Determination of Human Myosin III as a Motor Protein Having a Protein Kinase Activity

Abstract

The class III myosin is the most divergent member of the myosin superfamily, having a domain with homology to a protein kinase. However, the function of class III myosin at a molecular level is not known at all, and it has been questioned whether it is actually an actinbased motor molecule. Here, we showed that human myosin III has an ATPase activity that is significantly activated by actin (20-fold) with K actin of 112 M and V max of 0.34 s ؊1 , indicating the mechanoenzymatic activity of myosin III. Furthermore, we found that human myosin III has actin translocating activity (0.11 ؎ 0.05 m/s) using an in vitro actin gliding assay, and it moves toward the plus end of actin filaments. Myosin III containing calmodulin as the light chain subunit showed a protein kinase activity and underwent autophosphorylation. The autophosphorylation was the intramolecular process, and the sites were at the C-terminal end of the motor domain. Autophosphorylation significantly activated the kinase activity, although it did not affect the ATPase activity. The present study is the first report that clearly demonstrates that the class III myosin is an actin-based motor protein having a protein kinase activity.Myosin III is a member of the myosin superfamily, which consists of at least 18 classes (1-5). The class III myosin represents the most divergent member of the myosin superfamily. The motor domain of myosin III shows ϳ24 -27% sequence identity to myosin Is and ϳ22-26% sequence identity to myosin-IIs (6). Of particular interest is that myosin III has an amino terminus domain that resembles to protein kinases. This domain contains the characteristic motifs for protein kinases such as a glycine-rich loop, an invariable Lys residue required for ATP binding, and a catalytic loop. Given its high degree of divergence, if myosin III evolved at the same rate as other myosins, the myosin III lineage would predate the divergence of yeast. Class III myosin was originally found in Drosophila photoreceptor cells and subsequently found in vertebrates including human myosin III (7,8).The function of Myosin III is best studied in Drosophila photoreceptor cells. Each photoreceptor cell has a specialized organelle consisting of a stack of microvilli known as a rhabdomere. The phototransduction machinery is localized in the rhabdomere (9). Drosophila photoreceptors undergo a prolonged depolarization afterpotential that persists after cessation of the light stimulus. Prolonged depolarization afterpotential results from the stable conversion of rhodopsin to the light-activated form, metarhodopsin, in response to blue light. During a prolonged depolarization afterpotential, photoreceptor cells become refractory to subsequent prolonged depolarization afterpotential-inducing stimuli and are inactivated. Mutants that are defective for both inactivation and the prolonged depolarization afterpotential are known as neither inactivation nor afterpotential (nina) mutants (10, 11), and the myosin III was identified (12) as one of eight nina complementation gr...

Phototransduction and retinal degeneration in Drosophila

“…NINAC would be predicted to be required only for movement into the rhabdomeres, since it may be a plus-enddirected motor, based on analyses of a mammalian myosin III [190][191][192], and the barbed ends of the actin filaments are at the distal end of the rhabdomeres [193]. Consistent with this proposal, NINAC is required for translocation of the Gα q and Arr2 from the cell bodies to the rhabdomeres, but not for the movement out of the rhabdomeres [183,189].…”

Myosin 3A Kinase Activity Is Regulated by Phosphorylation of the Kinase Domain Activation Loop

“…Phosphorylation Sites in the Kinase Domain and Kinase Regulation-Our work and the work of others have demonstrated that class III myosins are capable of autophosphorylation (3,21,23,24,36). In this work, we determined the phosphorylation sites in the motor and kinase domain by mass spectrometry with and without undergoing in vitro autophosphorylation (Table 1).…”

Light-dependent subcellular translocation of Gqα in Drosophila photoreceptors is facilitated by the photoreceptor-specific myosin III NINAC

“…NINAC has been suggested to play a role in phototransduction (Porter et al, 1992;Wes et al, 1999a) as well as in linking the axial cytoskeleton to the microvillar membrane (Hicks et al, 1996;Hicks and Williams, 1992). Although mechanoenzymatic activity has not yet been demonstrated for Drosophila NINAC, a recent report by Komaba et al (Komaba et al, 2003) showed that human myosin III functions as an actin-based motor protein with a translocating activity of 0.11 µm second -1 , as determined in an in vitro actin gliding assay. Because human myosin III acts as a plus-end motor (Komaba et al, 2003) and microvilli consist of bundled actin filaments polarized with their plus ends oriented away from the cell body (Arikawa et al, 1990;Mooseker et al, 1982), we expected that NINAC might transport Gqα from the cell body to the rhabdomere.…”