Figure 1.

PhosphoSens assay development (A), enzyme titration and ATP Km determination (B). (A) Step 1: A library of ~1000 serine/threonine peptides of ~18 amino acid residues (based on known phosphorylation sites) was screened against STK17B kinase to identify the best substrate peptide (Ac-KAKTTKKRPQRATSN-C(Sox)-FS-amide). Step 2: Each amino acid position was mutated and evaluated. Step 3: Single mutations identified in the second step are combined in a matrix manner and again screened against STK17B kinase. The final optimized substrate peptide for STK17B has the sequence Ac-KKKKVKKRPQRADSD-C(Sox)-FA-amide. (B) With the optimized substrate peptide, enzyme titration of STK17B (labeled as GST-DRAK2) from SignalChem was performed from 80 nM top concentration with 2-fold dilution down (0, 1.25, 2.5, 5, 10, 20, 40, 80 nM) in the presence of 1 mM ATP. ATP Km was determined by measuring initial velocity (fluorescence intensity versus time) at 1-1024 µM ATP. The initial velocity was plotted against ATP concentration and fitted with the Michaelis−Menten equation to give Km of 6.5 µM for STK17B. ATP, adenosine triphosphate; DRAK2, DAP kinase-related apoptosis-inducing protein kinase 2; GST, glutathione S-transferase; RFU, relative fluorescence units; Sox, Sulfonamido-oxine; STK, serine/threonine kinase.