Intracellular distribution of gravin, a PKA and PKC binding protein, in vascular endothelial cells
Intracellular distribution of gravin, a PKA and PKC binding protein, in vascular endothelial cells
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DOI：
10.1159/000051043
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发表时间：
2001-03-01
影响因子：
1.7
通讯作者：
Bruchey, AK
中科院分区：
文献类型：
--
作者：
Grove, BD;Bruchey, AK
Gravin 是一种在组织和细胞中广泛表达的高分子量蛋白，在培养的内皮细胞中表达上调，表明它可能在伤口修复和血管发育中发挥作用。在当前的研究中，我们检查了 gravin 的细胞内分布，以确定它是否与细胞骨架或另一个细胞内区室相关。人脐静脉内皮细胞 (HUVEC) 的免疫荧光显微镜显示，gravin 具有点状染色分布，延伸至细胞边缘，并且似乎不与应力纤维、微管和中间丝共定位。此外，用细胞松弛素 D 或秋水仙碱破坏细胞骨架结构不会改变 gravin 分布。然而，共聚焦和免疫电子显微镜清楚地表明，gravin 集中在细胞边缘，与质膜紧密相连。对内皮细胞裂解物中的 gravin 进行免疫沉淀会导致蛋白激酶活性共沉淀，该蛋白激酶活性可以用 cAMP 从免疫沉淀物中洗脱，并且可以用 PKA 特异性抑制剂抑制。抗 PKA 催化亚基抗体与 cAMP 洗脱液免疫印迹上的 40 kD 条带发生反应，免疫沉淀物的免疫印迹进一步揭示 PKC α 与内皮细胞裂解物中的 gravin 共沉淀。这项研究表明，gravin 与质膜或膜骨架相关，可能通过将 PKA 和 PKC 靶向特定的膜相关位点并调节与内皮伤口愈合相关的 PKA/PKC 依赖性细胞活动，在内皮伤口愈合中发挥作用。版权所有 (C) 2001 S，Karger AG，巴塞尔。
Gravin, a high-molecular-weight protein expressed widely in tissues and cells, is upregulated in cultured endothelial cells under conditions which suggest that it may play a role in wound repair and vascular development. In the current study, we examined the intracellular distribution of gravin to determine if it is associated with the cytoskeleton or with another intracellular compartment. Immunofluorescence microscopy of human umbilical vein endothelial cells (HUVEC) revealed that gravin had a punctate staining distribution that extended to the cell margin and did not appear to colocalize with stress fibers, microtubules, and intermediate filaments. Moreover, disruption of the cytoskeletal structures with either cytochalasin D or colchicine did not alter gravin distribution. However, confocal and immunoelectron microscopy clearly revealed that gravin was concentrated at the cell margin in close association with the plasma membrane. Immunoprecipitation of gravin from endothelial cell lysates resulted in coprecipitation of protein kinase activity that could be eluted from the immunoprecipitates with cAMP and that was inhibitable with a PKA-specific inhibitor. An anti-PKA catalytic subunit antibody reacted with a 40-kD band on immunoblots of the cAMP eluate, Immunoblots of the immunoprecipitates further revealed that PKC alpha coprecipitated with gravin from endothelial cell lysates. This study indicates that gravin is associated with either the plasma membrane or the membrane skeleton and may play a role in endothelial wound healing by targeting PKA and PKC to specific membrane-associated sites and regulating PKA/PKC-dependent cellular activities associated with endothelial wound healing. Copyright (C) 2001 S, Karger AG, Basel.