Calcium Regulation of Calmodulin Binding to and Dissociation from the Myo1c Regulatory Domain
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- 作者：Slobodanka Manceva ; Tianming Lin ; Huy Pham ; John H. Lewis ; Yale E. Goldman ; E. Michael Ostap
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：October 23, 2007
- 年：2007
- 卷：46
- 期：42
- 页码：11718 - 11726
- 全文大小：339K
- 年卷期：v.46,no.42(October 23, 2007)
- ISSN：1520-4995
文摘
Myo1c is an unconventional myosin involved in cell signaling and membrane dynamics. Calciumbinding to the regulatory-domain-associated calmodulin affects myo1c motor properties, but the kineticdetails of this regulation are not fully understood. We performed actin gliding assays, ATPase measurements,fluorescence spectroscopy, and stopped-flow kinetics to determine the biochemical parameters that definethe calmodulin-regulatory-domain interaction. We found calcium moderately increases the actin-activatedATPase activity and completely inhibits actin gliding. Addition of exogenous calmodulin in the presenceof calcium fully restores the actin gliding rate. A fluorescently labeled calmodulin mutant (N111C) bindsto recombinant peptides containing the myo1c IQ motifs at a diffusion-limited rate in the presence andabsence of calcium. Measurements of calmodulin dissociation from the IQ motifs in the absence of calciumshow that the calmodulin bound to the IQ motif adjacent to the motor domain (IQ1) has the slowestdissociation rate (0.0007 s-1), and the IQ motif adjacent to the tail domain (IQ3) has the fastest dissociationrate (0.5 s-1). When the complex is equilibrated with calcium, calmodulin dissociates most rapidly fromIQ1 (60 s-1). However, this increased rate of dissociation is limited by a slow calcium-inducedconformational change (3 s-1). Fluorescence anisotropy decay of fluorescently labeled N111C bound tomyo1c did not depend appreciably on Ca2+. Our data suggest that the calmodulin bound to the IQ motifadjacent to the motor domain is rapidly exchangeable in the presence of calcium and is responsible forregulation of myo1c ATPase and motile activity.