ABSTRACT
Organelle transport by myosin-V is down-regulated during mitosis, presumably by myosin-V phosphorylation. We used mass spectrometry phosphopeptide mapping to show that the tail of myosin-V was phosphorylated in mitotic Xenopus egg extract on a single serine residue localized in the carboxyl-terminal organelle-binding domain. Phosphorylation resulted in the release of the motor from the organelle. The phosphorylation site matched the consensus sequence of calcium/calmodulin-dependent protein kinase II (CaMKII), and inhibitors of CaMKII prevented myosin-V release. The modulation of cargo binding by phosphorylation is likely to represent a general mechanism regulating organelle transport by myosin-V.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Calmodulin-Binding Proteins/metabolism , Melanosomes/metabolism , Mitosis , Molecular Motor Proteins/metabolism , Myosin Type V , Nerve Tissue Proteins/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Biological Transport , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Calmodulin-Binding Proteins/chemistry , Calmodulin-Binding Proteins/genetics , Cell Extracts , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Enzyme Inhibitors/pharmacology , Interphase , Mass Spectrometry , Melanophores/metabolism , Melanophores/ultrastructure , Molecular Sequence Data , Mutation , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Ovum , Peptides/pharmacology , Phosphopeptides/analysis , Phosphopeptides/metabolism , Phosphorylation , Phosphoserine/metabolism , Recombinant Fusion Proteins/metabolism , Transfection , XenopusABSTRACT
Previously, we have shown that melanosomes of Xenopus laevis melanophores are transported along both microtubules and actin filaments in a coordinated manner, and that myosin V is bound to purified melanosomes (Rogers, S., and V.I. Gelfand. 1998. Curr. Biol. 8:161-164). In the present study, we have demonstrated that myosin V is the actin-based motor responsible for melanosome transport. To examine whether myosin V was regulated in a cell cycle-dependent manner, purified melanosomes were treated with interphase- or metaphase-arrested Xenopus egg extracts and assayed for in vitro motility along Nitella actin filaments. Motility of organelles treated with mitotic extract was found to decrease dramatically, as compared with untreated or interphase extract-treated melanosomes. This mitotic inhibition of motility correlated with the dissociation of myosin V from melanosomes, but the activity of soluble motor remained unaffected. Furthermore, we find that myosin V heavy chain is highly phosphorylated in metaphase extracts versus interphase extracts. We conclude that organelle transport by myosin V is controlled by a cell cycle-regulated association of this motor to organelles, and that this binding is likely regulated by phosphorylation of myosin V during mitosis.
