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.