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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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Hpo Interacts with dRASSF and Controls Its Expression Levels(A) Hpo coimmunoprecipitates with dRASSF. Endogenous dRASSF was immunoprecipitated from Kc cells lysates with dRASSF antibodies or control (Myc antibodies). The Membrane was blotted with Hpo66 and dRASSF antibodies.(B) Hpo coimmunoprecipitates with dRASSF in Kc cells. Hpo-Flag and GFP-dRASSF were cotransfected in Kc cells. dRASSF or control (Myc antibodies) immunoprecipitates were blotted for GFP-dRASSF and Hpo-Flag.(C) Hpo lacking its SARAH domain (HpoΔC) does not interact with dRASSF. HpoΔC-Flag and GFP-dRASSF were cotransfected in Kc cells. Anti-dRASSF or control (anti-Myc) immunoprecipitates were blotted for GFP-dRASSF and Hpo-Flag.(D) Hpo controls dRASSF and Sav protein levels. Kc cells were treated with GFP, Hippo, Sav, or dRASSF dsRNAs. Lane 5 cells were treated with GFP dsRNA and 3 hr of Staurosporine (STS). Protein extracts were blotted with dRASSF, Hpo66, Sav, P-MST1, and tubulin antibodies. Hpo RNAi strongly reduces both Sav and dRASSF protein levels. dRASSF RNAi stabilizes Sav but is not sufficient to induce Hpo phosphorylation.(E) Hpo loss of function had no effect on dRASSF mRNA expression. RT-PCRs performed on Kc cell lysates treated with GFP or Hpo RNAi. Hpo, dRASSF, and Actin mRNA levels are shown.(F–F″) Hpo controls dRASSF protein levels in vivo. hpo mutant clones (marked by a lack of GFP) were generated in eye discs via the hpo42–48 allele. A robust reduction of dRASSF staining (in red [F′]) is observed in hpo clones.(G–G″) Ex does not affect dRASSF protein levels. Clones of ex mutant cells (marked by a lack of GFP) were generated in eye discs via the exe1 allele. dRASSF (in red [G′]) staining is unaffected in the clones.
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fig2: Hpo Interacts with dRASSF and Controls Its Expression Levels(A) Hpo coimmunoprecipitates with dRASSF. Endogenous dRASSF was immunoprecipitated from Kc cells lysates with dRASSF antibodies or control (Myc antibodies). The Membrane was blotted with Hpo66 and dRASSF antibodies.(B) Hpo coimmunoprecipitates with dRASSF in Kc cells. Hpo-Flag and GFP-dRASSF were cotransfected in Kc cells. dRASSF or control (Myc antibodies) immunoprecipitates were blotted for GFP-dRASSF and Hpo-Flag.(C) Hpo lacking its SARAH domain (HpoΔC) does not interact with dRASSF. HpoΔC-Flag and GFP-dRASSF were cotransfected in Kc cells. Anti-dRASSF or control (anti-Myc) immunoprecipitates were blotted for GFP-dRASSF and Hpo-Flag.(D) Hpo controls dRASSF and Sav protein levels. Kc cells were treated with GFP, Hippo, Sav, or dRASSF dsRNAs. Lane 5 cells were treated with GFP dsRNA and 3 hr of Staurosporine (STS). Protein extracts were blotted with dRASSF, Hpo66, Sav, P-MST1, and tubulin antibodies. Hpo RNAi strongly reduces both Sav and dRASSF protein levels. dRASSF RNAi stabilizes Sav but is not sufficient to induce Hpo phosphorylation.(E) Hpo loss of function had no effect on dRASSF mRNA expression. RT-PCRs performed on Kc cell lysates treated with GFP or Hpo RNAi. Hpo, dRASSF, and Actin mRNA levels are shown.(F–F″) Hpo controls dRASSF protein levels in vivo. hpo mutant clones (marked by a lack of GFP) were generated in eye discs via the hpo42–48 allele. A robust reduction of dRASSF staining (in red [F′]) is observed in hpo clones.(G–G″) Ex does not affect dRASSF protein levels. Clones of ex mutant cells (marked by a lack of GFP) were generated in eye discs via the exe1 allele. dRASSF (in red [G′]) staining is unaffected in the clones.

Mentions: In mammals, members of the RASSF family are known to interact with MST1 and thus to modulate its pro-apoptotic activity [7]. We therefore tested whether dRASSF can interact with Hpo. We performed coimmunoprecipitation (Co-IP) experiments in Drosophila Kc cells with dRASSF antibodies to immunoprecipitate endogenous protein. As expected, dRASSF robustly coimmunoprecipitated with Hpo (Figure 2A). The association between Hpo and Sav is mediated by these proteins' shared SARAH domains. Likewise, Hpo's SARAH domain is required for its association with dRASSF, as shown by the fact that a truncated form of Hpo (HpoΔC) [18] lacking this domain fails to bring down dRASSF (compare Figures 2B and 2C). Thus, the Hpo SARAH domain can associate with both Sav and dRASSF.


The Drosophila RASSF homolog antagonizes the hippo pathway.

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

Hpo Interacts with dRASSF and Controls Its Expression Levels(A) Hpo coimmunoprecipitates with dRASSF. Endogenous dRASSF was immunoprecipitated from Kc cells lysates with dRASSF antibodies or control (Myc antibodies). The Membrane was blotted with Hpo66 and dRASSF antibodies.(B) Hpo coimmunoprecipitates with dRASSF in Kc cells. Hpo-Flag and GFP-dRASSF were cotransfected in Kc cells. dRASSF or control (Myc antibodies) immunoprecipitates were blotted for GFP-dRASSF and Hpo-Flag.(C) Hpo lacking its SARAH domain (HpoΔC) does not interact with dRASSF. HpoΔC-Flag and GFP-dRASSF were cotransfected in Kc cells. Anti-dRASSF or control (anti-Myc) immunoprecipitates were blotted for GFP-dRASSF and Hpo-Flag.(D) Hpo controls dRASSF and Sav protein levels. Kc cells were treated with GFP, Hippo, Sav, or dRASSF dsRNAs. Lane 5 cells were treated with GFP dsRNA and 3 hr of Staurosporine (STS). Protein extracts were blotted with dRASSF, Hpo66, Sav, P-MST1, and tubulin antibodies. Hpo RNAi strongly reduces both Sav and dRASSF protein levels. dRASSF RNAi stabilizes Sav but is not sufficient to induce Hpo phosphorylation.(E) Hpo loss of function had no effect on dRASSF mRNA expression. RT-PCRs performed on Kc cell lysates treated with GFP or Hpo RNAi. Hpo, dRASSF, and Actin mRNA levels are shown.(F–F″) Hpo controls dRASSF protein levels in vivo. hpo mutant clones (marked by a lack of GFP) were generated in eye discs via the hpo42–48 allele. A robust reduction of dRASSF staining (in red [F′]) is observed in hpo clones.(G–G″) Ex does not affect dRASSF protein levels. Clones of ex mutant cells (marked by a lack of GFP) were generated in eye discs via the exe1 allele. dRASSF (in red [G′]) staining is unaffected in the clones.
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fig2: Hpo Interacts with dRASSF and Controls Its Expression Levels(A) Hpo coimmunoprecipitates with dRASSF. Endogenous dRASSF was immunoprecipitated from Kc cells lysates with dRASSF antibodies or control (Myc antibodies). The Membrane was blotted with Hpo66 and dRASSF antibodies.(B) Hpo coimmunoprecipitates with dRASSF in Kc cells. Hpo-Flag and GFP-dRASSF were cotransfected in Kc cells. dRASSF or control (Myc antibodies) immunoprecipitates were blotted for GFP-dRASSF and Hpo-Flag.(C) Hpo lacking its SARAH domain (HpoΔC) does not interact with dRASSF. HpoΔC-Flag and GFP-dRASSF were cotransfected in Kc cells. Anti-dRASSF or control (anti-Myc) immunoprecipitates were blotted for GFP-dRASSF and Hpo-Flag.(D) Hpo controls dRASSF and Sav protein levels. Kc cells were treated with GFP, Hippo, Sav, or dRASSF dsRNAs. Lane 5 cells were treated with GFP dsRNA and 3 hr of Staurosporine (STS). Protein extracts were blotted with dRASSF, Hpo66, Sav, P-MST1, and tubulin antibodies. Hpo RNAi strongly reduces both Sav and dRASSF protein levels. dRASSF RNAi stabilizes Sav but is not sufficient to induce Hpo phosphorylation.(E) Hpo loss of function had no effect on dRASSF mRNA expression. RT-PCRs performed on Kc cell lysates treated with GFP or Hpo RNAi. Hpo, dRASSF, and Actin mRNA levels are shown.(F–F″) Hpo controls dRASSF protein levels in vivo. hpo mutant clones (marked by a lack of GFP) were generated in eye discs via the hpo42–48 allele. A robust reduction of dRASSF staining (in red [F′]) is observed in hpo clones.(G–G″) Ex does not affect dRASSF protein levels. Clones of ex mutant cells (marked by a lack of GFP) were generated in eye discs via the exe1 allele. dRASSF (in red [G′]) staining is unaffected in the clones.
Mentions: In mammals, members of the RASSF family are known to interact with MST1 and thus to modulate its pro-apoptotic activity [7]. We therefore tested whether dRASSF can interact with Hpo. We performed coimmunoprecipitation (Co-IP) experiments in Drosophila Kc cells with dRASSF antibodies to immunoprecipitate endogenous protein. As expected, dRASSF robustly coimmunoprecipitated with Hpo (Figure 2A). The association between Hpo and Sav is mediated by these proteins' shared SARAH domains. Likewise, Hpo's SARAH domain is required for its association with dRASSF, as shown by the fact that a truncated form of Hpo (HpoΔC) [18] lacking this domain fails to bring down dRASSF (compare Figures 2B and 2C). Thus, the Hpo SARAH domain can associate with both Sav and dRASSF.

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