AFAP-110 AS A REGULATOR OF ANGIOGENESIS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 2008年旋转耶稣会子项目摘要 血管生成中的细胞信号传导涉及几种不同类型的信号分子的复杂相互作用，最终导致新血管的形成。 衔接蛋白在这些级联反应中连接不同的信号蛋白。 据文献记载，细胞迁移、粘附和微血管形成是血管生成中的必要事件，并且可能涉及通过细胞骨架的改变而发生的细胞形状的变化。 Src和蛋白激酶C（PKC）的激活已被证明是重要的血管内皮生长因子（VGEF）的途径，刺激血管生成的内皮细胞，虽然他们的作用知之甚少。 Src和PKC也通过与肌动蛋白丝相关蛋白（AFAP）相互作用影响肌动蛋白丝的变化。 AFAP是一种110千道尔顿的衔接蛋白，将信号分子PKC和Src连接到肌动蛋白丝。 我们的小组已经表明AFAP通过SH 3和SH 2相互作用与cSrc结合。 AFAP中的缺失（AFAP-110 Dlzip）影响蛋白质的构象变化并使其能够激活cSrc。 PKC激活剂，如PMA，指导AFAP的变化，使其能够激活cSrc。 阻断与cSrc相互作用（AFAP 71 A）或与PKC相互作用（AFAPDPH 1）的突变阻止AFAP激活cSrc以响应PKCa。 由于AFAP在内皮细胞中高水平表达，我们推测AFAP在血管生成中的作用。 我们将在四环素反应元件（TRE）的控制下创建野生型、显性阴性和显性阳性AFAP融合蛋白，并在我们创建的四环素诱导的SVEC 4-10细胞系中表达它们。 通过PCR扩增我们在pEFGP c3表达载体中的EGFP-AFAP融合蛋白并将扩增产物插入pTRE-Tight载体中来构建融合蛋白。 绿色荧光蛋白（GFP）是一个标签，它将使我们能够证明靶细胞已经吸收了重组蛋白。 我们先前已经证明，GFP-AFAP融合蛋白在COS细胞中是功能性的，并且在PKC结合和Src激活方面表现出与AFAP相同的行为。 使用内皮细胞系SVEC 4-10或人脐静脉内皮细胞测量重组融合蛋白刺激体外血管生成的能力。

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. 2008 Wheeling Jesuit Subproject Abstract Cell signaling in angiogenesis involves a complex interplay of several different types of signaling molecules that ultimately result in the formation of new blood vessels. Adaptor proteins serve to link different signaling proteins in these cascades. It is well documented that the cell migration, adhesion and microvessel formation are necessary events in angiogenesis and presumably involve changes in a cell's shape that occur through alterations in the cytoskeleton. Both Src and protein kinase C (PKC) activation have been shown to be important in vascular endothelial growth factor (VGEF) pathways that stimulate angiogenesis in endothelial cells, although their roles are poorly understood. Src and PKC also affect changes in actin filaments by interacting with the actin filament associated protein (AFAP). 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 via SH3 and SH2 interactions. Deletions in AFAP (AFAP-110Dlzip) affect a conformational change in the protein and enable it to activate cSrc. PKC activators, such as PMA, direct changes in AFAP that enable it to activate cSrc. Mutations that block interactions with cSrc (AFAP71A) or interactions with PKC (AFAPDPH1), prevent AFAP from activating cSrc in response to PKCa. As AFAP is expressed at high levels in endothelial cells, we hypothesize a role for AFAP in angiogenesis. We will create wild type, dominant negative and dominant positive AFAP fusion proteins under control of the tetracycle responsive element (TRE) and express them in a tetracycline-inducible SVEC 4-10 cell line that we have created. Fusion proteins will be constructed by PCR-amplifying the EGFP-AFAP fusion protein that we have in the pEFGP c3 expression vector and inserting the amplified product into the pTRE-Tight vector. The green fluorescent protein (GFP) is a tag that 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. The ability of the recombinant fusion proteins to stimulate angiogenesis in vitro will be measured using the endothelial cell line SVEC 4-10 or human umbilical vein endothelial cells.