Characterization of Synthetic Peptide Substrates for p34 cdc2 Protein Kinase

Abstract Synthetic peptide substrates for the cell division cycle regulated protein kinase, p34 cdc2 , have been developed and characterized. These peptides are based on the sequences of two known substrates of the enzyme, Simian Virus 40 Large T antigen and the human cellular recessive oncogene product, p53. The peptide sequences are H‐A‐D‐A‐Q‐H‐A‐T‐P‐P‐K‐K‐K‐R‐K‐V‐E‐D‐P‐K‐D‐F‐OH (T antigen) and H‐K‐R‐A‐L‐P‐N‐N‐T‐S‐S‐S‐P‐Q‐P‐K‐K‐K‐P‐L‐D‐G‐E‐Y‐ NH 2 (p53), and they have been employed in a rapid assay of phosphorylation in vitro. Both peptides show linear kinetics and an apparent K m of 74 and 120 μM, respectively, for the purified human enzyme. The T antigen peptide is specifically phosphorylated by p34 cdc2 and not by seven other protein serine/threonine kinases, chosen because they represent major classes of such enzymes. The peptides have been used in whole cell lysates to detect protein kinase activity, and the cell cycle variation of this activity is comparable to that measured with specific immune and affinity complexs of p34 cdc2 . In addition, the peptide phosphorylation detected in mitotic cells is depleted by affinity adsorption of p34 cdc2 using either antibodies to p34 cdc2 or by immobilized p13, a p34 cdc2 ‐binding protein. Purification of peptide kinase activity from mitotic HeLa cells yields an enzyme indistinguishable from p34 cdc2 . These peptides should be useful in the investigation of p34 cdc2 protein kinases and their regulation throughout the cell division cycle.