Glycogen synthase kinase-3β is activated by matrix metalloproteinase-2 mediated proteolysis in cardiomyoblasts
Glycogen synthase kinase-3β is activated by matrix metalloproteinase-2 mediated proteolysis in cardiomyoblasts
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DOI：
10.1093/cvr/cvp175
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发表时间：
2009-09-01
影响因子：
10.8
通讯作者：
Schulz, Richard
中科院分区：
文献类型：
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作者：
Kandasamy, Arulmozhi D.;Schulz, Richard
基质金属蛋白酶（MMP）-2通过降解心肌细胞中的肌节蛋白和细胞骨架蛋白参与心肌氧化应激损伤。糖原合成酶激酶（GSK）-3 β在氧化应激期间失调，易受蛋白水解裂解的影响。在此，我们确定GSK-3 β是否作为氧化应激的结果是MMP-2的底物。将MMP-2和GSK-3 β孵育，并通过免疫印迹和银染鉴定切割片段。对完整蛋白质及其初级切割片段进行胰蛋白酶消化，并通过LC-MS/MS分析所得肽。使用肽底物和[γ-P-32]-ATP测量GSK-3 β激酶活性。用H2 O2诱导H9 c2心肌细胞氧化应激，测定MMP-2和GSK-3 β的水平和活性。47 kDa GSK-3 β与MMP-2的孵育导致GSK-3 β的时间和浓度依赖性切割，如通过类似于30 kDa片段的出现所见。MS分析和Mascot数据库搜索产生了具有GSK-3 β的氨基酸序列的肽，其缺少N-末端区域。GSK-3 β激酶活性在与MMP-2孵育后显著增加，MMP-2被MMP抑制剂GM-6001消除。H2 O2刺激H9 c2心肌细胞后，MMP-2的活性和水平显著升高，GSK-3 β的水平显著降低，GSK-3 β激酶活性显著升高。MMP抑制剂降低了完整GSK-3 β的损失及其激酶活性的增加。H9 c2细胞裂解物中的MMP-2下拉测定显示MMP-2与GSK-3 β的缔合。GSK-3 β可能是MMP-2的靶点，MMP-2对其的切割增强其激酶活性。MMP-2可以切割GSK-3 β的N-末端，在该处发生丝氨酸-9的抑制性磷酸化。MMP-2介导的GSK-3 β激酶活性增强可能导致氧化应激增强导致的心脏损伤。
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.