Fig_4.tif (1.53 MB)

LATS1-mediated phosphorylation of CDC26 T7 inhibits the interaction between CDC26 and APC6.

figure

posted on 2015-02-27, 02:47 authored by Kenta Masuda, Tatsuyuki Chiyoda, Naoyuki Sugiyama, Aldo Segura-Cabrera, Yasuaki Kabe, Arisa Ueki, Koji Banno, Makoto Suematsu, Daisuke Aoki, Yasushi Ishihama, Hideyuki Saya, Shinji Kuninaka

(A–C) Pull-down assays showing the interaction of wild-type and mutant CDC26 with APC6. (A) The interaction of T7-phosphorylated CDC26 with APC6. Recombinant CDC26 and or distilled water (Ctrl) was incubated with 0 or 2 μl (400 ng) of GST-LATS1 in a cold in vitro kinase assay and then incubated with GST-tagged APC6 bound to glutathione-Sepharose beads. The input fraction and the bead-bound proteins were subjected to immunoblot analyses with the indicated antibodies. (B) The interaction of the CDC26 T7A and T7D mutants with APC6. Lysates from HeLa cells expressing FLAG alone (FLAG-mock) or FLAG-tagged wild-type, T7A-mutated, or T7D-mutated CDC26 were incubated with GST-tagged APC6 bound to glutathione-Sepharose beads. The bead-bound proteins and 3% of the input fraction were subjected to immunoblot analyses using the indicated antibodies. (C) HeLa cells expressing FLAG alone (FLAG-mock) or FLAG-tagged wild-type, T7A-mutated, T7D-mutated, or T7E-mutated CDC26 were transfected with an expression vector harboring HA-tagged APC6. Lysates of the transfected cells were immunoprecipitated with control IgG and an anti-FLAG antibody. The immunoprecipitates and 3% of the input fractions were analyzed by immunoblotting with anti-HA and anti-FLAG antibodies.