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Centrosomes are primary microtubule (MT)-organizing centers (MTOCs). During mitosis, they dramatically increase their size and MT-nucleating activity and participate in spindle assembly from spindle poles. These events require the serine/threonine kinase, Aurora A (AurA), and the centrosomal protein of 192 kDa (Cep192)/spindle defective 2 (Spd-2), but the underlying mechanism remains unclear. We have found that Cep192, unlike targeting protein for Xklp2 (TPX2), a known MT-localizing AurA activator, is an AurA cofactor in centrosome-driven spindle assembly. Cep192, through a direct interaction, targets AurA to mitotic centrosomes where the locally accumulating AurA forms homodimers or oligomers. The dimerization of endogenous AurA, in the presence of bound Cep192, triggers potent kinase activation that, in turn, drives MT assembly. Depletion of Cep192 or specific interference with AurA-Cep192 binding did not prevent AurA oligomerization on MTs but abrogated AurA recruitment to centrosomes and its activation by either sperm nuclei or anti-AurA antibody (αAurA)-induced dimerization. In these settings, MT assembly by both centrosomes and αAurA-coated beads was also abolished or severely compromised. Hence, Cep192 activates AurA by a mechanism different from that previously described for TPX2. The Cep192-mediated mechanism maximizes AurA activity at centrosomes and appears essential for the function of these organelles as MTOCs.
Fig. 1. AurA activation and MT assembly promoted in egg extract by sperm nuclei and αAurA beads. (A) Localization of AurA and Cep192 during MT assembly promoted by sperm nuclei-associated centrosomes (time course) and RanGTP. (Scale bars: 5 μm.) (B) W-blots showing the time course of AurA activation in extract by sperm nuclei (4 × 104/μL). (C) Extract was driven into interphase by the addition of calcium and supplemented with demembranated sperm nuclei (2 × 103/μL). Extract aliquots were withdrawn at the indicated times after sperm addition and analyzed by W-blotting using the indicated antibodies. (D) W-blots of extracts supplemented with XB buffer, nocodazole (50 μM), RanT24N (30 μM), Ran(Q69L)GTP (30 μM), or DNA beads (0.1 μL/μL). Where indicated, reaction mixtures contained sperm nuclei (4 × 104/μL). (E and F) Beads coated with nonimmune IgG (N/Imm), αAurA, or αAurA-Fab were incubated in rhodamine-tubulin+Ran(Q69L)GTP-supplemented extract and analyzed by fluorescence microscopy (E) and W-blotting (F). (Scale bar: 10 μm.) (G) W-blots of extract incubated with 1.7 μM goat anti-rabbit IgG, IgG fraction (αIgG) and increasing concentrations of αAurA-Fab [detected as IgG light chain (IgG L.C.)]. The concentration of AurA in egg extract is ≈0.35 μM.
Fig. 2. Cep192 is a cofactor of AurA at centrosomes. (A) Schematic diagram of Xenopus Cep192. The AurA-BD and the three ASH (ASPM, SPD-2, Hydin) domains (40) are shown. The numbers denote aa. The underlying gray lines indicate the polypeptides used for antibody production. (B) W-blot of Cep192 and TPX2IPs from extract. IgG H.C., IgG heavy chain. Note that the nonimmune IgG, even when used in approximately a twofold excess over αCep192-C and αTPX2 (as assessed by the intensity of IgG H.C.), brings down only a small, background amount of AurA. (C) Binding of recombinant Xenopus AurA to glutathione Sepharose-immobilized glutathione S-transferase (GST), GST-Cep521–757, and GST-TPX2-NT. (D) W-blot of αAurA beads retrieved from extracts incubated with 3 μM of the indicated GST polypeptides. (E) W-blot of mock-treated, Cep192-, and TPX2-depleted extracts incubated with 4 × 104/μL sperm nuclei (30 min) or with αAurA (0.3 μM) for the indicated times. (F) MT structures assembled in extracts supplemented with sperm nuclei, αAurA beads, or Ran(Q69L)GTP. (Scale bars: 10 μm.) (G) P-AurA and AurA detected by W-blot of αAurA beads retrieved from extracts in Fii. (H) W-blot showing partial rescue of the AurA activation defect by rCep192 in Cep192-depleted, fivefold diluted, extract. (I) Visualization of AurA oligomers by BiFC in extracts and transfected HeLa cells. BiFC complexes are shown in green; MTs (columns 1–3) and CFP (which denotes transfected cells; column 4, pseudocolor) in red; and DNA (DAPI; row 1) in blue. (Scale bars: 10 μm.)
Fig. 3. Cep192-mediated AurA activation is essential for MTOC function. (A and D) Localization of AurA and Cep192 on centrosomal asters assembled in extracts supplemented with 3 μM GST or Cep521–757 (A) or with 0.7 μM nonimmune IgG or αCep192-N (D). (Scale bars: 2.5 μm.) (B and E) Quantitation of MT aster surface area in A and D, respectively. The results obtained in the presence of 0.2 μM αCep192-N are also shown for comparison (E). Error bars, SD. *P < 0.0001 compared with the corresponding control extract, as determined by a two-tailed Student's t test. The number of asters analyzed is shown in parentheses. (C) W-blot of extracts incubated with XB buffer, Cep521–757 (3 μM), or TPX2-NT (6 μM) in the absence or presence of sperm nuclei (4 × 104/μL) or αAurA (0.6 μM). (F) W-blots and images of αAurA beads that were added to extract before (iii and iv) or after (i and ii) its incubation with 3 μM GST or Cep521–757 on ice. (Scale bar: 10 μm.) (G) In vitro kinase assay of endogenous AurA complexes isolated from egg extract incubated with XB buffer or Cep521–757 (3 μM). The reaction products were resolved by SDS/PAGE and transferred onto a nitrocellulose membrane, which was subjected to autoradiography (32PO4-histone H3 row) followed by W-blotting (the remaining rows). (H) W-blots of extracts incubated with soluble αAurA (0.5 μM) added either before (lanes 5–8) or after (lanes 1–4) incubation of extracts with 3 μM GST or Cep521–757 on ice. (I) Quantitation of MT-nucleating αAurA beads in F, Lower.
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