Use of peptide substrates for affinity purification of protein-serine kinases.
The ability of protein kinases to phosphorylate synthetic peptides corresponding to identified protein phosphorylation sites has previously been used to determine primary structural requirements and has helped define distinct "recognition sequences" for a variety of enzymes. Here, we have used an immobilized synthetic peptide derived from glycogen synthase to specifically purify two protein kinases. In the case of one, glycogen synthase kinase-3, the peptide is only a substrate if previously phosphorylated at a distinct site by another protein kinase, casein kinase-II. This prerequisite is reflected in the differential affinity of glycogen synthase kinase-3 for the immobilized phospho- and dephosphopeptide. This difference in binding has been exploited to effect purification of glycogen synthase kinase-3 as well as casein kinase-II. The general applicability of peptide-based affinity chromatography is discussed.
Woodgett
J R
JR
Ludwig Institute for Cancer Research, London, England.
D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D047388:Casein Kinases; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D038341:Glycogen Synthase Kinases; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000302:isolation & purification*; D017346:Protein Serine-Threonine Kinases; D011817:Rabbits; D013379:Substrate Specificity
D011494:Protein Kinases; D038341:Glycogen Synthase Kinases; D047388:Casein Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases