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Figure 2. TPX2 physically interacts with Aurora-A and is phosphorylated on serine. (A, top) Immobilized recombinant Aurora-A WT and KD proteins were incubated for 1 h at 4°C in mitotic HeLa cell extract, washed in LS buffer, and analyzed by SDS-PAGE followed by Coomassie blue staining (lanes 1 and 2). Controls show the results of incubating extract with beads alone (lane 3) and of loading WT Aurora-A protein without extract incubation (lane 4). Arrowheads indicate the prominent 100-kD protein that was identified as TPX2 by Western blotting. (A, bottom) Western blots were performed on the same samples using antibodies against TPX2 and Aurora-A. (B) In vitro binding of recombinant TPX2 (IN) to immobilized GST-tagged Aurora-A WT and KD proteins, or GST for control. (C) Mapping the interaction domains on Aurora-A and TPX2. Fragments of Aurora-A and TPX2, as indicated schematically, were produced by IVT, and binding to His6-TPX2 (top) or GST-Aurora-A (bottom) was assayed. Protein complexes were recovered on Ni-Sepharose beads and glutathione beads, respectively; Ni-Sepharose beads or GST-coated glutathione beads were used for control. Autoradiographs show corresponding amounts of input samples and proteins recovered in protein complexes. (D) In vitro kinase assay was performed with recombinant GST-tagged Aurora-A and recombinant human TPX2 in the presence of [γ-32P]ATP. Both the Coomassie blue–stained gel (left) and the autoradiograph (right) are shown. (D, bottom) Phosphoamino acid analysis of 32P-labeled TPX2 (right); ninhydrin staining (left) shows the migration of phosphoamino acid standards. Image published in: Kufer TA et al. (2002) Copyright © 2002, 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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