Glycogen synthase kinase-3β is activated by matrix metalloproteinase-2 mediated proteolysis in cardiomyoblasts

Matrix metalloproteinase (MMP)-2 contributes to myocardial oxidative stress injury by degrading sarcomeric and cytoskeletal proteins in cardiomyocytes. Glycogen synthase kinase (GSK)-3 beta is dysregulated during oxidative stress and is susceptible to proteolytic cleavage. Here we determined whether GSK-3 beta is a MMP-2 substrate as a result of oxidative stress. MMP-2 and GSK-3 beta were incubated and the cleavage fragments were identified by immunoblotting and silver stain. The intact protein and its primary cleavage fragment were subjected to trypsin digestion and the resultant peptides were analysed by LC-MS/MS. GSK-3 beta kinase activity was measured using a peptide substrate and [gamma-P-32]-ATP. Oxidative stress in H9c2 cardiomyoblasts was induced by H2O2 and the levels and activities of MMP-2 and GSK-3 beta were measured. Incubation of 47 kDa GSK-3 beta with MMP-2 resulted in the time- and concentration-dependant cleavage of GSK-3 beta as seen by appearance of an similar to 30 kDa fragment. MS analysis and Mascot database search yielded a peptide with an amino acid sequence of GSK-3 beta lacking the N-terminal region. GSK-3 beta kinase activity was significantly increased upon incubation with MMP-2 which was abrogated by the MMP inhibitor GM-6001. H2O2 challenge of H9c2 cardiomyoblasts significantly increased the activity and level of MMP-2, reduced the level of GSK-3 beta, and significantly increased GSK-3 beta kinase activity. Both the loss of intact GSK-3 beta and increase in its kinase activity were reduced with MMP inhibitors. MMP-2 pull-down assays in H9c2 cell lysates showed the association of MMP-2 with GSK-3 beta. GSK-3 beta may be a target of MMP-2 and its cleavage by MMP-2 enhances its kinase activity. MMP-2 may cleave off the N-terminal of GSK-3 beta where the inhibitory phosphorylation of serine-9 occurs. MMP-2-mediated augmentation of GSK-3 beta kinase activity may contribute to cardiac injury resulting from enhanced oxidative stress.