Arpc1b interacts with Aurora kinase A. (A) ZR-75 cells synchronized by double-thymidine block and released for 6 h were analyzed for centrosome numbers. α-Tubulin (green) is used to indicate spindle morphology and pericentrin (red) is used to indicate centrosomes. DNA is in blue. N, number of centrosomes per cell. Quantitation for the centrosome numbers in the different stable cell lines generated in ZR-75 cells is shown below panel A. Bar, 20 µm. (B) ZR-75 cell lysates were incubated with either GST or GST-Arpc1b and the protein complex precipitated was subjected to Western blot analysis with the antibodies. Ponceau-stained blot shows the quality of GST proteins used for the study. (C) GST control or GST-Aurora A was incubated with purified Arp2/3 complex and the immunoprecipitates were subjected to Western blot analysis using an anti-Arpc1b or anti-Arpc2 antibody. (D) ZR-75 cell extracts were fractionated onto 3–30% sucrose gradients by velocity sedimentation, and then subjected to Western blot analysis with the indicated antibodies. (E) GST-tagged deletion mutants of human Aurora kinase A. Aurora A, 1–403 is the full-length protein. In vitro–translated ³⁵S-labeled Arpc1b was used to study binding of different GST-Aurora A deletion mutants. The extent of binding was estimated by measuring signal intensity. Ponceau S staining shows equal quantity of GST and GST-Aurora A deletions used in the reaction. Asterisk denotes the GST-fused protein of interest; black arrows indicate the interacting region of Aurora kinase A with Arpc1b. (F) Asynchronized cells or cells blocked with thymidine and released for various time points were used for immunoprecipitation (IP). Transfected T7-Arpc1b was immunoprecipitated using T7 antibody and the blot was probed for Aurora A. (G) Asynchronized cells or cells blocked with thymidine and released for various time points were subjected to the sequential IP/Western blot with the indicated antibodies. (H) Protein extract from ZR-75 cells released for 0 or 8 h after double-thymidine block were subjected to reciprocal immunoprecipitation assays with an anti-Aurora A or anti-Arpc1b antibody, followed by Western blot analysis with the indicated antibodies. kD, kilodaltons.

Arpc1b stimulates activity of Aurora A. (A) GST alone or GST-Arpc1b was used in an in vitro kinase assay with GST-Aurora A or GST-Aurora A KD protein. Phosphorylated histone H3 was visualized by autoradiography. Ponceau-stained blot shows equal amount of histone H3 used for both reactions. Western blot shows equal amount of Aurora A was used in all the reactions. (B) GST, GST-Arpc1b, or GST-Arp3 was used in an in vitro kinase assay with recombinant Aurora A using Histone H3 as a substrate. Phosphorylated histone H3 was visualized by autoradiography (top panel). Ponceau-stained blot shows equal amount of GST proteins and histone H3 used for all the reactions. Asterisk denotes the GST-fused protein of interest. (C) Aurora A kinase activity was studied using histone H3 as a substrate in the presence or absence purified Arp2/3 complex. Ponceau-stained blot shows equal amount of histone H3 and the Western blot shows equal amount of Aurora A used in the reactions. (D) GST or GST-Arpc1b was incubated with phosphatase (PP1)-treated Aurora A for 20 min in presence of [³²P]γATP. Samples were analyzed for Aurora A phosphorylation (Thr-288, top panel) and total Aurora A (last panel). Fold change was expressed with respect to lane 1. (E) ZR-75 cells transfected with either control siRNA or Arpc1b siRNA were subjected to in vivo ³²P-labeling. Protein extracts were subjected to IP with an anti-Aurora A antibody, and phosphorylated Aurora A was visualized by autoradiography. Knockdown of Arpc1b was verified by Western blot with an anti-Arpc1b antibody (bottom panel). (F) Immunoblots showing levels of phospho-Aurora A (Thr288), phospho-plk1 (Thr 210), and phospho-histone H3 (Ser 10) in control and Arpc1b siRNA-treated ZR-75 cell lysates. kD, kilodaltons.

Phosphorylation of Arpc1b on Thr 21 regulates centrosome numbers in ZR-75 cells. (A) Expression of T7-Arpc1b (top) and endogenous Arpc1b (bottom) in stable cell lines generated in ZR-75 cells for indicated point mutations of Arpc1b. Quantitation for the centrosome numbers in the different stable cell lines generated in ZR-75 cells are shown below panel A. Synchronized cells released for 6 h after G1-S arrest were used for immunostaining. Representative images showing the distribution of γ-tubulin (green, B); Aurora A (green, C), and DNA (blue) in the stable cell lines. γ-Tubulin was used as a centrosome marker. Bars, 5 µm. (D) Synchronized cells released for 7 h after G1-S arrest were subjected to immunoprecipitation using agarose-tagged Aurora A antibody. The resulting precipitates were used for an in vitro kinase assay with histone H3 as substrate. Autoradiograph shows Aurora A, phosphorylated histone H3, and total histone H3. (E) ³²P-labeled ZR-75 cells expressing either T7-Arpc1b or T7-Arpc1bT21A were treated with 100 ng/ml of EGF for 5 min. Arpc1b was immunoprecipitated using an anti-T7 antibody (second panel) and the in vivo phosphorylation of Arpc1b was analyzed using autoradiography (first panel). The same blot was probed for Aurora A (third panel). WT, wild type; kD, kilodaltons.

Role of Pak1 in Arpc1b-mediated centrosome amplification. (A) Western blot analysis showing expression of different Pak isoforms in the Pak1^+/+ or Pak1^−/− MEFs. Vinculin was used as an internal control. (B) Western blot analysis showing expression of T7-Arpc1b and endogenous Arpc1b levels in stable cell lines generated in the Pak1^+/+ or Pak1^−/− MEFs for indicated point mutations of Arpc1b. Actin was used as an internal control. Quantitation of the centrosome numbers in the different stable cell lines generated in the Pak1^+/+ or Pak1^−/− MEFs cells are shown below panel B. (C and D) Representative images showing distribution of γ-tubulin (green) and DNA (blue) in the stable cell lines. γ-Tubulin was used as a centrosome marker. Circle marks centrosome location. Bar, 5 µm. kD, kilodaltons.

Arpc1b localizes to the centrosome. (A) Synchronized ZR-75 cells were released for 6 h and analyzed for endogenous Arpc1b colocalization with centrosomal proteins Aurora A and γ-tubulin. Aurora A and γ-tubulin (green); endogenous Arpc1b (red); DNA (blue). Circle marks centrosome location. Bar, 5 µm. (B) Equal amount of cell extract was immunoprecipitated with control IgG or γ-tubulin antibody from the synchronized cells (top panel). The same blot was probed for Arpc1b (second last panel) and Arp3 (last panel). The blot was stripped and probed for Aurora A (second panel). (C) Equal amount of cell extract was immunoprecipitated with control IgG or Aurora A antibody (top panel). The same blot was probed for γ-tubulin (second panel) and Arp3 (last panel). Input, cell lysate used as positive control. (D) Xenopus egg extract was treated with either GST (control) or GST-WASP-CA (Arp3 depletion) and used for IP experiments. The Western blot shows Arp3 depletion in GST-WASP-CA–treated egg extract (top panel) and the GST-Aprc1b (second panel) used for the study. Aurora A was immunoprecipitated from the treated egg extract and incubated with GST-Arpc1b protein. The Western blot shows immunoprecipitated Aurora A and GST-Arpc1b or endogenous Arpc1b in complex with Aurora A in control and Arp3-depleted egg extract. (E) Aurora A was immunoprecipitated from the treated egg extract. Western blot shows immunoprecipitated Aurora A and endogenous Arpc1b in complex with Aurora A in control and Arp3-depleted egg extract. kD, kilodaltons.

Arpc1b is essential for progression to G2-M phase of cell cycle. (A) Synchronized ZR-75 cells transfected with control, Arpc1b, or Arp3 siRNA and released for 7 h after G1-S arrest were immunostained with antibodies to Aurora A (green) or phospho-Aurora A T288 (red) as indicated. Insets represent higher magnification images of centrosomes. Bar plot shows percentage of cells with Aurora A–positive centrosomes (B), and distribution of phospho-Aurora A on centrosomes (C) on transfecting ZR-75 cells with control, Arpc1b, or Arp3 siRNA. Staining for phospho-Aurora A on both centrosomes: arrowheads in bar graph = absent from both centrosomes; diagonal lines = present on only one centrosome; black = reduced staining for both centrosomes. Bar, 5 µm. (D) ZR-75 cells were transfected with control siRNAs, or specific siRNAs targeting Arpc1b, Arp3, Arpc1a, or Aurora A, and protein extracts were subjected to Western blot analysis with the indicated antibodies. (E and G) Synchronized ZR-75 cells transfected with control or specific siRNAs targeting Arpc1b, Arpc1a, or Arp3 (E) or Aurora A (G) were scored for percentage of cells in G2-M phase at the indicated time points after release from G1-S arrest using FACS analysis; n = 3. (F and H) Synchronized ZR-75 cells transfected with either control or Arpc1b siRNA (F) or Aurora A siRNA (H) were scored for percentage of phospho-H3–positive cells at the indicated time points after release from G1-S arrest using FACS analysis; n = 3. (I and J) Synchronized ZR-75 cells stably expressing wild-type Arpc1b (ZR-75/T7-Arpc1b) or Arpc1bT21A (ZR-75/T7-Arpc1b T21A) were scored for percentage of cells in G2-M phase (I) or for percentage of phospho-H3–positive cells (J) at the indicated time points after release from G1-S arrest using FACS analysis; n = 3. kD, kilodaltons.

Phosphorylation of Arpc1b on Thr 21 regulates its interaction with Aurora A. (A) In vitro–translated ³⁵S-labeled Aurora A was used to study its binding with GST, GST-Arpc1b, or GST-Arpc1b T21A. The extent of binding was estimated by measuring signal intensity. Ponceau-stained blot shows equal quantity of GST-tagged proteins used for each reaction. (B) GST, GST-Arpc1b, and GST-Arpc1b T21A were used in an in vitro kinase assay with recombinant Aurora A protein. Phosphorylated histone H3 was visualized by autoradiography. Ponceau-stained blot shows equal amount of histone H3 and GST-tagged proteins used for the study. Circle marks GST-fused protein of interest. (C) The 1C1 monoclonal antibody to Xenopus Aurora A (Eg2) was used to immunoprecipitate Eg2 from CSF egg extract supplemented with GST control, GST-TPX2 (1–126), GST-Arpc1b, or GST-Arpc1b T21A and the bound proteins were analyzed by Western blotting using anti-GST antibodies. Asterisk denotes the GST-fused protein of interest. Bar plot shows fold change quantitated from three experiments. (D) A phospho-specific antibody that recognizes phospho-Thr 295 of Eg2 was used to study the extent of activation of Aurora A (top panel). Bottom panel shows equal amount of Aurora A used in all the reactions. Bar plot shows fold change quantitated from three experiments. kD, kilodaltons.

Arpc1b: a new Aurora A substrate. (A) Kinase assay for GST-Arpc1b and GST-Arpc1b T21A proteins in presence of Aurora A. Aurora A in the middle panel indicates comparable amounts of protein loading. Ponceau-stained blot shows equal quantity of GST-tagged proteins used for the study. Asterisk denotes the GST-fused protein of interest. (B) In vivo phosphorylation of T7-tagged Arpc1b in the Pak1^−/− MEFs treated with control or Arpc1b siRNA. (Top to bottom): [³²P]T7-Arpc1b, T7-Arpc1b, Aurora A, and vinculin. (C) Western blot showing extent of Arpc2 immunoprecipitated with Arpc1b from Pak1^+/+ or Pak1^−/− cells after Aurora A knockdown (top panel), effective Arpc1b pull-down (second panel), and effective knockdown of Aurora A (third panel). Vinculin was used as an internal control. (D) Western blot showing extent of Arpc1b immunoprecipitated with Arpc2 from the Pak1^−/− cells after Aurora A knockdown (second panel), effective Arpc2 pull-down (top panel), and effective knockdown of Aurora A (third panel). Vinculin was used as an internal control. (E) Pak1^−/− cells (synchronized in G1-S phase) transfected with either control or Arpc1b siRNAs were scored for the percentage of cells in G2-M phase at the indicated time points after release from G1-S arrest using FACS analysis; n = 3. (F) Proposed model explaining the role for threonine 21 phosphorylation on Arpc1b in Aurora A activation leading to centrosome amplification. WB, Western blot; IP, immunoprecipitation; kD, kilodaltons.