AFAP-110 AS A REGULATOR OF ANGIOGENESIS

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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.