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 se veral metabolic enzymes and transcription factors in response to extra cellular signals. Here we report the use of a synthetic peptide derive d from the sequence of the cyclic AMP responsive element binding prote in (CREB) as a specific substrate for GSK-3 isoforms. The 13-amino aci d 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 cr eates a phosphorylation site for GSK-3 since GSK-3 recognizes the cons ensus motif -S-X-X-X-S(P)-. Although the COOH-terminal serine of the p eptide can be phosphorylated by PHA and several other kinases, the pho spho-CREB peptide is specific for GSK-3 with K(m)s of 140 and 200 mu M for GSK-3 alpha and GSK-3 beta isoforms, respectively. Using the phos pho-CREB peptide, we have successfully purified GSK-3 activity from ra bbit skeletal muscle and Escherichia coli cells transformed with a GSK -3 expression vector. The assay described provides a convenient and sp ecific determination of GSK-3 activity. (C) 1994 Academic Press, Inc.