Peptide substrates suitable for assaying glycogen synthase kinase-3 in crude cell extracts.
In this study we describe the characterization and use of new peptide substrates for assaying glycogen synthase kinase-3 (GSK-3) which are based on the sequence around the single GSK-3 phosphorylation site in the translation factor eIF2B. The new peptides offer important advantages over previous substrates, which were based on the sequence around the multiple GSK-3 phosphorylation sites in glycogen synthase (GS), for the assay of GSK-3 in cell extracts. In particular, decreases in GSK-3 activity following, e.g., insulin treatment, are partially or completely masked when the GS-based peptides are used but are readily measured using the new, eIF2B-based, peptides. The new peptides, unlike those based on GS, are therefore suitable for the assay of changes in GSK-3 activity in cell extracts without the need for prior immunoprecipitation or ion-exchange chromatography.
Welsh
G I
GI
Department of Biosciences, University of Kent at Canterbury, United Kingdom.
Patel
J C
JC
Proud
C G
CG
D016475:3T3 Cells; D017667:Adipocytes; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis* / Q000737:chemistry; D002457:Cell Extracts / Q000737:chemistry; D006224:Cricetinae; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics; D010766:Phosphorylation
D002457:Cell Extracts; D007328:Insulin; D010455:Peptides; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3