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 re gulation are not understood. The present study clarified mammalian myosin V I motor function and regulation at a molecular level. Myosin VI ATPase acti vity was highly activated by actin with K-actin of 9 mum. A predominant amo unt 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, suggestin g that myosin VI has a weak affinity or slow binding for ATP. On the other hand, ADP markedly inhibited the actin-activated ATPase activity, suggestin g a high affinity for ADP. These results suggested that myosin VI is predom inantly in a strong actin binding state during the ATPase cycle. p21-activa ted kinase 3 phosphorylated myosin VI, and the site was identified as Thr(4 06). The phosphorylation of myosin VI significantly facilitated the actin-t ranslocating activity of myosin VI. On the other hand, Ca2+ diminished the actin-translocating activity of myosin VI although the actin-activated ATPa se activity was not affected by Ca2+. Calmodulin was not dissociated from t he heavy chain at high Ca2+, suggesting that a conformational change of cal modulin upon Ca2+ binding, but not its physical dissociation, determines th e inhibition of the motility activity. The present results revealed the dua l regulation of myosin VI by phosphorylation and Ca2+ binding to calmodulin light chain.