Phosphorylation of ICAP1 inhibits its nuclear accumulation to modulate integrin and CCM signaling

ICAP1 的磷酸化抑制其核积累，从而调节整合素和 CCM 信号传导

• 批准号: 9761278
• 负责人: Valerie Liao Su
• 金额: $ 2.97万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9761278
• 关键词: Address; Affect; Automobile Driving; Binding; Binding Proteins; Biochemistry; Biological Assay; Biology; Blood Vessels; Cell Adhesion; Cell physiology; Cellular biology; Complex; Cytoplasm; Cytoplasmic Tail; Data; Defect; Disease; Dysplasia; Endothelial Cells; Flow Cytometry; Health; Hemorrhage; Human; In Vitro; Integrins; Knockout Mice; Knowledge; Lesion; Life; Link; Mediating; Migration Assay; Molecular; Morus; Myocardial Ischemia; Neurologic; Nuclear; Nuclear Localization Signal; Pathogenesis; Pathway interactions; Permeability; Phenotype; Phosphorylation; Phosphotransferases; Population; Process; Proteins; ROCK1 gene; Regulation; Research; Role; Seizures; Serine; Serine Phosphorylation Site; Signal Transduction; Stimulus; Techniques; Testing; Thrombosis; Vascular Diseases; Work; acute coronary syndrome; adhesion receptor; angiogenesis; cell motility; cerebral cavernous malformations; in vivo; insight; leukocyte homing; loss of function; monolayer; neurovascular; nucleocytoplasmic transport; prevent; protein purification; response; shear stress; stroke risk; vascular abnormality

Project Summary/Abstract Integrin cytoplasmic domain associated protein-1 (ICAP1) is associated with two processes essential for vascular health: integrin signaling and cerebral cavernous malformation (CCM) signaling. Consistent with this, ICAP1 null mice display vascular abnormalities like excessive vessel branching and dilation. ICAP1 negatively regulates integrin adhesion receptors, which mediate essential vascular processes like angiogenesis, leukocyte homing, and cell migration. Therefore, aberrant integrin signaling can result in vascular disorders like acute coronary syndromes, myocardial ischemia, and thrombosis. ICAP1 also binds Krev/Rap1 Interaction Trapped-1 (KRIT1), a protein whose loss of function causes CCM, a neurovascular dysplasia affecting up to 0.5% of the human population. CCM is characterized by leaky mulberry-like lesions that result in a life-long risk of stroke, hemorrhages, and seizures. To elucidate how perturbations in integrin and CCM signaling result in vascular disease, the underlying molecular regulators (e.g., ICAP1) of both pathways must be critically examined. Both ICAP1 and KRIT1 undergo nucleocytoplasmic shuttling and have functional nuclear localization signals. However, the Calderwood lab recently established that ICAP1 directs KRIT1 nuclear localization. Furthermore, because cytoplasmic ICAP1 enhances its ability to suppress integrin activation and cytoplasmic KRIT1 may in part prevent the leaky vascular phenotype observed in CCM, it is likely that ICAP1 localization regulates both integrin and CCM signaling. However, pertinent gaps in knowledge remain including: 1) the signals that regulate nuclear accumulation of ICAP1 and the ICAP1/KRIT1 complex and 2) the consequences of specific subcellular localization of ICAP1 and the ICAP1/KRIT1 complex. The proposed research strategy addresses these issues by: investigating phosphorylation as a regulator of ICAP1 nuclear localization (Aim 1), evaluating the impact of ICAP1 phosphorylation on integrin and endothelial cell function (Aim 2), and examining the role of the ICAP1/KRIT1 complex localization in CCM-relevant processes (Aim 3). To tackle these aims, techniques from fields ranging from vascular biology (e.g., monolayer permeability and network formation assays) to cell biology (e.g., flow cytometry and cell migration assays) to biochemistry (e.g., protein purification and phosphorylation assays) will be harnessed. Combined, these aims will test the hypothesis that phosphorylation of ICAP1 drives both ICAP1 and the ICAP1/KRIT1 complex to subcellular compartments necessary for proper integrin and CCM signaling. As such, the proposal will elucidate ICAP1’s molecular role in integrin biology and CCM pathogenesis to inform the link between aberrant integrin/CCM signaling and vascular disease.

项目概要/摘要 整合素胞质结构域相关蛋白-1 (ICAP1) 与两个必需的过程相关 血管健康：整合素信号传导和脑海绵状血管瘤（CCM）信号传导。与此相一致的是， ICAP1缺失小鼠表现出血管异常，例如血管过度分支和扩张。 ICAP1 阴性 调节整合素粘附受体，介导重要的血管过程，如血管生成、白细胞 归巢和细胞迁移。因此，异常的整合素信号传导可导致血管疾病，如急性血管疾病 冠状动脉综合征、心肌缺血和血栓形成。 ICAP1 还结合 Krev/Rap1 相互作用 Trapped-1 (KRIT1)，一种蛋白质，其功能丧失会导致 CCM，这是一种神经血管发育不良，影响高达 0.5% 的神经血管发育不良。 人口。 CCM 的特点是漏桑样病变，导致终生中风风险， 出血和癫痫发作。阐明整合素和 CCM 信号传导的扰动如何导致血管 疾病，必须严格检查这两种途径的潜在分子调节因子（例如 ICAP1）。 ICAP1 和 KRIT1 均经历核质穿梭并具有功能性核定位 信号。然而，Calderwood 实验室最近确定 ICAP1 指导 KRIT1 核定位。 此外，由于细胞质 ICAP1 增强了其抑制整合素激活和细胞质 ICAP1 的能力。 KRIT1 可能部分阻止 CCM 中观察到的渗漏血管表型，ICAP1 定位很可能 调节整合素和 CCM 信号传导。然而，相关的知识差距仍然存在，包括：1） 调节 ICAP1 和 ICAP1/KRIT1 复合物核积累的信号，以及 2) 的后果 ICAP1 和 ICAP1/KRIT1 复合物的特异性亚细胞定位。 拟议的研究策略通过以下方式解决这些问题： 研究磷酸化作为调节剂 ICAP1 核定位（目标 1），评估 ICAP1 磷酸化对整合素和内皮细胞的影响 细胞功能（目标 2），并检查 ICAP1/KRIT1 复合体定位在 CCM 相关过程中的作用 （目标 3）。为了实现这些目标，血管生物学等领域的技术（例如单层渗透性 和网络形成测定）到细胞生物学（例如流式细胞术和细胞迁移测定）到生物化学 （例如，蛋白质纯化和磷酸化测定）将被利用。这些目标结合起来将考验 假设 ICAP1 磷酸化驱动 ICAP1 和 ICAP1/KRIT1 复合物进入亚细胞 正确的整合素和 CCM 信号传导所必需的隔室。因此，该提案将阐明 ICAP1 在整合素生物学和 CCM 发病机制中的分子作用，可揭示异常之间的联系 整合素/CCM 信号传导和血管疾病。