Fig. 2. Autophosphorylation is the oldest and uniformly conserved allosteric activation mechanism in all Aurora kinases.

(A) Additive differences in primary sequence between AurA_human and Aur_ANC4 (82.4% identity), Aur_ANC3 (80.1% identity), Aur_ANC2 (72.7% identity) and Aur_ANC1 (69.5% identity) are shown in cyan, green, orange and red spheres, respectively. (B) Corresponding k_cat (left) and K_M (middle) for dephosphorylated (deP) and T288-phosphorylated (P) Aurora kinases show that phosphorylation increases catalytic efficiency by increasing k_cat and decreasing K_M (see also Fig. S4). To best illustrate this combined effect, the fold increase in k_obs at 1 mM Lats2 upon Aurora’s T288 phosphorylation is shown on the right. Phosphorylation of Lats2 peptide was monitored using the ADP/NADH coupled assay with 1 μM dephosphorylated or 0.05 μM phosphorylated Aurora and 5 mM ATP, 20 mM MgCl₂ at 25°C. Error bars represent the standard error for the estimate of k_cat or K_M through the Michaelis-Menten equation and are a measure of the goodness of fit of the data. Error bars for increase in k_obs are calculated using jackknifing and error propagation.