title: Calpain regulates N-terminal interaction of GSK-3β with 14-3-3ζ, p53 and PKB but not with axin.
abstract: Calpain produces a truncation of GSK3β 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-3β and calpain-cleaved GSK-3β. Commercial GSK-3β 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-3β. Using axin GST-tagged, we pulled down the protein in the presence of full length GSK-3β and calpain-cleaved GSK-3β. Western blot analyses showed full length GSK-3β in the pellet as well GSK-3β cleaved by calpain. Thus axin was able to bind GSK-3β without the N-terminal end. When the same experiment was carried out with GST-tagged 14-3-3ζ, p53 and PKB, full length GSK-3β was observed in the pellet, but GSK-3β truncated by calpain was not pulled down demonstrating that GSK-3β N-terminal end is necessary to interact with these three proteins. Our data demonstrate that N-terminal end is necessary for 14-3-3ζ, p53 and PKB interaction. However, the interaction of GSK3β with axin is not altered by calpain. These data support a physiological role for GSK3β truncation mediated by calpain.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC/UAM, Fac. Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
authors.forename: Paloma
authors.initials: P
authors.lastname: Goñi-Oliver
authors.forename: Jesús
authors.initials: J
authors.lastname: Avila
authors.forename: Félix
authors.initials: F
authors.lastname: Hernández
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry / Q000378:metabolism*; D060466:Axin Protein / Q000378:metabolism*; D015153:Blotting, Western; D002154:Calpain / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D038362:Glycogen Synthase Kinase 3 / Q000737:chemistry / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D011485:Protein Binding; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D060466:Axin Protein; D016159:Tumor Suppressor Protein p53; D000071679:Glycogen Synthase Kinase 3 beta; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; D002154:Calpain