Fig. 1. Aurora-A phosphorylates CDC25B on serine 353 in vitro and in vivo. (A) MS/MS spectrum of the monophosphorylated peptide, 353-SVTPPEEQQEAEEPK-367 (doubly charged precursor ion, MH2²⁺, at m/z 889.38) displays series of b- and y-ions [according to Biemann's nomenclature (Biemann, 1990)], intense doubly charged y13 (at m/z 756,4) together with weak mono-charged b2 (at m/z 266.9) and indicating that the serine 353 residue is phosphorylated and not threonine 355. (B) CDC25B alignment with Aurora-A consensus phosphorylation site. (C) Recombinant proteins were incubated with purified recombinant Aurora-A at 37°C for 30 minutes. The samples were analysed by w estern blotting with the 35C1 monoclonal anti-Aurora-A or affinity-purified polyclonal anti-serine 353 phosphorylated epitope – SE96 or monoclonal anti-maltose binding protein (MBP). MBP-CDC25B migrated as a doublet because of the presence of a degradation product. (D) Recombinant MBP-CDC25B and MBP-CDC25B S353A mutant were phosphorylated or not by Aurora-A as in C. Western blot analysis was performed with SE96 and anti-CDC25B antibodies. (E) Western blot analysis of CDC25B affinity-purified (15 ng/lane) from human U2OS cells expressing polyHis-tagged CDC25B, with SE96 antibody and anti-CDC25B polyclonal antibody was performed in the presence of 1000x molar excess of the phosphorylated peptide (lane 2), the unphosphorylated peptide (lane 3), or after prior incubation of the sample for 60 minutes at 30°C in the presence of phosphatase.