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

整合素是一类跨膜蛋白，可将细胞固定在细胞外基质内。在血细胞上表达的一些整合素与其他细胞（如内皮细胞）的表面受体结合，使白细胞通过血管壁粘附和转运进入组织。整合素通过增加其配体结合亲和力和组装成与细胞骨架紧密相连的复杂粘附结构，介导其与配体的强结合。这些功能依赖于两个关键的整合素调节蛋白，talin和kindlin，它们结合到整合素的细胞质结构域并控制整合素的构象和组织成粘附复合物。破译talin和kindlin在整合素调控中的重要作用是过去10-20年来整合素研究的中心焦点。近年来，这两种整合素调控因子的调控机制开始得到更详细的阐明。特别是，通过翻译后修饰对点燃的调节一直知之甚少，这是本文提出的研究项目的核心。kindlin-3是kindlin家族的成员，仅在造血细胞中形成，质谱研究使我们能够确定几个磷酸化位点。在这里，我们计划研究它们在不同造血细胞群中调节整合素功能的作用。在初步工作中，我们已经在静态和动态粘附研究中表明丝氨酸8的磷酸化增加了整合素介导的细胞粘附。为此，我们产生了转基因Hoxb8细胞系，其中kindin -3蛋白的丝氨酸8被丙氨酸或谷氨酸取代。在从这些细胞系分化出来的中性粒细胞和巨噬细胞的综合生化和细胞生物学研究中，我们旨在研究这种修饰对(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的形成的影响。（ii）此外，我们已经产生了两个携带kindin -3基因突变的转基因小鼠系，以研究丝氨酸8磷酸化在体内调节整合素介导的过程中的相关性。（iii）此外，我们将阐明kindin -3丝氨酸8磷酸化导致整合素依赖性细胞粘附增强的机制。（iv）伴随着这项工作，我们将描述我们在kindin -3蛋白中发现的其他磷酸化位点，以及它们对Hoxb8细胞系统中整合素功能调节的影响。

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.