Use of a synthetic peptide as a selective substrate for glycogen synthase kinase 3.
Glycogen synthase kinase 3 (GSK-3) is involved in the regulation of several metabolic enzymes and transcription factors in response to extracellular signals. Here we report the use of a synthetic peptide derived from the sequence of the cyclic AMP responsive element binding protein (CREB) as a specific substrate for GSK-3 isoforms. The 13-amino acid peptide, KRREILSRRPSYR, was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (PKA) and purified on a C18 cartridge. Phosphorylation of the COOH-terminal serine of the peptide by PKA creates a phosphorylation site for GSK-3 since GSK-3 recognizes the consensus motif -S-X-X-X-S(P)-. Although the COOH-terminal serine of the peptide can be phosphorylated by PKA and several other kinases, the phospho-CREB peptide is specific for GSK-3 with Kms of 140 and 200 microM for GSK-3 alpha and GSK-3 beta isoforms, respectively. Using the phospho-CREB peptide, we have successfully purified GSK-3 activity from rabbit skeletal muscle and Escherichia coli cells transformed with a GSK-3 expression vector. The assay described provides a convenient and specific determination of GSK-3 activity.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
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D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange / Q000379:methods; D016384:Consensus Sequence; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004926:Escherichia coli; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D017362:Cyclic AMP Response Element-Binding Protein; D009842:Oligopeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D011494:Protein Kinases; D038341:Glycogen Synthase Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3