STK25 phosphorylates PRKAR1A to regulate PKA signaling

STK25 磷酸化 PRKAR1A 来调节 PKA 信号传导

• 批准号: 10736399
• 负责人: Barry M. Fine
• 金额: $ 56.23万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736399
• 关键词: A kinase anchoring protein; Adrenergic Agents; Affinity; Animal Model; Anterior; Arteries; Binding; Biochemical; Biological Models; Calcium; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cardiovascular system; Catalytic Domain; Catecholamines; Cause of Death; Cell Death; Cell Line; Cell Survival; Chronic; Complex; Contracts; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Development; Disease; Dryness; Echocardiography; Fibrosis; Heart; Heart failure; Histologic; Human; Human Cell Line; In Vitro; Incidence; Knock-in; Knock-in Mouse; Knock-out; Knockout Mice; Lead; Left; Ligation; Loss of Heterozygosity; Macromolecular Complexes; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Mediating; Medical; Metabolic; Metabolism; Modality; Modeling; Mus; Mutation; Myocardial Infarction; Outcome; Pathway interactions; Phenotype; Phosphorylation; Phosphorylation Inhibition; Phosphotransferases; Physiology; Prevalence; Proliferating; Protein Dephosphorylation; Protein Inhibition; Protein Phosphorylation Inhibition; Protein Subunits; Proteins; Proteomics; Publishing; Regulation; Renin-Angiotensin-Aldosterone System; Role; Second Messenger Systems; Serum Markers; Signal Pathway; Signal Transduction; Specificity; Surveys; Survival Rate; System; Testing; Therapeutic; Time; Transgenic Mice; Work; conditional knockout; drug development; experimental study; fighting; follow-up; heart function; improved; improved outcome; in vitro activity; in vivo; induced pluripotent stem cell; induced pluripotent stem cell derived cardiomyocytes; inhibitor; insight; mimetics; mortality; mouse model; new therapeutic target; novel; novel therapeutics; pharmacologic; pressure; prevent; protein kinase A kinase; recruit; release of sequestered calcium ion into cytoplasm; response; therapeutic target; translational potential

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.

项目摘要 心力衰竭的新疗法的开发是迫切需要的，并且该领域中的当前药物开发是必要的。 不足以跟上这一疾病日益增加的发病率和死亡率。新的治疗靶点 需要更接近于人类心脏生理学的模型系统。蛋白激酶A (PKA)是一种cAMP敏感性激酶，是心肌细胞β-肾上腺素能刺激的中枢中继 收缩和钙流。由于底物广泛，信号传导的特异性依赖于仔细的调节 cAMP代谢和激酶活性。PKA的调节亚基既抑制激酶活性， 募集效应物和大分子结合伴侣以协调激酶信号传导。I型调节亚单位 被广泛表达，但它们的功能是如何调节的还不是很清楚。在最近出版的作品中，我们 证明I型α调节亚基（PRKAR 1A）被激酶STK 25磷酸化。研究中 在人诱导多能干细胞衍生的心肌细胞（iPSC-CM）中进行， PRKAR 1A导致PKA激酶活性的抑制和下游信号传导对cAMP的响应， 增加与催化亚基的结合。小鼠中Stk 25的敲除研究证实了其在体内抑制肿瘤生长的作用。 PKA活性。在进一步的初步数据中，Stk 25敲除与治疗后的结果改善相关。 心肌梗死伴纤维化减少和心功能增加。在一套符合逻辑和可行的 目的，我们提出测试的假设，STK 25磷酸化PRKAR 1A导致抑制PKA STK 25和PRKAR 1A之间的这种信号传导具有治疗潜力。在目标1中，我们使用 基因修饰的iPSC-CM的研究磷酸化如何导致增加的机制， 抑制PKA活性以及表征大分子PKA复合物响应的变化 PRKAR 1A的磷酸化。在目标2中，利用具有敲入突变的转基因小鼠来研究 体内Prkar 1a磷酸化及其对PKA活性的调节。在第三个目标中，一个诱导条件句 在小鼠中产生Stk 25的敲除，并用于探索在施用Stk 25后的改善的潜在机制。 Stk 25缺失引起的心肌梗死。我们将检查心肌梗死后Stk 25的丢失 是否赋予任何益处以及Prkar 1a磷酸化的减少是否介导这种改善。我们也 将研究一种新的抑制剂，以探索靶向这一途径的药理学潜力， 体内梗死。我们认为，这一建议将对我们理解PKA产生重大影响 在心肌细胞中的调节，并建立PRKAR 1A的磷酸化作为一种潜在的治疗 心肌梗死和心力衰竭的治疗方式。