This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.2008 Wheeling Jesuit Subproject AbstractCell 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.
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