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 Ca2+ (catch treatment) in the presence of MgATP at a low free Ca2+ concentration (the catch state). Catch treatment with >50μm okadaic acid, >1μm microcystin LR, 1 μm cyclosporin A, 1 μm 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. 32P 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. “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 Ca2+ (catch treatment) in the presence of MgATP at a low free Ca2+ concentration (the catch state). Catch treatment with >50μm okadaic acid, >1μm microcystin LR, 1 μm cyclosporin A, 1 μm 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. 32P 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 (1Rüegg J.C. Physiol. Rev. 1971; 51: 201-248Crossref PubMed Scopus (246) Google Scholar, 2Watabe S. Hartshorne D.J. Comp. Biochem. Physiol. B. 1990; 96: 639-646Crossref PubMed Scopus (59) Google Scholar). This high tension state, called the “catch state,” is terminated by a neurotransmitter, 5-hydroxytryptamine (3Twarog B.M. J. Cell. Comp. Physiol. 1954; 44: 141-163Crossref PubMed Scopus (139) Google Scholar), activating cAMP-dependent protein kinase (PKA) 1The abbreviations used are: PKA, cAMP-dependent protein kinase; PP2B, protein phosphatase 2B; DTT, dithiothreitol; PIPES, 1,4-piperazinediethanesulfonic acid; TRITC, tetramethylrhodamine isothiocyanate. 1The abbreviations used are: PKA, cAMP-dependent protein kinase; PP2B, protein phosphatase 2B; DTT, dithiothreitol; PIPES, 1,4-piperazinediethanesulfonic acid; TRITC, tetramethylrhodamine isothiocyanate. 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 (4Pfitzer G. Rüegg J.C. J. Comp. Physiol. 1982; 147: 137-142Crossref Scopus (55) Google Scholar). 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 (5Benian G.M. Kiff J.E. Neckelmann N. Morerman D.G. Waterston R.H. Nature. 1989; 342: 45-50Crossref PubMed Scopus (263) Google Scholar, 6Benian G.M. Ayme-Southgate A. Tinley T.L. Rev. Physiol. Biochem. Pharmacol. 1999; 138: 235-268Crossref PubMed Google Scholar), it is also called “mini-titin” and is abundantly present in molluscan muscles (7Vibert P. Edelstein S.M. Castellani L. Elliott Jr., B.W. J. Muscle Res. Cell Motil. 1993; 14: 598-607Crossref PubMed Scopus (73) Google Scholar, 8Vibert P. York M.L. Castellani L. Edelstein S. Elliott B. Nyitray L. Adv. Biophys. 1996; 33: 199-209Crossref PubMed Scopus (8) Google Scholar). 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 (9Siegman M.J. Funabara D. Kinoshita S. Watabe S. Hartshorne D.J. Butler T.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5383-5388Crossref PubMed Scopus (83) Google Scholar). The catalytic subunit of PKA phosphorylates 2–3 serine residues of the mussel catch muscle twitchin (10Funabara D. Kinoshita S. Watabe S. Siegman M.J. Butler T.M. Hartshorne D.J. Biochemistry. 2001; 40: 2087-2095Crossref PubMed Scopus (39) Google Scholar, 11Funabara D. Watabe S. Mooers S.U. Narayan S. Dudas C. Hartshorne D.J. Siegman M.J. Butler T.M. J. Biol. Chem. 2003; 278: 29308-29316Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). 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 still not clear. Castellani and Cohen (12Castellani L. Cohen C. FEBS Lett. 1992; 309: 321-326Crossref PubMed Scopus (34) Google Scholar) reported that skinned catch muscles, from which endogenous phosphatase was extensively washed out, restored their ability to maintain catch tension following addition of exogenous bovine calcineurin (12Castellani L. Cohen C. FEBS Lett. 1992; 309: 321-326Crossref PubMed Scopus (34) Google Scholar). Calcineurin is a Ca2+-calmodulin-dependent phosphatase (13Tonks N.K. Cohen P. Biochim. Biophys. Acta. 1983; 747: 191-193Crossref PubMed Scopus (34) Google Scholar) classified as a Ser/Thr protein phosphatase 2B (PP2B) (14Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. 1988; 61: 149-200PubMed Google Scholar). Thus, calcineurin-like phosphatases might be involved in the regulation of the catch, and they could dephosphorylate the target protein, i.e. twitchin. As Castellani and Cohen (12Castellani L. Cohen C. FEBS Lett. 1992; 309: 321-326Crossref PubMed Scopus (34) Google Scholar) describe in their report, however, “a more complicated scheme involving a cascade of kinases/phosphatases is, of course, also possible.” It has not yet been determined whether calcineurin-like phosphatase directly or indirectly dephosphorylates twitchin in catch muscles. By using thick and thin filaments isolated from catch muscles of the mussel Mytilus galloprovincialis, our group a assay of the binding of thin filaments to thick filaments in vitro to the catch state A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (55) Google Scholar). These in vitro are more using muscle fibers to essential for the catch state and to cascades signals onward since the catch state be with purified and is of other our group showed that the essential for the catch state are myosin, twitchin, and actin, other such as A. L. J. Biol. PubMed Scopus Google Scholar), and are not A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (55) Google Scholar). By this in vitro reconstitution have the the of twitchin phosphorylation and the binding of actin filaments to thick filaments, and have determined the of phosphatase involved in the initiation of the catch state the of of its initiation by phosphatase inhibitors. In purified twitchin is dephosphorylated by PP2B, and this is with the initiation of the catch state in this with the A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (55) Google Scholar), have the by using only the purified and characterized myosin, twitchin, actin, PKA, PP2B, and and the regulation scheme of the catch contraction. soluble protein fraction was from the byssus retractor and smooth adductor muscles of The muscles in mm mm mm mm and mm at for the was extensively A, and the soluble protein fraction was as the of the and twitchin as A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (55) Google Scholar). thick filaments from the of the muscle homogenate after the of the soluble protein The was by using mm mm mm mm DTT, and mm at for of added to the The filaments by the and the was in the thick filaments by of this to be in either the or catch state as thick filaments in the state, the filaments with the soluble protein fraction in the presence of 1 mm ATP and cAMP. 1 at the filaments by at for and in A. thick filaments in the catch state, the filaments and the soluble protein fraction in the presence of free 1 at an of was added to the free Ca2+ concentration to and the filaments by and in A. and twitchin purified from the muscles as A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (55) Google Scholar). filaments by twitchin in the catch state (the dephosphorylated it was with the soluble protein fraction in the presence of free and after 1 the was added as The was to a with A. Twitchin was by in the and A. In of Catch vitro reconstitution assay was as reported A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (55) Google Scholar). of actin filaments to thick filaments in the presence of MgATP at a low free Ca2+ concentration was a filaments in mm mm mm mm mm DTT, and mm and with actin filaments of muscle with at the was a with an and an filaments and actin filaments with an and with a Actin filaments bound to thick filaments in the catch state they not in the state. The following “catch was used to thick filaments from the to catch state. with either the soluble protein fraction of muscle 0.2 or 0.2 μm bovine calcineurin and μm bovine calmodulin in the presence of free at an of was added to the the following was used to thick filaments from the catch to state. with either the soluble protein fraction or bovine PKA in the presence of and mm In autoradiography was added in the relaxation The was at for The of kinase and phosphatase used in this from the okadaic microcystin cyclosporin calcineurin autoinhibitory peptide and the peptide of PKA and was the Nature. PubMed Scopus Google Scholar). the was it was to an with at for and of Catch our the catch state was by using thick filaments, and of not to actin filaments in the catch state A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (55) Google Scholar). This of the thick filaments to of the results of the in vitro catch an assay that to the state more and thick filaments after high of the of the muscle homogenate for of the of thick filaments twitchin, and A. L. J. Biol. PubMed Scopus Google Scholar), and with a of actin as of long filaments for with a When they pretreated with the soluble protein fraction of muscles and relaxation for they not to actin filaments in the presence of MgATP at a low free Ca2+ concentration state). treatment with the soluble protein fraction and Ca2+ catch they bound to actin filaments catch and relaxation treatment of actin filaments the These results are the as for the thick filaments A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (55) Google Scholar), indicating that the reproduced the catch and of they not the in the thick bound to actin filaments in the catch state and bound to almost in the state. The the catch and was thus more with the thick filaments in a of with of A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (55) Google Scholar). Therefore, used thick filaments for our in vitro studies of the catch and of the catch state of the thick filaments using the soluble protein the thick filaments in the state with actin filaments, they not in the presence of MgATP at a low free Ca2+ concentration catch treatment with the soluble protein fraction of they bound to actin filaments and relaxation treatment from actin filaments the the catch treatment was with the soluble protein fraction to indicating the of the soluble protein the of binding the of the soluble protein the soluble protein fraction was no the as of a and a in the of and the μm in and μm in of phosphatase the initiation of the catch state. Catch treatment of the thick filaments with the soluble protein fraction was in the presence of of okadaic and microcystin and in the presence of 1 μm cyclosporin 1 μm and 0.2 mm calcineurin autoinhibitory peptide the the binding of actin filaments to thick filaments in the presence of MgATP at a low free Ca2+ concentration was of as in and the μm in of of Catch the of the catch state in the intracellular concentration and PKA is (4Pfitzer G. Rüegg J.C. J. Comp. Physiol. 1982; 147: 137-142Crossref Scopus (55) Google Scholar). studies with skinned muscle fibers (9Siegman M.J. Funabara D. Kinoshita S. Watabe S. Hartshorne D.J. Butler T.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5383-5388Crossref PubMed Scopus (83) Google Scholar) and in vitro reconstitution A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (55) Google Scholar) that phosphorylation of twitchin the catch state. These studies also that twitchin is dephosphorylated by protein phosphatases during the initiation of the catch state. In our in vitro assay the initiation of the catch state during the catch treatment A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (55) Google Scholar), of twitchin have during the catch which types of phosphatases during the initiation of the catch state using the in vitro assay studied the of several types of phosphatase during catch treatment the binding of actin filaments to thick phosphatase such as okadaic and microcystin LR, types of phosphatases at The of okadaic where phosphatase is to are for protein phosphatase for protein phosphatase and μm for PP2B C. A. Biochem. J. 1988; PubMed Scopus Google Scholar). The of microcystin are for protein phosphatase for protein phosphatase and for PP2B C. S. Cohen P. FEBS Lett. 1990; PubMed Scopus Google Scholar). These not protein phosphatase Thus, the of where the the of phosphatase involved. When the catch treatment was with a of soluble protein actin filaments bound to the thick filaments When the phosphatase was only a actin filaments bound to thick The catch treatment was in the presence of of either okadaic or microcystin a actin filaments bound the catch treatment was in the presence of μm okadaic or 1 μm microcystin Thus, the for okadaic and microcystin to be and These results indicate that PP2B is involved in the initiation of the catch state. the of PP2B, studied the of for and to and PP2B in the presence of J. Jr., J. Cell. Full Text PDF PubMed Scopus Google Scholar). Calcineurin autoinhibitory peptide is a for PP2B J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). In our actin filaments not to thick filaments after the catch treatment in the presence of 1 μm cyclosporin A, 1 μm FK506, or 0.2 mm calcineurin autoinhibitory peptide These results indicate that present in the Mytilus catch muscles, and PP2B was involved in the initiation of the catch state. The that Ca2+ is required for the initiation of the catch state A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (55) Google Scholar) is with since PP2B is a protein phosphatase (13Tonks N.K. Cohen P. Biochim. Biophys. Acta. 1983; 747: 191-193Crossref PubMed Scopus (34) Google Scholar). It be that after the catch treatment phosphatase actin filaments bound to thick filaments in the presence of not The results indicate that not the binding of actin filaments in the catch state the initiation of the catch state. the results indicate that PP2B is involved in the initiation of the catch state, it is not whether PP2B and its activator calmodulin (13Tonks N.K. Cohen P. Biochim. Biophys. Acta. 1983; 747: 191-193Crossref PubMed Scopus (34) Google Scholar) are the only in the soluble protein fraction of muscles essential for the initiation of the catch state. thus the catch treatment with bovine calcineurin and calmodulin instead of the soluble protein that actin filaments bound to thick relaxation treatment with bovine PKA terminated the catch state Thus, PP2B and calmodulin are essential in the soluble protein fraction for the initiation of the catch state, and PKA is essential for its It be that the initiation of the catch state by PP2B and calmodulin not Phosphorylation during and of Catch protein phosphorylation during the and initiation of the catch state, thick filaments in the catch state. The state was identified by the binding of actin filaments to the filaments in the presence of MgATP at a low free Ca2+ concentration These thick filaments to be in the dephosphorylated state. relaxation treatment with the soluble protein fraction actin filaments from the thick filaments The catch state was restored by the following catch treatment with the soluble protein in the binding of actin filaments to the thick filaments The results with bovine PKA in the relaxation treatment and with bovine calcineurin and calmodulin in the catch treatment instead of the soluble protein which protein the relaxation in the presence of and that twitchin, and the not and Twitchin was the in which is with the results for skinned catch muscle fibers (9Siegman M.J. Funabara D. Kinoshita S. Watabe S. Hartshorne D.J. Butler T.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5383-5388Crossref PubMed Scopus (83) Google Scholar). the relaxation treatment in the presence of catch treatment was that still in the during this catch and autoradiography that twitchin was the other and that the not and These results indicate that of twitchin is to the initiation of the catch state and that the phosphorylation of other such as myosin, and are not involved in the regulation of the catch state. of and the of a of PKA to the of kinase in the soluble protein fraction that is for phosphorylation of twitchin. When a peptide of PKA L. S.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar) was added during relaxation actin filaments not to thick filaments not showed that in the presence of this twitchin was not and to some The of and thus to other types of of twitchin during catch treatment was by μm okadaic acid, μm microcystin LR, 1 μm cyclosporin A, 1 μm FK506, or 0.2 mm calcineurin autoinhibitory peptide the catch treatment with to initiate the catch state in the in vitro reconstitution the results are with the that of twitchin initiates the catch state. It be that twitchin was in the The of to be a in its thick filaments showed the catch and as of Catch and Phosphorylation with and reported A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (55) Google Scholar), actin filaments to thick filaments of purified and twitchin in the catch state. autoradiography with purified to of other that the thick Twitchin was with the soluble protein fraction and Ca2+ and was using This treatment dephosphorylated twitchin. When filaments with this twitchin, they bound actin filaments in the presence of MgATP at a low free Ca2+ concentration relaxation treatment with the soluble protein only a actin filaments bound to the thick filaments catch treatment with the soluble protein fraction restored the binding of actin filaments to thick filaments PKA was for the soluble protein fraction in the relaxation treatment catch treatment with bovine calcineurin and calmodulin in binding of actin filaments to the thick filaments the was added during the relaxation showed that twitchin was during the relaxation treatment and dephosphorylated during the catch treatment These results the as with thick filaments and phosphorylation and of twitchin was also of the to the showed that the either with or twitchin was by the soluble protein no phosphorylation of the was with with the bovine This that the phosphorylation of the was to other PKA and that this phosphorylation was not involved in the regulation of the catch state. It be that purified twitchin was and that the of in the after the catch treatment with the soluble protein fraction The was of in the soluble was not in the catch treatment with bovine calcineurin and calmodulin The using filaments The in vitro reconstitution that they bound to no actin filaments after the catch treatment A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (55) Google Scholar). The was by treatment with the soluble protein fraction and was not with the bovine and not at in of the not of Catch with this used thick filaments for the catch state instead of thick filaments, of which not to actin filaments in the catch state A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (55) Google Scholar). This might have been of the of thick filaments or in the thick filaments by of the of muscle they also to be might be other for the of the in the of the thick filaments, it was to thin filaments from the muscle and thin filaments might have some of the of the thick filaments in the catch state. The present thick filaments only a of actin more actin filaments with could to the thick filaments to the it is well that thick filaments of catch muscles a of in their Cohen C. J. J. Biol. 1971; PubMed Scopus Google Scholar, B. J. Cell Biol. PubMed Scopus Google Scholar). In the present thick filaments of and and as is not involved in the catch state. The of in the thick filaments be in the have ability to to actin filaments in the catch state. In they to and to with a These indicate that reconstitution assay of the catch state with thick filaments is more that with of in other types of muscles thus thick filaments PP2B Twitchin in Catch and Cohen (12Castellani L. Cohen C. FEBS Lett. 1992; 309: 321-326Crossref PubMed Scopus (34) Google Scholar) reported that chemically skinned catch muscles their calcineurin-like phosphatase and their ability to maintain catch tension with the ability is restored by bovine that calcineurin-like phosphatase is required for catch contraction. the catch state is terminated by PKA (4Pfitzer G. Rüegg J.C. J. Comp. Physiol. 1982; 147: 137-142Crossref Scopus (55) Google Scholar), its reverse reaction, is required for the initiation of the catch state. with skinned muscles suggested that phosphorylation of twitchin is with the relaxation of the catch state (9Siegman M.J. Funabara D. Kinoshita S. Watabe S. Hartshorne D.J. Butler T.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5383-5388Crossref PubMed Scopus (83) Google Scholar) and that purified twitchin is by PKA (10Funabara D. Kinoshita S. Watabe S. Siegman M.J. Butler T.M. Hartshorne D.J. Biochemistry. 2001; 40: 2087-2095Crossref PubMed Scopus (39) Google Scholar, 11Funabara D. Watabe S. Mooers S.U. Narayan S. Dudas C. Hartshorne D.J. Siegman M.J. Butler T.M. J. Biol. Chem. 2003; 278: 29308-29316Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). These results indicate that the target of the PKA is twitchin, the phosphorylation of which the catch state of molluscan muscles. As for the reverse reaction, calcineurin-like phosphatase PP2B dephosphorylate twitchin in a no has been that twitchin is the target for more complicated phosphatase cascade such as in muscles was also In muscles, the of protein phosphatase 1 is by a protein This protein is by PKA and dephosphorylated by PP2B, and its protein phosphatase Thus, PP2B dephosphorylates 1 activating protein phosphatase which dephosphorylates other such as a Cohen P. 1983; PubMed Scopus Google Scholar). In the present that low of okadaic or microcystin LR, to protein phosphatase not the initiation of the catch state and This that the phosphatase cascade of PP2B and protein phosphatase as in muscles Cohen P. 1983; PubMed Scopus Google Scholar), not initiate the catch state. also that to PP2B such as cyclosporin A, FK506, and calcineurin autoinhibitory peptide of twitchin and initiation of the catch state. This that phosphatases other PP2B not dephosphorylate twitchin, the catch state. In in the catch state with myosin, twitchin, actin, bovine calcineurin and its activator calmodulin. In this twitchin was and this These results indicate that twitchin is the target of PP2B and that its initiates the catch state. by PKA and in vitro studies have not only the protein essential for the catch state and its regulation also their during the of the These are actin, myosin, twitchin, PKA, PP2B, and and their are in Twitchin is with filaments A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (55) Google Scholar). In the state, twitchin is and actin filaments are from the When the muscle is by the intracellular free Ca2+ concentration and the Ca2+ not only also the Thus, the active PP2B directly dephosphorylates twitchin during active of the It be that the present showed no other component is required for this of the results in a in the intracellular free Ca2+ which the of the twitchin is in the dephosphorylated state at this actin filaments to the filaments (the catch state). Phosphorylation of twitchin by the active PKA causes of the actin filaments from the filaments, in the of the catch state (the state). The and have the in of the regulation of the catch state. they increase in catch muscle phosphorylation and of twitchin are in the of ATP In bovine PKA phosphorylates and that cAMP. When the intracellular Ca2+ concentration PP2B dephosphorylates and the in a in the concentration Cell. Biochem. Scopus Google Scholar). it is whether such a also in catch muscles, it could be for the catch and the of ATP of Twitchin in vitro reconstitution have that twitchin is essential for the catch state A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (55) Google Scholar). or is a of the present in (5Benian G.M. Kiff J.E. Neckelmann N. Morerman D.G. Waterston R.H. Nature. 1989; 342: 45-50Crossref PubMed Scopus (263) Google Scholar, 6Benian G.M. Ayme-Southgate A. Tinley T.L. Rev. Physiol. Biochem. Pharmacol. 1999; 138: 235-268Crossref PubMed Google Scholar) and (7Vibert P. Edelstein S.M. Castellani L. Elliott Jr., B.W. J. Muscle Res. Cell Motil. 1993; 14: 598-607Crossref PubMed Scopus (73) Google Scholar, 8Vibert P. York M.L. Castellani L. Edelstein S. Elliott B. Nyitray L. Adv. Biophys. 1996; 33: 199-209Crossref PubMed Scopus (8) Google Scholar, J. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The also and that they protein kinase that has a to that of smooth muscle kinase their (5Benian G.M. Kiff J.E. Neckelmann N. Morerman D.G. Waterston R.H. Nature. 1989; 342: 45-50Crossref PubMed Scopus (263) Google Scholar, 6Benian G.M. Ayme-Southgate A. Tinley T.L. Rev. Physiol. Biochem. Pharmacol. 1999; 138: 235-268Crossref PubMed Google Scholar, D. Kinoshita S. Watabe S. Siegman M.J. Butler T.M. Hartshorne D.J. Biochemistry. 2001; 40: 2087-2095Crossref PubMed Scopus (39) Google Scholar, 11Funabara D. Watabe S. Mooers S.U. Narayan S. Dudas C. Hartshorne D.J. Siegman M.J. Butler T.M. J. Biol. Chem. 2003; 278: 29308-29316Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). of this and twitchin kinase phosphorylates J. A. J. Biochem. PubMed Scopus (34) Google Scholar), their are to be In our in vitro reconstitution however, no of 32P the during either the relaxation or the catch treatment Thus, the kinase of twitchin, it is not to the regulation of the catch state. The of its kinase to be