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West Virginia IDeA Network of Biomedical
Research Excellence |
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Robert Shurina
Wheeling Jesuit UniversityAFAP-110
as a Regulator of Angiogenesis
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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.
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