Figure 1.

Dbf2-Mob1 peptide substrate requires arginine at position -3. (A) Synthetic peptides based on the predicted optimal substrate of Dbf2-Mob1. The underlined residues represent the predicted preferred amino acids for Dbf2-Mob1 substrate specificity; the asterik denotes the single amino acid substitution. (B) The various peptides denoted in (A) at a concentration of 250 μM were treated with ~13 ng of ^FHHDbf2 bound to ^H6Mob1^TM9. Aliquots of the kinase reaction were quenched at the indicated timepoints to determine the amount of phosphorylation by liquid scintillation. The Optimal peptide was also treated with the kinase-inactive ^FHHDbf2(N305A)-^H6Mob1^TM9 complex, as denoted by D2M. Similar results were obtained in 4 independent experiments. (C) Using the conditions in (B), K_m and V_max was determined for each peptide with the exception of R-3K, R-3A, negative control, and the Optimal peptide treated with kinase-inactive ^FHHDbf2(N305A)-^H6Mob1^TM9, due to low phosphorylation.