Dual regulation of mammalian myosin VI motor function

Myosin VI is expressed in a variety of cell types and is thought to play a role in membrane trafficking and endocytosis, yet its motor function and regulation are not understood. The present study clarified mammalian myosin VI motor function and regulation at a molecular level. Myosin VI ATPase activity was highly activated by actin with K-actin of 9 mum. A predominant amount of myosin VI bound to actin in the presence of ATP unlike conventional myosins. K-ATP was much higher than those of other known myosins, suggesting that myosin VI has a weak affinity or slow binding for ATP. On the other hand, ADP markedly inhibited the actin-activated ATPase activity, suggesting a high affinity for ADP. These results suggested that myosin VI is predominantly in a strong actin binding state during the ATPase cycle. p21-activated kinase 3 phosphorylated myosin VI, and the site was identified as Thr(406). The phosphorylation of myosin VI significantly facilitated the actin-translocating activity of myosin VI. On the other hand, Ca2+ diminished the actin-translocating activity of myosin VI although the actin-activated ATPase activity was not affected by Ca2+. Calmodulin was not dissociated from the heavy chain at high Ca2+, suggesting that a conformational change of calmodulin upon Ca2+ binding, but not its physical dissociation, determines the inhibition of the motility activity. The present results revealed the dual regulation of myosin VI by phosphorylation and Ca2+ binding to calmodulin light chain.