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.

项目概要 心力衰竭新疗法的开发是一个迫切需要，目前该领域的药物开发正在推进 不足以跟上这种疾病发病率和死亡率不断上升的步伐。 需要更接近人类心脏生理学的模型系统。 (PKA) 是一种 cAMP 敏感激酶，是心肌细胞 β-肾上腺素能刺激的中枢继电器 收缩和钙通量具有广泛的底物，信号传导的特异性依赖于仔细的调节。 cAMP 代谢和激酶活性的调节亚基都抑制激酶活性并有帮助。 招募效应子和大分子结合伙伴来协调 I 型调节亚基。 被广泛表达，但它们的功能如何调节尚不清楚。 研究表明 Iα 调节亚基 (PRKAR1A) 被激酶 STK25 磷酸化。 在人诱导多能干细胞衍生的心肌细胞（iPSC-CM）中进行，磷酸化 PRKAR1A 通过抑制 PKA 激酶活性和响应 cAMP 的下游信号传导 小鼠中 Stk25 与催化亚基的结合增加证实了其体内抑制作用。 在进一步的初步数据中，Stk25 敲除与术后结果的改善相关。 心肌梗塞与纤维化减少和心脏功能增强在一组逻辑和可行的。 目的，我们建议检验 PRKAR1A 的 STK25 磷酸化导致 PKA 抑制的假设 活性，并且 STK25 和 PRKAR1A 之间的这种信号传导具有治疗潜力。 对 iPSC-CM 进行基因改造，以研究磷酸化如何导致增加的机制 PKA 活性的抑制以及表征响应中大分子 PKA 复合物的变化 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。 Prkar1a 磷酸化及其体内 PKA 活性的调节第三个目标是诱导条件。 在小鼠中产生 Stk25 敲除，用于探索 Stk25 敲除后改善的机制 Stk25 缺失引起的心肌梗塞 我们将检查心肌梗塞后 Stk25 是否缺失。 带来任何好处以及 Prkar1a 磷酸化的减少是否会介导这种改善。 将研究一种新型抑制剂，以探索心肌梗塞后靶向该通路的药理学潜力 我们相信这一提议将对我们对 PKA 的理解产生重大影响。 心肌细胞中的调节并确定 PRKAR1A 磷酸化作为潜在的治疗方法 心肌梗塞和心力衰竭的治疗方式。