Purification of GSK-3 by affinity chromatography on immobilized axin

Glycogen synthase kinase 3 (GSK-3), an element of the Wnt signalling pathwa y, plays a key role in numerous cellular processes including cell prolifera tion, embryonic development, and neuronal functions. It is directly involve d in diseases such as cancer (by controlling apoptosis and the levels of be ta -catenin and cyclin D1), Alzheimer's disease (tau hyperphosphorylation), and diabetes las a downstream element of insulin action, GSK-3 regulates g lycogen 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-3 bet a, beta -catenin, phosphatase 2A, and APC. A polyhistidine-tagged;ed axin p eptide (residues 419 - 672) was produced in Escherichia coli and either imm obilized on Ni-NTA agarose beads or purified and immobilized on CNBr-activa ted 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 ac tivity. This single step purification method provides a convenient tool to follow the status of GSK-3 (protein level, phosphorylation state, kinase ac tivity) 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. (C) 2000 Academic Press.