|dc.description.abstract||Rho GTPases are molecular switches that mediate the formation of dendritic spines – actin-enriched protrusions that make excitatory synapses with nearby neurons. Once activated by guanine nucleotide exchange factors (GEFs), GTPases regulate actin dynamics within spines. The roles of several Rho GTPases are known in spines; the functions of many GEFs, however, are unclear. Here, we show that the Rac GEF Asef2 is important for the development of dendritic spines. Endogenous Asef2 localizes to spines, and spine and synapse density can be increased or decreased by expression or knockdown of Asef2, respectively. This effect is GEF activity-dependent, since mutation of Asef2’s GEF domain blocks spine formation. Also, knockdown of Rac blocks Asef2-mediated spine formation, suggesting that Asef2 activates Rac to promote spine development. Finally, the actin-binding protein spinophilin also regulates Asef2 function by targeting Asef2 to spines.
In a related project, we investigated the regulation of Asef2 activity by identifying putative phosphorylation sites. Six phosphorylation sites were identified by mass spectrometry. Most of these sites cluster around the autoinhibitory region of Asef2, suggesting that they may regulate Asef2 activity. To test this, one of the residues – serine 106 – was mutated to alanine (S106A, non-phosphorylatable analogue) or to aspartic acid (S106E, phosphomimetic analogue), and the effect of these mutations on cell migration (another actin-dependent process) was assessed. The S106A mutant inhibited Asef2-mediated Rac activation, cell migration, and adhesion turnover (a component of cell migration), while the S106E mutant promoted Rac activation but did not influence cell migration compared to wild-type Asef2. These results suggest that phosphorylation is an important mechanism for regulating Asef2 activity and function.||