STK25 phosphorylates PRKAR1A to regulate PKA signaling

项目摘要 开发治疗心力衰竭的新疗法是一项迫切的需要，目前该领域的药物开发是 不足以跟上这种疾病日益增长的发病率和死亡率。新的治疗靶点 更接近人类心脏生理学的模型系统也是必需的。蛋白激酶A 蛋白激酶A(PKA)是一种cAMP敏感的激酶，是β-肾上腺素能兴奋心肌细胞的中枢继电器 收缩和钙流动。由于底物种类繁多，信号的特异性依赖于仔细的调节。 对cAMP代谢和激酶活性的影响。PKA的调节亚基既能抑制激酶活性，又能帮助 招募效应器和大分子结合伙伴来协调激酶信号。第I类调节亚基 被广泛表达，但其功能是如何调节的还不是很清楚。在最近出版的著作中，我们 证明I型α调节亚基(PRKAR1a)被激酶STK25磷酸化。在研究中 在人诱导多能干细胞来源的心肌细胞(IPSC-CM)中进行，磷酸化 PRKAR1A通过cAMP途径抑制PKA激酶活性和下游信号转导 增加与催化亚单位的结合。小鼠体内STK25基因敲除研究证实其体内抑制作用 PKA活性。在进一步的初步数据中，STK25基因敲除与预后的改善有关 心肌梗死后纤维化减轻，心功能增强。在一套合乎逻辑和可行的 旨在验证PRKAR1A的STK25磷酸化导致PKA抑制的假设 STK25和PRKAR1A之间的这种信号转导具有治疗潜力。在目标1中，我们使用 转基因IPSC-CM研究磷酸化导致增加的机制 抑制PKA活性以及表征作为响应的大分子PKA复合体的变化 与PRKAR1a的磷酸化有关。在目标2中，利用具有敲入突变的转基因小鼠来研究 Prkar1a的磷酸化及其对体内PKA活性的调节。在第三个目标中，一个诱导性的条件 STK25基因敲除是在小鼠体内产生的，并用于探索在 STK25缺失引起的心肌梗死。我们将检查心肌梗死后STK25的丢失 是否有任何益处以及Prkar1a磷酸化水平的降低是否介导了这种改善。我们也 将研究一种新的抑制剂，以探索在心肌梗死后靶向这一途径的药理学潜力 活体内梗塞。我们相信，这一建议将对我们对PKA的理解产生重大影响 对心肌细胞的调节和建立PRKAR1A的磷酸化作为一种潜在的治疗方法 心肌梗死和心力衰竭的方式。

Project Summary The development of new therapies in heart failure is a critical need and current drug development in this field is not sufficient to keep pace with the increasing incidence and mortality of this disease. Novel therapeutic targets are required as are model systems that more closely resemble human cardiac physiology. Protein kinase A (PKA) is a cAMP sensitive kinase that is the central relay for beta-adrenergic stimulation of cardiomyocyte contraction and calcium flux. With a broad set of substrates, specificity of signaling relies on careful regulation of both cAMP metabolism and kinase activity. Regulatory subunits of PKA both inhibit kinase activity and help recruit effectors and macromolecular binding partners to orchestrate kinase signaling. Type I regulatory subunits are broadly expressed yet how their function is modulated is not well known. In recently published work, we demonstrate that the Type Iα regulatory subunit (PRKAR1A) is phosphorylated by the kinase STK25. In studies performed in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CM), phosphorylation of PRKAR1A led to inhibition of PKA kinase activity and downstream signaling in response to cAMP through increased binding to the catalytic subunit. Knockout studies of Stk25 in mice confirmed its in vivo role of inhibiting PKA activity. In further preliminary data, the Stk25 knockout was associated with improved outcomes after myocardial infarction with decreased fibrosis and increased cardiac function. In a set of logical and feasible aims, we propose to tests the hypothesis that STK25 phosphorylation of PRKAR1A leads to inhibition of PKA activity and that this signaling between STK25 and PRKAR1A has therapeutic potential. In Aim 1, we use genetically modified iPSC-CM’s to investigate the mechanism of how phosphorylation leads to increased inhibition of PKA activity as well as characterize the changes to the macromolecular PKA complex in response to phosphorylation of PRKAR1A. In Aim 2, transgenic mice with knock-in mutations are utilized to investigate Prkar1a phosphorylation and its regulation of PKA activity in vivo. In the third aim, an inducible conditional knockout of Stk25 is generated in mice and is used to explore the mechanism underlying the improvement after myocardial infarction in response to loss of Stk25. We will examine if loss of Stk25 after a myocardial infarction imparts any benefit and whether a decrease in Prkar1a phosphorylation mediates this improvement. We also will investigate a novel inhibitor to explore the pharmacologic potential of targeting this pathway after myocardial infarction in vivo. We believe that this proposal will have significant impact on our understanding of PKA regulation in cardiomyocytes and establish the phosphorylation of PRKAR1A as a as a potential therapeutic modality in myocardial infarction and heart failure.