AFAP-110 as a Regulator of Angiogenesis

Abstract: Cell signaling in angiogenesis involves a complex interplay of several different types of signaling molecules that culminate 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 lab has shown that AFAP activates Src by binding to its SH3 domain, thereby loosening up the structure and enabling the enzyme to bind ATP and to phosphorylate substrates. PKC activators, such as PMA, direct changes in AFAP that enable it to activate cSrc. Deletions in AFAP (AFAP-110Dlzip ) affect a conformational change in the protein and 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 that can cross the cell membrane and become functional in the transfected cell. Fusion proteins will be constructed by inserting an Antennepedia sequence in frame with GFP-AFAP. 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. Antennapedia sequences have received much attention recently for their ability to serve as "Trojan horses" that can deliver cargo proteins across cell membranes, presumably by triggering endocytosis in the target cell. The ability of the recombinant fusion proteins to stimulate angiogenesis in vitro will be measured using endothelial cells or rat aortic ring explant cultures.