PMID:7978284
| Citation |
Wang, QM, Roach, PJ and Fiol, CJ (1994) Use of a synthetic peptide as a selective substrate for glycogen synthase kinase 3. Anal. Biochem. 220:397-402 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1006/abio.1994.1356 |
| Keywords |
Amino Acid Sequence; Animals; Calcium-Calmodulin-Dependent Protein Kinases/analysis; Calcium-Calmodulin-Dependent Protein Kinases/isolation & purification; Calcium-Calmodulin-Dependent Protein Kinases/metabolism; Chromatography, Ion Exchange/methods; Consensus Sequence; Cyclic AMP Response Element-Binding Protein/metabolism; Cyclic AMP-Dependent Protein Kinases/metabolism; Escherichia coli; Glycogen Synthase Kinase 3; Glycogen Synthase Kinases; Kinetics; Molecular Sequence Data; Muscles/enzymology; Oligopeptides/chemical synthesis; Oligopeptides/metabolism; Peptide Fragments/metabolism; Phosphorylation; Protein Kinases/metabolism; Rabbits; Recombinant Proteins/analysis; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Sensitivity and Specificity; Substrate Specificity |
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