Fig. 4. Disruption of CKIα function by RNAi induces a naked cuticle, Arm protein elevation and expansion of the Engrailed expression domain. Cuticle preparation (A and B), immunostaining patterns of Arm (C and D) and Engrailed (E and F) in the embryos injected with LacZ- (A, C and E) or CKIα-dsRNA (B, D and F).

Fig. 5. Epistatic analysis showing dsh and zw3 have little effect on CKIα-RNAi-mediated Arm elevation. To cultures of S2R+ cells, 7.5 µg of LacZ-, Dsh-, ZW3- or CKIα-dsRNA were added in combinations, so that the total amount of dsRNA per culture was equal to 15 µg. After a 3 day incubation, cellular extracts were subjected to western blot analysis.

Fig. 3. CKIα-RNAi stabilizes Arm but not the 155 kDa Cubitus interruptus (Ci) in clone-8 cells. Lysates from cells incubated with dsRNA were subjected to western blot analysis with anti-Arm antibodies or an antibody against the N-terminal portion of Ci. Cells incubated with or without 20 µM of lactacystin for 6 h were used to demonstrate that both Arm and the 155 kDa Ci are processed by proteasomes. An arrow and an arrowhead indicate the full-length Ci (155 kDa) and the proteolytically processed 75 kDa form, respectively.

Fig. 2. CKIα-RNAi stabilizes Arm protein but does not affect the rate of Arm protein synthesis. (A) Fluorogram showing incorporation of radioactivity into total cellular proteins or immunoprecipitated Arm. (B) Pulse–chase analysis of Arm protein in S2R+ cells treated with LacZ- or CKIα-dsRNA. Fluorograms of anti-Arm immunoprecipitates sampled at the indicated chase time are shown. (C) Kinetics of Arm turnover in S2R+ cells incubated with LacZ- (open squares) or CKIα-dsRNA (filled squares). Pulse–chase data for labeled Arm were quantified by a BAS-2000, with time = 0 set as 100%.

Fig. 10. Model depicting CKI function in the down-regulation of Arm protein. In this figure, CKI and ZW3 are shown to play redundant roles in the phosphorylation of Arm, which causes its ubiquitylation and rapid degradation. It is also possible that CKI phosphorylates Arm in the ubiquitylation machinery and thus strengthens Arm–Slimb interaction, which ensures Arm ubiquitylation. ZW3-mediated Arm phosphorylation is suppressed by Wg signaling via Dsh. The effect of Wg signaling on CKI-induced phosphorylation of Arm in vivo remains elusive. Direct association of CKI with Dsh is indicated in this figure, because overexpression of CKIα induced hyper-phosphorylation of Dsh (the function of this phosphorylation remains unclear, Figure 6A) and direct binding of these two proteins has been shown in vertebrates.

Fig. 1. RNAi-mediated disruption of Drosophila CKI gene expression leads to accumulation of Arm protein in Schneider S2R+ cells. (A) Western blot analysis of the lysates of cells incubated with dsRNA. In the bottom blot, lysates from the pMK-CKIε-HA transfectant induced with (+) or without (–) CuSO₄ were used to demonstrate that the antibody against human CKIε recognizes Drosophila CKIε. (B) Specificity of CKIα- and CKIε-RNAi. The CKIα-HA or CKIε-HA transfectants were incubated with dsRNA for 60 h, and induced with CuSO₄ for a further 12 h, before being subjected to western blotting. (C) Northern blot analysis showing specific degradation of the target mRNA by individual dsRNA.

Fig. 7. CKIα target sequence in Arm protein. (A) Stable S2R+ transfectants expressing various myc-tagged Arm proteins were incubated with 15 µg of LacZ- or CKIα-dsRNA for 60 h, and then incubated for a further 12 h in the presence of CuSO₄. The cell lysates were then subjected to western blot analysis with antibody against the myc-epitope or Dα-catenin. The pMK-Arm-myc constructs used to establish cell lines are shown above the panels. (B) Amino acid sequence of Arm from codon 37 to 75 is shown. Ser and Thr residues mutated in some constructs are shown in bold. The box indicates a stretch of acidic amino acids.

Fig. 8. The Arm ED to QN mutant protein is stable, compared with the wild-type. (A) Transient expression experiment: 0.2 µg of pAcLacZ, together with 0.2 µg of pMK33 vector or various pMK-Arm-myc constructs were introduced into S2R+ cells, and after 36 h CuSO₄ was added for 12 h. Expression levels of myc-tagged Arm were analyzed by western blotting. Note relative β-galactosidase activities in the cell lysates were almost the same. (B) Stable expression experiments. Stably transfected cells (5 × 10⁶) were plated and expression of various myc-tagged Arm was induced with CuSO₄. Expression levels of myc-tagged Arm and Dα-catenin were analyzed (upper two panels). Total RNAs from these cells were subjected to northern blot analysis with Arm or RP49 probe (lower two panels).

Fig. 9. In vitro kinase experiment to determine the major phosphorylation site for CKIα and ZW3 in the N-terminal region of Arm. Various GST–Arm fusion proteins were phosphorylated by CKIα-HA (upper panel) and ZW3-HA (middle panel). HA-immunoprecipitate from naive S2R+ cells was used as a negative control (the right lane in each panel). The same amount (10 µg) of GST or GST–Arm fusion proteins were used for each reaction (50 µl) and reaction mixtures were incubated at 30°C for 10 min, before 10 µl of each was subjected to SDS–PAGE. The bottom panel shows the staining profiles of GST–Arm fusion proteins in the dried gel, from which the autoradiogram shown in the top panel was generated. The arrows and the arrowheads indicate the migration positions of plain GST and GST–Arm fusion proteins, respectively. The open arrow heads show the migration positions of a GST–Arm protein with the ED to QN mutation, which has a higher electrophoretic mobility. The amount of ³²P incorporated into each GST–Arm fusion protein was expressed as a percentage of the amount incorporated into the protein containing the wild-type Arm sequence.

Fig. 6. CKIα phosphorylates Arm in vivo and in vitro. (A) Overexpression of CKIα leads to the hyper-phosphorylation of Arm and Dsh protein. S2R+ transfectants that overexpressed the wild-type or kinase-negative form of HA-tagged CKIα or HA-tagged wild-type ZW3 were cultured in the presence or absence of CuSO₄ for 14 h. Then the cells were incubated for a further 6 h in the presence or absence of lactacystin (20 µM). Cell lysates were subjected to western blot analysis. Hyper-phosphorylated forms of Arm and Dsh are indicated by an open arrow and a closed arrow, respectively. The arrowhead indicates a marked increase in the phosphorylated forms of Arm upon CKIα induction, and this was detected only in the presence of lactacystin. (B) CKIα-RNAi decreased the amount of highly modified forms of Arm. The S2R+ cell cultures preincubated with LacZ- or CKIα-dsRNA for 30 h, were further incubated for 6 h in the presence or absence of 20 µM lactacystin. Western blot of the cell lysates are shown. (C) In vitro kinase assay for CKIα. HA-tagged CKIα immunoprecipitated from cells treated with or without Wg was incubated with His-tagged Arm or a CKI substrate peptide. The upper panel is the autoradiogram showing the kinetcs of Arm phosphorylation. The lower panel shows the kinetics of the substrate peptide phosphorylation with the immunoprecipitates from Wg-treated (filled squares) and non-treated (open squares) cells.