Enzymatic Characterization and Functional Domain Mapping of Brain Myosin-V

doi:10.1074/jbc.271.29.17561

Enzymatic Characterization and Functional Domain Mapping of Brain Myosin-V*

From the Departments of ^‡ Biology,
^∥ Cell Biology, and
^# Pathology, Yale University, New Haven, Connecticut 06511 and
^§ Department of Biochemistry, School of Medicine at Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil 14049-900

^∥ ∥ To whom correspondence should be addressed:
Dept. of Biology, Yale University, P. O. Box 208103, New Haven, CT 06520-8103.
Tel.: 203-432-3469; Fax: 203-432-6161; E-mail: Mooseker{at}minerva.cis.Yale.edu

↵^∥ Present address: Dept. of Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

Abstract

The actin binding and ATPase properties, as well as the functional domain structure of chick brain myosin-V, a two-headed, unconventional myosin, is reported here. Compared to conventional myosin from skeletal muscle, brain myosin-V exhibits low K-EDTA- and Ca-ATPase activities (1.8 and 0.8 ATP/s per head). The physiologically relevant Mg-ATPase is also low (∼0.3 ATP/s), unless activated by the presence of both F-actin and Ca²⁺ (V_max of 27 ATP/s). Ca²⁺ stimulates the actin-activated Mg-ATPase over a narrow concentration range between 1 and 3 μM. In the presence of saturating Ca²⁺ and 75 mM KCl, surprisingly low concentrations of F-actin activate the Mg-ATPase in a hyperbolic manner (K_ATPase of 1.3 μM). Brain myosin-V also binds with relatively high affinity (compared to other known myosins) to F-actin in the presence of ATP, as assayed by cosedimentation. Digestion of brain myosin-V with calpain yielded a 65-kDa head domain fragment that cosediments with actin in an ATP-sensitive manner and a 80-kDa tail fragment that does not interact with F-actin. The 80-kDa fragment results from cleavage one residue beyond the proline-, glutamate-, serine-, threonine-rich region. Our data indicate that the Mg-ATPase cycle of brain myosin-V is tightly regulated by Ca²⁺, probably via direct binding to the calmodulin light chains in the neck domain, which like brush border myosin-I, results in partial (∼30%) dissociation of the calmodulin associated with brain myosin-V. The effect of Ca²⁺ binding, which appears to relieve suppression by the neck domain, can be mimicked by calpain cleavage near the head/neck junction.

Footnotes

↵^¶ Received stipend support from Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior.
↵” Supported by the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico.
↵^‴ Supported by a National Institutes of Health training grant to the Yale Liver Center.
↵* This work was supported by grants from Fundacao de Amparo a Pesquisa do Estado de São Paulo and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (to R. E. L.) and by the following grants (to M. S. M.): a basic research grant from the Muscular Dystrophy Association, National Institutes of Health Program Project Grant DK38979, and National Institutes of Health Grant DK25387. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

BM-V

brain myosin-V

CaM

calmodulin

BB

brush border

PEST

proline, glutamate, serine, threonine rich

DTT

dithiothreitol

PAGE

polyacrylamide gel electrophoresis.
↵² F. Espindola, unpublished data.
↵³ S. Tauhata, unpublished data.
- Received September 26, 1995.
- Revision received May 1, 1996.