Journal: The Journal of Biological Chemistry

Article Title: Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity *

doi: 10.1074/jbc.M110.144220

Figure Lengend Snippet: ASAP and Plk1 co-localize at centrosomes and interact in vivo during mitosis. A and B, asynchronous U-2 OS cells were grown on glass coverslips and fixed in PAF/MTSB (A) or F/PHEM/methanol (B) (see “Materials and Methods”) and labeled with the polyclonal anti-ASAP (green), or the monoclonal anti-Plk1 antibodies (red) and stained with Hoechst 33258 (blue) without (A) or after (B) depolymerization of MTs by incubation on ice for 60 min. The different stages of the cell cycle are shown from prophase (A) or interphase (B) to telophase (scale bar, 10 μm). C, U-2 OS cell lysates were immunoprecipitated (IP) with rabbit IgG, rabbit anti-ASAP, or anti-Myc antibodies, and the precipitates were analyzed by immunoblotting (IB) with antibodies against ASAP or Plk1. Left, asynchronous cells were co-transfected with FLAG-ASAP and Myc-Plk1 (Input, 10% of protein extracts; immunoprecipitated with 200 μg of protein extracts). Right, endogenous proteins were co-immunoprecipitated from synchronized mitotic cells (see “Materials and Methods”) (Input, 10% of protein extracts; immunoprecipitated with 2 mg of protein extracts). A higher exposure is shown for Plk1 immunoblot. See supplemental Fig. S1A for uncropped gels. D, asynchronous U-2 OS cells were transfected with FLAG-ASAP and either Myc-Plk1-WT (left) or Myc-Plk1-PBD mutant (mut) (right) and immunoprecipitated with rabbit IgG or rabbit anti-ASAP antibody. Precipitates were analyzed by immunoblotting with antibodies against ASAP or Plk1. See supplemental Fig. S1B for uncropped gel. E, U-2 OS cells were transfected with FLAG-ASAP, and 24 h later, cell lysates were immunoprecipitated with rabbit IgG or rabbit anti-ASAP antibodies (Input, 10% of protein extracts immunoprecipitated with 200 μg of protein extracts). Precipitates were analyzed by immunoblotting with antibodies against ASAP, Plk1, or Aurora A. See supplemental Fig. S1C for uncropped gel. F, FLAG-ASAP-transfected cells were arrested at the G1/S boundary by thymidine block (2 mm for 24 h) and then released into fresh medium. Samples harvested at the indicated time points were analyzed by immunoblotting using antibodies against ASAP, Plk1, or Aurora A. Inputs are shown on the left and immunoprecipitates on the right. α-Tubulin was used as a loading control.

Article Snippet: The Plk1 inhibitor ZK-thiazolidinone (TAL) was described in Ref. 30 and kindly provided by Bayer Schering Pharma AG.

Techniques: In Vivo, Labeling, Staining, Incubation, Immunoprecipitation, Western Blot, Transfection, Mutagenesis, Blocking Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity *

doi: 10.1074/jbc.M110.144220

Figure Lengend Snippet: Plk1 promotes ASAP localization to centrosomes and spindles through the NEDD1-γ-tubulin pathway. A, asynchronous U-2 OS cells were transfected with Luciferase GL2 (control), ASAP, or Plk1 siRNAs. Cell extracts were immunoblotted (IB) and probed with anti-ASAP, Plk1, and α-actin (loading control) antibodies. For immunofluorescence analysis, asynchronous U-2 OS cells grown on coverslips were transfected with GL2 or Plk1 siRNAs or incubated with 1 μm TAL overnight. Cells were fixed in PAF/MTSB (left panel) or F/PHEM/methanol (right panel), probed with the polyclonal anti-ASAP (green) and the monoclonal anti-Plk1 antibody (red), and stained with Hoechst 33258 (blue) without (left panel) or after (right panel) MT depolymerization by incubation on ice for 60 min (scale bar, 10 μm). The percentage of mitosis with aberrant localization of ASAP is shown in the table. B, asynchronous U-2 OS cells were transfected with GL2 or γ-tubulin siRNAs. Cell extracts were immunoblotted and probed with the anti-ASAP, anti-γ-tubulin, and α-actin (loading control) antibodies. For immunofluorescence analysis, cells were fixed in F/PHEM/methanol, probed with the polyclonal anti-ASAP (green) and the monoclonal anti-γ-tubulin antibody (red), and stained with Hoechst 33258 (blue) without (left panel) or after (right panel) MT depolymerization by incubation on ice for 60 min (scale bar, 10 μm). The percentage of mitosis with aberrant localization of ASAP is shown in the table. C, synchronized mitotic cells were immunoprecipitated (IP) with rabbit IgG or rabbit anti-ASAP antiserum, and precipitates were analyzed by immunoblotting with antibodies against ASAP and γ-tubulin (Input, 10% of protein extracts immunoprecipitated with 2 mg of protein extracts). D, centrosomes were isolated and enriched from U-2 OS cells using a discontinuous sucrose gradient (see “Materials and Methods”). Left panel, anti-ASAP and γ-tubulin antibodies were used for Western blot analysis. Right panel, centrosomes from fraction 2 were spun down onto coverslips and analyzed by immunofluorescence using the polyclonal anti-ASAP (green) and the monoclonal anti-γ-tubulin antibodies (red).

Article Snippet: The Plk1 inhibitor ZK-thiazolidinone (TAL) was described in Ref. 30 and kindly provided by Bayer Schering Pharma AG.

Techniques: Transfection, Luciferase, Control, Immunofluorescence, Incubation, Staining, Immunoprecipitation, Western Blot, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity *

doi: 10.1074/jbc.M110.144220

Figure Lengend Snippet: Plk1 phosphorylates ASAP on S289 in vitro. A, purified, recombinant GST-ASAP, α-casein, or GST was incubated with purified His6-tagged wild-type Plk1 (His6-Plk1-WT) or the kinase-dead mutant (N281A) (His6-Plk1-KD) and [γ-32P]ATP. The kinase reaction mixtures were then analyzed by SDS-PAGE and autoradiography (upper panel); the gel was also stained with Coomassie Blue (lower panel). B, purified GST, GST-ASAP fragments G1–G6 or GST-ASAP were used in a kinase assay with His6-Plk1-WT as described in A and analyzed by SDS-PAGE and autoradiography (upper panel); the Coomassie Blue-stained gel is shown in the lower panel. C, in vitro kinase assays were performed using purified GST-ASAP, GST-ASAP mutants (S289A or S369A/S370A), or GST as described in A and analyzed by SDS-PAGE and autoradiography (upper panel); the Coomassie Blue-stained gel is shown in the lower panel.

Article Snippet: The Plk1 inhibitor ZK-thiazolidinone (TAL) was described in Ref. 30 and kindly provided by Bayer Schering Pharma AG.

Techniques: In Vitro, Purification, Recombinant, Incubation, Mutagenesis, SDS Page, Autoradiography, Staining, Kinase Assay

Journal: The Journal of Biological Chemistry

Article Title: Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity *

doi: 10.1074/jbc.M110.144220

Figure Lengend Snippet: Phosphorylation of ASAP at Ser-289 by Plk1 in vivo occurs at centrosomes during mitosis. A, U-2 OS cell lysates transfected with FLAG-ASAP-WT or FLAG-ASAP-S289A were immunoblotted (IB) with the anti-ASAP-P-S289 (left) and anti-ASAP (right) antibodies. NT, nontransfected cells. α-Tubulin is shown as a loading control. B, asynchronous U-2 OS cells were grown on glass coverslips, fixed in F/PHEM/methanol, probed with the anti-ASAP-Ser(P)-289 (green) and anti-γ-tubulin (red) antibodies, and stained with Hoechst 33258 (blue); the different stages of the cell cycle are shown from G2 to telophase (scale bar, 10 μm). C, U-2 OS cells were synchronized by double thymidine block and transfected with FLAG-ASAP during the first release, as indicated on the schematic. At the indicated time points after the second release, cells lysates were analyzed by immunoblotting using the indicated antibodies. α-Actin was used as a loading control. D, U-2 OS cells were transfected with FLAG-ASAP-WT, synchronized by thymidine block, and released in the presence of either dimethyl sulfoxide (DMSO) (control) or 1 μm TAL for 13 h. Cell extracts were immunoblotted with the anti-ASAP-Ser(P)-289, anti-FLAG, anti-Plk1, and anti-H3-Ser(P)-10 antibodies. α-Actin is shown as a loading control. E, U-2 OS cells were grown on glass coverslips, transfected with YFP-ASAP-WT (left panels) or YFP-ASAP-S289A mutant (right panels), fixed in PAF/MTSB (upper panels) or in F/PHEM/methanol (lower panels), and probed with anti-ASAP (green) and anti-α-tubulin (red) (top), anti-γ-tubulin (red) (bottom) and stained with Hoechst 33258 (blue) (scale bar, 10 μm).

Article Snippet: The Plk1 inhibitor ZK-thiazolidinone (TAL) was described in Ref. 30 and kindly provided by Bayer Schering Pharma AG.

Techniques: Phospho-proteomics, In Vivo, Transfection, Control, Staining, Blocking Assay, Western Blot, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity *

doi: 10.1074/jbc.M110.144220

Figure Lengend Snippet: Plk1-dependent phosphorylation of ASAP on serine 289 does not require a priming phosphorylation by Cdk1/Cyclin B in vivo. A, purified GST-ASAP, histone H1 (positive control), or GST-SIP (negative control) was incubated with purified Cdk1/Cyclin B complex and [γ-32P]ATP. The kinase reaction mixtures were then analyzed by SDS-PAGE and autoradiography (upper panel); the gel was also stained with Coomassie Blue (lower panel). B, purified GST-ASAP-WT or GST-ASAP-S289A mutant (negative control) was first incubated or not with Cdk1/Cyclin B complex for 30 min, treated with the CDK1 inhibitor RO-3306 for 10 min, and finally incubated with Plk1 for 30 min (see “Materials and Methods”). Reaction products were analyzed by SDS-PAGE and immunoblotted (IB) with anti-ASAP-Ser(P)-289, anti-ASAP, anti-Cyclin B, and anti-Plk1 antibodies as indicated. C, U-2 OS cells were transfected with FLAG-ASAP-WT, synchronized by thymidine block, and released for 16 h in the presence of nocodazole. Then, mitotic cells were recovered by mitotic shake-off and treated for 30 min with dimethyl sulfoxide (DMSO) (control), 50 μm roscovitine (CDK inhibitor), or 2 μm RO-3306 (CDK1 inhibitor). Cell extracts were immunoblotted with the anti-ASAP-Ser(P)-289, anti-FLAG, anti-Plk1, anti H3-Ser(P)-10 (mitotic marker) and anti-Cdc27 antibodies. The phosphoshift band of anti-Cdc27 is shown as a marker of Cdk activity. α-Actin was used as a loading control.

Article Snippet: The Plk1 inhibitor ZK-thiazolidinone (TAL) was described in Ref. 30 and kindly provided by Bayer Schering Pharma AG.

Techniques: Phospho-proteomics, In Vivo, Purification, Positive Control, Negative Control, Incubation, SDS Page, Autoradiography, Staining, Mutagenesis, Transfection, Blocking Assay, Control, Marker, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity *

doi: 10.1074/jbc.M110.144220

Figure Lengend Snippet: Model for the role of Plk1-dependent ASAP phosphorylation in maintaining centrosome integrity. Plk1 is a master regulator of centrosome maturation through phosphorylation and targeting of different PCM components. These various phosphorylations and interactions promote the recruitment of NEDD1, γ-tubulin, and ASAP to the centrosome. Plk1 binds to ASAP via its PBD (not shown) and phosphorylates ASAP on Ser-289. In the absence of Plk1-driven Ser-289 phosphorylation of ASAP, numeral and structural centrosome abnormalities occur, leading to abnormal mitotic spindle formation.

Article Snippet: The Plk1 inhibitor ZK-thiazolidinone (TAL) was described in Ref. 30 and kindly provided by Bayer Schering Pharma AG.

Techniques: Phospho-proteomics