Cardiovascular Research Advance Access [Accepted Manuscript] published online on June 3, 2009
Cardiovascular Research, doi:10.1093/cvr/cvp175
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Glycogen synthase kinase-3β is activated by matrix metalloproteinase-2 mediated proteolysis in cardiomyoblasts
Departments of Pediatrics and Pharmacology, Cardiovascular Research Group, University of Alberta, Edmonton, Alberta, Canada
Correspondence: Dr. Richard Schulz, Cardiovascular Research Group, University of Alberta, 4-62 Heritage Medical Research Centre, Edmonton, Alberta, Canada T6G 2S2. E-mail: richard.schulz{at}ualberta.ca. Tel: +1-780-492-6581; Fax: +1-780-492-9753
Aims: Matrix metalloproteinase (MMP)-2 contributes to myocardial oxidative stress injury by degrading sarcomeric and cytoskeletal proteins in cardiomyocytes. Glycogen synthase kinase (GSK)-3β activity is dysregulated during oxidative stress and is susceptible to proteolytic cleavage. Here we determined whether GSK-3β is a MMP-2 substrate as a result of oxidative stress.
Methods: MMP-2 and GSK-3β 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-3β kinase activity was measured using a peptide substrate and [
-32P]-ATP. Oxidative stress in H9c2 cardiomyoblasts was induced by H2O2 and the levels and activities of MMP-2 and GSK-3β were measured.
Results: Incubation of 47 kDa GSK-3β with MMP-2 resulted in the time- and concentration-dependant cleavage of GSK-3β as seen by appearance of an 
30 kDa fragment. MS analysis and Mascot database search yielded a peptide with an amino acid sequence of GSK-3β lacking the N-terminal region. GSK-3β 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-3β and significantly increased GSK-3β kinase activity. Both the loss of intact GSK-3β 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-3β.
Conclusions: GSK-3β 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-3β where the inhibitory phosphorylation of serine-9 occurs. MMP-2 mediated augmentation of GSK-3β kinase activity may contribute to cardiac injury resulting from enhanced oxidative stress.
Time for primary review: 33 Days
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  A. D. Kandasamy, A. K. Chow, M. A.M. Ali, and R. Schulz Matrix metalloproteinase-2 and myocardial oxidative stress injury: beyond the matrix Cardiovasc Res, August 20, 2009; (2009) cvp268v2. [Abstract] [Full Text] [PDF]
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