Ca2+-dependent Regulation of the Motor Activity of Myosin V

Kazuaki Homma,Junya Saito,Reiko Ikebe,Mitsuo Ikebe

doi:10.1074/jbc.m003132200

Kazuaki Homma, Junya Saito + Show 2 more

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https://doi.org/10.1074/jbc.m003132200

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Abstract

Mouse myosin V constructs were produced that consisted of the myosin motor domain plus either one IQ motif (M5IQ1), two IQ motifs (M5IQ2), a complete set of six IQ motifs (SHM5), or the complete IQ motifs plus the coiled-coil domain (thus permitting formation of a double-headed structure, DHM5) and expressed in Sf9 cells. The actin-activated ATPase activity of all constructs except M5IQ1 was inhibited above pCa 5, but this inhibition was completely reversed by addition of exogenous calmodulin. At the same Ca(2+) concentration, 2 mol of calmodulin from SHM5 and DHM5 or 1 mol of calmodulin from M5IQ2 were dissociated, suggesting that the inhibition of the ATPase activity is due to dissociation of calmodulin from the heavy chain. However, the motility activity of DHM5 and M5IQ2 was completely inhibited at pCa 6, where no dissociation of calmodulin was detected. Inhibition of the motility activity was not reversed by the addition of exogenous calmodulin. These results indicate that inhibition of the motility is due to conformational changes of calmodulin upon the Ca(2+) binding to the high affinity site but is not due to dissociation of calmodulin from the heavy chain.

Highlights

Serves as a regulatory component of myosin V
Since the two-headed structure is critical for the regulation of both conventional smooth muscle and non-muscle myosin motor function, it is plausible that the two-headed structure may play some role in the regulatory mechanisms of myosin V
It was found that calmodulin dissociation is responsible for inhibition of actinactivated ATPase activity at high Ca2ϩ, inhibition of motility occurs at lower Ca2ϩ where calmodulin is not dissociated from myosin V heavy chain

Summary

Introduction

Serves as a regulatory component of myosin V. For naturally isolated myosin V, motility activity is inhibited at high Ca2ϩ, whereas actin-activated ATPase activity markedly increases in the presence of Ca2ϩ [7]. The aim of the present study is to clarify the regulation of myosin V motor function by Ca2ϩ To address these questions, we have produced a two-headed myosin V construct, a singleheaded construct having the entire neck domain (six IQ motifs), and a truncated single-headed construct containing only two IQ motifs. We have produced a two-headed myosin V construct, a singleheaded construct having the entire neck domain (six IQ motifs), and a truncated single-headed construct containing only two IQ motifs These myosin V constructs were expressed, purified, and examined for motor function. It was found that calmodulin dissociation is responsible for inhibition of actinactivated ATPase activity at high Ca2ϩ, inhibition of motility occurs at lower Ca2ϩ where calmodulin is not dissociated from myosin V heavy chain

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