Abstract
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 microm. 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, Ca(2+) diminished the actin-translocating activity of myosin VI although the actin-activated ATPase activity was not affected by Ca(2+). Calmodulin was not dissociated from the heavy chain at high Ca(2+), suggesting that a conformational change of calmodulin upon Ca(2+) 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 Ca(2+) binding to calmodulin light chain.
Highlights
Myosin, a motor protein that translocates actin filaments upon hydrolysis of ATP, constitutes a superfamily with 18 classes based upon phylogenetic sequence comparisons of the motor domains [1,2,3,4]
The present results revealed the dual regulation of myosin VI by phosphorylation and Ca2؉ binding to calmodulin light chain
It should be noted that functional myosin VI was only obtained with co-infection of calmodulin virus, in contrast to myosin V in which functional protein can be obtained without calmodulin co-infection [23]
Summary
A motor protein that translocates actin filaments upon hydrolysis of ATP, constitutes a superfamily with 18 classes based upon phylogenetic sequence comparisons of the motor domains [1,2,3,4]. The head domain is itself divided into a globular motor domain and a neck domain containing a light chain binding region. The role of the IQ motif and bound calmodulin serving as a regulatory component of unconventional myosins was first studied for mammalian myosins I For both brush border myosin I [17, 18] and myosin I (19 –21), high Ca2ϩ inhibits motor activity due to its binding to the calmodulin light chain. While calmodulin can be dissociated from its heavy chain at high Ca2ϩ, the motility activity of myosin V is abolished at pCa 6, where no calmodulin dissociation takes place, suggesting that a Ca2ϩ binding-induced conformational change of the bound cal-
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