Figure 3.

dNek2 Is An APC/C^Fzr Substrate

(A) Schematic representations of human Nek2A and dNek2 proteins: Nek2A has two functional APC/C recognition motifs, KEN box (gray box) and MR motif (red box). dNek2 has five D box-like motifs (green boxes) and one MR motif (red box).

(B and C) Immunoblotting of D.mel-2 cell lysates stably expressing dNek2-GFP. RNAi of fzr or apc4, but not kanR, accumulated dNek2-GFP and the canonical APC/C substrate, CycA. dNek2-GFP band intensities (relative to kanR) were quantified and the mean values are presented in a bar graph (C) (n = 3, error bars represent SEM).

(D–F) In vitro destruction assays of CycB3 and dNek2, labeled with sulfur-35, in interphase extracts in the presence or absence of the recombinant Xenopus Fzr (X-Fzr) or Drosophila Fzr (D-Fzr). The samples were analyzed in autoradiographs (D). Similar to CycB3 (D) (left panel) dNek2 was efficiently degraded upon the addition of X-Fzr or D-Fzr (D) (right panel). The relative CycB3 (E) or dNek2 (F) band intensities (relative to time 0) were quantified and the mean values are presented in line graphs (n = 3, error bars represent SEM). See also Figure S4.

(G–L) The control (ey-Gal4) and ey>cdc16RNAi1 eye discs carrying pUbq-dNek2-GFP (G–H₁) (green) or UAS-dNek2-GFP (I–J₁) (green), stained for DNA (blue) and ELAV (red). Anterior to the left. Weak dNek2-GFP signals (green) were detected in both the anterior and posterior regions to the MF in the control carrying pUbq-dNek2-GFP (G, G₁) and ey>cdc16RNAi accumulated dNek2-GFP in the posterior region of the eye imaginal discs (H, H₁). In the control eye disc inducing dNek2-GFP by ey-Gal4 (control) (I, I₁), dNek2-GFP signals were detected anteriorly to the MF, but absent in the posterior region in the early L3 stage. ey>cdc16RNAi caused robust accumulation of dNek2-GFP signals in the posterior region of the eye discs (J, J₁). Asterisks mark the MF. Scale bars, 50 μm. dNek2-GFP signal intensities were quantified and their mean values are presented in bar graphs, (K) and (L), respectively. Error bars indicate SEM (n = 8–12, ^∗∗∗p < 0.0001, ^∗p < 0.05).

(M) Immunoblot analysis of eye-antenna disc extracts of the control (ey-Gal4) or ey>cdc16RNAi1 carrying UAS-dNek2-GFP. dNek2-GFP was accumulated upon cdc16RNAi. α-Tubulin was used as a loading control.