Abstract
Daam1 is a mammalian formin that, like other formins, has a catalytic FH2 domain. However, unlike other formins, the linker region differs in that it contains a small antiparallel beta sheet structure that partially occludes the two actin binding sites of the FH2 domain. This feature makes unmodified Daam1 a poor actin nucleator in vitro, as compared to most other formins. Here, we use total internal reflection fluorescence (TIRF) microscopy and bulk fluorescence assembly assays to demonstrate that Src kinase can phosphorylate Daam1 in vitro to increase its nucleation activity. This effect is dampened in a point mutant of Daam1 lacking the critical tyrosine at residue 652. We propose that phosphorylation by Src kinase in vivo serves as a regulatory mechanism in controlling Daam1 function by releasing the protein from its novel autoinhibited conformation to stimulate actin assembly. Experiments in NIH3T3 cells support this hypothesis by demonstrating that inhibiting Src family kinases reduce the number of filopodia protruding from the cells. The biological importance of this mechanism will continue to be elucidated using in vivo experiments on Daam1 mutants in live cells.