Purification of GSK-3 by affinity chromatography on immobilized axin.
Glycogen synthase kinase 3 (GSK-3), an element of the Wnt signalling pathway, plays a key role in numerous cellular processes including cell proliferation, embryonic development, and neuronal functions. It is directly involved in diseases such as cancer (by controlling apoptosis and the levels of beta-catenin and cyclin D1), Alzheimer's disease (tau hyperphosphorylation), and diabetes (as a downstream element of insulin action, GSK-3 regulates glycogen and lipid synthesis). We describe here a rapid and efficient method for the purification of GSK-3 by affinity chromatography on an immobilized fragment of axin. Axin is a docking protein which interacts with GSK-3ss, beta-catenin, phosphatase 2A, and APC. A polyhistidine-tagged axin peptide (residues 419-672) was produced in Escherichia coli and either immobilized on Ni-NTA agarose beads or purified and immobilized on CNBr-activated Sepharose 4B. These "Axin-His6" matrices were found to selectively bind recombinant rat GSK-3 beta and native GSK-3 from yeast, sea urchin embryos, and porcine brain. The affinity-purified enzymes displayed high kinase activity. This single step purification method provides a convenient tool to follow the status of GSK-3 (protein level, phosphorylation state, kinase activity) under various physiological settings. It also provides a simple and efficient way to purify large amounts of active recombinant or native GSK-3 for screening purposes.
Primot
A
A
Station Biologique, CNRS, BP 74, 29682 Roscoff cedex, Bretagne, France.
Baratte
B
B
Gompel
M
M
Borgne
A
A
Liabeuf
S
S
Romette
J L
JL
Jho
E H
EH
Costantini
F
F
Meijer
L
L
D000595:Amino Acid Sequence; D000818:Animals; D060466:Axin Protein; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D006801:Humans; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012097:Repressor Proteins; D014407:Tumor Cells, Cultured
D060466:Axin Protein; D011506:Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3