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

整合素是跨膜蛋白家族，其将细胞锚在其周围细胞外基质内。在血细胞上表达的一些整合素结合到其他细胞（例如内皮细胞）的表面受体，使得白细胞能够粘附并穿过血管壁迁移到组织中。整合素通过增加其配体结合亲和力和通过其组装成与细胞骨架强连接的复杂粘附结构来介导与其配体的强结合。这些功能依赖于两个关键的整合素调节蛋白，talin和kindlin，其结合到整合素的胞质结构域，并控制整合素的构象和组织成粘附复合物。解读talin和kindlin在整合素调节中的重要作用是过去10-20年来整合素研究的中心焦点。最近，这两种整联蛋白调节剂的调节已经开始被更详细地阐明。特别是，kindlin的翻译后修饰的调节一直知之甚少，是这里提出的研究项目的核心。kindlin-3是专门在造血细胞中形成的kindlin家族的成员，其质谱研究使我们能够鉴定出几个磷酸化位点。在这里，我们计划调查他们的作用，在不同的造血细胞群调节整合素功能。在初步工作中，我们已经证明，在静态和动态粘附研究中，丝氨酸8的磷酸化增加了整合素介导的细胞粘附。为此，我们已经产生了遗传修饰的Hoxb 8细胞系，其中kindlin-3蛋白的丝氨酸8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的全面生物化学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、整联蛋白功能和活性的调节以及整联蛋白粘附位点的形成的影响。(ii)此外，我们已经产生了两个基因修饰的小鼠品系携带这些突变的kindlin-3基因，研究丝氨酸8磷酸化的相关性，在体内调节整合素介导的过程。(iii)此外，我们将阐明整合素依赖的细胞粘附由于kindlin-3丝氨酸8磷酸化增强的机制。(iv)伴随着这项工作，我们将表征额外的磷酸化位点，我们已经确定在kindlin-3蛋白质方面的影响，在Hoxb 8细胞系统中的整合素功能的调节。

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.