Abstract
To understand the domain requirements of phosphorylation-dependent regulation, we prepared three recombinant constructs of nonmuscle heavy meromyosin IIB containing 1) two complete heads, 2) one complete head and one head lacking the motor domain, and 3) one complete head and one head lacking both motor and regulatory domains. Steady-state ATPase measurements showed that phosphorylation did not alter the affinity for actin by more than a factor of 2 for any construct. Phosphorylation increased V(max) by a factor of 10 for construct 1 and 1.5-3 for construct 2 but had no effect for construct 3. Single turnover measurements, a better measure of slow rates inherent to unphosphorylated regulated myosins, showed that the single-headed construct 2, like construct 3 retains less than 1% of the regulatory properties of the double-headed construct 1 (300-fold activation). Therefore, a complete head cannot be down-regulated by a regulatory domain (without the motor domain) on the partner head. Two motor domains are required for regulation. This result is predicted by a structural model (Wendt, T., Taylor, D., Messier, T., Trybus, K. M., and Taylor, K. A. (1999) J. Cell Biol. 147, 1385-1390) showing interaction between the motor domains for unphosphorylated smooth muscle myosin, if motor-motor interaction is the basis for down-regulation.
Highlights
The actin-activated MgATPase activity and motor properties of smooth muscle and nonmuscle myosins are regulated by phosphorylation of the regulatory light chain (RLC) [1,2,3]
The class of light chains that binds nearest to the motor domain is termed the essential light chain (ELC)1 whereas the class that binds nearest to the coiled-coil rod is termed the regulatory light chain (RLC)
This interaction between the motor domains was not evident in phosphorylated HMM. It is not known whether this motor-motor domain interaction is a requirement for down-regulated kinetic properties, but if it is, a prediction of the mechanism is that a myosin lacking one full head would not be regulated, which is what has been observed
Summary
Vol 276, No 44, Issue of November 2, pp. 41465–41472, 2001 Printed in U.S.A. Phosphorylation-dependent Regulation Is Absent in a Nonmuscle Heavy Meromyosin Construct with One Complete Head and One Head Lacking the Motor Domain*. Reconstructions of images of unphosphorylated HMM showed an asymmetrical structure with an interaction between the actin-binding region of the motor domain of one head and the converter region of the motor domain of the partner head. This interaction between the motor domains was not evident in phosphorylated HMM. It is not known whether this motor-motor domain interaction is a requirement for down-regulated kinetic properties, but if it is, a prediction of the mechanism is that a myosin lacking one full head would not be regulated, which is what has been observed. This strongly suggests that the motor domains of both heads of nonmuscle myosin IIB are required for the down-regulated kinetic behavior inherent to the unphosphorylated state of a regulated myosin
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