The N-terminal Region of Twitchin Binds Thick and Thin Contractile Filaments

Abstract

Catch is a mechanical state present in some invertebrate smooth muscles in which force output and high resistance to muscle lengthening are maintained for long periods of time with very low energy utilization. In the catch state, intracellular [Ca 2ϩ ] is close to basal concentration (1), and redevelopment of force following unloading of the muscle is absent (2). A hypothesis to explain the long term maintenance of catch force was based on a slowing of cycling of myosin attached to actin when activation waned (see Lowy and Millman (3)). On the other hand, Ruegg proposed that catch was maintained by an independent linkage among thick filaments, possibly mediated by paramyosin (4, 5). As will be discussed below, much of the recent evidence supports the idea that catch results from an independent linkage between thick and thin filaments.Catch force is rapidly relaxed by stimulation of serotonergic nerves (6) that results in an increase in cAMP (7) and subsequent activation of cyclic AMP-dependent protein kinase (PKA) 2 (8). The protein that is phosphorylated by PKA during relaxation of catch force is twitchin, and it is the phosphorylation state of this protein that determines the presence or absence of the catch state at basal [Ca 2ϩ ] (9, 10). Twitchin is so named because Caenorhabditis elegans mutants that lack the protein show nearly constant twitching of body wall muscles (11). Twitchin is a mini-titin that is composed of Ig and fibronectin domains as well as a kinase domain near the C terminus (11). The domain organization of twitchin from Mytilus (12) is shown in Fig. 1. The central portion of the molecule shares domain homology with part of the A band portion of titin, and twitchin is known to be associated with thick filaments in catch muscle (13,14). In vitro, twitchin is phosphorylated by PKA with a stoichiometry of about 3 mol of phosphate/mol of twitchin (15). The D1 phosphorylation site is located between the 7th and 8th Ig domain in the N-terminal part of the molecule, and the D2 site is near the C terminus between the 21st and 22nd Ig domain (12,15). Another site of phosphorylation shares a seven-amino acid sequence with the D1 site, is located in the same Ig linker region, and is referred to as DX (16). Also present in the same linker region as D1 and DX is a DFRXXL actin-binding motif (17) similar to those that mediate the binding of smooth muscle myosin light chain kinase to the thin filament (18) and myosin IIIA tail interactions with the thin filament (19).Using an in vitro motility assay, Yamada et al. (20) showed that mechanical aspects of the catch state can be demonstrated using thick filaments reconstituted from purified myosin, F-actin, and twitchin. They found, however, that little if any binding of twitchin to actin occurred in co-sedimentation experiments. On the other hand, Shelud'ko et al. (21) described significant twitchin-actin interactions that were greatly decreased when twitchin was phosphorylated. Their

Earning stripes: myosin binding protein-C interactions with actin

“…In the tri-helix bundle a highly conserved sequence was found that bears homology to the actin binding sequences of troponin I, twitchin, and other actin binding proteins [8, 11, 23]. Missense variants associated with hypertrophic cardiomyopathy also affected actin binding, further supporting the idea that tri-helix bundle mediates actin binding of the motif [5].…”

Myosin Binding Protein‐C: A Regulator of Actomyosin Interaction in Striated Muscle

“…Additionally, twitchin, a protein similar to titin, characterized in C. elegans as a thick filament regulatory protein, interacts with both myosin and actin in a phosphorylation-dependent manner to modulate cross-bridges formation [78, 79]. However, current models (as described above) to explain the mechanism of contractile regulation through dynamic binding to myosin and actin are lacking.…”

Molecular Cloning and Tissue Distribution of Troponin I from the Japanese Pearl Oyster, <i>Pinctada fucata</i>

“…Once Ca 2+ concentrations decrease to resting levels, catch muscle enters the high-tension catch state, which is able to be maintained for long periods of time. Twitchin, a giant myosin-associated protein, is thought to tether together the thin and thick filaments through its phosphorylation sites [2] [3] [4].…”