Fig 2: LATS1/2 phosphorylates the T7 residue of CDC26 in vivo.(A, B) The effects of in vitro incubation with LATS1 on T7-phosphorylation of CDC26. (A) Recombinant GST-tagged wild-type CDC26 was incubated with or without active GST-LATS1 and cold ATP, and then subjected to immunoblot analyses with the indicated antibodies. The control reaction (lane 1) included an equivalent amount of distilled water instead of kinase. (B) GST alone, GST-tagged recombinant wild-type (WT) or T7A-mutated CDC26 (left panel), and thrombin-cleaved WT or T7A-mutated CDC26 (right panel) were incubated with active GST-LATS1 and cold ATP for 30 min and then analyzed as described in (A). GST-CDC26 WT and other minor bands at lower molecular weight (that are possible degradation products) are observed (B, left panel). (C) Immunoblot analyses of Ser909 or Thr1079-phosphorylated LATS1 and T7-phosphorylated CDC26 levels in mitotic HeLa cells. The cells were treated with nocodazole for 16 h and then collected at the indicated times after the release from synchronization at prometaphase. (D) The effects of lambda phosphatase (λ-PPase) on the levels of Ser909-phosphorylated LATS1 and T7-phosphorylated CDC26 in extracts of HeLa cells that were grown asynchronously (Async) or treated with nocodazole (noc) or taxol for 16 h. The cell lysates were incubated with or without λ-PPase for 30 min at 30°C and then subjected to immunoblot analyses with the indicated antibodies. (E) The effects of siRNA-mediated knockdown of LATS1 and/or LATS2 on the level of T7-phosphorylated CDC26 in HeLa cells. The cells were transfected with a control, LATS1-specific, or LATS1/LATS2-specific siRNA, treated with nocodazole for 16 h to activate endogenous LATS1, and then subjected to immunoblot analyses using the indicated antibodies. (F) The effect of in vitro incubation with LATS2 on T7-phosphorylation of CDC26. Active GST-LATS1 and LATS2 were incubated with GST-tagged wild-type CDC26 for 30 min in the presence of cold ATP and then subjected to immunoblot analysis with the indicated antibodies. Ponceau S staining was used as a loading control.

Fig 3: 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.

Fig 4: The N-terminus of CDC26 is highly conserved and contains LATS1 consensus motif.(A) Phosphorylation of CDC26 by immunopurified LATS1. HEK293T cells were transfected with a control vector or a vector expressing HA-tagged wild-type (WT) or kinase-dead (KD) LATS1, and then treated with 100 nM okadaic acid for 2 h. Lysates of the cells were immunoprecipitated with an anti-HA antibody. The immunoprecipitates were incubated with GST alone or GST-tagged CDC26 or GST-cleaved CDC26 in an in vitro kinase assay for 30 min at 30°C and then subjected to SDS-PAGE followed by autoradiography (left panel) and Coomassie Blue staining (right panel). (B) Schematic illustration of the human CDC26 deletion mutants and the amino acid sequences of the N-terminal region (N), C-terminal region (C), and mid-region (M) of the protein. Candidate Ser/Thr phosphorylation targets are shown in red. (C) Mapping of the region of CDC26 phosphorylated by LATS1. Active GST-LATS1 (200 ng) was incubated with GST alone or GST-tagged wild-type (WT) CDC26 or the deletion mutants shown in (B). The samples were then subjected to SDS-PAGE followed by autoradiography and Coomassie Blue staining. Asterisks depict the phosphorylated CDC26 deletion mutants. Open arrowhead indicates autophophorylated GST-LATS1. Filled arrowhead depicts non-specifically phosphorylated bands. (D) Consensus LATS1 phosphorylation motifs identified by in vitro kinase assays combined with phosphoproteomic approaches. (E) ClustalW alignment of the amino acid sequences of CDC26 from various species. Consensus sequences are indicated by asterisks. Double and single dots indicate the strong (>0.5) and weak (<0.5) groups of amino acids, all of which scored positively in the PAM250 matrix (Gonnet). The T7 residue matched the Arg-x-x-Ser/Thr (RxxT) LATS1 consensus motif.

Fig 5: LATS1 phosphorylates the conserved N-terminal T7 residue of CDC26 in vitro.(A, C) Phosphorylation of CDC26 by recombinant LATS1. GST alone and GST-tagged wild-type (WT) and T7A-mutated CDC26 were incubated with the indicated amount (200ng/μl) of GST-LATS1 at 30°C for 30 min (A) or 100ng GST-LATS1 at the indicated time (C). The samples were then subjected to SDS-PAGE followed by autoradiography and Coomassie Blue staining. To adjust for differences in the reaction volume in (A), distilled water was added to the reactions in lanes 1, 5, and 9. (B) Quantitative analysis of the autoradiography data shown in (A). The intensity of each band was quantified and normalized to the intensity of the corresponding band on the Coomassie Blue-stained gel. Data are shown as the relative intensities compared with that in the reaction containing 1 μl (200 ng) of WT CDC26 and represent the mean ± standard deviation of three independent experiments. (D) GST-tagged wild-type (WT) and T7A-mutated CDC26-N (Fig. 1B) were incubated with the indicated amount (200ng/μl) of GST-LATS1 at 30°C for 30 min. The samples were then subjected to SDS-PAGE followed by autoradiography and Coomassie Blue staining. Open arrowhead indicates autophophorylated GST-LATS1.