) Sav competes with dRASSF to bind Hpo. Kc cell lysates expressing, respectively, HpoKD-Flag, HA-dRASSF, Sav-HA (200 ng), and Sav-HA (400 ng) were mixed, incubated overnight, and immunoprecipitated with Flag antibody. Blots were probed with HA and Flag antibodies. Increasing the amount of Sav displaced dRASSF from HpoKD (compare lanes 5 and 6).(D) The dRASSF phenotype is sensitive to hpo loss of function. The histogram represents the total body weight as a percent of control flies (white). The reduction in body size in dRASSF flies can be partially rescued by removal of one copy of hpo (hpo42–48 allele). ∗p < 0.05 (white n = 80, dRASSFX16/X16 n = 80, FRT42D, hpo42–48/+ n = 80, and FRT42D, hpo42–48/+; dRASSFX16/X36 n = 80).(E–E″′) Sav controls dRASSF protein level. GFP (in green) and Sav were expressed in the posterior half of the wing disc by the engrailed-GAL4 (en-GAL4) driver. A robust reduction of dRASSF staining (in red [E′]) was observed in the en domain. [E″′] shows Sav overexpression in a separate disc.(F–I) dRASSF reduces apoptosis induced by sav and wts coexpression. Shown are scanning electron micrographs of Drosophila heads from GMR::Gal4 control animals (F) or from GMR::Gal4/UAS::dRASSF (G), GMR::Gal4;GMR::sav+wts (H), or (I) GMR::Gal4/UAS::dRASSF;GMR::sav+wts (I). Overexpression of dRASSF inhibits the rough-eye phenotype generated by coexpression of Sav and Wts.See Supplemental Experimental Procedures for exact genotypes. Error bars correspond to standard deviations.
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fig3: dRASSF Antagonizes Hpo Activity(A) Hpo/Sav and Hpo/dRASSF are two distinct complexes. Hpo-Myc, Sav-HA, and GFP-dRASSF were cotransfected in Kc cells. Anti-Myc, anti-HA, anti-dRASSF, or control (anti-Flag) immunoprecipitates were blotted for GFP-dRASSF, Sav-HA, Hpo-Myc, and phospho-MST1. Hpo interacts with Sav and dRASSF (lane 2). Sav and dRASSF interact only with Hpo and not with each other (lanes 3 and 4). The Hpo fraction bound to Sav is highly phosphorylated (lane 3), and that bound to dRASSF is not (lane 4).(B) dRASSF inhibits Hpo phosphorylation. Kc cells were treated for 4 days with eGFP, dRASSF, or Hpo dsRNAi. The addition of STS 3 hr prior to lysis induced Hpo phosphorylation (lane 1). Western blots were probed with dRASSF, Hpo34, and phospho-MST1 antibodies. In the presence of dRASSF dsRNAi, Hpo phosphorylation increased (lane 2). As expected, the Hpo band disappeared upon Hpo dsRNAi treatment (lane 3).(C) Sav competes with dRASSF to bind Hpo. Kc cell lysates expressing, respectively, HpoKD-Flag, HA-dRASSF, Sav-HA (200 ng), and Sav-HA (400 ng) were mixed, incubated overnight, and immunoprecipitated with Flag antibody. Blots were probed with HA and Flag antibodies. Increasing the amount of Sav displaced dRASSF from HpoKD (compare lanes 5 and 6).(D) The dRASSF phenotype is sensitive to hpo loss of function. The histogram represents the total body weight as a percent of control flies (white). The reduction in body size in dRASSF flies can be partially rescued by removal of one copy of hpo (hpo42–48 allele). ∗p < 0.05 (white n = 80, dRASSFX16/X16 n = 80, FRT42D, hpo42–48/+ n = 80, and FRT42D, hpo42–48/+; dRASSFX16/X36 n = 80).(E–E″′) Sav controls dRASSF protein level. GFP (in green) and Sav were expressed in the posterior half of the wing disc by the engrailed-GAL4 (en-GAL4) driver. A robust reduction of dRASSF staining (in red [E′]) was observed in the en domain. [E″′] shows Sav overexpression in a separate disc.(F–I) dRASSF reduces apoptosis induced by sav and wts coexpression. Shown are scanning electron micrographs of Drosophila heads from GMR::Gal4 control animals (F) or from GMR::Gal4/UAS::dRASSF (G), GMR::Gal4;GMR::sav+wts (H), or (I) GMR::Gal4/UAS::dRASSF;GMR::sav+wts (I). Overexpression of dRASSF inhibits the rough-eye phenotype generated by coexpression of Sav and Wts.See Supplemental Experimental Procedures for exact genotypes. Error bars correspond to standard deviations.

Mentions: Because Hpo, Sav, and dRASSF all contain a SARAH domain, we speculated that dRASSF might also bind Sav. To test this, we investigated whether dRASSF interacts with Sav by co-IP but repeatedly failed to detect such an interaction (Figure S2C and data not shown). Because the possibility of a ternary complex had been raised by Scheel and Hofmann [13], we then tested whether the three proteins could be found in the same complex. We coexpressed Hpo, Sav, and dRASSF in cultured Kc cells. As expected, Hpo was able to bind Sav and dRASSF (Figure 3A). However, Sav immunoprecipitates only contained Hpo and not dRASSF, and dRASSF immunoprecipitates contained Hpo but not Sav (Figure 3A). We obtained identical results with endogenous IPs by using dRASSF and Sav antibodies (Figure S2C). These data support the notion that Sav and dRASSF are not present in the same complex but are in two different Hpo complexes.


The Drosophila RASSF homolog antagonizes the hippo pathway.

Polesello C, Huelsmann S, Brown NH, Tapon N - Curr. Biol. (2006)

dRASSF Antagonizes Hpo Activity(A) Hpo/Sav and Hpo/dRASSF are two distinct complexes. Hpo-Myc, Sav-HA, and GFP-dRASSF were cotransfected in Kc cells. Anti-Myc, anti-HA, anti-dRASSF, or control (anti-Flag) immunoprecipitates were blotted for GFP-dRASSF, Sav-HA, Hpo-Myc, and phospho-MST1. Hpo interacts with Sav and dRASSF (lane 2). Sav and dRASSF interact only with Hpo and not with each other (lanes 3 and 4). The Hpo fraction bound to Sav is highly phosphorylated (lane 3), and that bound to dRASSF is not (lane 4).(B) dRASSF inhibits Hpo phosphorylation. Kc cells were treated for 4 days with eGFP, dRASSF, or Hpo dsRNAi. The addition of STS 3 hr prior to lysis induced Hpo phosphorylation (lane 1). Western blots were probed with dRASSF, Hpo34, and phospho-MST1 antibodies. In the presence of dRASSF dsRNAi, Hpo phosphorylation increased (lane 2). As expected, the Hpo band disappeared upon Hpo dsRNAi treatment (lane 3).(C) Sav competes with dRASSF to bind Hpo. Kc cell lysates expressing, respectively, HpoKD-Flag, HA-dRASSF, Sav-HA (200 ng), and Sav-HA (400 ng) were mixed, incubated overnight, and immunoprecipitated with Flag antibody. Blots were probed with HA and Flag antibodies. Increasing the amount of Sav displaced dRASSF from HpoKD (compare lanes 5 and 6).(D) The dRASSF phenotype is sensitive to hpo loss of function. The histogram represents the total body weight as a percent of control flies (white). The reduction in body size in dRASSF flies can be partially rescued by removal of one copy of hpo (hpo42–48 allele). ∗p < 0.05 (white n = 80, dRASSFX16/X16 n = 80, FRT42D, hpo42–48/+ n = 80, and FRT42D, hpo42–48/+; dRASSFX16/X36 n = 80).(E–E″′) Sav controls dRASSF protein level. GFP (in green) and Sav were expressed in the posterior half of the wing disc by the engrailed-GAL4 (en-GAL4) driver. A robust reduction of dRASSF staining (in red [E′]) was observed in the en domain. [E″′] shows Sav overexpression in a separate disc.(F–I) dRASSF reduces apoptosis induced by sav and wts coexpression. Shown are scanning electron micrographs of Drosophila heads from GMR::Gal4 control animals (F) or from GMR::Gal4/UAS::dRASSF (G), GMR::Gal4;GMR::sav+wts (H), or (I) GMR::Gal4/UAS::dRASSF;GMR::sav+wts (I). Overexpression of dRASSF inhibits the rough-eye phenotype generated by coexpression of Sav and Wts.See Supplemental Experimental Procedures for exact genotypes. Error bars correspond to standard deviations.
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Related In: Results  -  Collection

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fig3: dRASSF Antagonizes Hpo Activity(A) Hpo/Sav and Hpo/dRASSF are two distinct complexes. Hpo-Myc, Sav-HA, and GFP-dRASSF were cotransfected in Kc cells. Anti-Myc, anti-HA, anti-dRASSF, or control (anti-Flag) immunoprecipitates were blotted for GFP-dRASSF, Sav-HA, Hpo-Myc, and phospho-MST1. Hpo interacts with Sav and dRASSF (lane 2). Sav and dRASSF interact only with Hpo and not with each other (lanes 3 and 4). The Hpo fraction bound to Sav is highly phosphorylated (lane 3), and that bound to dRASSF is not (lane 4).(B) dRASSF inhibits Hpo phosphorylation. Kc cells were treated for 4 days with eGFP, dRASSF, or Hpo dsRNAi. The addition of STS 3 hr prior to lysis induced Hpo phosphorylation (lane 1). Western blots were probed with dRASSF, Hpo34, and phospho-MST1 antibodies. In the presence of dRASSF dsRNAi, Hpo phosphorylation increased (lane 2). As expected, the Hpo band disappeared upon Hpo dsRNAi treatment (lane 3).(C) Sav competes with dRASSF to bind Hpo. Kc cell lysates expressing, respectively, HpoKD-Flag, HA-dRASSF, Sav-HA (200 ng), and Sav-HA (400 ng) were mixed, incubated overnight, and immunoprecipitated with Flag antibody. Blots were probed with HA and Flag antibodies. Increasing the amount of Sav displaced dRASSF from HpoKD (compare lanes 5 and 6).(D) The dRASSF phenotype is sensitive to hpo loss of function. The histogram represents the total body weight as a percent of control flies (white). The reduction in body size in dRASSF flies can be partially rescued by removal of one copy of hpo (hpo42–48 allele). ∗p < 0.05 (white n = 80, dRASSFX16/X16 n = 80, FRT42D, hpo42–48/+ n = 80, and FRT42D, hpo42–48/+; dRASSFX16/X36 n = 80).(E–E″′) Sav controls dRASSF protein level. GFP (in green) and Sav were expressed in the posterior half of the wing disc by the engrailed-GAL4 (en-GAL4) driver. A robust reduction of dRASSF staining (in red [E′]) was observed in the en domain. [E″′] shows Sav overexpression in a separate disc.(F–I) dRASSF reduces apoptosis induced by sav and wts coexpression. Shown are scanning electron micrographs of Drosophila heads from GMR::Gal4 control animals (F) or from GMR::Gal4/UAS::dRASSF (G), GMR::Gal4;GMR::sav+wts (H), or (I) GMR::Gal4/UAS::dRASSF;GMR::sav+wts (I). Overexpression of dRASSF inhibits the rough-eye phenotype generated by coexpression of Sav and Wts.See Supplemental Experimental Procedures for exact genotypes. Error bars correspond to standard deviations.
Mentions: Because Hpo, Sav, and dRASSF all contain a SARAH domain, we speculated that dRASSF might also bind Sav. To test this, we investigated whether dRASSF interacts with Sav by co-IP but repeatedly failed to detect such an interaction (Figure S2C and data not shown). Because the possibility of a ternary complex had been raised by Scheel and Hofmann [13], we then tested whether the three proteins could be found in the same complex. We coexpressed Hpo, Sav, and dRASSF in cultured Kc cells. As expected, Hpo was able to bind Sav and dRASSF (Figure 3A). However, Sav immunoprecipitates only contained Hpo and not dRASSF, and dRASSF immunoprecipitates contained Hpo but not Sav (Figure 3A). We obtained identical results with endogenous IPs by using dRASSF and Sav antibodies (Figure S2C). These data support the notion that Sav and dRASSF are not present in the same complex but are in two different Hpo complexes.

Bottom Line: Wts phosphorylates Yki and thus inhibits its activity and reduces expression of Yki target genes such as the caspase inhibitor DIAP1 and the micro RNA bantam.In this study, we show that the Drosophila RASSF ortholog (dRASSF) restricts Hpo activity by competing with Sav for binding to Hpo.In addition, we observe that dRASSF also possesses a tumor-suppressor function.

View Article: PubMed Central - PubMed

Affiliation: Apoptosis and Proliferation Control Laboratory, Cancer Research UK, London Institute, 44 Lincoln's Inn Fields, London, United Kingdom.

ABSTRACT
Correct organ size is determined by the balance between cell death and proliferation. Perturbation of this delicate balance leads to cancer formation . Hippo (Hpo), the Drosophila ortholog of MST1 and MST2 (Mammalian Sterile 20-like 1 and 2) is a key regulator of a signaling pathway that controls both cell death and proliferation . This pathway is so far composed of two Band 4.1 proteins, Expanded (Ex) and Merlin (Mer), two serine/threonine kinases, Hpo and Warts (Wts), the scaffold proteins Salvador (Sav) and Mats, and the transcriptional coactivator Yorkie (Yki). It has been proposed that Ex and Mer act upstream of Hpo, which in turn phosphorylates and activates Wts. Wts phosphorylates Yki and thus inhibits its activity and reduces expression of Yki target genes such as the caspase inhibitor DIAP1 and the micro RNA bantam. However, the mechanisms leading to Hpo activation are still poorly understood. In mammalian cells, members of the Ras association family (RASSF) of tumor suppressors have been shown to bind to MST1 and modulate its activity . In this study, we show that the Drosophila RASSF ortholog (dRASSF) restricts Hpo activity by competing with Sav for binding to Hpo. In addition, we observe that dRASSF also possesses a tumor-suppressor function.

Show MeSH
Related in: MedlinePlus