Activation of the beta-3 integrins: Role of the Kindlins

摘要 该提案旨在确定kindlin-3在生理和病理生理反应中的作用。 Kindlin-3（FERMT 3）是FERM结构域胞内衔接蛋白家族的三个成员之一， 具有>20个结合配偶体，从而控制多种细胞类型中的多种反应。我们的贡献 kindlin领域的研究包括证明它们在整合素活化中的重要作用; 它们与整联蛋白β亚基相互作用的分子基础;一种疾病，LADIII， 与严重出血、免疫力低下、感染易感性增加以及 由kindlin-3缺乏引起的骨硬化症，证明不仅是整联蛋白依赖性的，而且是 kindlin的整合素独立功能（例如，扰动止血和血管通透性）;以及 kindlin在癌症生物学中的核心作用的含义。增加kindlin-3重要性的是， 它并不局限于造血细胞，而是存在于并有助于造血细胞的生物反应。 内皮细胞和癌细胞，特别是乳腺癌的进展和转移。尽管 kindlin-3在血管生物学和病理学中的明确意义，正是这种kindlin特别被认为是 由于缺乏细胞和体内模型来了解其广泛性， 生物功能。我们已经解决了其中一些障碍，现在准备解决关键问题 关于Kindlin-3我们已经开发了检测方法和小鼠品系，其中Kindlin-3的分子基础 功能可以分解。具体而言，我们手头有小鼠品系，其中整合素依赖性和 可以区分独立的应答（整联蛋白结合位点失活）和组织特异性敲除 Kindlin-3以将其从单核细胞/巨噬细胞和红系细胞中删除。我们还开发了CRISPR/cas9 这项技术使我们能够敲除或取代癌细胞中的kindlin-3。重要的是我们有 在kindlin-3中发现了一个独特的磷酸化位点，该位点在其他两种kindlin中并不保守， 表明这种翻译后事件在造血和乳腺癌细胞中具有功能意义 并区分Kindlin-3结合配偶体。我们的具体目标是：1）确定整合素的作用- 在乳腺癌进展和转移中的依赖性和非依赖性功能; 2）确定 Kindlin-3磷酸化在血管和癌细胞中的重要性;和3）确定Kindlin-3如何 影响红细胞生成和红系细胞形状。 总之，这些研究将建立kindlin-3发挥其已知功能的基本机制， 确定了该分子以前未被认识到的功能，并可能确定Kindlin-3是否可以作为 生物标志物和作为治疗靶点。

Abstract This proposal seeks to establish the role of kindlin-3 in physiological and pathophysiological responses. Kindlin-3 (FERMT3) is one of the three member family of FERM domain intracellular adapter proteins, which has >20 binding partners and thereby control numerous responses in numerous cells types. Our contributions to the kindlin field have included the demonstration of their essential role in integrin activation; delineation of the molecular basis for their interaction with integrin beta subunits; identification of a disease, LADIII, associated with severe bleeding, compromised immunity, increased susceptibility to infections and osteopetrosis arising from a deficiency of kindlin-3, demonstration of not only integrin dependent, but also integrin independent functions of kindlins (e.g. perturbed hemostasis and vascular permeability); and the implication of a central role of the kindlins in cancer biology. Adding to the importance of kindlin-3 is evidence that it is not restricted to hematopoietic cells, but is present in and contributes to the biological responses of endothelial cells and cancer cells, particularly to the progression and metastasis of breast cancer. Despite the clear significance of kindlin-3 in vascular biology and pathology, it is this kindlin that has been particularly resistant to analysis arising from the lack of cellular and in vivo models to understand the breadth of its biological functions. We have resolved some of these barriers and are now poised to resolve key questions regarding kindlin-3. We have developed assays and mouse strains in which the molecular basis of kindlin-3’s function can be dissected. Specifically, we have in hand mouse strains in which the integrin-dependent and independent responses can be distinguished (integrin binding site disabled) and tissue specific knockouts of kindlin-3 to delete it from monocyte/macrophages and erythroid cells. We have also developed CRISPR/cas9 technology which has allowed us to knockout or replace kindlin-3 in cancer cells. Importantly, we have identified a unique phosphorylation site in kindlin-3 that is not conserved in the other two kindlins, and have shown that this post-translational event is of functional significance in hematopoietic and breast cancer cells and distinguishes between kindlin-3 binding partners. Our specific aims are: 1) identify the role of integrin- dependent and -independent functions in breast cancer progression and metastasis; 2) determine the importance of phosphorylation of kindlin-3 in vascular and cancer cells; and 3) determine how kindlin-3 influences erythropoiesis and erythroid cell shape. Overall, these studies will establish basic mechanisms by which kindlin-3 exerts its known functions, may identify previously unappreciated functions of this molecule and may establish if kindlin-3 can serve as a biomarker and as a therapeutic target.