Myosin 1b (Myo1b), a class I myosin, is a widely expressed, single-headed, actin-associated molecular motor. Transient kinetic and single-molecule studies indicate that it is kinetically slow and responds to tension. Localization and subcellular fractionation studies indicate that Myo1b associates with the plasma membrane and certain subcellular organelles such as endosomes and lysosomes. Whether Myo1b directly associates with membranes is unknown. We demonstrate here that fulllength rat Myo1b binds specifically and with high affinity to phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and phosphatidylinositol 3,4,5-triphosphate (PIP 3 ), two phosphoinositides that play important roles in cell signaling. Binding is not Ca 2؉ -dependent and does not involve the calmodulin-binding IQ region in the neck domain of Myo1b. Furthermore, the binding site is contained entirely within the C-terminal tail region, which contains a putative pleckstrin homology domain. Single mutations in the putative pleckstrin homology domain abolish binding of the tail domain of Myo1b to PIP 2 and PIP 3 in vitro. These same mutations alter the distribution of Myc-tagged Myo1b at membrane protrusions in HeLa cells where PIP 2 localizes. In addition, we found that motor activity is required for Myo1b localization in filopodia. These results suggest that binding of Myo1b to phosphoinositides plays an important role in vivo by regulating localization to actin-enriched membrane projections.Class I myosins are single-headed members of the myosin superfamily that bind actin filaments and produce mechanical force by hydrolyzing ATP. Class I myosins consist of an N-terminal head or motor domain containing the ATP-and actin-binding sites, a neck region containing repeats of a light chain-binding region known as an IQ domain, and a C-terminal tail domain. Class I myosins are widely expressed in protozoans and metazoans. In mammals, there are eight class I myosins, Myo1a-h 2 (1), which play roles in diverse cellular events such as membrane trafficking, formation of membrane protrusions, cell migration, and transcription in the nucleus (2).The myosin I tail domain, a basic region referred to as the TH1 domain, is involved in membrane binding. Acanthamoeba myosin IC binds phosphatidylserine and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and colocalizes with PIP 2 in dynamic regions of the plasma membrane, including pseudopods, endocytic cups, and the base of filopodia (3). Vertebrate Myo1a, abundant in the brush border of the small intestine, also binds phosphatidylserine and PIP 2 (4), suggesting that Myo1a tethers the core bundles of actin filaments in the microvilli directly to the membrane (5). The mammalian myosin I Myo1c, which mediates GLUT4 transport in adipocytes (6, 7) and adaptation in the specialized hair cells of the inner ear (8, 9), associates with phosphoinositides having phosphates at positions 4 and 5 of the inositol ring (10).Vertebrate Myo1b is widely expressed in tissues such as the brain, heart, lung, kidney, and liver (11). Myo1...

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...

Myosin IIIA is expressed in photoreceptor cells and thought to play a critical role in phototransduction processes, yet its function on a molecular basis is largely unknown. Here we clarified the kinetic mechanism of the ATPase cycle of human myosin IIIA. The steady-state ATPase activity was markedly activated ϳ10-fold with very low actin concentration. The rate of ADP off from actomyosin IIIA was 10 times greater than the overall cycling rate, thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form was very slow, but the rate was markedly accelerated by actin binding. The dissociation constant of the ATP-bound form of myosin IIIA from actin is submicromolar, which agrees well with the low K actin . These results indicate that ATP hydrolysis predominantly takes place in the actin-bound form for actomyosin IIIA ATPase reaction. The obtained K actin was much lower than the previously reported one, and we found that the autophosphorylation of myosin IIIA dramatically increased the K actin , whereas the V max was unchanged. Our kinetic model indicates that both the actin-attached hydrolysis and the P i release steps determine the overall cycle rate of the dephosphorylated form. Although the stable steady-state intermediates of actomyosin IIIA ATPase reaction are not typical strong actin-binding intermediates, the affinity of the stable intermediates for actin is much higher than conventional weak actin binding forms. The present results suggest that myosin IIIA can spend a majority of its ATP hydrolysis cycling time on actin.Class III myosin was originally found in Drosophila photoreceptor cells (1), and a majority of the cell biological work related to class III myosin has been done with the Drosophila system. Class III myosin was subsequently identified from humans (2, 3), striped bass (4), and Limulus (5). In vertebrate, two isoforms of class III myosin, myosin IIIA and myosin IIIB, have been cloned (2, 3). Among them, most studies have been done with myosin IIIA. Both isoforms are highly expressed in the retina. Myosin IIIA is also expressed in inner ear hair cells, and it is responsible for progressive nonsyndromic hearing loss in humans (6). Immunohistochemical studies revealed that myosin IIIA is concentrated in the distal ends of rod and cone ellipsoid and colocalizes with the plus-distal ends of inner segment actin filament bundles, where actin forms the microvilli-like calycal processes (4). Interestingly, the transfection of green fluorescent protein-myosin IIIA into HeLa cells revealed that myosin IIIA localizes at the tip of filopodia (7), suggesting that myosin IIIA accumulates at the plus end of actin bundles. The major cytoskeletal structure of filopodia is the actin bundles, and the plus ends of the actin filaments are localized at the tip; therefore, the localization of myosin IIIA at the tip of filopodia suggests that this myosin traveled on actin filaments and accumulated at the end of the actin track. This is consistent with our result that my...

Previous findings suggested that the motor activity of human myosin IIIA (HM3A) is influenced by phosphorylation [Kambara, T., et al. (2006) J. Biol. Chem. 281, 37291–37301]; however, how phosphorylation controls the motor activity of HM3A is obscure. In this study, we clarify the kinetic basis of the effect of phosphorylation on the ATP hydrolysis cycle of the motor domain of HM3A (huM3AMD). The affinity of human myosin IIIA for filamentous actin in the presence of ATP is more than 100-fold decreased by phosphorylation, while the maximum rate of ATP turnover is virtually unchanged. The rate of release of ADP from acto-phosphorylated huM3AMD is 6-fold greater than the overall cycle rate, and thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form is markedly increased by phosphorylation by 30-fold. The dissociation constant for dissociation of the ATP-bound form of huM3AMD from actin is greatly increased by phosphorylation, and this result agrees well with the significant increase in the Kactin value of the steady-state ATPase reaction. The rate constant of the Pi off step is greater than 60 s−1, suggesting that this step does not limit the overall ATP hydrolysis cycle rate. Our kinetic model indicates that phosphorylation induces the dissociation of huM3AMD from actin during the ATP hydrolysis cycle, and this is due to the phosphorylation-dependent marked decrease in the affinity of huM3AMD· ATP for actin and the increase in the ATP hydrolysis rate of huM3AMDin the actin-dissociated state. These results suggest that the phosphorylation of myosin IIIA significantly lowers the duty ratio, which may influence the cargo transporting ability of the native form of myosin IIIA that contains the ATP-independent actin binding site in the tail.