Clin Res Cardiol 100, Suppl 1, April 2011

P642 - Glycogen synthase kinase 3 beta (GSK3β) forms regulated protein complexes with Proteinkinase A
 
S. Hamer1, M. Reinartz1, M. Gerlach1, A. Gödecke1
 
1Institut für Molekulare Kardiologie, Düsseldorf;
 
Glycogen Synthase Kinase 3 beta (GSK3β) is a serine/threonine protein kinase, which is inhibited by phosphorylation on serine 9, by e.g. Akt or PKC. Recent results in transgenic mice demonstrated that cardiac specific expression of a constitutively active GSK3β antagonised  the development of cardiac hypertrophy in response to chronic β-adrenergic stimulation, suggesting antagonism of GSK3β and PKA-mediated signal transduction.
To systematically analyze GSK3β dependent signalling we aimed to identify GSK-associated protein complexes via Tandem Affinity Purification (TAP). Therefore the cDNA of GSK3β was C-terminally fused to a TAP-Tag consisting of a Flag/Strep II Tag. This enabled us to perform a two-step purification of the recombinant GSK3β and its interacting partners under native conditions. Lentiviral transduction of HEK293 cells and the cardiomyocyte cell line HL-1 was used to generate cell lines expressing GSK-tag constructs. To analyse whether the protein  interactions depended on the catalytic activity of GSK3β we constructed a Serine 9 mutant of the GSK3β-TAP-Tag (Ser9 -> Ala9), which cannot be phosphorylated and thereby is constitutively active. We also generated a mutant with mutated Lysines 85, 86 in the ATP-binding domain to Alanines which leads to an inactive, kinase-dead mutant.
Tandem affinity purification of GSK3β TAP-TAG and subsequent identification of copurified proteins by mass spectrometry resulted in the identification of multiple GSK3β associated protein complexes. In HEK293 cells the active GSK3β binds to AKAP220 and to Axin-1 whereas the inactive form was linked to DNA-dependent protein kinase catalytic subunit, CAD protein and Tubulin β. This demonstrated that GSK3β associates with different proteins depending on its catalytic activity. Similar results were obtained for HL-1 cells. Here we found complexes of the active GSK3β with Acetyl-CoA-carboxylase, Junction plakoglobin and Axin. In contrast, Calnexin and HSP90co-chaperone cdc37 coeluted only with the inactive GSK3β.
In addition, to the proteins described above, the regulatory and catalytic subunit of Proteinkinase A I alpha coeluted with GSK3β. Comparison of WT and mutant GSK3β demonstrated that the association with PKA was substantially reduced in the inactive GSK3β mutant. Moreover, stimulation of PKA with 8-Br- cAMP (2mM) in cells and in isolated GSK3β/PKA complexes led to a predominant release of the catalytic subunit from the PKA-GSK3β complex. In contrast, PKA inihibition by H-89 (10µM) did not alter the ratio of PKA subunits in the GSK3β associated complex. P-GSK3β levels in 8-Br-cAMP stimulated cells increased in comparison to H-89 treated cells, demonstrating that GSK3β is a substrate of PKA.
Conclusion: GSK3β modulates multiple biological processes via formation of stable protein complexes depending on its catalytic activity. A regulated interaction with the catalytic and regulatory subunits of PKA may form the basis for the observed antagonism of PKA and GSK3β.
 
Clin Res Cardiol 100, Suppl 1, April 2011
Zitierung mit Vortrags- oder Posternummer s.o.
DOI 10.1007/s00392-011-1100-y

http://www.abstractserver.de/dgk2011/ft/abstracts/P642.htm