Molecular Regulation of the Diaphanous-related Formins
Diaphanous-related formins (DRFs) are a conserved family of proteins that are found in a wide variety of species, ranging from slime mold to yeast to humans. DRFs are known to play an essential role in the movement and division of cells, as well as in the maintenance of cellular shape. Because these processes are so important to cell integrity, it is imperative to understand the mechanism in which these proteins are regulated in cells. It has been shown that DRFs normally exist in a “closed” inactive state, facilitated by the binding of two regions of the protein, the Diaphanous Autoregulatory Domain (DAD) and Diaphanous Inhibitory Domain (DID). The binding of an important cellular signaling protein, Rho GTPase, serves to “open” and activate the DRF protein. However, recent studies have shown that, while necessary to the regulation of DRFs, Rho GTPase binding is not sufficient to fully activate the protein. Our laboratory has hypothesized that phosphorylation, a widespread method of cellular signaling, may be required to fully activate the protein. In the search for potential DRF phosphorylation sites, undergraduate students in the laboratory identified 8 possible amino acid residues that have a high probability of being phosphorylated. Using site-directed mutagenesis, we have generated mutations that would mimic both “on” and “off” phosphorylation states at these specific sites. In addition, we have discovered that the DRF protein is phosphorylated by the specific kinase, p21-activated kinase (PAK). Through the use of in vitro kinase assays, our results show that PAK phosphorylation occurs at the DRF amino acid serine-150. We have also used cellular microinjection to monitor the expression of the mutant DRF proteins in a human cervical cancer cell line to fully visualize the effect of the activated and inactive proteins. This is the first time that it has been definitively shown that any DRF is phosphorylated; however, further studies are needed to address whether the phosphorylation of serine-150 by PAK plays an integral role in DRF regulation.
Faculty Mentor: Brad Wallar
Kate presented at the Annual Meeting of the American Society for Biochemistry and Molecular Biology April 6-9, 2008 in San Diego, CA.
Page last modified July 14, 2009