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First published online 6 February 2008
doi: 10.1242/dev.015255

Development 135, 1081-1088 (2008)
Published by The Company of Biologists 2008
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In vivo regulation of Yorkie phosphorylation and localization

Hyangyee Oh and Kenneth D. Irvine*

Howard Hughes Medical Institute, Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers The State University of New Jersey, Piscataway, NJ 08854, USA.


Figure 1
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  		Fig. 1. Yki is predominantly cytoplasmic. Panels show regions of wing imaginal discs; in this and subsequent figures, panels marked ' show separate channels of the stain on the left. (A,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,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,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,D') ykiB5 mutant clones, marked by absence of lacZ (green), and stained for DNA (DAPI, blue) and Yki (red). (E,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 apicobasal aspect of these cells, but is low in nuclei (e.g. as highlighted by asterisks to the right of nuclei).

 

Figure 2
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  		Fig. 2. Yki is phosphorylated by Wts in vivo. (A) Western blot of lysates of wing imaginal discs from wild-type (wt) animals treated with CIP, or untreated wt, exe1, ftG-rv, exe1 ftG-rv and wtsP2, as indicated, run on a conventional 7.5% PAGE gel, probed with anti-Yki and, as a loading control, anti-{alpha}-Actin. (B) Western blot of lysates of wing imaginal discs from wt or wtsP2 animals treated with CIP, or untreated wt, exe1, ftG-rv, exe1 ftG-rv and wtsP2 as indicated, run on a Phos-Tag gel (25 µM Phos-Tag), probed with anti-Yki and, as a loading control, anti-Actin. Arrow indicates 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-{alpha}-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.

 

Figure 3
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  		Fig. 3. Fat and Hippo signaling regulate the nuclear localization of Yki. (A-H') High magnification views 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 and (H) fatG-rv. Arrows indicate clone edges. (I-J') High magnification views 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.

 

Figure 4
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  		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 panels 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 under-represented 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 amino acids.

 

Figure 5
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  		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) and 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; and (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-{alpha}-Tubulin as a loading control. (F,G) Wing imaginal discs form sd-Gal4 UAS-yki:GFP (F) and sd-Gal4 UAS-yki-S168:GFP (G). Images were collected in parallel with same confocal settings. 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.

 

Figure 6
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  		Fig. 6. Influence of Warts on clone growth and survival. (A-F) Wing imaginal discs from animals in which clones of cells expressing UAS transgenes under the 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 and (F) UAS-hpo UAS-wts UAS-yki-S168A:GFP. GFP expression is green, nuclei are in blue.

 

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