doi: 10.1016/j.ydbio.2009.08.026.
Epub 2009 Sep 3.
Phosphorylation-independent repression of Yorkie in Fat-Hippo signaling
Affiliations
- PMID: 19733165
- PMCID: PMC2774787
- DOI: 10.1016/j.ydbio.2009.08.026
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Phosphorylation-independent repression of Yorkie in Fat-Hippo signaling
Dev Biol.
.
Abstract
The Fat-Hippo signaling pathway plays an important role in the regulation of normal organ growth during development, and in pathological growth during cancer. Fat-Hippo signaling controls growth through a transcriptional co-activator protein, Yorkie. A Fat-Hippo pathway has been described in which Yorkie is repressed by phosphorylation, mediated directly by the kinase Warts and indirectly by upstream tumor suppressors that promote Warts kinase activity. We present here evidence for an alternate pathway in which Yorkie activity is repressed by direct physical association with three other pathway components: Expanded, Hippo, and Warts. Each of these Yorkie repressors contains one or more PPXY sequence motifs, and associates with Yorkie via binding of these PPXY motifs to WW domains of Yorkie. This direct binding inhibits Yorkie activity independently from effects on Yorkie phosphorylation, and does so both in vivo and in cultured cell assays. These results emphasize the importance of the relative levels of Yorkie and its upstream tumor suppressors to Yorkie regulation, and suggest a dual repression model, in which upstream tumor suppressors can regulate Yorkie activity both by promoting Yorkie phosphorylation and by direct binding.
Figures
A–C) Horizontal (upper left) and vertical (bottom and right) sections of wing discs with flip-out clones expressing Yki:V5 (wild-type control) or Yki:V53SA (activated Yki), identified by elevated Yki (red), and with nuclei labeled by DAPI (cyan). A) act>y+>Gal4; UAS-yki:V53SA. B) act>y+>Gal4; UAS-Myc:wts.2;UAS-yki:V53SA UAS-ex[ex-3]/UAS-hpo[dMst.3]. In the presence of elevated Wts, Hpo, and Ex, Yki:V53SA-expressing cells become apoptotic and drop basally (arrows). C) act>y+>Gal4 UAS-yki:V5. D-L) Eye discs expressing Yki:V5 (UAS-yki:V5) or Yki:V53SA (UAS-yki:V53SA) under GMR-Gal4 control, stained for elevated Yki (red), and with nuclei labeled by DAPI (cyan). E) Expression of Yki:V53SA results in extensive overgrowth, which is mostly taken up in folds of tissue. F-L) Co-expression of UAS-ex[ex-3], UAS-Myc:wts.2 or UAS-hpo[dMst.3], or combinations thereof, with Yki:V53SA, as indicated. M-T) Close-ups of Yki:V53SA expression in eye discs under GMR-Gal4 control to show nuclear localization, using same genotypes as in E-L.
A) Schematic structures of Ex, Wts, Hpo, and Yki, depicting locations of FERM domain, PPXY motifs, kinase domains, WW domains, Sd binding region of Yki (NH) and Wts phosphorylation sites on Yki (P). B–F) Co-immunprecipitation experiments on proteins expressed in S2 cells. Upper panels (Input) show Western blots on lysates, lower panels (IP) show Western blots on material precipitated on anti-V5 or anti-FLAG beads, as indicated. B) Co-precipitation of tagged Yki and Ex (left) or Yki and Hpo (right) is eliminated or reduced by mutation of the WW domains (−WW) but is not affected by mutation of the Yki phosphorylation sites (3SA). 3SA-WW is a Yki isoform that includes both WW domain and phosphorylation site mutations. C) Co-precipitation of tagged Hpo, Wts, and Ex with Yki. When all four proteins are mixed together, Yki co-precipitates Hpo, Wts, or Ex, and this association is eliminated or reduced by mutation of the WW domains (−WW) but not by mutation of Yki phosphorylation sites (3SA). D) Binding between Yki and Wts requires the PPXY motifs, but not the kinase activity of Wts (Wts-KD). Wts kinase activity, but not the PPXY motifs, promotes phosphorylation of Yki, as evidenced by blotting with phospho-specific antisera (Yki-S168P)(Dong et al., 2007) and by Phos-tag gel analysis (Oh and Irvine, 2008). E) Upper panel shows that FLAG-tagged Hpo can co-precipitate endogenous Yki; lower panel shows that V5 tagged Yki can co-precipitate endogenous Hpo. GFP control proteins are not visible here because their mobilities are distinct from Hpo and Yki. F) Co-precipitation of HpoY591A,(PPXA), Wts, and Yki in S2 cells. Yki-Hpo binding requires the PPXY motif of Hpo, and is not affected by exogenous Wts; Wts-Hpo binding does not require the Hpo PPXY motif.
Expression from a UAS-luciferase reporter is indicated by luciferase activity in lysates of S2 cells transfected to express wild-type (red) or 3SA mutant (green) Gal4-DBD:Yki:V5 fusions (Yki) (or a control Gal4-DBD protein, blue). In A, B, where indicated, Yki transgenes were co-expressed with Flag:Hpo (H), Ex:HA (E), Myc:Wts (W), or Myc:WtsKD or Myc:Wts5xPPxA mutants, using equal amounts of DNA, or, where indicated (5x), a five-fold excess of DNA. Histograms depict the average values from triplicate experiments; error bars indicate standard deviation. The results depicted in A and B are from separate experiments. C) Western blot on lysates from samples in B, showing similar expression of Wts. D) Upper panel shows luciferase activity, lower panel shows Western blot on the same cell lysates. The first three experimental (Yki) samples employed HA-tagged Ex and Gal4-DBD:Yki, and the last three employed V5-tagged Wts and Gal4-DBD:Yki, as indicated. The proteins are distinguished on the Western blots by their mobilities, Gal4-DBD:Yki runs as a doublet.
Portions of wing discs, panels marked by prime symbols show individual channels of immunofluorescent staining. A,B) Expression of UAS-yki:V5 (A) or UAS-yki:V5−WW (B) under ptc-Gal4. C,D) ykiB5 mutant clones in the wing imaginal disc, marked by absence of Yki (green) and stained for Ex (magenta). yki clones are shown to emphasize that the staining in wild-type cells is not background, and that in wild-type Yki co-localization with Ex was not discernible. C) Shows a vertical section, D) Shows a horizontal section, because this is a thin section and the discs are not flat Ex staining is in focus in only part of the image; Ex is also down-regulated within yki mutant clones. E,F) Horizontal (E) and vertical (F) sections through a clone co-expressing Ex, Hpo and Yki:V53SA; (AyGal4; UAS-yki:V5 UAS-hpo[dMst.3]/UAS-ex[ex-3]) a fraction of Yki (green/white) co-localizes with Ex (magenta/white) at the sub-apical membrane, in the absence of Ex over-expression no specific accumulation of Yki at the membrane was detected. G,H) Wing imaginal discs with MARCM clones (marked by presence of GFP, green) of cells mutant for wtsX1 and stained for expression of Diap1 (red). Clones in H also over-express Ex from a UAS-ex transgene. Upregulation of Diap1 (arrows), clone size, and clone shape are similar in the presence and absence of exogenous Ex. I-L) Overlap of Ex (magenta/white) and Yki (green/white) at the sub-apical membrane in wtsX1 mutant clones. I,K show horizontal sections, J,L show vertical sections. Clones in I,J over-express endogenous Ex due to mutation of wts (Hamaratoglu et al., 2006), clones in K,L also over-express Ex from a UAS-ex transgene; at the confocal settings used to detect this elevated Ex, endogenous Ex in wild-type cells is not detected. By contrast to the situation in wild-type, where Ex levels are elevated, a distinct accumulation of Yki could be identified over-lapping Ex. Examples of this are highlighted by the yellow boxes.
A) In the Yki “off” state, Wts is active, and levels of Wts and Ex are high (thick outline). Active Warts phosphorylates Yki, which inhibits Yki by promoting its association with 14-3-3 proteins in the cytoplasm, thereby excluding it from the nucleus. In addition, Wts and Ex can directly bind Yki to exclude it from the nucleus B) In the Yki “on” state, Wts is inactive, and levels of Wts and/or Ex are lower (dashed outline). Components of the Hippo kinase cassette are unphosphorylated, and interactions between them are reduced. Yki is not phosphorylated, and enters the nucleus where it complexes with Sd to promote the transcription of downstream target genes.
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