Aurora B phosphorylates Ska1 and Ska3 in vitro. (A) In vitro kinase assay with Aurora B alone or Aurora B plus ZM on the Ska complex or mitotic centromere-associated kinesin (MCAK; as a positive control; Andrews et al., 2004; Lan et al., 2004). A Coomassie-stained gel and autoradiogram (left and right, respectively) are shown. Asterisks indicate unspecific bands in the mitotic centromere-associated kinesin preparation. Molecular mass is indicated in kilodaltons. (B) Sequence alignment of the potential Aurora B sites in Ska1 and Ska3 from the indicated species; alignments were performed with CLUSTALW (network protein sequence analysis). Potential Aurora B sites conserved across species are marked with a rectangle. (C) A table summarizing collected information for the seven potential Aurora B sites mutated in Ska1 and Ska3. (D) In vitro Aurora B kinase assays of Ska1^WT-His and His-Ska3^WT and the corresponding nonphosphorylatable mutants. An autoradiogram and Coomassie-stained gel (top and bottom, respectively) are shown.