Liu Qinghang, Wilkins Benjamin J, Lee Yong J, Ichijo Hidenori, Molkentin Jeffery D
Department of Pediatrics, University of Cincinnati, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave., MLC7020, Cincinnati Ohio 45229-3039, USA.
Mol Cell Biol. 2006 May;26(10):3785-97. doi: 10.1128/MCB.26.10.3785-3797.2006.
The calcium-calmodulin-activated protein phosphatase calcineurin functions as a key mediator of diverse biologic processes, including differentiation, apoptosis, growth, and adaptive responses, in part through dephosphorylation and activation of nuclear factor of activated T-cell (NFAT) transcription factors. Apoptosis signal-regulating kinase 1 (ASK1) is an upstream component of the mitogen-activated protein kinases that serves as a pivotal regulator of cytokine-, oxidative-, and stress-induced cell death. Here, we performed a yeast two-hybrid screen with calcineurin B as bait, which identified ASK1 as a direct physical interacting partner. The C-terminal 218 amino acids of ASK1 were sufficient to mediate interaction with calcineurin B in yeast, as well as in mammalian cell lysates. Importantly, endogenous calcium binding B subunit (CnB) protein interacted with endogenous ASK1 protein in cardiomyocytes at baseline, suggesting that the interaction observed in yeast was of potential biologic relevance. Indeed, calcineurin directly dephosphorylated ASK1 at serine 967 using purified proteins or mammalian cell lysates. Dephosphorylation of ASK1 serine 967 by calcineurin promoted its disassociation from 14-3-3 proteins, resulting in ASK1 activation. Calcineurin and ASK1 cooperatively enhanced cardiomyocyte apoptosis, while expression of a dominant negative ASK1 blocked calcineurin-induced apoptosis. Mouse embryonic fibroblasts deficient in ask1 were also partially resistant to calcineurin- or ionomycin-induced apoptosis. Finally, ASK1 negatively regulated calcineurin-NFAT signaling indirectly through c-Jun NH2-terminal kinase (JNK)- and p38-mediated phosphorylation of NFAT, which blocked calcineurin- and agonist-dependent hypertrophic growth of cardiomyocytes. Thus, ASK1 and calcineurin-NFAT constitute a feedback regulatory circuit in which calcineurin positively regulates ASK1 through direct dephosphorylation, while ASK1 negatively regulates calcineurin-NFAT signaling through p38- and JNK-mediated NFAT phosphorylation.
钙调神经磷酸酶是一种钙调蛋白激活的蛋白磷酸酶，在多种生物学过程中发挥关键介导作用，包括分化、凋亡、生长和适应性反应，部分是通过使活化T细胞核因子（NFAT）转录因子去磷酸化并激活来实现的。凋亡信号调节激酶1（ASK1）是丝裂原活化蛋白激酶的上游成分，是细胞因子、氧化应激和应激诱导的细胞死亡的关键调节因子。在此，我们以钙调神经磷酸酶B为诱饵进行了酵母双杂交筛选，鉴定出ASK1是直接的物理相互作用伴侣。ASK1的C末端218个氨基酸足以介导其在酵母以及哺乳动物细胞裂解物中与钙调神经磷酸酶B的相互作用。重要的是，内源性钙结合B亚基（CnB）蛋白在基线时与心肌细胞中的内源性ASK1蛋白相互作用，这表明在酵母中观察到的相互作用具有潜在的生物学相关性。实际上，使用纯化蛋白或哺乳动物细胞裂解物时，钙调神经磷酸酶可直接使ASK1的丝氨酸967去磷酸化。钙调神经磷酸酶使ASK1丝氨酸967去磷酸化促进了它与14-3-3蛋白的解离，导致ASK1激活。钙调神经磷酸酶和ASK1协同增强心肌细胞凋亡，而显性负性ASK1的表达则阻断了钙调神经磷酸酶诱导的凋亡。缺乏ask1的小鼠胚胎成纤维细胞对钙调神经磷酸酶或离子霉素诱导的凋亡也有部分抗性。最后，ASK1通过c-Jun氨基末端激酶（JNK）和p38介导的NFAT磷酸化间接负调节钙调神经磷酸酶-NFAT信号传导，这阻断了钙调神经磷酸酶和激动剂依赖性的心肌细胞肥大生长。因此，ASK1和钙调神经磷酸酶-NFAT构成了一个反馈调节回路，其中钙调神经磷酸酶通过直接去磷酸化正向调节ASK1，而ASK1通过p38和JNK介导的NFAT磷酸化负调节钙调神经磷酸酶-NFAT信号传导。
