Glycogen Synthase Kinase 3 beta (GSK3ß) is a multifunctional serine/threonine protein kinase, which is part of the PI3Kinase/Akt-, Wnt- and Hedgehog-pathway. Hence it has important roles in the regulation of gene transcription, protein synthesis and apoptosis. GSK3ß is implicated in the development of cardiac hypertrophy, type 2 diabetes and Alzheimers' disease. Besides glycogen synthase, several substrates like eIF2B, NFAT and Axin have already been identified.

To systematically analyze GSK3ß dependent signal transduction we aimed at identification of GSK3ß's interacting partners via Tandem Affinity Purification (TAP). W created a recombinant TAP-tagged GSK3ß (GSK3ß-TAP-Tag) carrying a Flag/Strep-Tag at the C-terminal end. Hereby we were able to perform a clean two-step purification of the recombinant GSK3ß and its interacting partners under native conditions.

We found a similar behaviour of the recombinant GSK3ß-TAP-Tag compared to the endogenously expressed GSK3ß concerning subcellular localization (confocal microscopy) and its phosphorylation by Protein kinase B (Akt). Using a synthetic peptide of Glycogen synthase as a substrate we could demonstrate that the tagged GSK3ß also had enzymatic activity. Gel filtration analyses showed that both enzymes, GSK3ß and GSK3ß-TAP-Tag, formed high molecular weight complexes in cells.

To identify protein interactions of GSK3ß transfected HEK293 cells were treated with Ly294002 (Inhibitor of PI3Kinase) to stimulate GSK3ß activity. Purification of the recombinant protein led to the copurification of protein complexes, which were identified via mass spectrometry. Besides GSK3ß-TAP-Tag, we found Acetyl-CoA-carboxylase, ATP-citrate synthase and Axin, which are known to be phosphorylated by GSK3ß. Furthermore, the following proteins were identified as interacting enzymes of GSK3ß: Alanyl-tRNA synthetase, Annexin A2, Elongation factor 1, Elongation factor 2, Filamin, Glyceraldehyde-3-phosphate dehydrogenase, Heat shock cognate 71 protein, HSP A5 Protein, HSP 90-beta, Nuclear migration protein nudC, Pyruvate kinase M1, Transketolase.

Conclusion: 

TAP is a powerful method to identify interacting proteins of GSK3ß. Several known interacting enzymes of GSK3ß were identified, also some new interacting partners of GSK3ß were found. Our results point to a connection of GSK3ß to important cellular processes like cell metabolism, cytoskeletal organization and subcellular transport mechanisms.