Phosphorylation of a Single Head of Smooth Muscle Myosin Activates the Whole Molecule

Abstract

Regulatory light chain (RLC) phosphorylation activates smooth and non-muscle myosin IIs, but it has not been established if phosphorylation of one head turns on the whole molecule. Baculovirus expression and affinity chromatography were used to isolate heavy meromyosin (HMM) containing one phosphorylated and one dephosphorylated RLC (1-P HMM). Motility and steady-state ATPase assays indicated that 1-P HMM is nearly as active as HMM with two phosphorylated heads (2-P HMM). Single turnover experiments further showed that both the dephosphorylated and phosphorylated heads of 1-P HMM can be activated by actin. Singly-phosphorylated full-length myosin was also an active species with two cycling heads. Our results suggest that phosphorylation † This work was supported by NIH Grants AR47906 and HL38113 to KMT

Smooth Muscle Heavy Meromyosin Phosphorylated on One of Its Two Heads Supports Force and Motion

“…The construct that contained a single-phosphorylated head was prepared as described previously (1), with the exception that the HMM heavy chain contained the biotin tag described above but with no FLAG tag after it. His-tagged wild-type and FLAG-tagged T18A/S19A regulatory light chains were co-infected with this heavy chain construct followed by sequential affinity columns to isolate a homogeneous population of heavy meromyosin containing one wild-type light chain and one T18A/S19A light chain.…”

“…His-tagged wild-type and FLAG-tagged T18A/S19A regulatory light chains were co-infected with this heavy chain construct followed by sequential affinity columns to isolate a homogeneous population of heavy meromyosin containing one wild-type light chain and one T18A/S19A light chain. The tags on the light chains were removed by thrombin cleavage, and the preparation was phosphorylated with myosin light chain kinase before functional analysis as described previously (1). SDS and charge gel electrophoresis were used to confirm that equal amounts of the two light chains were present and that the final preparation was 50% phosphorylated as described in detail in Rovner et al (1).…”

“…Recently, the approach to this problem has been improved by employing various methods that allow isolation of a singlephosphorylated species (1,19,20). Single-phosphorylated heavy meromyosin (HMM) had much less than half the hydrolytic and mechanical activity of double-phosphorylated HMM when prepared using light chain exchange or stripping protocols (19,20).…”

“…Single-phosphorylated heavy meromyosin (HMM) had much less than half the hydrolytic and mechanical activity of double-phosphorylated HMM when prepared using light chain exchange or stripping protocols (19,20). Using differential tagging of constructs expressed in Sf9 cells followed by sequential affinity columns, the single-phosphorylated HMM (HMM-1P) had more than half the ATPase activity and actin filament speeds in the in vitro motility assay that were similar to doublephosphorylated HMM (1).…”

“…An optical trap assay was used to show that the unitary step size and attachment time of an expressed single HMM-1P molecule was indistinguishable from that of double-phosphorylated HMM (HMM-2P) (1), suggesting that at least one of the heads of HMM-1P is equivalent to a head of HMM-2P. The optical trap was further used to characterize the force-velocity relationship for a small ensemble of HMM-1P molecules (21).…”