Effects and regulation of phosphatase inhibitor-1 in the heart and its therapeutic potential in heart failure

蛋白质磷酸化/去磷酸化的调控是细胞内信号转导的重要组成部分。磷酸酶抑制因子-1(I-1)是一种典型的蛋白水解酶和磷酸酶相互作用的介导物。活化的cAMP依赖的蛋白激酶(PKA)在Thr-35处磷酸化I-1，将其转化为磷酸酶-1的有效抑制物。抑制磷酸酶-1随后增强了PKA介导的蛋白质磷酸化。我们发现，I-1的腺病毒过表达确实能放大成年大鼠心肌细胞的肌电激动剂反应。这与磷蛋白(PLB)的磷酸化增加有关，PLB是心脏功能的关键调节因子。相反，在衰竭的人类心脏样本中，我们观察到Thr-35处I-1的下调和去磷酸化与PLB的磷酸化降低有关。在最近的一项研究中，据报道，Ser-67上I-1的磷酸化降低了它抑制磷酸酶-1的能力。有趣的是，细胞内Ca~(2+)的升高可以通过相同的Ca~(2+)激活的磷酸酶2B(钙调神经磷酸酶)触发两个磷酸化位点的去磷酸化。本项目的目的是(I)测试I-1过表达是否对不同的PKA底物(例如pLb与兰诺定受体相反)的磷酸化有不同的影响，(Ii)评价I-1在心肌细胞中的激酶/磷酸酶对I-1的S调节，(Iii)鉴定I-1过表达的小鼠(心脏特异的I-1TG)，(Iv)在可调节启动子(Tet-Off系统)的控制下建立和鉴定过表达突变的I-1的转基因小鼠(在I-1敲除背景上被Ala取代的Ser-67)，和(V)将I-1TG小鼠与收缩功能障碍的小鼠模型杂交。这一结果有望扩大我们对I-1在心肌细胞信号转导中的作用的理解，并有助于回答I-1是否可能成为治疗衰竭心脏功能抑制的潜在靶点的问题。

Control of protein phosphorylation/dephosphorylation is an integral component of intracellular signal transduction. Phosphatase inhibitor-1 (I-1) is a prototypical mediator of cross-talk between kinases and phosphatases. Activated cAMP-dependent protein kinase (PKA) phosphorylates I-1 at Thr-35, converting it into a potent inhibitor of phosphatase-1. Inhibition of phosphatase-1 subsequently enhances PKA-mediated protein phosphorylation. We showed that adenovirally overexpression of I-1 does indeed amplify ß-agonist responses in adult rat cardiac myocytes. This was associated with increased phosphorylation of phospholamban (PLB), a key regulator of cardiac function. Conversely, in samples from failing human hearts we observed that downregulation and dephosphorylation of I-1 at Thr-35 was associated with decreased phosphorylation of PLB. In a recent study, phosphorylation of I-1 at Ser-67 has been reported to reduce its ability to inhibit phosphatase-1. Interestingly, a rise in intracellular Ca2+ could trigger the dephosphorylation of both phosphorylation sites by the same Ca2+-activated phosphatase 2B (calcineurin). The aim of this project is (i) to test whether overexpression of I-1 has different effects on phosphorylation of various PKA substrates (e.g. PLB as opposed to the ryanodine receptor), (ii) to evaluate I-1´s regulation by kinases/phosphatases in cardiac myocytes, (iii) to characterize I-1 overexpressing mice (cardiac specific I-1 TG), (iv) to generate and to characterize transgenic mice overexpressing mutated I-1 (Ser-67 replaced by Ala on an I-1 knockout background) under control of a regulatable promoter (Tet-Off system), and (v) to cross-breed I-1 TG mice with mice models with contractile dysfunction. The results are expected to extend our understanding of I-1´s role in cardiac myocyte signaling and contribute to answering the question whether I-1 may constitute a potential target in treating the depressed function of failing hearts.