Identification and functional characterization of kindlin-3 phosphorylation and its role in integrin regulation in mice

kindlin-3磷酸化的鉴定和功能表征及其在小鼠整合素调节中的作用

• 批准号: 529848534
• 负责人: Privatdozent Dr. Markus Moser
• 金额: --
• 依托单位: Institute for Experimental Hematology
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/529848534?language=en
• 关键词: Identification; functional; characterization; kindlin; phosphorylation

Integrins are a family of transmembrane proteins that anchor cells within their ambient extracellular matrix. Some integrins expressed on blood cells bind to surface receptors of other cells such as endothelial cells, enabling leukocyte adhesion and transmigration through the vascular wall into the tissue. Integrins mediate strong binding to their ligands by increasing their ligand binding affinity and by their assembly into complex adhesion structures that are strongly linked to the cytoskeleton. These functions depend on the two key integrin regulatory proteins, talin and kindlin, which bind to the cytoplasmic domain of integrins and control both integrin conformation and organization into adhesion complexes. Deciphering the essential role of talin and kindlin in integrin regulation has been a central focus of integrin research over the past 10-20 years. Recently, the regulation of these two integrin regulators has begun to be elucidated in more detail. In particular, the regulation of kindlins by post-translational modifications has been poorly understood and is central to the research project proposed here. Mass spectrometry studies of kindlin-3, the member of the kindlin family formed exclusively in hematopoietic cells, have allowed us to identify several phosphorylation sites. Here, we plan to investigate their role in regulating integrin function in different hematopoietic cell populations. In preliminary work, we have shown that phosphorylation of serine 8 increases integrin-mediated cell adhesion in static and dynamic adhesion studies. To this end, we have generated genetically modified Hoxb8 cell lines in which the serine 8 of the kindlin-3 protein has been replaced by alanine or glutamic acid. In comprehensive biochemical and cell biological studies on neutrophils and macrophages differentiated from these cell lines, we aim to investigate the impact of this modification on (i) cell adhesion, regulation of integrin function and activity, and formation of integrin adhesion sites. (ii) In addition, we have already generated two genetically modified mouse lines carrying these mutations in the kindlin-3 gene to investigate the relevance of serine 8 phosphorylation for the regulation of integrin-mediated processes in vivo. (iii) Furthermore, we will elucidate the mechanism underlying the enhanced integrin-dependent cell adhesion due to kindlin-3 serine 8 phosphorylation. (iv) Accompanying this work, we will characterize additional phosphorylation sites we have identified in the kindlin-3 protein with respect to their impact on the regulation of integrin function in the Hoxb8 cell system.

整合素是跨膜蛋白家族，将细胞锚定在周围的细胞外基质内。血细胞上表达的一些整合素与其他细胞（例如内皮细胞）的表面受体结合，使白细胞能够粘附并通过血管壁迁移到组织中。整联蛋白通过增加其配体结合亲和力以及组装成与细胞骨架牢固连接的复杂粘附结构来介导与其配体的强结合。这些功能取决于两个关键的整合素调节蛋白：talin 和 kindlin，它们与整合素的细胞质结构域结合，并控制整合素构象和组织成粘附复合物。破译talin和kindlin在整合素调节中的重要作用一直是过去10-20年整合素研究的中心焦点。最近，这两种整合素调节剂的调节已开始得到更详细的阐明。特别是，人们对翻译后修饰对kindlins的调节知之甚少，这也是本文提出的研究项目的核心。 kindlin-3（仅在造血细胞中形成的 kindlin 家族成员）的质谱研究使我们能够识别几个磷酸化位点。在这里，我们计划研究它们在调节不同造血细胞群整合素功能中的作用。在初步工作中，我们已经表明，在静态和动态粘附研究中，丝氨酸 8 的磷酸化可增加整合素介导的细胞粘附。为此，我们生成了基因修饰的 Hoxb8 细胞系，其中 kindlin-3 蛋白的丝氨酸 8 已被丙氨酸或谷氨酸取代。在对从这些细胞系分化的中性粒细胞和巨噬细胞进行的综合生化和细胞生物学研究中，我们的目的是研究这种修饰对（i）细胞粘附、整合素功能和活性的调节以及整合素粘附位点形成的影响。 (ii) 此外，我们已经生成了两种在 kindlin-3 基因中携带这些突变的转基因小鼠品系，以研究丝氨酸 8 磷酸化与体内整合素介导过程调节的相关性。 (iii) 此外，我们将阐明由于 kindlin-3 丝氨酸 8 磷酸化而增强整合素依赖性细胞粘附的机制。 (iv) 伴随这项工作，我们将表征我们在 kindlin-3 蛋白中鉴定的其他磷酸化位点及其对 Hoxb8 细胞系统中整合素功能调节的影响。