p150^glued is an aurora A substrate in vitro. (A) Coomassie blue–stained gel of the total embryonic extract (left) and the MAPs fraction obtained after sedimentation of taxol-polymerized microtubules (right). The strong band corresponds to the tubulins (arrowhead). (B) A kinase assay with (+) or without (−) aurora A–(His)6 was performed using 20 µg MAPs preparation. (left) The proteins were separated by SDS-PAGE and stained by Coomassie blue (CB). (right) The discrete phosphorylated band (P32) was excised and identified by mass spectrometry as p150^glued. The white line indicates that intervening lanes have been spliced out. (C, left) Extracts from S2 cells stably expressing 3xFlag–aurora A were subjected to IP with preimmune (PI) or affinity-purified immune (I) anti-p150^glued antibodies. (right) Extracts from wild-type S2 cells (control) or S2 cells stably expressing 3xFlag–aurora A were subjected to anti-Flag IP. The precipitates were revealed with anti-p150^glued (top) or anti-Flag antibodies (bottom). Note the presence of p150^glued in 3xFlag–aurora A precipitates and, conversely, the presence of aurora A in p150^glued immunoprecipitates. (D) Scheme of the p150^glued fusion proteins used in the kinase assay. N- and C-terminal fragments of p150^glued are displayed in green and blue, respectively. (E) Recombinant MBP, MBP-Ct-Gl, and MBP-Nt-Gl were used for in vitro kinase assays using (+) or not using (−) aurora A–(His)6 protein kinase in the presence of radio-labeled γ-[³²P]ATP. The position of the aurora A–(His)6 band is indicated by arrows (+). MBP and MBP-Ct-Gl, indicated by open and closed arrowheads, respectively, are not phosphorylated, whereas MBP-Nt-Gl (asterisks) is strongly phosphorylated by aurora A. The Coomassie blue–stained gel (left) and the corresponding autoradiography (right) are shown. (F) Position of the eight phosphorylated Ser residues (yellow) in the p150^glued MBD (amino acids 0–200).