Phosphorylation of tropomyosin extends cooperative binding of myosin beyond a single regulatory unit

Abstract

Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tm phosphorylation in modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tm phosphorylation is necessary for longrange cooperative activation of myosin binding. We used a novel optical trapping assay to measure the isometric stall force of an ensemble of myosin molecules moving actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. The data show that the thin filament is cooperatively activated by myosin across regulatory units when Tm is phosphorylated. When Tm is dephosphorylated, this "long-range" cooperative activation is lost and the filament behaves identically to bare actin filaments. However, these effects are not due to dissociation of dephosphorylated Tm from the reconstituted thin filament. The data suggest that end-to-end interactions of adjacent Tm molecules are strengthened when Tm is phosphorylated, and that phosphorylation is thus essential for long range cooperative activation along the thin filament.

Force Spectroscopy Reveals Multiple “Closed States” of the Muscle Thin Filament

“…2). These Hill coefficients are consistent with our previous evidence suggesting that phosphorylated Tm extends cooperative activation beyond one thin filament regulatory unit (11).…”

“…In the ␣ isoform of Tm found in striated muscle, there is a phosphorylation site near the carboxyl terminus at Ser-283 (10). This phosphorylation site is in the head-to-tail overlap between neighboring Tm dimers, and we showed previously that Tm phosphorylation is necessary to extend cooperative activation beyond one regulatory unit (11). We also found that actin filaments decorated with phosphorylated Tm (AϩTm.P) produced greater isometric force at intermediate densities of myosin than bare actin filaments or actin filaments decorated with dephosphorylated Tm (AϩTm.DP) (11).…”

“…However, steric hindrance in and of itself cannot explain the force enhancements (11,23) and increased ATPase rates (38) that have been observed with the addition of Tm. Thus Tm must have other effects on single cross-bridge kinetics to account for those changes and the data presented here.…”

“…F-actin was prepared from rat skeletal muscle according to Pardee and Spudich (14), biotinylated with N-hydroxysuccinimide biotin according to Rao et al (11), and stabilized with phalloidin. Tm was also prepared from rat hind leg skeletal muscle as described by Smillie (15) and reconstituted with F-actin according to Rao et al (11). The level of Tm phosphorylation * This work was supported, in whole or in part, by National Institutes of Health NSF Graduate Training Grant MCB0718430 (to the University of Virginia), National Institutes of Health Biophysics Graduate Training Grant GM080186 (to the University of Virginia), and National Institutes of Health Cardiovascular Graduate Training Grant HL07284 (to the University of Virginia).…”

Actin regulation by tropomodulin and tropomyosin in neuronal morphogenesis and function

“…Phosphorylation of Tpm does not cause measurable changes in direct interactions with actin but alters the recruitment of myosins onto thin filaments (Rao et al, 2009). Structural studies indicate that this phosphorylation causes an increase in stiffness of the Tpm-Tpm binding junction (Lehman et al, 2015).…”