Myosin Loop 2 Is Involved in the Formation of a Trimeric Complex of Twitchin, Actin, and Myosin

Funabara, Daisuke; Osawa, Rika; Ueda, Miki; Kanoh, Satoshi; Hartshorne, David J.; Watabe, Shugo

doi:10.1074/jbc.m109.016485

Cited by 24 publications

(24 citation statements)

References 20 publications

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“…A phosphorylation-dependent binding of the C-terminal IGD2IG fragment to actin and myosin has been shown previously (26), and its possible participation as a mechanical linkage in catch has been discussed (26,27). Overall, these results suggest that each end of the twitchin molecule acts as an independent tether between thick and thin filaments.…”

Section: Discussionsupporting

confidence: 58%

“…Insight regarding the portion of twitchin that is involved in possible tethering of thick and thin filaments was provided by the finding that a fragment of twitchin containing the D2 phosphorylation site and flanking Ig domains bound to both actin and myosin when D2 was unphosphorylated but not when phosphorylated (26). This and further studies (27) showed that this fragment of twitchin forms a phosphorylation-sensitive heterotrimeric complex with a portion of the myosin loop 2 region and actin subdomain 1. The implications of such a complex in mediating a connection of thick and thin filaments in catch are obvious.…”

mentioning

confidence: 95%

“…This raises the possibility that a protein as small as DXD1 (consisting of 143 amino acids) may be sufficient to tether thick and thin filaments in a phosphorylation-dependent manner as has been shown for the IGD2IG portion of twitchin (27). Importantly, the phosphorylation-dependent increase in force with the addition of IGDXD1IG is consistent with the idea that the linkage of thick and thin filaments through this part of twitchin can result in the continued maintenance of some of the force generated by the myosin cross-bridge when it subsequently detaches from actin (see Ref.…”

Section: Binding Of the Recombinant Proteins Igdxd1ig And Igd2ig To Tmentioning

confidence: 99%

“…Synthetic Peptide D2-Funabara et al (26,27) have shown that unphosphorylated IGD2IG co-sediments with both thick and thin filaments. Because the binding of the N-terminal segment of IGDXD1IG to actin seems to be mediated by sequences immediately surrounding the phosphorylation sites, we tested whether the same was true for IGD2IG.…”

Section: Binding Of the Recombinant Proteins Igdxd1ig And Igd2ig To Tmentioning

confidence: 99%

“…In order to confirm the binding of IGD2IG to the thin filament and to compare it with IGDXD1IG, a solid phase binding assay modified from that described by Funabara et al (26,27) was used. In this case, wells in a plastic immunoplate were coated for 45 min with the recombinant proteins in a solution similar to that used for filament isolation except for the inclusion of 100 mM imidazole.…”

Section: Peptide Synthesis and Recombinant Protein Expression Andmentioning

confidence: 99%

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

Butler

Mooers

Narayan

et al. 2010

Journal of Biological Chemistry

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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

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Section: Discussionsupporting

confidence: 58%

mentioning

confidence: 95%

Section: Binding Of the Recombinant Proteins Igdxd1ig And Igd2ig To Tmentioning

confidence: 99%

Section: Binding Of the Recombinant Proteins Igdxd1ig And Igd2ig To Tmentioning

confidence: 99%

Section: Peptide Synthesis and Recombinant Protein Expression Andmentioning

confidence: 99%

See 3 more Smart Citations

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

Butler

Mooers

Narayan

et al. 2010

Journal of Biological Chemistry

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Survey of the year 2009: applications of isothermal titration calorimetry

Falconer

Collins

2010

J. Mol. Recognit.

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Isothermal titration calorimetry (ITC) is now an established and invaluable method for determining the thermodynamic constants, association constant and stoichiometry of molecular interactions in aqueous solutions. The technique has become widely used by biochemists to study protein interaction with other proteins, small molecules, metal ions, lipids, nucleic acids and carbohydrates; and nucleic acid interaction with small molecules. The drug discovery industry has utilized this approach to measure protein (or nucleic acid) interaction with drug candidates. ITC has been used to screen candidates, guide the design of potential drugs and validate the modelling used in structure-based drug design. Emerging disciplines including nanotechnology and drug delivery could benefit greatly from ITC in enhancing their understanding and control of nano-particle assembly, and drug binding and controlled release.

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A force-activated kinase in a catch smooth muscle

Butler

Siegman

2011

J Muscle Res Cell Motil

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Permeabilized anterior byssus retractor muscles (ABRM) from Mytilus edulis were used as a simple system to test whether there is a stretch dependent activation of a kinase as has been postulated for titin and the mini-titin twitchin. The ABRM is a smooth muscle that shows catch, a condition of high force maintenance and resistance to stretch following stimulation when the intracellular Ca++ concentration has diminished to sub-maximum levels. In the catch state twitchin is unphosphorylated, and the muscle maintains force without myosin crossbridge cycling through what is likely a twitchin mediated tether between thick and thin filaments. In catch, a small change in length results in a large change in force. The phosphorylation state of an added peptide, a good substrate for molluscan twitchin kinase, with the sequence KKRAARATSNVFA was used as a measure of kinase activation. We find that there is about a two-fold increase in phosphorylation of the added peptide with a 10% stretch of the ABRM in catch. The increased phosphorylation is due to activation of a kinase rather than to an inhibition of a phosphatase. The extent of phosphorylation of the peptide is decreased when twitchin is phosphorylated and catch force is not present. However, there is also a large increase in peptide phosphorylation when the muscle is activated in pCa 5, and the catch state does not exist. The force-sensitive kinase activity is decreased by ML-9 and ML-7 which are inhibitors of twitchin kinase, but not by the Rho kinase inhibitor Y-27632. There is no detectable phosphorylation of myosin light chains, but the phosphorylation of twitchin increases by a small, but significant extent with stretch. It is possible that twitchin senses force output resulting in a force-sensitive twitchin kinase activity that results in autophosphorylation of twitchin on site(s) other than those responsible for relaxation of catch.

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Myosin Loop 2 Is Involved in the Formation of a Trimeric Complex of Twitchin, Actin, and Myosin

Cited by 24 publications

References 20 publications

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

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

Survey of the year 2009: applications of isothermal titration calorimetry

A force-activated kinase in a catch smooth muscle

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