13] Use of synthetic peptides mimicking phosphorylation sites for affinity purification of protein-serine kinases

Abstract

This chapter utilizes the affinity of two protein-serine kinases for a synthetic peptide substrate to effect their purification. One of the kinases, glycogen synthase kinase 3 (GSK-3), exhibits an unusual specificity requirement. Certain substrates must be prephosphorylated by a distinct protein kinase before they are recognized by GSK-3. A purification strategy is developed to exploit this unusual prerequisite using a peptide derived from glycogen synthase, which exhibits high-affinity binding to GSK-3 only when prephosphorylated by casein kinase II. The utility of synthetic peptides for the affinity purification of other protein kinases is also discussed. The initial steps in the purification of casein kinase II and GSK-3 are similar, permitting their simultaneous isolation. Skeletal muscle from the hind limbs and back of female New Zealand White rabbits, killed by intravenous injection of a lethal dose of sodium pentabarbitone, is rapidly excised and placed into ice. The muscle is coarsely ground and homogenized in 2.5 vol of 4 m M EDTA, 15 m M 2-mercaptoethanol, 0.1 m M phenylmethylsulfonyl fluoride, 1 m M benzamidine, pH 7.0, for two 30-sec low-speed pulses in a Waring commercial blender.