Mechanisms for regulation of cell adhesion and migration

描述（由申请人提供）：受调节的细胞粘附和迁移对多种生物过程至关重要，包括早期胚胎发育、神经元路径寻找、炎症反应、伤口愈合和组织完整性、细胞介导的免疫以及肿瘤侵袭和转移。整合素跨膜受体是细胞迁移的关键调节因子，并通过与整合素细胞质域相关的调节蛋白的复杂组装起作用，其中许多蛋白起分子支架的作用。本研究的目的是确定新的调节途径，其中支架蛋白调节细胞粘附和迁移通过蛋白质伴侣的调节结合。在之前的工作中，我们表征了LIM结构域支架PINCH，它是整合素依赖性细胞粘附、迁移和信号转导所必需的。PINCH与Integrin-linked kinase （ILK）和Ras suppressor 1 （RSU1）具有高亲和力结合，RSU1是一种富含亮氨酸的重复蛋白，可抑制表达活化Ras的培养细胞的转化，Ras是30%的人类肿瘤中存在的致癌基因。我们提出，PINCH支架作为一个节点，通过调节ILK和RSU1等伙伴的募集，耦合到其他信号通路，来调节整合素信号传导。通过在果蝇中进行的一系列实验，我们特异性地破坏了与PINCH的结合相互作用，并改变了PINCH复合物组分的亚细胞定位，我们将剖析在执行PINCH的整合素依赖功能时ILK和RSU1结合的要求。我们的初步数据表明，与ILK的结合将PINCH定位在细胞膜上的整合素相关复合物中，并调节PINCH向细胞核的穿梭。我们将确定PINCH是否在细胞核中具有基本功能，以及它是否在细胞质中与细胞核中具有重叠的结合伙伴补体。其他数据表明，RSU1通过与PINCH的相互作用被隔离在整合素相关复合物中。我们将研究PINCH-RSU1结合是如何调节的，并确定独立于PINCH的RSU1的伙伴和活动。对PINCH如何通过其支架活性调节细胞粘附和迁移的分子理解可能最终为转移性癌症等人类疾病的预防和治疗干预提供策略，在这些疾病中，异常细胞迁移起着突出作用。公共卫生相关性：细胞粘附和迁移是许多生物过程的关键组成部分，包括胚胎发育、炎症和免疫反应、伤口愈合和肿瘤侵袭和转移。细胞的粘附和迁移是由一个复杂的调控电路网络控制的。这项研究将定义新的分子机制，通过蛋白质相互作用调节粘附和迁移，最终目标是确定由异常细胞迁移引起的疾病的潜在治疗靶点，如肿瘤的恶性进展。

DESCRIPTION (provided by applicant): Regulated cell adhesion and migration are critical for a variety of biological processes including early embryonic development, neuronal path-finding, inflammatory response, wound healing and tissue integrity, cell-mediated immunity, and tumor invasion and metastasis. Integrin trans-membrane receptors are key regulators of cell migration, and act through an intricate assembly of regulatory proteins associated with the integrin cytoplasmic domain, many of which function as molecular scaffolds. The goal of this research is to identify novel regulatory pathways by which scaffolding proteins modulate cell adhesion and migration via regulated binding of protein partners. In previous work, we characterized the LIM domain scaffold PINCH, which is required for integrin-dependent cell adhesion, migration and signaling. PINCH binds with high affinity to both Integrin-linked kinase (ILK) and Ras suppressor 1 (RSU1), a Leucine-rich repeat protein that suppresses transformation of cultured cells expressing activated Ras, an oncogene present in 30% of all human tumors. We propose that the PINCH scaffold serves as a node for modulating integrin signaling by regulated recruitment of partners like ILK and RSU1, coupled to other signaling pathways. Through a series of experiments in Drosophila in which we specifically disrupt binding interactions with PINCH and alter the subcellular localization of components of the PINCH complex, we will dissect the requirement for ILK and RSU1 binding in carrying out the integrin-dependent functions of PINCH. Our preliminary data indicates that binding to ILK localizes PINCH within integrin-associated complexes at the cell membrane, and regulates the shuttling of PINCH to the nucleus. We will determine if PINCH has essential functions in the nucleus, and whether it has an overlapping complement of binding partners in the cytosol versus the nucleus. Additional data suggest that RSU1 is sequestered in integrin-associated complexes by its interaction with PINCH. We will examine how PINCH-RSU1 binding is regulated, and identify partners and activities for RSU1 that are independent of PINCH. A molecular understanding of how PINCH regulates cell adhesion and migration through its scaffolding activity may ultimately suggest strategies for prevention and therapeutic intervention in human pathologies like metastatic cancer, in which aberrant cell migration plays a prominent role. Public Health Relevance: Cell adhesion and migration are critical components of many biological processes including embryonic development, inflammatory and immune response, wound healing, and tumor invasion and metastasis. Cell adhesion and migration are controlled by an intricate network of regulatory circuits. This research will define new molecular mechanisms by which protein interactions regulate adhesion and migration, with the ultimate goal of identifying potential therapeutic targets for diseases arising from aberrant cell migration, such as the malignant progression of tumors.