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1.
FIGURE 2.

FIGURE 2. From: KIBRA Protein Phosphorylation Is Regulated by Mitotic Kinase Aurora and Protein Phosphatase 1.

Aurora kinases phosphorylate KIBRA in vitro. A, schematic diagram of truncated GST-KIBRA constructs. The purified proteins were stained with Coomassie Blue. B, GST-KIBRA-N, -M, and -C proteins were used as substrates for in vitro kinase assays as described under “Experimental Procedures.” Autoradiography shows the 32P incorporation, and Western blot (WB) shows the substrate loading. C, in vitro kinase assays with Xenopus Aurora-A kinases (KD, K169R). D, in vitro kinase assays with Xenopus Aurora-B kinase.

Ling Xiao, et al. J Biol Chem. 2011 October 21;286(42):36304-36315.
2.
FIGURE 7.

FIGURE 7. From: KIBRA Protein Phosphorylation Is Regulated by Mitotic Kinase Aurora and Protein Phosphatase 1.

Phosphorylation of KIBRA Ser539 modulates NF2-KIBRA interaction. A, HEK293T cells were transfected with FLAG-KIBRA and treated with VX680 and/or nocodazole as indicated before immunoprecipitation (IP). Immunoprecipitates and total protein lysates were subjected to Western blot analysis with the indicated antibodies. B, HEK293T cells were co-transfected with FLAG-KIBRA and control siRNA (ctrl) or siRNA against Aurora-A and -B (A/B). Cells were treated with nocodazole as indicated before immunoprecipitation. Immunoprecipitates and total protein lysates were subjected to Western blot analysis with the indicated antibodies. C, HEK293T cells were transfected with FLAG-KIBRA or KIBRA S539A mutant. The transfected cells were treated with or without nocodazole before immunoprecipitation. Immunoprecipitates and total protein lysates were subjected to Western blot analysis with the indicated antibodies. D, HEK293T cells were transfected with various DNAs as indicated. After 24 h, the cells were treated with or without nocodazole, and total protein lysates were subjected to Western blot analysis with the indicated antibodies. E, transfection, nocodazole treatment, and Western blotting were done as in D.

Ling Xiao, et al. J Biol Chem. 2011 October 21;286(42):36304-36315.
3.
FIGURE 5.

FIGURE 5. From: KIBRA Protein Phosphorylation Is Regulated by Mitotic Kinase Aurora and Protein Phosphatase 1.

PP1 dephosphorylates KIBRA in vitro. A, a simplified procedure for phosphatase assays (left panel). In vitro kinase assays used Aurora-A kinase to phosphorylate GST-KIBRA-M with or without V481A/F483A (VF) mutations (right panel). B, in vitro dephosphorylation assays using Aurora-A-phosphorylated GST-KIBRA as substrate as described under “Experimental Procedures.” Data from three independent experiments are shown. Error bars represent S.D. C, in vitro dephosphorylation assays using phosphatases PP1 and PP2A. The graph represents the percentage of dephosphorylation from three independent experiments. Error bars represent S.D. Inset, HEK293T cells were transfected with HA-Akt. Cells were lysed and immunoprecipitated (IP) with HA antibody after a 20-min insulin stimulation. The same amount of PP2A was used to dephosphorylate immunoprecipitated HA-Akt. WB, Western blot.

Ling Xiao, et al. J Biol Chem. 2011 October 21;286(42):36304-36315.
4.
FIGURE 4.

FIGURE 4. From: KIBRA Protein Phosphorylation Is Regulated by Mitotic Kinase Aurora and Protein Phosphatase 1.

Aurora-A kinase associates with KIBRA. A, HEK293T cells were treated with nocodazole. The cells were lysed and immunoprecipitated (IP) with IgG (control) and Aurora-A (Aur-A) antibody. The immunoprecipitates were probed with anti-KIBRA to check for the presence of endogenous KIBRA. Total cell lysates before immunoprecipitation were used as input. HC, heavy chain. B, schematic diagram of KIBRA with various deletions. Boxes 1 and 2 represent WW domains. The binding ability of truncated KIBRA is indicated by “+” (binding) or “−” (no binding). C, HEK293T cells were transfected with various DNAs as indicated. At 2 days post-transfection, cells were lysed and immunoprecipitated with FLAG antibody. The immunoprecipitates were probed with anti-Aurora-A. Total cell lysates before immunoprecipitation were also probed with GFP antibody to show the Aurora-A expression levels. SE, short exposure; LE, long exposure. D, HEK293T cells were transfected with various DNAs as indicated. At 2 days post-transfection, cells were lysed and immunoprecipitated with FLAG antibody. The immunoprecipitates were probed with anti-GFP. Total cell lysates before immunoprecipitation were also analyzed. E, schematic diagram of Aurora-A with various deletions. The binding ability of truncated Aurora-A is indicated by + (binding) or − (no binding). F, HEK293T cells were transfected with various DNAs as indicated. At 2 days post-transfection, cells were lysed and immunoprecipitated with GFP antibody. The immunoprecipitates were probed with anti-FLAG to check for the presence of FLAG-tagged KIBRA protein. Total cell lysates before immunoprecipitation were also analyzed by Western blot analysis with FLAG and GFP antibodies.

Ling Xiao, et al. J Biol Chem. 2011 October 21;286(42):36304-36315.
5.
FIGURE 8.

FIGURE 8. From: KIBRA Protein Phosphorylation Is Regulated by Mitotic Kinase Aurora and Protein Phosphatase 1.

Phosphorylation of KIBRA Ser539 regulates cell cycle progression. A, characterization of Tet-On-inducible MCF-7 cells expressing WT KIBRA or KIBRA S539A mutant. The cell lines were established as described under “Experimental Procedures” and treated with doxycycline (Dox; 1 μg/ml) as indicated. Total protein lysates were subjected to Western blot analysis with the indicated antibodies. B, the cell lines in A were first induced by addition of doxycycline for 24 h, and cells were further treated with (+Noc) or without (−Noc) nocodazole (100 ng/ml for 8 h). Cells were stained with Alexa Fluor 488-conjugated phospho-H3 (p-H3) Ser10 (Cell Signaling Technology), and the percentage of H3 Ser10-positive cells was analyzed by flow cytometry. Data are averages ± S.D. from three independent experiments. Error bars represent S.D. C, the cell lines in A were treated with nocodazole, and mitotic cells were collected by mechanic shake-off. Mitotic cells were replated in normal medium and harvested at the indicated time points. Total cell lysates were analyzed by Western blotting with the indicated antibodies.

Ling Xiao, et al. J Biol Chem. 2011 October 21;286(42):36304-36315.
6.
FIGURE 6.

FIGURE 6. From: KIBRA Protein Phosphorylation Is Regulated by Mitotic Kinase Aurora and Protein Phosphatase 1.

PP1 interacts with and dephosphorylates KIBRA in vivo. A, HEK293T cells were treated with OA at the indicated concentrations (50 nm for 1 h and 1 μm for 30 min) before harvesting. Total protein lysates were subjected to Western blot analysis with the indicated antibodies. B, HEK293T cells were treated with OA (1 μm for 30 min) or vehicle only. Total protein lysates were subjected to Western blot analysis with the indicated antibodies. C, MCF-7 (lanes 1 and 2) or HeLa (lanes 3 and 4) cells were transfected with control (lanes 1 and 3) or siRNA for human PP1c-α, -β, and -γ (lanes 2 and 4). At 36 h post-transfection, endogenous KIBRA was immunoprecipitated (IP). The immunoprecipitates and total protein lysates were subjected to Western blot analysis with the indicated antibodies. We could not detect PP1c-β in either HeLa or MCF-7 cells (data not shown). D, HEK293T cells were transfected with various DNAs as indicated. At 2 days post-transfection, total cell lysates were subjected to Western blot analysis with antibodies as indicated. DN, dominant negative (D64N); VF, V481A/F483A mutant. E, HEK293T cells were transfected with various DNAs as indicated. At 2 days post-transfection, cells were lysed and immunoprecipitated with FLAG antibody. The immunoprecipitates were probed with anti-HA to check for the presence of PP1 protein. Total cell lysates before immunoprecipitation were also analyzed by Western blotting with HA antibody to show the expression of HA-PP1. LC, light chain. F, HEK293T cells were transfected with various DNAs with or without siRNA against KIBRA as indicated. Immunoprecipitation and Western blot analysis were done as in E. KIBRA siRNAs 1 and 2 have been described previously (39).

Ling Xiao, et al. J Biol Chem. 2011 October 21;286(42):36304-36315.
7.
FIGURE 3.

FIGURE 3. From: KIBRA Protein Phosphorylation Is Regulated by Mitotic Kinase Aurora and Protein Phosphatase 1.

Aurora kinases phosphorylate KIBRA at Ser539 both in vitro and in vivo. A, schematic diagram of truncated GST-KIBRA-M constructs. KIBRA Ser539 resides in a highly conserved Aurora phosphorylation consensus ((R/K)X(pS/pT)(I/L/V)). B, in vitro kinase assays with Aurora-A kinase. Autoradiography shows the 32P incorporation, and Western blot (WB) shows the substrate loading. C, in vitro kinase assays using Aurora-A kinase to phosphorylate GST-KIBRA-M2 with or without S539A mutation. D, various DNA constructs were transfected into HEK293T cell as indicated. At 2 days after transfection, cells were subjected to metabolic labeling in the presence of 32P as described under “Experimental Procedures.” Immunoprecipitation (IP), autoradiography, and Western blot analysis were done as in Fig. 1B. E, in vitro kinase assays with Aurora-A kinase without 32P. The samples were probed with a phosphospecific antibody against KIBRA Ser539. F, KIBRA was immunoprecipitated from HeLa cells treated with Taxol or vehicle only and treated with λ-phosphatase as indicated. The samples were probed with phospho-Ser539 and total KIBRA antibodies. G, HeLa cells were transfected with control siRNA (ctrl) or siRNA against Aurora-A and -B (A/B). At 2 days after transfection, cells were treated with DMSO or Taxol, and VX680 was added to the cells for 2 h before harvesting as indicated. The samples were analyzed by Western blotting with the indicated antibodies. H, various DNA constructs were transfected into HEK293T cells as indicated. At 2 days after transfection, cells were lysed and subjected to immunoprecipitation with FLAG antibody. The precipitates were immunoblotted with the indicated antibodies. Total lysates before immunoprecipitation were also subjected to Western blot analysis with the indicated antibody.

Ling Xiao, et al. J Biol Chem. 2011 October 21;286(42):36304-36315.
8.
FIGURE 1.

FIGURE 1. From: KIBRA Protein Phosphorylation Is Regulated by Mitotic Kinase Aurora and Protein Phosphatase 1.

Phosphorylation of KIBRA is regulated by cell cycle in Aurora-dependent manner. A, various cell lysates were treated with or without λ-phosphatase (λ PPase) (see “Experimental Procedures”) and probed with anti-KIBRA antibody. B, FLAG-tagged KIBRA was transfected into HEK293T cells. At 2 days after transfection, cells were either kept under normal culture or metabolically labeled in the presence of 32P (see “Experimental Procedures”). Cells were then lysed, and proteins were immunoprecipitated (IP) using anti-FLAG antibody. Immunoprecipitated products were separated by SDS-PAGE and transferred onto PVDF membrane followed by autoradiography and Western blot analysis. C, HeLa and MCF-7 cells were synchronized at the indicated cell phase. Total cell lysates were subjected to Western blot analysis with the indicated antibodies. Polyclonal anti-KIBRA (39) was used in this assay. Asy, asynchronized. D, HeLa cells were synchronized using the double thymidine method. Total cell lysates were harvested at the indicated time points after release and subjected to Western blot analysis with the indicated antibodies. E, nocodazole-arrested (G2/M) HeLa cells were treated with VX680 (1 μm for 4 h) or DMSO. Total lysates were then subjected to Western blot analysis with the indicated antibodies. Activation of Aurora kinases was judged by T-loop autophosphorylation (phospho-Thr288 in Aurora-A and phospho-Thr232 in Aurora-B). H3 is a substrate for Aurora-B. F, FLAG-KIBRA and GFP-Aurora constructs were transfected into HEK293T cells as indicated. At 2 days after transfection, cells were lysed and treated with or without λ-phosphatase. The lysates were subjected to Western blot analysis with the indicated antibodies. KD, K162R for Aurora-A and K109R for Aurora-B; Aur, Aurora.

Ling Xiao, et al. J Biol Chem. 2011 October 21;286(42):36304-36315.

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