Glycogen synthase kinase-3beta is activated by matrix metalloproteinase-2 mediated proteolysis in cardiomyoblasts.
OBJECTIVE
Matrix metalloproteinase (MMP)-2 contributes to myocardial oxidative stress injury by degrading sarcomeric and cytoskeletal proteins in cardiomyocytes. Glycogen synthase kinase (GSK)-3beta is dysregulated during oxidative stress and is susceptible to proteolytic cleavage. Here we determined whether GSK-3beta is a MMP-2 substrate as a result of oxidative stress.


RESULTS
MMP-2 and GSK-3beta 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-3beta kinase activity was measured using a peptide substrate and [gamma-(32)P]-ATP. Oxidative stress in H9c2 cardiomyoblasts was induced by H(2)O(2) and the levels and activities of MMP-2 and GSK-3beta were measured. Incubation of 47 kDa GSK-3beta with MMP-2 resulted in the time- and concentration-dependant cleavage of GSK-3beta as seen by appearance of an approximately 30 kDa fragment. MS analysis and Mascot database search yielded a peptide with an amino acid sequence of GSK-3beta lacking the N-terminal region. GSK-3beta kinase activity was significantly increased upon incubation with MMP-2 which was abrogated by the MMP inhibitor GM-6001. H(2)O(2) challenge of H9c2 cardiomyoblasts significantly increased the activity and level of MMP-2, reduced the level of GSK-3beta, and significantly increased GSK-3beta kinase activity. Both the loss of intact GSK-3beta 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-3beta.


CONCLUSIONS
GSK-3beta 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-3beta where the inhibitory phosphorylation of serine-9 occurs. MMP-2-mediated augmentation of GSK-3beta kinase activity may contribute to cardiac injury resulting from enhanced oxidative stress.
Kandasamy
Arulmozhi D
AD
Department of Pediatrics, Cardiovascular Research Centre, University of Alberta, 4-62 Heritage Medical Research Centre, Edmonton, AB, Canada T6G 2S2.
Schulz
Richard
R
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002154:Calpain / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004151:Dipeptides / Q000494:pharmacology; D004789:Enzyme Activation; D038362:Glycogen Synthase Kinase 3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D066298:In Vitro Techniques; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors; D008969:Molecular Sequence Data; D032386:Myoblasts, Cardiac / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D004151:Dipeptides; D061965:Matrix Metalloproteinase Inhibitors; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3; D002154:Calpain; D020778:Matrix Metalloproteinase 2