This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Podosomes are actin-rich projections of the cell membrane that are associated with cancer cell migration and metastases. Their formation requires rearrangements of the actin cytoskeleton, which are mediated through the interaction of a host of actin-binding proteins. Tumor-promoting phorbol esters cause actin cytoskeletal rearrangements that result in podosome formation in a variety of cell types;including vascular smooth muscle cells, osteoclasts, macrophages, endothelial cells, neural cells myoblasts and transformed fibroblasts. Tumor-promoting phorbol esters function by activating one or more PKC isoforms, resulting in the reorganization of the actin cytoskeleton and src activation AFAP-110, an adaptor protein that cross-links actin filaments, activates src in response to phosphorylation by PKC-a and is involved in podosome formation. Increased levels of AFAP-110 are correlated with increases in podosome lifetime in A7r5 tumor cells and prostate cancer. Although the role of AFAP-110 as a src activator is well established, the mechanism by which activated AFAP-110 is itself regulated remains incompletely characterized. Scaffolding proteins, such as Tks5, recruit AFAP-110 and other signaling proteins to podosomes. Homer2 is a scaffolding protein of the Vesl/Homer family that interacts with both actin filaments and activated Rho GTPases in mouse cerebellar cells and can prevent podosome formation in cdc42-activated HeLa cells. Homer2 has been identified as a binding partner for AFAP-110 in two independent yeast two-hybrid studies. We hypothesize that Homer2 is a binding partner and regulator for AFAP-110. In this proposal we will address the mechanism by which Homer2 abrogates podosome formation.
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