New protein kinase A and G-dependent signaling pathways and networks in the regulation of platelet activation

血小板活化调节中的新蛋白激酶 A 和 G 依赖性信号通路和网络

• 批准号: 261249434
• 负责人: Professorin Dr. Kerstin Jurk
• 金额: --
• 依托单位: Centrum für Thrombose und Hämostase (CTH)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/261249434?language=en
• 关键词: New; protein; kinase; dependent; signaling

Platelet function is tightly regulated by activatory and inhibitory signaling pathways. Cyclic nucleotide-dependent protein kinases A (PKA) and G (PKG) represent major key players in inhibition of important platelet activation steps, including adhesion, shape change, integrin activation, granule release, aggregation and pro-coagulant activity. However, our understanding of the complex network of PKA/PKG-dependent inhibitory signaling that modulates distinct platelet functions is incomplete. Based on our recently published data with phospho-CalDAG-GEFI and integrin regulation [A9, A4] we hypothesize that the identification and functional characterization of novel cAMP/PKA and cGMP/PKG substrates will identify important mechanisms of platelet inhibition. Furthermore, these studies may indicate attractive candidates for novel diagnostic and /or therapeutic targets for selective platelet activation or inhibition.Based on preliminary phosphoproteome data we intend to investigate the role of newly identified PKA/PKG-specific and PKA/PKG-common substrate proteins in inhibitory platelet function by using established biochemical and functional in vitro methods that enable analysis of phospho-deficient/-mimetic megakaryocytic mutants and knockdowns as well as phosphoprotein ligands in platelets. Functional characterization of the novel PKA/PKG-common substrate endosulfine-alpha (ENSA) will be additionally addressed by generation of a megakaryocyte- and platelet-specific ENSA-deficient mouse model. Quantitative LC-MS-based phosphoproteomic and advanced platelet function analysis will be used to comprehensively elucidate the signaling crosstalk between PKA- or PKG-mediated inhibiting, and thrombin- or collagen-mediated activating pathways and its consequences for platelet function. Based on bioinformatics analysis of quantitative phosphoproteomic data we aim to identify relevant inhibitory downstream signatures and to develop a dynamic platelet inhibitory model.

血小板功能受激活和抑制信号通路的严格调控。环核苷酸依赖性蛋白激酶A(PKA)和环核苷酸依赖性蛋白激酶G(PKG)是抑制血小板黏附、变形、整合素活化、颗粒释放、聚集和促凝活性等重要活化步骤的关键分子。然而，我们对调控不同血小板功能的PKA/PKG依赖的抑制信号的复杂网络的了解是不完整的。根据我们最近发表的关于磷酸化CalDAG-GEFI和整合素调控的数据[A9，A4]，我们推测，新的cAMP/PKA和cGMP/PKG底物的鉴定和功能表征将识别重要的血小板抑制机制。此外，这些研究可能为选择性激活或抑制血小板的新的诊断和/或治疗靶点提供有吸引力的候选。基于初步的磷蛋白质组数据，我们打算通过建立的体外生化和功能方法来研究新发现的PKA/PKG特异性和PKA/PKG共同底物蛋白在抑制血小板功能中的作用，这些方法使得能够分析血小板中磷缺乏/模拟的巨核细胞突变体和敲除以及磷蛋白配体。新的PKA/PKG-共同底物Enosuline-Alpha(ENSA)的功能特征将通过建立巨核细胞和血小板特异性ENSA缺陷小鼠模型来解决。基于LC-MS的磷酸蛋白质定量分析和先进的血小板功能分析将全面阐明PKA或PKG介导的抑制和凝血酶或胶原介导的激活途径之间的信号串扰及其对血小板功能的影响。基于对定量磷蛋白质组数据的生物信息学分析，我们的目标是识别相关的抑制下游信号，并建立动态的血小板抑制模型。

项目成果

Proteome-wide detection of S-nitrosylation targets and motifs using bioorthogonal cleavable-linker-based enrichment and switch technique

• DOI: 10.1038/s41467-019-10182-4
• 发表时间: 2019-05-16
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Mnatsakanyan, Ruzanna;Markoutsa, Stavroula;Zahedi, Rene P.
• 通讯作者: Zahedi, Rene P.

Platelets and Renal Disorders

• DOI: 10.1007/978-3-319-47462-5_79
• 发表时间: 2017
• 作者: J. Lutz;K. Jurk