Ca 2+ Independent and Tail Dependent Regulation of the Motor Activity of Myosin X

Abstract

Myosin X is involved in the actin cytoskeletal reorganization and protrusion of filopodia. Here we studied the molecular mechanism of regulation of myosin X. The actin-activated ATPase activity of M10Full was Ca2+ independent and significantly lower than that of M10HMM. The tail domain significantly inhibited the actin-activated ATPase activity of M10HMM regardless of Ca2+. The inhibition showed significant dependence on salt concentration, suggesting that the inhibition is dependent on ionic interaction between the tail domain and the head/neck domain of myosin X. The in vitro actin gliding velocity was markedly inhibited (4 fold) by the tail. These results suggest that the tail domain functions as an intra-molecular inhibitor of the myosin X motor function. The deletion of FERM domain abolished the inhibitory activity of the tail. On the other hand, deletion of the N-terminal PEST domain did not affect the inhibitory activity. Further truncation of the PH domain abolished the inhibitory activity of the tail. These results suggest that both the PH and FERM domains of the tail are required for the inhibition. On the other hand, the elimination of both IQ domains and the SAH/coiled-coil domain showed no effect on the tail induced inhibition. Furthermore, M10IQo co-immunoprecipitated with M10PH-FERM. The result indicated that the tail domain (PH-FERM) directly interacts with the motor domain to inhibit the motor activity. Electron microscopy revealed that the full-length myosin X molecules were monomeric, showing the wider molecules in low salt with ATP, while narrow molecules, similar head shapes to the M10HMM, in high salt. Our observation suggested that the tail domain folds backward to the head, such that it appeared to interact with the motor domain, and thus inhibits the motor activity of myosin X.(Supported by NIH).