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 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.
Yoshimura
M
M
Department of Physiology, University of Massachusetts Medical School, 55 Lake Ave., Worcester, MA 01655-0127, USA.
Homma
K
K
Saito
J
J
Inoue
A
A
Ikebe
R
R
Ikebe
M
M
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D054462:p21-Activated Kinases
D000199:Actins; D002147:Calmodulin; D020409:Molecular Motor Proteins; D011994:Recombinant Proteins; C089785:myosin VI; D013912:Threonine; D000255:Adenosine Triphosphate; C515912:Pak3 protein, mouse; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D000251:Adenosine Triphosphatases; D018995:Myosin Heavy Chains; D002118:Calcium