Mimicking phosphorylation of αB-crystallin on serine-59 is necessary and sufficient to provide maximal protection of cardiac myocytes from apoptosis
Mimicking phosphorylation of αB-crystallin on serine-59 is necessary and sufficient to provide maximal protection of cardiac myocytes from apoptosis
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DOI：
10.1161/01.res.0000052989.83995.a5
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发表时间：
2003-02-07
影响因子：
20.1
通讯作者：
Glembotski, CC
中科院分区：
文献类型：
--
作者：
Morrison, LE;Hoover, HE;Glembotski, CC
α B-晶体蛋白（alphaBC），一种在心脏中高水平表达的小的热休克蛋白，在应激后在Ser-19、45和59上磷酸化。然而，尚不清楚alphaBC磷酸化是否直接影响细胞存活。在本研究中，制备了编码在位置19、45和59处具有Ser至Ala（阻止磷酸化）或Ser至Glu（模拟磷酸化）突变的alphaBC形式的构建体。评价了每种形式对培养的心肌细胞在高渗或缺氧应激后凋亡的影响。与对照组相比，表达在所有三个位置具有Ser至Ala取代的alphaBC的细胞，alphaBC（AAA），表现出更多的应激诱导的凋亡。表达alphaBC（AAE）或（EEE）的细胞的细胞凋亡比表达alphaBC（AAA）的细胞少3倍，这表明Ser-59的磷酸化具有保护作用。已知alphaBC结合半胱天冬酶原-3并降低半胱天冬酶-3活化。与表达alphaBC（AAA）的细胞相比，表达alphaBC（AAE）的细胞中caspase-3的活化降低了3倍。这些结果表明，模拟Ser-59上的alphaBC的磷酸化对于赋予半胱天冬酶-3抑制和保护心肌细胞免受高渗或缺氧应激是必要的和足够的。这些发现提供了直接证据，证明alphaBC（S59 P）有助于在生理相关应激（如短暂缺氧）后观察到的心脏保护作用。确定alphaBC（S59 P）的靶点将揭示这种小热休克蛋白的细胞保护作用机制的重要细节。
alphaB-Crystallin (alphaBC), a small heat shock protein expressed in high levels in the heart, is phosphorylated on Ser-19, 45, and 59 after stress. However, it is not known whether alphaBC phosphorylation directly affects cell survival. In the present study, constructs were prepared that encode forms of alphaBC harboring Ser to Ala (blocks phosphorylation) or Ser to Glu (mimics phosphorylation) mutations at positions 19, 45, and 59. The effects of each form on apoptosis of cultured cardiac myocytes after hyperosmotic or hypoxic stress were assessed. Compared with controls, cells that expressed alphaBC with Ser to Ala substitutions at all three positions, alphaBC(AAA), exhibited more stress-induced apoptosis. Cells expressing either alphaBC(AAE) or (EEE) exhibited 3-fold less apoptosis than cells expressing alphaBC(AAA), indicating that phosphorylation of Ser-59 confers protection. alphaBC is known to bind to procaspase-3 and to decrease caspase-3 activation. Compared with cells expressing alphaBC(AAA), the activation of caspase-3 was decreased by 3-fold in cells expressing alphaBC(AAE). These results demonstrate that mimicking the phosphorylation of alphaBC on Ser-59 is necessary and sufficient to confer caspase-3 inhibition and protection of cardiac myocytes against hyperosmotic or hypoxic stress. These findings provide direct evidence that alphaBC(S59P) contributes to the cardioprotection observed after physiologically relevant stresses, such as transient hypoxia. Identifying the targets of alphaBC(S59P) will reveal important details about the mechanism underlying the cytoprotective effects of this small heat shock protein.