Cooperation between the Two Heads of Smooth Muscle Myosin Is Essential for Full Activation of the Motor Function by Phosphorylation

Abstract

The motor function of smooth muscle myosin (SmM) is regulated by phosphorylation of the regulatory light chain (RLC) bound to the neck region of the SmM heavy chain. It is generally accepted that unphosphorylated RLC induces interactions between the two heads and between the head and the tail, thus inhibiting the motor activity of SmM, whereas phosphorylation of RLC interrupts those interactions, thus reversing the inhibition and restoring the motor activity to the maximal value. One assumption of this model is that single-headed SmM is fully active regardless of phosphorylation. To re-evaluate this model, we produced a number of SmM constructs with coiled coils of various lengths and examined their structure and regulation. With these constructs we identified the segment in the coiled-coil key for the formation of a stable double-headed structure. In agreement with the current model, we found that the actin-activated ATPase activity of unphosphorylated SmM increased with shortening of the coiled-coil. However, contrary to the current model, we found that the actin-activated ATPase activity of phosphorylated SmM decreased with shortening coiled-coil and only the stable double-headed SmM was fully activated by phosphorylation. These results indicate that single-headed SmM is neither fully active nor fully inhibited. Based on our findings, we propose that cooperation between the two heads is essential, not only for the inhibition of unphosphorylated SmM, but also for the activation of phosphorylated SmM.

Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load

“…These data suggest ADP release is rate-limiting, so acto·M10HMM·ADP will be the predominant steady-state intermediate, making this a high duty ratio motor. Regarding the difference between the current results with HMM and the earlier results with subfragment-1 (S1), it is worth noting that a recent paper has produced evidence of smooth muscle myosin-2 S1 has a much lower steady-state actinactivated ATPase activity than HMM (67).…”

Mouse Myosin-19 Is a Plus-end-directed, High-duty Ratio Molecular Motor

“…We found that both RLC12b and RLC9 in Myo19 can be phosphorylated by MLCK, but this phosphorylation has little effect on the motor activity of Myo19-truncated constructs. It is known that phosphorylation of RLC by MLCK regulates the motor activity of smooth muscle myosin but not the truncated smooth muscle myosin having the coiled-coil tail completely deleted (22). Thus, it is possible that MLCK regulates the motor activity of full-length Myo19, but not the truncated Myo19.…”

“…Oligonucleotides were synthesized by Invitrogen. Actin, CaM, and MLCK (myosin light chain kinase) were prepared as described previously (22,23). Two mouse myosin-5a constructs, i.e.…”

“…At the indicated time, 110 l of reaction solution was mixed with 10% TCA (final concentration) and the denatured protein was precipitated by centrifugation at 12,000 ϫ g for 5 min. The precipitated protein was solubilized in 20 l of sample solution (8 M urea, 10 mM DTT, and 0.005% bromphenol blue) and analyzed by urea/glycerol PAGE as described previously (22).…”

Effects of Mutations in the Phenamacril-Binding Site of Fusarium Myosin-1 on Its Motor Function and Phenamacril Sensitivity

“…Myosin-5a HMM, rabbit skeletal muscle, and actin were prepared as described previously. 14 , 27 , 28 …”