Effect of Phosphorylation in the Motor Domain of Human Myosin IIIA on Its ATP Hydrolysis Cycle

Abstract

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.

Intermolecular Autophosphorylation Regulates Myosin IIIa Activity and Localization in Parallel Actin Bundles

“…They report that the motor-only construct exhibits an extremely high affinity for actin, suggesting that this construct behaves very differently than the construct in our studies. The kinetic analysis of this motor-only construct following phosphorylation with an external kinase domain resulted in a 100-fold reduction in actin affinity of the motor (3,12). The maximal ATPase results of two reports from the same group (2,3,12) are contradictory, as the earlier study reported a 3-fold lower maximal rate in the dephosphorylated state, whereas the more recent report suggests no change.…”

“…The Ikebe group (3,12) has reported the kinetic analysis of a motor-only human Myo3A construct with both the kinase and neck domains removed. They report that the motor-only construct exhibits an extremely high affinity for actin, suggesting that this construct behaves very differently than the construct in our studies.…”

“…The Ikebe group (3) reported similar results for a motor-only construct, with differences that may be attributed to their removal of the lever arm and use of lower salt concentrations in assays (5). In addition, it was demonstrated that phosphorylation can reduce the actin affinity of the motor-only construct 100-fold without reducing maximal ATPase activity (3,12). These results imply that kinase activity may be associated with down-regulation of the myosin motor.…”

“…Additionally, motor function may be regulated intracellularly by factors such as calcium concentration, the presence or absence of cargo, and/or phosphorylation of the heavy chain motor or tail or on light chains (8 -10). It is postulated that autophosphorylation of the Myo3A motor may act as a means for its regulation in photoreceptors and inner ear hair cells under specific cellular conditions (5,11,12).…”

Mouse class III myosins: kinase activity and phosphorylation sites

“…The functions of kinase/myosins are of interest because of their importance for sensory cell function and survival. The kinase/myosins of vertebrates are molecular motors, and the motor and actin binding properties of human MYO3A are being studied in detail (Komaba et al. 2003, 2010; Kambara et al.…”