Brittany N. Stropich
Protein Interactions in Diaphanous-related Formins: defining specific biochemical interactions involved in the regulation of important cellular processes
The cytoskeleton plays a crucial role in the maintenance of cell shape, cell movement, and cellular division. Our laboratory focuses on a conserved family of proteins, the Diaphanous-related formins (DRFs), that act as "molecular switches" in signaling to the cytoskeleton. Since the DRFs are an important focal point in many cellular processes, it is vital that these proteins are tightly regulated. Normally, DRFs exist in an inactive state ("closed") by an intramolecular interaction between the Diaphanous inhibitory domain (DID) and the Dia-autoregulatory domain (DAD). In response to external stimuli, DRFs are activated by Rho GTPases, which bind to the GTPase Binding Domain (GBD) and "open" the protein. Once activated, the DRF can send signals to other cellular pathways. Here, we have focused on the regulation of a specific DRF by probing the DID-DAD interactions and determining a potential phosporylation site that may act as a mechanism to activate the DRF. Using a combination of site-directed mutagenesis and cell biology, we have generated mutations in the DID region that mimic a proposed constantly activated and inactivated state of the DRF. As a result, we have found a mutation that causes filopodia forming actin in cells. In order to determine if the mutations disrupt the DID-DAD binding, we have future plans to employ fluorescence anisotropy to calculate the affinity of purified GBD-DID protein constructs to fluorescein-labeled DAD peptides. Thus, we have the ability to be able to "link" the affinity of specific GBD-DID interactions with functional effects in the cell.
Mentor: Dr. Brad Wallar
Page last modified July 14, 2009