Figure 3. Slimb regulates Kibra (Kib) abundance via a consensus degron.

(A–A’) Depletion of Slimb in the dorsal compartment of the wing imaginal disc results in increased Kib-GFP levels both apically (A) and basally (A’). (B) Alignment of the fly, mouse, and human Kib protein sequences showing the conservation of the putative Slimb degron motif DSGXXS (underlined). The vertical lines indicate conserved residues. (C) Immunoblot showing that depletion of Slimb in S2 cells decreases Kib ubiquitination. (D) Co-IP experiments showing that Kib forms a complex with Slimb in S2 cell lysates in a degron-dependent manner. (E) Ubiquitination of the degron mutant, Kib^S677A, is diminished and is insensitive to Hippo pathway inactivation. Asterisks indicate non-specific bands. (F–G) Widefield fluorescence images of wing discs expressing either UASp-Kib-GFP (F) or UASp-Kib^S677A-GFP (G) with the nub>Gal4 driver; images were taken using identical settings. (H) Immunoblot of wing disc cell lysates (20 discs each) of UASp-Kib-GFP or UASp-Kib^S677A-GFP expressed with the nub>Gal4 driver. (I–J) Ectopic expression of Kib^S677A-GFP in the wing results in stronger growth suppression than expression of wild-type Kib-GFP. Quantification of wing sizes in (I) is represented as mean ± SEM relative to the control; n=number of wings (one wing per fly). Statistical comparison was performed using the one-way analysis of variance (ANOVA) test followed by Tukey’s Honestly Significant Difference (HSD) test.

Figure 3—figure supplement 1. Effect of different E3 ubiquitin ligases involved in the Hippo (Hpo) pathway on Kibra (Kib) levels.

(A–C) Depletion of SCF^Slimb E3 ubiquitin ligase components Slimb (A), Cul1 (B), or SkpA (C) in the dorsal compartment of the wing imaginal disc results in increased Ubi>Kib-GFP levels. (D) Depletion or overexpression of other E3 ubiquitin ligases known to regulate Hpo pathway components has no effect on Ubi>Kib-GFP levels. Yellow dashed line represents the dorsal–ventral boundary, with dorsal side up (for ap>Gal4); yellow arrows indicate the anterior–posterior boundary, with posterior to the right (for hh and en>Gal4). (E–G’) Ubi>Kib-GFP levels are elevated upon depletion of Slimb alone or co-depletion of Slimb and Expanded (Ex), but not when Ex alone is depleted in the posterior compartment of the wing imaginal disc. (H–I’) Confocal images of Kib-GFP or Kib^S677A-GFP expressed under UASp control with nub>Gal4. Kib^S677A-GFP shows similar localization to Kib-GFP apically but Kib^S677A-GFP forms foci basally. Note that images in (I) and (I’) were taken at lower gain than images in (H) and (H’) to avoid saturation. Scale bars=10 μm.