Figure 2
Identification of phosphorylation sites in the Ataxin-10 proteins.
(a) GST-Ataxin-10-WT, S12A and His-Aurora B were purified from E. coli cells and incubated in kinase buffers containing 32P-ATP with or without ZM. Coomassie blue (CBB) staining showed input Ataxin-10 proteins and autoradiography detected phosphorylated GST-Ataxin-10. (b) Mass spectrum identified an Ataxin-10 peptide phosphorylated at S12. From this collision-induced dissociation spectrum, a phosphorylated peptide RL(pS)GVMVPAPIQDLEAL of Ataxin-10 was identified following incubation with Aurora B in an IVK reaction. “b” and “y” ion series represented fragment ions containing the N- and C-termini of the peptide, respectively. (c) A comparison of S12 and Aurora B phosphorylation consensus motif. The residues in red fit the consensus motif. (d) Recombinant GST-tagged wild-type Ataxin-10 or the S12A mutant were purified from E. coli and incubated with recombinant His-Aurora B, or His-Aurora B with ZM. The samples were then analyzed by Western blot with anti-pS12 antibodies. (e) FLAG-tagged wild-type Ataxin-10 or the S12A mutant were transfected into HeLa cells and the cell lysates were subject to IP and IB analysis using the antibodies indicated. (f) HeLa cells were synchronized in cytokinetic phase as described in Methods and treated with 4 mM ZM447439 (Aurora kinase inhibitor) or 1 μm BI2356 (Plk inhibitor) for 2 hrs, then blotted with antibodies towards Ataxin-10, pS12 or pS77. Uncropped images of blots were shown in supplemental Figure S1.
