Biochemical and Functional Characterization of the Interaction of Myo1c with 14-3-3

Abstract

Myosin-IC (Myo1c) and 14-3-3, an adaptor protein proposed to associate with Myo1c, have been implicated in regulating the delivery of GLUT4-containing compartments to the plasma membrane in response to insulin. However, molecular and functional details of the Myo1c-14-3-3 interaction have yet to be described. It has been proposed that 14-3-3 binding to Myo1c is phosphorylation-dependent with a binding site at S701, near the first calmodulin-associated, IQ-motif of the motor. Here we show that 14-3-3 binding to Myo1c increases with increasing calcium concentration. However, 14-3-3 binding does not appear to displace calmodulin from Myo1c at concentrations where calcium-bound calmodulin normally remains associated. 14-3-3 induces dissociation of actin from coverslip-bound Myo1c in the in vitro motility assay, resulting in the inhibition of directional actin gliding. Surprisingly, phosphorylation of S701 with CAM kinase-II, which has been suggested to regulate 14-3-3 binding, has little effect on the Myo1c-14-3-3 interaction or the ability of 14-3-3 to inhibit motility. Our data suggest that 14-3-3 is able to interact with Myo1c and inhibit its activity in a calcium dependent, but phosphorylation independent manner.