LATS1-mediated phosphorylation of CDC26 modulates the ubiquitination of PLK1 in vivo.

(A) The effects of wild-type (WT) and kinase-dead (KD) LATS1 on the ubiquitination of PLK1 in vivo. HEK293T cells were co-transfected with FLAG-tagged PLK1, Myc-tagged ubiquitin and a control vector (mock) or WT or KD LATS1, and then PLK1 was immunoprecipitated using an anti-FLAG antibody. The immunoprecipitates and input fractions were analyzed by immunoblotting with antibodies against c-Myc or FLAG. (B) The effect of a phosphor-mimic mutant of CDC26 (T7D) on the ubiquitination of PLK1 in vivo. HeLa cells were treated with a siRNA targeting the 3’UTR of endogenous CDC26 and then induced to express exogenous wild-type, T7A-mutated, or T7D-mutated CDC26 by adding doxycycline to the culture medium. The cells were then co-transfected with FLAG-tagged PLK1 and Myc-tagged ubiquitin and treated with the proteasome inhibitor MG132 (10 μM) for 12 h prior to cell collection. PLK1 was immunoprecipitated using an anti-FLAG antibody, and the immunoprecipitates and input fractions were analyzed by immunoblotting with antibodies against c-Myc or FLAG.