Use of a synthetic peptide as a selective substrate for glycogen synthase kinase 3

Q. May Wang, Peter J. Roach, Carol J. Fiol

Research output: Contribution to journalArticle

36 Scopus citations

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 μM for GSK-3α and GSK-3β 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.

Original languageEnglish (US)
Pages (from-to)397-402
Number of pages6
JournalAnalytical biochemistry
Volume220
Issue number2
DOIs
StatePublished - Aug 1 1994

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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