Calpain regulates N-terminal interaction of GSK-3β with 14-3-3ζ, p53 and PKB but not with axin

Calpain produces a truncation of GSK3 beta that removes the N-terminal inhibitory domain. Here we analyze the effect of that truncation on protein-protein interaction. We pulled down GST-tagged proteins in the presence of full length GSK-3 beta and calpain-cleaved GSK-3 beta. Commercial GSK-3 beta was first incubated with calpain for 2.5 min in vitro, and then with GST-tagged proteins in the presence of calpeptin, a synthetic calpain inhibitor. Western blot analyses were performed to determine if there is an interaction between these GST-tagged proteins and truncated GSK-3 beta. Using axin GST-tagged, we pulled down the protein in the presence of full length GSK-3 beta and calpain-cleaved GSK-3 beta. Western blot analyses showed full length GSK-3 beta in the pellet as well GSK-3 beta cleaved by calpain. Thus axin was able to bind GSK-3 beta without the N-terminal end. When the same experiment was carried out with GST-tagged 14-3-3 zeta, p53 and PKB, full length GSK-3 beta was observed in the pellet, but GSK-3 beta truncated by calpain was not pulled down demonstrating that GSK-3 beta N-terminal end is necessary to interact with these three proteins. Our data demonstrate that N-terminal end is necessary for 14-3-3 zeta p53 and PKB interaction. However, the interaction of GSK3 beta with axin is not altered by calpain. These data support a physiological role for GSK3 beta truncation mediated by calpain. (C) 2011 Elsevier B.V. All rights reserved.