Dephosphorylation of Tonic and Phasic Smooth Muscle Myosin in the In Vitro Motility Assay Exhibits Different Kinetics

In this paper we review some of our recent work on the structural and biochemical characterization of isoforms of the heavy chain of vertebrate smooth muscle myosin II. There exist both amino-terminal and carboxyl-terminal alternatively spliced isoforms of the smooth muscle myosin heavy chain (MHC). mRNA splicing at the 3′ end generates two MHCs, which differ in length and amino acid sequence in the carboxyl terminus. We will refer to the longer, 204-kDa isoform as MHC204 and the shorter, 200-kDa isoform as MHC200. We found that MHC204, but not MHC200, can be phosphorylated by casein kinase II on a serine near the carboxyl terminus, suggesting that these isoforms may be differentially regulated. The physiological significance of this phosphorylation is not known. However, as demonstrated in this paper, phosphorylation does not appear to affect filament formation, velocity of movement of actin filaments by myosin in an in vitro motility assay, actin-activated Mg2+ ATPase activity, or myosin conformation. Our results also show that MHC204 and MHC200 form homodimers, but not heterodimers. Purified MHC204 and MHC200 homodimers are not enzymatically different, at least as measured using an in vitro motility assay. The amino-terminal spliced MHC204 and MHC200 isoforms are the result of the specific insertion or deletion of seven amino acids near the ATP-binding region in the myosin head. We refer to these isoforms as inserted (MHC204-I; MHC200-I) or noninserted (MHC204; MHC200), respectively. In contrast to the carboxyl-terminal spliced isoforms, the amino-terminal spliced inserted and noninserted myosin heavy chain isoforms are enzymatically different. The inserted isoform, which is expressed in intestinal, phasic-type smooth muscle, has a higher actin-activated Mg ATPase activity and moves actin filaments at a greater velocity in an in vitro motility assay than the noninserted MHC isoform, which is expressed in tonic-type vascular smooth muscle. The results presented in this review suggest that the alternative splicing of smooth muscle mRNA results in at least four different isoforms of the myosin heavy chain molecule. The potential relevance of these molecular isoforms to smooth muscle function is discussed.Key words: myosin, heavy chain isoforms.

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Affinity for MgADP and force of unbinding from actin of myosin purified from tonic and phasic smooth muscle

AJP Cell Physiology ◽

10.1152/ajpcell.00100.2008 ◽

2008 ◽

Vol 295 (3) ◽

pp. C653-C660 ◽

Cited By ~ 9

Author(s):

Renaud Léguillette ◽

Nedjma B. Zitouni ◽

Karuthapillai Govindaraju ◽

Laura M. Fong ◽

Anne-Marie Lauzon

Keyword(s):

Smooth Muscle ◽

Alternative Hypothesis ◽

In Vitro Motility Assay ◽

In Vitro Motility ◽

Tonic Muscle ◽

Latch State ◽

Muscle Myosin ◽

Phasic Smooth Muscle

Smooth muscle is unique in its ability to maintain force at low MgATP consumption. This property, called the latch state, is more prominent in tonic than phasic smooth muscle. Studies performed at the muscle strip level have suggested that myosin from tonic muscle has a greater affinity for MgADP and therefore remains attached to actin longer than myosin from phasic muscle, allowing for cross-bridge dephosphorylation and latch-bridge formation. An alternative hypothesis is that after dephosphorylation, myosin reattaches to actin and maintains force. We investigated these fundamental properties of smooth muscle at the molecular level. We used an in vitro motility assay to measure actin filament velocity (νmax) when propelled by myosin purified from phasic or tonic muscle at increasing [MgADP]. Myosin was 25% thiophosphorylated and 75% unphosphorylated to approximate in vivo conditions. The slope of νmax versus [MgADP] was significantly greater for tonic (−0.51 ± 0.04) than phasic muscle myosin (−0.15 ± 0.04), demonstrating the greater MgADP affinity of myosin from tonic muscle. We then used a laser trap assay to measure the unbinding force from actin of populations of unphosphorylated tonic and phasic muscle myosin. Both myosin types attached to actin, and their unbinding force (0.092 ± 0.022 pN for phasic muscle and 0.084 ± 0.017 pN for tonic muscle) was not statistically different. We conclude that the greater affinity for MgADP of tonic muscle myosin and the reattachment of dephosphorylated myosin to actin may both contribute to the latch state.

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Smooth muscle myosin cross-bridge interactions modulate actin filament sliding velocity in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.111.2.453 ◽

1990 ◽

Vol 111 (2) ◽

pp. 453-463 ◽

Cited By ~ 190

Author(s):

D M Warshaw ◽

J M Desrosiers ◽

S S Work ◽

K M Trybus

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Myosin ◽

Copolymer Composition ◽

Weakly Bound ◽

In Vitro Motility Assay ◽

In Vitro Motility ◽

Chemically Modified ◽

Cross Bridges ◽

Muscle Myosin

Although it is generally believed that phosphorylation of the regulatory light chain of myosin is required before smooth muscle can develop force, it is not known if the overall degree of phosphorylation can also modulate the rate at which cross-bridges cycle. To address this question, an in vitro motility assay was used to observe the motion of single actin filaments interacting with smooth muscle myosin copolymers composed of varying ratios of phosphorylated and unphosphorylated myosin. The results suggest that unphosphorylated myosin acts as a load to slow down the rate at which actin is moved by the faster cycling phosphorylated cross-bridges. Myosin that was chemically modified to generate a noncycling analogue of the "weakly" bound conformation was similarly able to slow down phosphorylated myosin. The observed modulation of actin velocity as a function of copolymer composition can be accounted for by a model based on mechanical interactions between cross-bridges.

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Thiophosphorylation independently activates each head of smooth muscle myosin in vitro

AJP Cell Physiology ◽

10.1152/ajpcell.1995.269.5.c1160 ◽

1995 ◽

Vol 269 (5) ◽

pp. C1160-C1166 ◽

Cited By ~ 8

Author(s):

D. E. Harris ◽

C. J. Stromski ◽

E. Hayes ◽

D. M. Warshaw

Keyword(s):

Smooth Muscle ◽

Myosin Light Chain ◽

Smooth Muscle Myosin ◽

Mechanical Activity ◽

In Vitro Motility Assay ◽

In Vitro Motility ◽

Measured Activity ◽

Coated Surface ◽

Muscle Myosin

To determine whether thiophosphorylation of the 20-kDa myosin light chain activates each head of smooth muscle myosin independently of the head with which it is paired, chicken gizzard smooth muscle myosin was randomly thiophosphorylated, producing a mixture of unphosphorylated and singly and doubly thiophosphorylated myosin. Thiophosphorylation levels were measured by glycerol-urea gels, and the activity of this myosin was determined by actin-activated adenosinetriphosphatase measurements and in an in vitro motility assay, where the velocity of actin filaments moving over a myosin-coated surface is measured. Activity at each thiophosphorylation level was similar to that previously observed for mixtures of unphosphorylated and doubly thiophosphorylated myosin (D. E. Harris, S. S. Work, R. K. Wright, N. R. Alpert, and D. M. Warshaw. J. Muscle Res. Cell Motil. 15: 11-19, 1994). All doubly thiophosphorylated myosin was then formed into filaments and removed from randomly thiophosphorylated myosin by centrifugation. The remaining myosin (mixture of unphosphorylated and singly phosphorylated myosin), which could not polymerize because of their conformation, retained approximately 70% activity compared with mixtures of unphosphorylated and doubly thiophosphorylated myosin. Thus a thiophosphorylated smooth muscle myosin head can produce substantial biochemical and mechanical activity, even when it is paired with an unphosphorylated partner.

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