(A) Mass spectrometry data of heavily phosphorylated (P) and dephosphorylated (deP) Aurora A. The dephosphorylated protein was obtained after treatment of heavily phosphorylated, Escherichia coli-produced Aurora A with λ-protein phosphatase (λPP). (B) AP phosphorylation by dephosphorylated (, 0.01 ± 0.005 s−1) or T288V mutant Aurora A (, 0.05 ± 0.002 s−1) is increased by up to 50-fold (, 0.5 ± 0.1 s−1) and 25-fold (, 1.2 ± 0.1 s−1), respectively, in the presence of TPX21−45. This rate is comparable to the kinetics of phosphorylated Aurora A in the absence of TPX21−45 (, 1.0 ± 0.2 s−1). Phosphorylated Aurora A shows up to a twofold increase in AP kinetics in the presence of TPX21−45 (, 2.3 ± 0.2 s−1). Reactions are carried out in the presence of 1 μM protein, 50 μM TPX21−45, 5 mM ATP, and 1 mM AP in assay buffer (20 mM TrisHCl, 200 mM NaCl, 20 mM MgCl2, 3% (vol/vol) glycerol, 1 mM TCEP, pH 7.50) at 25°C. Phosphorylated peptide production was monitored by reverse phase-high performance liquid chromatography (RP-HPLC).