Brain myosin-V, a calmodulin-carrying myosin, binds to calmodulin-dependent protein kinase II and activates its kinase activity.
Myosin-V, an unconventional myosin, has two notable structural features: (i) a regulatory neck domain having six IQ motifs that bind calmodulin and light chains, and (ii) a structurally distinct tail domain likely responsible for its specific intracellular interactions. Myosin-V copurifies with synaptic vesicles via its tail domain, which also is a substrate for calmodulin-dependent protein kinase II. We demonstrate here that myosin-V coimmunoprecipitates with CaM-kinase II from a Triton X-100-solubilized fraction of isolated nerve terminals. The purified proteins also coimmunoprecipitate from dilute solutions and bind in overlay experiments on Western blots. The binding region on myosin-V was mapped to its proximal and medial tail domains. Autophosphorylated CaM-kinase II binds to the tail domain of myosin-V with an apparent Kd of 7.7 nM. Surprisingly, myosin-V activates CaM-kinase II activity in a Ca2+-dependent manner, without the need for additional CaM. The apparent activation constants for the autophosphorylation of CaM-kinase II were 10 and 26 nM, respectively, for myosin-V versus CaM. The maximum incorporation of 32P into CaM-kinase II activated by myosin-V was twice that for CaM, suggesting that myosin-V binding to CaM-kinase II entails alterations in kinetic and/or phosphorylation site parameters. These data suggest that myosin-V, a calmodulin-carrying myosin, binds to and delivers CaM to CaM-kinase II, a calmodulin-dependent enzyme.
Costa
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MC
Department of Biochemistry, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo 14049-900, Brazil.
Mani
F
F
Santoro
W
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Espreafico
E M
EM
Larson
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RE
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D002645:Chickens; D004789:Enzyme Activation; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D024701:Myosin Type V; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D050765:Qa-SNARE Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; C078829:chicken brain myosin-V p190; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D024701:Myosin Type V