Abstract
Myosin-IC (Myo1c) has been proposed to function in delivery of glucose transporter type 4 (GLUT4)-containing vesicles to the plasma membrane in response to insulin stimulation. Current evidence suggests that, upon insulin stimulation, Myo1c is phosphorylated at Ser701, leading to binding of the signaling protein 14-3-3β. Biochemical and functional details of the Myo1c-14-3-3β interaction have yet to be described. Using recombinantly expressed proteins and mass spectrometry-based analyses to monitor Myo1c phosphorylation, along with pulldown, fluorescence binding, and additional biochemical assays, we show here that 14-3-3β is a dimer and, consistent with previous work, that it binds to Myo1c in the presence of calcium. This interaction was associated with dissociation of calmodulin (CaM) from the IQ motif in Myo1c. Surprisingly, we found that 14-3-3β binds to Myo1c independent of Ser701 phosphorylation in vitro Additionally, in contrast to previous reports, we did not observe Myo1c Ser701 phosphorylation by Ca2+/CaM-dependent protein kinase II (CaMKII), although CaMKII phosphorylated four other Myo1c sites. The presence of 14-3-3β had little effect on the actin-activated ATPase or motile activities of Myo1c. Given these results, it is unlikely that 14-3-3β acts as a cargo adaptor for Myo1c-powered transport; rather, we propose that 14-3-3β binds Myo1c in the presence of calcium and stabilizes the calmodulin-dissociated, nonmotile myosin.