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

项目摘要/摘要 整合素胞浆域相关蛋白-1(ICAP1)与两个过程有关 血管健康：整合素信号和脑海绵状血管畸形信号。与此一致的是， ICAP1基因缺失的小鼠表现出血管异常，如过度的血管分支和扩张。ICAP1为负 调节整合素黏附受体，它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，异常的整合素信号可导致急性等血管疾病 冠脉综合征、心肌缺血和血栓形成。ICAP1还结合Krev/Rap1相互作用-1 (KRIT1)，一种功能丧失导致CCM的蛋白质，这是一种神经血管发育不良，影响高达0.5%的 人类人口。CCM的特点是桑叶样渗漏的病变会导致终身中风的风险， 出血和癫痫发作。阐明整合素和CCM信号的扰动如何导致血管 对于疾病，必须严格检查这两个途径的潜在分子调控因子(如ICAP1)。 ICAP1和KRIT1都经历了核质穿梭，并具有功能性的核定位 信号。然而，考德伍德实验室最近证实，ICAP1指导着KRIT1的核定位。 此外，由于细胞质ICAP1增强了其抑制整合素激活和细胞质的能力 KRIT1可以部分阻止CCM中观察到的泄漏血管表型，ICAP1的定位可能是 调节整合素和CCM信号。然而，相关的知识差距仍然存在，包括：1) 调控ICAP1和ICAP1/KRIT1复合体核积累的信号和2) ICAP1和ICAP1/KRIT1复合体的亚细胞定位。 拟议的研究策略通过以下方式解决这些问题：将磷酸化作为一种调节因素进行研究 ICAP1的核定位(目标1)，评估ICAP1的磷酸化对整合素和内皮的影响 细胞功能(目标2)，并研究ICAP1/KRIT1复合体定位在CCM相关过程中的作用 (目标3)。为了达到这些目的，从血管生物学(例如，单层渗透性)等领域的技术 和网络形成分析)到细胞生物学(例如，流式细胞术和细胞迁移分析)到生物化学 (例如，蛋白质纯化和磷酸化分析)将得到利用。这些目标加在一起，将考验 假设ICAP1的磷酸化驱动ICAP1和ICAP1/KRIT1复合体进入亚细胞 正确的整合素和CCM信号所需的间隔。因此，该提案将澄清 ICAP1‘S分子在整合素生物学中的作用与CCM发病机制的联系 整合素/CCM信号与血管疾病。

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.