Protein Phosphatase 2B Dephosphorylates Twitchin, Initiating the Catch State of Invertebrate Smooth Muscle

Abstract

"Catch" is the state where some invertebrate muscles sustain high tension for long periods at low ATP hydrolysis rates. Physiological studies using muscle fibers have not yet fully provided the details of the initiation process of the catch state. The process was extensively studied by using an in vitro reconstitution assay with several phosphatase inhibitors. Actin filaments bound to thick filaments pretreated with the soluble protein fraction of muscle homogenate and Ca 2؉ (catch treatment) in the presence of MgATP at a low free Ca 2؉ concentration (the catch state). Catch treatment with >50 M okadaic acid, >1 M microcystin LR, 1 M cyclosporin A, 1 M FK506, or 0.2 mM calcineurin autoinhibitory peptide fragment produced almost no binding of the actin filaments, indicating protein phosphatase 2B (PP2B) was involved. Use of bovine calcineurin (PP2B) and its activator calmodulin instead of the soluble protein fraction initiated the catch state, indicating that only PP2B and calmodulin in the soluble protein fraction are essential for the initiation process. The initiation was reproduced with purified actin, myosin, twitchin, PP2B, and calmodulin. 32 P autoradiography showed that only twitchin was dephosphorylated during the catch treatment with either the soluble protein fraction or bovine calcineurin and calmodulin. These results indicate that PP2B directly dephosphorylates twitchin and initiates the catch state and that no other component is required for the initiation process of the catch state.Invertebrate muscles such as the smooth adductor and byssus retractor muscles of bivalves maintain high tension with little energy expenditure after active contractions (1, 2). This high tension state, called the "catch state," is terminated by a neurotransmitter, 5-hydroxytryptamine (3), activating cAMPdependent protein kinase (PKA) 1 through an increase in the intracellular concentration of cAMP. When a catalytic subunit of PKA was added to chemically demembranated catch muscles, the passive tension of the catch state diminished (4). Phosphorylation of a target protein(s) by this kinase thus causes relaxation of the catch tension.Twitchin is a candidate for this target protein. Originally identified in the nematode Caenorhabditis elegans (5, 6), it is also called "mini-titin" and is abundantly present in molluscan muscles (7,8). Studies with the anterior byssus retractor muscle of the mussel suggested that its phosphorylation by PKA was involved in the relaxation of the catch state (9). The catalytic subunit of PKA phosphorylates 2-3 serine residues of the mussel catch muscle twitchin (10, 11). In contrast to the relaxation of the catch state, the regulation of the reverse reaction, i.e. initiation of the catch state, has not been well characterized yet, since the reaction in vivo seems to occur in parallel with the activation of muscle contraction. Therefore, how initiation signals are relayed onward and amplified in cascades and which types of phosphatases dephosphorylate twitchin to initiate the catch state are stil...

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

“…As suggested previously (11), the formation of the high force state is thought to displace the twitchin interactions with myosin and actin and thus prevent formation of catch. This is consistent with observations made with reconstituted fibers (18). Thus, during an active contraction, the competition between the actin-myosin interaction sites and the sites on actin subdomain I and on myosin loop 2 for twitchin would be lessened by formation of the high force state.…”

Effects of vanadate, phosphate and 2,3-butanedione monoxime (BDM) on skinned molluscan catch muscle

“…However, this is unlikely in view of a very recent finding that the Ca 2+ -dependent protein phosphatase 2B is involved in dephosphorylation of twitchin [52]. In our experiments, [Ca 2+ ] was below 10 À8 M when cAMP was added so that the activity of this phosphatase is presumably very low under our experimental conditions.…”

Unphosphorylated twitchin forms a complex with actin and myosin that may contribute to tension maintenance in catch

“…It should be emphasized that a direct role for Ca 2+ in binding of the D2 peptide to either actin or myosin is not suggested and there is no evidence from our data to implicate a Ca 2+ sensitive step, other than the regulation of actin-activated ATPase by Ca 2+ binding to myosin. Dephosphorylation of twitchin by calcineurin (Castellani and Cohen, 1992;Yamada et al, 2004) would be coincident with initiation of contraction since both events are Ca 2+ dependent, and thus for most of the contractile phase twitchin is dephosphorylated and theoretically (Siegman et al, 1998). A competition between twitchin and myosin might explain the in vitro inhibition of Mg 2+ ATPase activity of actomyosin by relatively high concentrations of TWD2-S.…”