AFAP-110 AS A REGULATOR OF ANGIOGENESIS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。AFAP-110作为血管生成的调节因子血管生成中的细胞信号涉及一系列信号通路的相互作用，最终导致新血管的形成。细胞迁移、黏附和微血管形成是血管生成过程中的必要事件，涉及细胞S形状的变化，这些变化是通过细胞骨架的变化而发生的。SRC和PKC通过与肌动蛋白细丝相关蛋白(AFAP)相互作用来影响肌动蛋白细丝的变化。Src和蛋白激酶C(PKC)的激活在刺激血管生成的血管内皮生长因子(Vgef)途径中都是重要的，尽管它们的作用尚不清楚。AFAP是一个110 kDa的接头蛋白，它将信号分子PKC和Src连接到肌动蛋白细丝上。我们的小组已经证明，AFAP与中国证监会绑定并激活它。AFAP中的删除使其能够激活中国证监会。此外，PKC激活器指示AFAP中的更改，使其能够激活中国证监会。阻止与CSRC或与PKC相互作用的突变阻止AFAP激活CSRC以响应PKC？由于AFAP在血管内皮细胞中高水平表达，我们假设AFAP在血管生成中起作用。我们通过为AFAP的野生型、显性阴性和显性阳性形式创建细胞可渗透的融合蛋白来评估其在血管生成中的作用。融合蛋白将通过在带有GFP-AFAP的框架内插入触角序列来构建。绿色荧光蛋白(GFP)将使我们能够证明目标细胞已经摄取了重组蛋白。我们先前已经证明GFP-AFAP融合蛋白在COS细胞中具有功能，并且在PKC结合和Src激活方面表现出与AFAP相同的行为。天线序列已被证明是特洛伊木马？可以通过细胞膜运送货物蛋白质。重组融合蛋白在体外刺激血管生成的能力将通过内皮细胞或大鼠主动脉环外植体培养来衡量。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. AFAP-110 as a regulator of angiogenesis Cell signaling in angiogenesis involves an interplay of signaling pathways that culminate in the formation of new blood vessels. Cell migration, adhesion and microvessel formation are necessary events in angiogenesis, and involve changes in a cell?s shape that occur through alterations in the cytoskeleton. Src and PKC affect changes in actin filaments by interacting with the actin filament associated protein (AFAP). Both Src and protein kinase C (PKC) activation are important in vascular endothelial growth factor (VGEF) pathways that stimulate angiogenesis, although their roles are poorly understood. AFAP is a 110-kilodalton-adaptor protein that links the signaling molecules PKC and Src to actin filaments. Our group has shown that AFAP binds to cSrc and activates it. Deletions in AFAP enable it to activate cSrc. Also, PKC activators direct changes in AFAP that enable it to activate cSrc. Mutations that block interactions with cSrc or interactions with PKC prevent AFAP from activating cSrc in response to PKC?. Since AFAP is expressed at high levels in endothelial cells, we hypothesize a role for AFAP in angiogenesis. We assess its role in angiogenesis by creating cell permeable fusion proteins for the wild type, dominant negative and dominant positive forms of AFAP. Fusion proteins will be constructed by inserting an Antennepedia sequence in frame with GFP-AFAP. The green fluorescent protein (GFP) will allow us to demonstrate that the target cell has taken up the recombinant protein. We have previously shown that the GFP-AFAP fusion protein is functional in COS cells and exhibits identical behavior to AFAP regarding PKC binding and Src activation. Antennapedia sequences have been shown to serve as ?Trojan horses? that can deliver cargo proteins across cell membranes. The ability of the recombinant fusion proteins to stimulate angiogenesis in vitro will be measured using endothelial cells or rat aortic ring explant cultures.