High affinity binding of brain myosin-Va to F-actin induced by calcium in the presence of ATP.
Brain myosin-Va consists of two heavy chains, each containing a neck domain with six tandem IQ motifs that bind four to five calmodulins and one to two essential light chains. Previous studies demonstrated that myosin-Va exhibits an unusually high affinity for F-actin in the presence of ATP and that its MgATPase activity is stimulated by micromolar Ca(2+) in a highly cooperative manner. We demonstrate here that Ca(2+) also induces myosin-Va binding to and cosedimentation with F-actin in the presence of ATP in a similar cooperative manner and calcium concentration range as that observed for the ATPase activity. Neither hydrolysis of ATP nor buildup of ADP was required for Ca(2+)-induced cosedimentation. The Ca(2+)-induced binding was inhibited by low temperature or by 0.6 m NaCl, but not by 1% Triton X-100. Tight binding between myosin-Va and pyrene-labeled F-actin in the presence of ATP and Ca(2+) was also detected by quenching of the pyrene fluorescence. Negatively stained preparations of actomyosin-Va under Ca(2+)-induced binding conditions showed tightly packed F-actin bundles cross-linked by myosin-Va. Our data demonstrate that high affinity binding of myosin-Va and F-actin in the presence of ATP or 5'-O-(thiotriphosphate) is induced by micromolar concentrations of Ca(2+). Since Ca(2+) regulates both the actin binding properties and actin-activated ATPase of myosin-Va over the same concentration range, we suggest that the calcium signal may regulate the mechanism of processivity of myosin Va.
Tauhata
S B
SB
Department of Cellular and Molecular Biology, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil, 14049-900.
dos Santos
D V
DV
Taylor
E W
EW
Mooseker
M S
MS
Larson
R E
RE
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D000205:Actomyosin / Q000648:ultrastructure; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001921:Brain; D002118:Calcium / Q000494:pharmacology*; D002645:Chickens; D006358:Hot Temperature; D018995:Myosin Heavy Chains / Q000378:metabolism* / Q000648:ultrastructure; D024701:Myosin Type V / Q000378:metabolism* / Q000648:ultrastructure; D009419:Nerve Tissue Proteins / Q000378:metabolism* / Q000648:ultrastructure; D017830:Octoxynol / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D012965:Sodium Chloride / Q000494:pharmacology
D000199:Actins; D009419:Nerve Tissue Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D017830:Octoxynol; D000205:Actomyosin; D024701:Myosin Type V; D018995:Myosin Heavy Chains; D002118:Calcium