Kinetic Mechanism of Human Myosin IIIA

Abstract

Myosin IIIA is specifically expressed in photoreceptors and cochlea and is important for the phototransduction and hearing processes. In addition, myosin IIIA contains a unique N-terminal kinase domain and C-terminal tail actin-binding motif. We examined the kinetic properties of baculovirus expressed human myosin IIIA containing the kinase, motor, and two IQ domains. The maximum actin-activated ATPase rate is relatively slow (k cat ‫؍‬ 0.77 ؎ 0.08 s ؊1 ), and high actin concentrations are required to fully activate the ATPase rate (K ATPase ‫؍‬ 34 ؎ 11 M). However, actin co-sedimentation assays suggest that myosin III has a relatively high steady-state affinity for actin in the presence of ATP (K actin ϳ 7 M). The rate of ATP binding to the motor domain is quite slow both in the presence and absence of actin (K 1 k ؉2 ‫؍‬ 0.020 and 0.001 M ؊1 ⅐s ؊1 , respectively). The rate of actin-activated phosphate release is more than 100-fold faster (85 s ؊1 ) than the k cat , whereas ADP release in the presence of actin follows a two-step mechanism (7.0 and 0.6 s ؊1 ). Thus, our data suggest a transition between two actomyosin-ADP states is the rate-limiting step in the actomyosin III ATPase cycle. Our data also suggest the myosin III motor spends a large fraction of its cycle in an actomyosin ADP state that has an intermediate affinity for actin (K d ϳ 5 M). The long lived actomyosin-ADP state may be important for the ability of myosin III to function as a cellular transporter and actin crosslinker in the actin bundles of sensory cells.

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

“…Protein Expression and Purification-The MYO3A kinase and MYO3A 2IQ constructs were expressed with the baculovirus/SF9 cell system and purified as described (3,23,24). Protein purity was assessed by Coomassie-stained SDS-polyacrylamide gels, and protein concentration was determined by the Bradford assay using BSA as a standard or by absorbance using the predicted extinction coefficients (3,23,24).…”

“…Kinase Activity Assays-Autophosphorylation of MYO3A 2IQ was detected by kinase assay using [ 32 P]ATP or anti-phosphothreonine Western blotting (3,23,24). Myosin at specified concentrations was allowed to react with 200 M [ 32 P]ATP at room temperature (ϳ22°C) for specific time periods ranging from 0 to 60 min.…”

“…Protein purity was assessed by Coomassie-stained SDS-polyacrylamide gels, and protein concentration was determined by the Bradford assay using BSA as a standard or by absorbance using the predicted extinction coefficients (3,23,24). Actin was purified from rabbit skeletal muscle using an acetone powder method (25).…”

“…Reagents-ATP and ADP were prepared from powder (22 (3,23,24) was modified with site-directed mutagenesis to introduce the phosphorylation site mutations (T178A and T178D; T184A and T184E) and a mutation to abolish motor activity (E722A). The T184E and T184A mutations were also introduced in the full-length GFP-tagged version of MYO3A for expression in COS7 cells (3).…”