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.
Highlights
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
Our results show that in vitro binding of dimeric 14-3-3 to Myo1c is independent of phosphorylation and that it is enhanced by calcium-mediated dissociation of calmodulin from the motor’s lever arm
We found that 14-3-3 does not affect the ATPase activity of Myo1c in the presence of calcium and that 14-3-3 bound to myosin in the presence of calcium does not support actin filament gliding
Summary
CaM, calmodulin; CaMKII, Ca2ϩ/calmodulin-dependent protein kinase II; TFP, trifluoperazine; ChREBP, carbohydrate response element– binding protein. Mutation of serine to glutamic acid does not always mimic phosphorylation [17], we found in both assays that both mutants bound 14-3-3, with enhanced binding in the presence of calcium We treated both mutants with 0.5 mM TFP and again found 14-3-3 binding (Fig. 3C), supporting the finding that phosphorylation is not required for 14-3-3 binding. The peptide–14-3-3 complex has a higher fluorescence level than the sum of the individual proteins (Fig. 4A) In both cases, we found that two peptides bind one 14-3-3 dimer, which suggests a tight affinity with dissociation constants well below experimental protein concentrations. Titrations showed that both IQ1-WT and IQ1-pSer701 bound 14-3-3 with a similar stoichiometry of two peptides per 14-3-3 dimer (Fig. 4B) This result differs from 14-3-3 binding experiments with Myo1c-3IQ, which suggests that one Myo1c-3IQ binds one 14-3-3 dimer, which may be the result of steric constraints. Micromolar concentrations of calmodulin are able to restore actin gliding in the presence of calcium [13], 10 M 14-3-3 was not able to rescue the inhibited motility of Myo1c (Fig. 5B)
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