Exploring phosphoregulation of MYO3A using quantitative fluorescence image analysis in COS7 cells.

Myosin IIIA is an unconventional myosin that contains a kinase domain, and is involved in the formation of hair-cell stereocilia. To investigate its regulatory roles, we mimicked phosphorylation in mchr-MYO3AΔK constructs and assayed their ability to influence filopodial properties in COS7 cells. The phosphomimics generated fewer filopodia. Coexpression of mchr-MYO3AΔK with a GFP-construct containing only the MYO3A kinase domain also resulted in generation of fewer filopodia. Structural predictions suggest that the phosphorylation sites inhibit actin/MYO3A interactions. Taken together, these analyses link MYO3A phosphorylation with the regulation of its ability to create actin protrusions such as filopodia and stereocilia.