Use of a Synthetic Peptide as a Selective Substrate for Glycogen Synthase Kinase 3

Q. M. Wang, Peter Roach, C. J. Fiol

Research output: Contribution to journalArticle

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

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Glycogen Synthase Kinase 3
Peptides
Substrates
Cyclic AMP
Carrier Proteins
Phosphorylation
Protein Kinases
Serine
Protein Isoforms
Protein-Serine-Threonine Kinases
Cyclic AMP-Dependent Protein Kinases
Escherichia coli
Muscle
Assays
Catalytic Domain
Skeletal Muscle
Transcription Factors
Phosphotransferases
Rabbits

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry

Cite this

Use of a Synthetic Peptide as a Selective Substrate for Glycogen Synthase Kinase 3. / Wang, Q. M.; Roach, Peter; Fiol, C. J.

In: Analytical Biochemistry, Vol. 220, No. 2, 01.08.1994, p. 397-402.

Research output: Contribution to journalArticle

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