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    Awarded Grants
    Scaffolding Proteins and Rho Signaling in Breast Cancer

    Scientific Abstract:
    Background: Rho-family GTPases control many aspects of cell behavior, including cell migration, cell-adhesion, cell proliferation and gene expression. There is now considerable evidence that aberrant regulation of Rho-GTPases plays a significant role in cancer development. Unlike Ras, there are no reports of mutated forms of Rho proteins in tumors, suggesting the mechanisms involving Rho-GTPase deregulation are indirect and involve alterations in Rho expression and activation. Rho activation is mediated by guanine-nucleotide exchange factors (GEFs), which catalyze the exchange of GDP for GTP. Many Rho-GEFs were originally isolated as constitutively active mutants capable of inducing transformation and are likely candidates for aberrant GTPase activation in human cancer. How these GEFs are regulated is still mostly unknown. Sequence analysis shows that approximately half of the Rho-GEF family members contain putative binding motifs for PDZ domains in their C-terminus. PDZ domains are protein-protein interaction domains that act as scaffolds to concentrate signaling molecules at specialized regions in the cell. Our preliminary results show that these motifs in Rho-GEFs are functional and specifically bind to PDZ-containing proteins. In addition, we find that some PDZ proteins are able to inhibit the activity of RhoGEFs upon binding. Hypothesis: We propose that the interaction between Rho-GEFs and PDZ proteins is important for Rho-GEFs targeting and regulation and may play a role in the abnormal regulation of Rho-GTPases observed in breast cancer. Specific Aims: 1) Identification of novel interactions between Rho-GEFs and PDZ-containing proteins in breast cancer cells. 2) To determine the function of the interactions between RhoGEFs and PDZ-containing proteins. Study Design. We selected 6 RhoGEFs whose expression varies significantly in breast tumors. Proteins that specifically bind to the C-terminal tails of the selected Rho-GEFs will be isolated from model human mammary cell lines using synthetic peptides and identified using mass spectrometry. The effect of these novel interactions on Rho-GEF targeting, activity and Rho-GTPase signaling will be analyzed using a combination of biochemical and cell biological techniques. Potential Outcomes and Benefits: Elucidation of the mechanisms that result in Rho overexpression and activation will provide insight into the role of these proteins in breast cancer progression and metastasis and has the potential to uncover new targets for drug discovery and therapeutic intervention.

    Lay Abstract:
    Background: The Rho-GTPases comprise a family of proteins that act as molecular switches, controlling signaling pathways related to cell-motility, cell-adhesion, cell proliferation and gene expression. It has been previously shown that abnormal regulation of Rho-GTPases is associated with breast cancer and in some cases correlated with a poor prognosis. In contrast to Ras-GTPases, there are no reported mutations found associated with cancer in the Rho family, suggesting their role in cancer is likely to involve alterations in Rho expression and activation. Rho proteins need to be activated by a family of proteins called Rho-guanine nucleotide exchange factors (GEFs). Studies showing that some Rho-GEF mutations are capable of induce cellular transformation suggest the Rho-GEFs are likely to play a significant role in the deregulation of RhoGTPases found in human cancers. However, little is known about how RhoGEFs are regulated in the cell. We performed a bioinformatics analysis of the Rho-GEF family and found that several of its members contain a recognition sequence for a common protein interaction domain termed PDZ. PDZ domains are usually found in proteins that functions as scaffolds, recruiting different components of a certain pathway to the right location in the cell. In our preliminary results we characterized the interactions between some of these RhoGEFs and PDZ containing proteins in different tissues. We also found that some of these PDZ proteins were able to target the RhoGEFs to the plasma membrane and also altered their activity upon binding. Our primary goal is to identify novel proteins that regulate Rho-GEFs in breast cancer cells and to characterize their function. We propose that the interaction between Rho-GEFs and PDZ proteins is important for Rho-GEFs targeting and regulation and may play a role in the deregulation of Rho-GTPases observed in breast cancer. We have developed an approach to identify binding partners for proteins containing a conserved PDZ binding sequence. We will use this approach to identify PDZ containing proteins that bind to Rho-GEFs that are differentially expressed in breast tumors. We will analyze the role of these interactions in the context of Rho-GTPase activation, cell-adhesion, proliferation and motility utilizing biochemical and cell biological approaches. In summary, our studies will contribute in the understanding of the role of the Rho proteins in breast cancer progression and have the potential to uncover new pathways that can be targeted in breast cancer therapies.