Phosphorylation of Smooth Muscle Myosin Heads Regulates the Head-induced Movement of Tropomyosin

Graceffa, Philip

doi:10.1074/jbc.m001979200

Cited by 17 publications

(36 citation statements)

References 50 publications

(39 reference statements)

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“…From modeling of this cooperativity, it was concluded that the binding of each myosin head activated seven actin protomers (44). The movement of tropomyosin by a double-headed thio-phosphorylated myosin fragment also takes place with a cooperativity of seven actin protomers (39). Consequently, a strong relationship between the myosin-induced tropomyosin movement and myosin phosphorylation-dependent force is reasonable.…”

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confidence: 93%

“…Myosin was prepared from rabbit skeletal muscle and its single-head subfragment 1 (S1) by chymotryptic digestion (38). Actin was labeled at Cys-374 with the FRET acceptors DAB-maleimide (39) or DDP-maleimide (50), which are not fluorescent. The Tm ␣␤-heterodimer was specifically labeled at either Cys-36 of the ␤-chain or Cys-190 of the ␣-chain with the fluorescence probes IAE-DANS, acrylodan, or pyrene-maleimide (38).…”

Section: Methodsmentioning

confidence: 99%

“…movement of smooth muscle tropomyosin to a new position on actin (38 -40). This movement was more efficient for phosphorylated smooth muscle heads than for unphosphorylated ones (39). Furthermore, tropomyosin's enhancement of ATPase activity increases from a very low value (under some conditions, inhibition (41, 42)) up to a plateau level as the number of myosin heads binding to actin increases (41)(42)(43).…”

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confidence: 99%

“…This led to the postulation that smooth muscle regulation includes the myosin-induced movement of tropomyosin to a position on actin where it can enhance the actomyosin ATPase activity (39). Furthermore, the myosin must be phosphorylated to promote such a change more easily (39). In this way, myosin phosphorylation and tropomyosin movement would act together to switch on smooth muscle contraction.…”

mentioning

confidence: 99%

“…Furthermore, tropomyosin's enhancement of ATPase activity increases from a very low value (under some conditions, inhibition (41, 42)) up to a plateau level as the number of myosin heads binding to actin increases (41)(42)(43). This led to the postulation that smooth muscle regulation includes the myosin-induced movement of tropomyosin to a position on actin where it can enhance the actomyosin ATPase activity (39). Furthermore, the myosin must be phosphorylated to promote such a change more easily (39).…”

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confidence: 99%

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Effect of Caldesmon on the Position and Myosin-induced Movement of Smooth Muscle Tropomyosin Bound to Actin

Graceffa

Mazurkie

2005

Journal of Biological Chemistry

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It is known that the actin-binding protein caldesmon inhibits actomyosin ATPase activity and might in this way take part in the thin filament regulation of smooth muscle contraction. Although the molecular mechanism of this inhibition is unknown, it is clear that the presence of actin-bound tropomyosin is necessary for full inhibition. Recent evidence also suggests that the myosin-induced movement of tropomyosin plays a key role in regulation. In this work, fluorescence studies provide evidence to show that caldesmon interacts with and alters the position of tropomyosin in a reconstituted actin thin filament and thereby limits the ability of myosin heads to move tropomyosin. Caldesmon interacts with the Cys-190 region in the COOH-terminal half of tropomyosin, resulting in the movement of this part of tropomyosin to a new position on actin. Additionally, this constrains the myosin-induced movement of this region of tropomyosin. On the other hand, caldesmon does not appear to interact with the Cys-36 region in the NH 2 -terminal half of tropomyosin and neither alters the position of nor significantly constrains the myosin-induced movement of this part of tropomyosin. The ability of caldesmon to limit the myosin-induced movement of tropomyosin provides a possible molecular basis for the inhibitory function of caldesmon. The different movements of the two halves of tropomyosin indicate that actin-bound tropomyosin moves as a flexible molecule and not as a rigid rod. Interestingly, caldesmon, which inhibits tropomyosin's potentiation of actomyosin ATPase activity, moves tropomyosin in one direction, whereas myosin heads, which enhance potentiation, move tropomyosin in the opposite direction.Muscle contraction takes place when myosin heads, projecting from the thick filament, cyclically interact with actin protomers in the thin filament, causing the filaments to slide past each other with a resulting muscle shortening. Actin-activated myosin hydrolysis of ATP provides the energy for this process. Tropomyosin, a long coiled-coil dimer, which binds end-to-end on each side of the thin filament, is involved in switching the muscle on. In skeletal muscle, contraction is switched on by Ca 2ϩ binding to actin-bound troponin, which results in the relocation of tropomyosin and thus the exposure of myosinbinding sites on actin (2-5). The fact that each tropomyosin molecule covers seven actin protomers and binds end-to-end to itself endows the switching process with cooperativity (for a review, see Refs. 6 -9).Smooth muscle does not contain troponin, and the main regulatory switch is the phosphorylation of myosin light chains (for a review, see Refs. 10 and 11). However, myosin phosphorylation does not account for all aspects of smooth muscle regulation, i.e. there can be a decoupling between force and myosin phosphorylation levels, and thus it has been put forth that the actin thin filament and its associated proteins also play a role in regulation (for a review, see Refs. 12 and 13). The actin-binding protein caldesmon has bee...

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confidence: 93%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Effect of Caldesmon on the Position and Myosin-induced Movement of Smooth Muscle Tropomyosin Bound to Actin

Graceffa

Mazurkie

2005

Journal of Biological Chemistry

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Different positions of tropomyosin isoforms on actin filament are determined by specific sequences of end‐to‐end overlaps

et al. 2011

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Tropomyosins are dimeric rod-like proteins which polymerize along actin filaments and regulate interactions with other actin-binding proteins. Homologous sequences responsible for the binding of tropomyosin to consecutive actin monomers repeat along tropomyosin and are called actin-binding periods. In this work, the localization of tropomyosin isoforms on actin alone and on actin–myosin complex was evaluated by measuring Förster resonance energy transfer (FRET) distances between a donor (AEDANS) attached to either the N-terminal actin-binding period 1 or to the central actin-binding period 5 and an acceptor (DABMI) bound to actin's Cys374. The recombinant -tropomyosin isoforms–TM2, TM5a, and TM1b9a, used in this study, had various amino acid sequences of the N- and C-termini forming the end-to-end overlap. Although the sequences of actin-binding period 5 of the three isoforms were identical, the donor–acceptor distances calculated for each isoform varied between 38.6 and 41.5 Å. Differences in FRET distances between the three tropomyosin isoforms labeled in actin-binding period 1 varied between 34.8 and 40.2 Å. Rigor binding of myosin heads to actin increased all measured distances. The degree and cooperativity of myosin-induced shift was different for each of the isoforms and actin-binding periods. The structural differences correlate with cooperative regulation of actin-activated S1 ATPase by the three tropomyosins. The results indicate that amino acid sequences of the end-to-end overlap determine specific orientation of tropomyosin isoform on actin. This can be important for steric and cooperative regulation of the actin filament and determine functional specificity of multiple tropomyosin isoforms present in eucaryotic cells.

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Direct observation of tropomyosin binding to actin filaments

2015

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Tropomyosin is an elongated α-helical coiled-coil that binds to seven consecutive actin subunits along the long-pitch helix of actin filaments. Once bound, tropomyosin polymerizes end-to-end and both stabilizes F-actin and regulates access of various actin binding proteins including myosin in muscle and non-muscle cells. Single tropomyosin molecules bind weakly to F-actin with millimolar Kd, whereas the end-to-end linked tropomyosin associates with about a one thousand-fold greater affinity. Despite years of study, the assembly mechanism of tropomyosin onto actin filaments remains unclear. In the current study, we used total internal reflection fluorescence (TIRF) microscopy to directly monitor the cooperative binding of fluorescently labeled tropomyosin molecules to phalloidin-stabilized actin filaments. We find that tropomyosin molecules assemble from multiple growth sites following random low affinity binding of single molecules to actin. As the length of the tropomyosin chain increases, the probability of detachment decreases, which leads to further chain growth. Tropomyosin chain extension is linearly dependent on tropomyosin concentration, occurring at approximately 100 monomers/(μM*s). The random tropomyosin binding to F-actin leads to discontinuous end-to-end association where gaps in the chain continuity smaller than the required seven sequential actin monomers are available. Direct observation of tropomyosin detachment revealed the number of gaps in actin-bound tropomyosin, the time course of gap annealing, and the eventual filament saturation process.

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Phosphorylation of Smooth Muscle Myosin Heads Regulates the Head-induced Movement of Tropomyosin

Cited by 17 publications

References 50 publications

Effect of Caldesmon on the Position and Myosin-induced Movement of Smooth Muscle Tropomyosin Bound to Actin

Effect of Caldesmon on the Position and Myosin-induced Movement of Smooth Muscle Tropomyosin Bound to Actin

Different positions of tropomyosin isoforms on actin filament are determined by specific sequences of end‐to‐end overlaps

Direct observation of tropomyosin binding to actin filaments

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