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. 2008 Mar;135(6):1081-8.
doi: 10.1242/dev.015255. Epub 2008 Feb 6.

In vivo regulation of Yorkie phosphorylation and localization

Hyangyee Oh  1 , Kenneth D Irvine
Affiliations

In vivo regulation of Yorkie phosphorylation and localization

Hyangyee Oh et al. Development. 2008 Mar.

Abstract

Yorkie (Yki), a transcription factor of the Fat and Hippo signaling pathways, is negatively regulated by the Warts kinase. Here, we use Phos-tag gels to characterize Warts-dependent phosphorylation of Yki in vivo, and show that Warts promotes phosphorylation of Yki at multiple sites. We also show that Warts inhibits Yki nuclear localization in vivo, and can promote binding of Yki to 14-3-3 proteins in cultured cells. In vivo assessment of the influence of individual upstream regulators of Warts reveals that some mutants (e.g. fat) have only partial effects on Yki phosphorylation, and weak effects on Yki localization, whereas other genotypes (e.g. ex fat double mutants) have stronger effects on both Yki phosphorylation and localization. We also identify serine 168 as a critical site through which negative regulation of Yki by Warts-mediated phosphorylation occurs, but find that this site is not sufficient to explain effects of Hippo signaling on Yki in vivo. These results identify modulation of subcellular localization as a mechanism of Yki regulation, and establish that this regulation occurs in vivo through multiple sites of Warts-dependent phosphorylation on Yki.

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Figures

Fig 1
Fig 1. Yki is predominantly cytoplasmic
Panels show portions of wing imaginal discs; in this and subsequent figures panels marked prime show separate channels of the stain to the left. A) ykiB5 mutant clones (black arrows), marked by absence of lacZ (magenta), with UAS-yki:GFP (green) expressed under ci-Gal4 control. Twin clones (white arrows) are visible in both anterior and posterior compartments. B) Yki:GFP (green) expression in posterior cells (right) under en-Gal4 control; nuclei are marked by DAPI stain (blue). Nuclear Yki:GFP is barely above background. C) Yki-S168A:GFP (green) expression in posterior cells (right) under en-Gal4 control. Levels of nuclear Yki-S168A:GFP are higher than for Yki:GFP, but it is still predominantly cytoplasmic. D) ykiB5 mutant clones, marked by absence of lacZ (green), and stained for DNA (Dapi, blue) and Yki (red). E) Vertical section through the disc shown in D. The center of the wing disc forms a pseudostratified epithelium, with nuclei in different focal planes. Yki staining is detected throughout the apical-basal aspect of these cells, but is low in nuclei (e.g. as highlighted by asterisks to the right of nuclei).
Fig 2
Fig 2. Yki is phosphorylated by Wts in vivo
A) Western blots of lysates of wing imaginal discs from animals of wild type, treated with CIP (wt – CIP), wild type (wt), exe1, ftG-rv, exe1 ftG-rv, wtsP2, as indicated, run on a conventional 7.5% PAGE gel, probed with anti-Yki, and, as a loading control, anti-α-Actin. B) Western blot of lysates of wing imaginal discs from animals of wild type, treated with CIP (wt – CIP), wild type (wt), exe1, ftG-rv, exe1 ftG-rv, wtsP2, wtsP2 treated with CIP, as indicated, run on a Phos-Tag gel (25 μM Phos-Tag), probed with anti-Yki, and, as a loading control, anti-Actin. Arrow points to band corresponding to unphosphorylated Yki. C) Conventional 4–15% PAGE gel, probed with anti-Yki (upper panel), and, as a loading control (lower panel) anti-α-Actin. Lane 1 Shows S2 cell lysate treated with ds RNA corresponding to GFP (control), Lane 2 shows lysate from S2 cells treated with ds RNA corresponding to yki. Yki protein (arrow) is reduced, but a faint background band (above) is unaffected.
Fig 3
Fig 3. Fat and Hippo signaling regulate the nuclear localization of Yki
Panels A-H show close-ups of imaginal discs, stained for Yki (red), nuclei (DAPI, blue), and containing mutant clones marked by absence of GFP (green). A) wtsX1 B) Mer4; exe1 C) fatG-rv D) exe1 E) exe1 fatG-rv F) hpo42-47 G) matse235 H) fatG-rv. Arrows point to clone edges. I, J Show closeups of imaginal discs expressing RNAi lines under ci-Gal4 control, stained for Yki (red), nuclei (DAPI, blue), and Engrailed (green). ci-Gal4 drives expression in anterior cells (yellow line), partially complementary to Engrailed. I) ci-Gal4 UAS-ex RNAi UAS-dcr2. J) ci-Gal4 UAS-fat RNAi UAS-dcr2.
Fig 4
Fig 4. Yki phosphorylation and 14-3-3 binding
A) Western blots (4–15% PAGE) of samples from S2 cells co-transfected with the indicated proteins. Bottom two panels show blot on material precipitated with anti-V5 beads, upper two panel shows input; GFP:V5 (control protein) and 14-3-3:V5 proteins have similar mobility. B) Western blots (using anti-V5 epitope) on lysates of S2 cells co-transfected to express the indicated proteins. Transfection of Wts, Hpo, and Sav promotes phosphorylation of Yki and Yki-S168A, as revealed both on conventional SDS-PAGE (top panels) and Phos-Tag gels (30 μM Phos-Tag, bottom panels), however a distinct mobility isoform (arrow), representing partially phosphorylated Yki, is observed with Yki-S168A mutant but not wild-type Yki on Phos-tag gels. Transfer of heavily phosphorylated proteins from Phos-Tag gels can be poor, hence they may be underrepresented on these blots. C) Western blots of lysates of wing imaginal discs from animals expressing Yki:GFP (lane 1) or Yki-S168A:GFP (lane 2) under sd-Gal4 control. Upper panel shows conventional SDS-PAGE, lower panel shows a Phos-tag gel (60 μM Phos-Tag) D) Western blot on lysates of S2 cells co-transfected to express the indicated proteins, run on a Phos-tag gel (60 μM Phos-Tag). Yki-N is an N terminal fragment of Yki, comprising the first 240 aa.
Fig 5
Fig 5. Yki-S168A is hyperactivated
A–D) Heads of adult flies from wild type (A), UAS-yki GMR-Gal4 (B), UAS-yki:GFP GMR-Gal4 (C), UAS-yki-S168A:GFP GMR-Gal4 (D). E) Western blot (4-15% gradient gel) with anti-Yki on lysates from wing imaginal discs of 1. Wild-type 2. sd-Gal4 UAS-yki:GFP 3. sd-Gal4 UAS-yki-S168:GFP. 4. sd-Gal4 UAS-yki. Upper left panel shows a lower exposure, endogenous Yki is barely visible; upper right panel shows a longer exposure of the same gel, endogenous Yki is now visible. Green arrow marks Yki:GFP, black arrow marks Yki. Lower left panel shows anti-α-Tubulin as a loading control. F,G) Wing imaginal discs form sd-Gal4 UAS-yki:GFP (F) and sd-Gal4 UAS-yki-S168:GFP. Images were collected in parallel with same confocal settings, note that Yki-S168A:GFP expression (green) is weaker than Yki:GFP expression but the disc is overgrown. H,I) Wing imaginal discs containing clones of cells expressing UAS-yki:GFP (H) or UAS-yki-S168:GFP (I) (green), and stained for expression of a th-lacZ reporter (red). th expression is induced by yki-S168:GFP (arrows), but not by yki:GFP. In I) th-lacZ expression in the center of the disc is out of the plane of focus.
Fig 6
Fig 6. Influence of Warts on clone growth and survival
A–F show wing imaginal discs from animals in which clones of cells expressing UAS transgenes under control of actin-Gal4 have been induced by Flip-out: A) UAS-yki:GFP B) UAS-yki-S168A:GFP C) UAS-hpo UAS-wts UAS-GFP D) UAS-yki:GFP UAS-hpo E) UAS-hpo UAS-yki-S168A:GFP F) UAS-hpo UAS-wts UAS-yki-S168A:GFP. GFP expression is green, nuclei are blue.
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References

    1. Basu S, Totty NF, Irwin MS, Sudol M, Downward J. Akt phosphorylates the Yes-associated protein, YAP, to induce interaction with 14-3-3 and attenuation of p73-mediated apoptosis. Mol Cell. 2003;11:11–23. - PubMed
    1. Bennett FC, Harvey KF. Fat Cadherin Modulates Organ Size in Drosophila via the Salvador/Warts/Hippo Signaling Pathway. Curr Biol. 2006;16:2101–10. - PubMed
    1. Chen HK, Fernandez-Funez P, Acevedo SF, Lam YC, Kaytor MD, Fernandez MH, Aitken A, Skoulakis EM, Orr HT, Botas J, et al. Interaction of Akt-phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type 1. Cell. 2003;113:457–68. - PubMed
    1. Cho E, Feng Y, Rauskolb C, Maitra S, Fehon R, Irvine KD. Delineation of a Fat tumor suppressor pathway. Nat Genet. 2006;38:1142–50. - PubMed
    1. Cho E, Irvine KD. Action of fat, four-jointed, dachsous and dachs in distal-to-proximal wing signaling. Development. 2004;131:4489–500. - PubMed

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