The Drosophila RASSF Homolog Antagonizes the Hippo Pathway

Summary Correct organ size is determined by the balance between cell death and proliferation. Perturbation of this delicate balance leads to cancer formation [1]. 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 2, 3. 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 bantam4, 5, 6. 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 [7]. 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.


Supplemental Experimental Procedures
Generation and Characterization of dRASSF Alleles GE23517 (from Genexel) and EY2800 P elements are viable without apparent phenotypes. Excisions of GE23517 were screened by PCR over the original P element with the following primers: 5 0 -CGATC GATTGTTTACGTTCCGCTGTGC-3 0 and 5 0 -GGGCAGCAGGAGATA TGGTGATTAGTCG-3 0 . Sequencing of the PCR products revealed deletions of 932 bp and 620 bp starting at the P element insertion for the dRASSF X16 and dRASSF X36 alleles, respectively. dRASSF X51 is a 1924 bp deletion removing sequences of both sides of the original P elements. dRASSF 44.2 was generated by mobilization of EY2800. To map the deletion of dRASSF 44.2 , we obtained genomic DNA from homozygous flies and amplified the dRASSF locus by using the primers 5 0 -CATCAACGCCCGGGCTTGCAGTGTA-3 0 and 5 0 -GTCAGCACGGTTTATCAGTGTTGG-3 0 . Sequencing of the PCR product revealed a deletion of 2120 bp including the start sites of transcription and translation. We furthermore confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) that the expression of the neighboring cenB-1A gene was not visibly affected.
Genomic Rescue Construct pWRpA-dRASSF and pAW-dRASSF A 7 kb Spe1-BamH1 fragment of the BAC 17P04 containing the CG4656 locus was cloned into pBluescript, verified, and subsequently cloned into the pWhiteRabbitpolyA transformation vector (N.H.B., unpublished data).

Standard Growth Conditions and Larval Irradiation
For each experiment, 50 L1 larvae (0-2 hr collections) were collected 24 hr after egg deposition (AED). Weight measurements of adult flies were performed as described [S7]. Relative wing and cell sizes were quantified, and cell number values were calculated as described [S8]. Images of adult flies were obtained with a Nikon DXM1200 digital camera on a Leica MZFL III microscope. Wing pictures were obtained with a Zeiss Axioplan microscope. Averages of triplicate and duplicate experiments are presented, respectively, in Figures 1 and 3. L3 larvae were irradiated with 40 Gy of g rays and reared in standard growth conditions.

Kc Cell Assays
In depletion experiments Drosophila embryonic Kc cells were treated with dRASSF, Hpo, or eGFP dsRNAs for 4 days. dsRNAs were synthesized with T7 Ribomax large-scale RNA production systems (Promega) and purified with the MEGAclear system (Ambion). Treating Kc cells with 1 mM staurosporine (Sigma) for 3 hr induced Hpo phosphorylation. Transfections were done with Effectene (Qiagen).

Genetics and Immunocytochemistry
Mosaic tissues were obtained with the FLP/FRT system with eyFLP drivers. Tissues were dissected in 13 PBS, fixed in 4% formaldehyde in PBS for 20 min at room temperature, washed in PBS containing 0.1% Triton X-100 (PBT), transferred in PBT 0.3% Triton for 30 min, and washed again in PBT. Tissues were then blocked for two hours in PBT containing 10% goat serum. Primary antibodies were incubated overnight at 4 C. dRASSF antibodies (59 and 60) were used at 1/200, Sav antibody at 1/100, and phospho-Histone H3 (Upstate) and activated Caspase3 (Cell Signaling) antibodies at 1/500. Secondary antibodies (Rhodamine Red X donkey anti rabbit from Jackson ImmunoResearch) were incubated for 2 hr at room temperature at 1/500. After washes, tissues were mounted in Vectashield (Vector). Fluorescence images were acquired on a Zeiss LSM510 Meta confocal laser scanning microscope (253 and 403 objective lenses). (E and E 0 ) Immunostaining with dRASSF antibody in eye discs containing dRASSF X36 mutant cells (GFP-negative cells). (F) Histogram representing survival rates (in percent) to adulthood after g-irradiation (40 Gy). dRASSF flies are less resistant to irradiation than controls. white n = 150, dRASSF X16 n = 400. n = number of L3 larvae irradiated. Error bars correspond to standard deviations. Figure S2. sav and wts Mutations Do Not Alter dRASSF Levels (A and B) Third-larval-instar eye-imaginal discs stained with anti-dRASSF (red). In sav 3 (A) and wts latsX1 (B) mutant cells (lack of GFP), dRASSF protein levels are not changed. (C) Sav/Hpo and dRASSF/Hpo are two distinct complexes. Kc cell lysates were immunoprecipitated with Myc, dRASSF, or Sav antibodies and blotted with dRASSF, Hpo66, and Sav antibodies. Whereas Hpo binds dRASSF and Sav, Sav did not bring down dRASSF, nor did dRASSF bring down Sav. Duplicate IPs are shown.
Clone Size and Mitotic-Index Quantification GFP-positive and -negative areas were separated with Image J; the same macro as that described in Colombani et al.
[S9] was used. Clones areas were measured with Image J histogram function and Histone H3-positive cells were quantified with the Image J cell counter.