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

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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.