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Figure 6. Phosphorylation of TACC3 by Aurora A is required for centrosomal localization of TACC3 and its microtubule-stimulating activity. (A) Localization of WT versus phosphorylation mutant of TACC3 on mitotic centrosomes. Microtubules around mitotic centrosomes were visualized by the addition of fluorescent tubulin in add-back experiments. Bar, 10 μm. Insets show immunolocalization of TACC3 on centrosomes in immunodepleted extracts with add back of WT versus the phosphorylation mutant of TACC3. Bar, 1 μm. (B) Quantification of immunostaining of TACC3 on centrosomes in A. Relative fluorescence intensity of TACC3 staining on centrosomes (within 1.079 μm radius) was measured. (C) Quantification of tubulin fluorescence in A. Relative fluorescence intensity of labeled tubulin around mitotic centrosomes (within 21.079 μm radius) was measured. (B and C) Values shown are the means plus SD. (D) Immunoblots of depleted extracts in add-back experiments. Mitotic extracts were immunodepleted with control IgG (lane 1; mock dep) or anti-TACC3 antibody (lanes 2–4; Δ TACC3) with adding back of control buffer (lanes 1 and 2; + buffer), TACC3-WT (lane 3; + TACC3-WT), or phosphorylation mutant TACC3 (lanes 4; + TACC3-3A). (E) Sedimentation assay to monitor XMAP215 affinity to microtubules in the presence of WT or phosphorylation mutant TACC3. 2.5 μM GMPCPP microtubules were mixed with 0.5 μM XMAP215 alone (lanes 1, 2, 7, and 8), 0.5 μM XMAP215 + 1.0 μm TACC3-WT (lanes 3, 4, 9, and 10), or 0.5 μM XMAP215 + 1.0 μm TACC3-3A (lanes 5, 6, 11, and 12) in the absence (lanes 1–6) or presence (lanes 7–12) of Aurora A. Mixtures were incubated for 38 min at 30°C and sedimented, and supernatants (S) and pellets (P) were subjected to SDS-PAGE. The gels were stained with Coomassie brilliant blue (top; CBB staining) or transferred onto nitrocellurose membrane for immunoblotting (IB) using the phosphospecific TACC3 antibody (antiphospho-Ser626; bottom). Image published in: Kinoshita K et al. (2005) Copyright © 2005, The Rockefeller University Press. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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