Site-specific Phosphorylation and Point Mutations of Telokin Modulate Its Ca2+-desensitizing Effect in Smooth Muscle

Forskolin and 8-bromoguanosine 3′-5′-cyclic monophosphate (8-Br-cGMP) induce phosphorylation of Ser-13 of telokin and relaxation of smooth muscle at constant calcium. Comparison with the effect of wild type with aspartate (D; to mimic phosphorylation) and alanine (A; non-phosphorylatable) mutants of telokin showed that the S13D mutant was more effective than wild type in relaxing smooth muscle at constant calcium. The efficacy of the Ser-13A, S12A, and S12D mutants was not significantly different from that of wild-type telokin. The effect of neither S13D nor Ser-13A was affected by 8-Br-cGMP, whereas the effect of wild type, S12A, and S12D was enhanced by 8-Br-cGMP, indicating the specificity of Ser-13 charge modification. Mutation of Ser-19 (a mitogen-activated protein kinase site) showed the S19A to be more effective than, and S19D to be not different from, wild-type telokin. The effect of both mutants was slightly enhanced by 8-Br-cGMP. A truncated (residues 1–142) form lacking the acidic C terminus had the same relaxant effect as wild-type telokin, whereas the C-terminal peptide (residues 142–155) had no effect. We conclude that site-specific modification of the N terminus modulates the Ca2+-desensitizing effect of telokin on force. Forskolin and 8-bromoguanosine 3′-5′-cyclic monophosphate (8-Br-cGMP) induce phosphorylation of Ser-13 of telokin and relaxation of smooth muscle at constant calcium. Comparison with the effect of wild type with aspartate (D; to mimic phosphorylation) and alanine (A; non-phosphorylatable) mutants of telokin showed that the S13D mutant was more effective than wild type in relaxing smooth muscle at constant calcium. The efficacy of the Ser-13A, S12A, and S12D mutants was not significantly different from that of wild-type telokin. The effect of neither S13D nor Ser-13A was affected by 8-Br-cGMP, whereas the effect of wild type, S12A, and S12D was enhanced by 8-Br-cGMP, indicating the specificity of Ser-13 charge modification. Mutation of Ser-19 (a mitogen-activated protein kinase site) showed the S19A to be more effective than, and S19D to be not different from, wild-type telokin. The effect of both mutants was slightly enhanced by 8-Br-cGMP. A truncated (residues 1–142) form lacking the acidic C terminus had the same relaxant effect as wild-type telokin, whereas the C-terminal peptide (residues 142–155) had no effect. We conclude that site-specific modification of the N terminus modulates the Ca2+-desensitizing effect of telokin on force. smooth muscle myosin light chain kinase smooth muscle myosin light chain phosphatase 20-kDa regulatory myosin light chain 8-bromoguanosine 3′-5′-cyclic monophosphate cGMP-dependent protein kinase intracellular free calcium concentration 1,4-piperazinediethanesulfonic acid wild type Contraction and relaxation of smooth muscle are dependent mainly upon the intracellular free Ca2+ concentration ([Ca2+]i ). The magnitude of contractile force developed depends on the level of myosin regulatory light chain (MLC20)1phosphorylation that, in turn, is determined by the relative activities of myosin light chain kinase (MLCK) and smooth muscle myosin light chain phosphatase (SMPP-1M). The Ca2+ sensitivity of contraction can be affected by any change in kinase and/or phosphatase activities that alters this ratio. At a fixed [Ca2+]i , both MLCK and SMPP-1M can be modulated by second messenger-mediated signaling pathways, resulting in a change in MLC20 phosphorylation and, consequently, in an increase or decrease in force developed (reviewed in Refs. 1Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1733) Google Scholar and 2Somlyo A.P. Somlyo A.V. J. Physiol. 2000; 522: 177-185Crossref PubMed Scopus (1080) Google Scholar). The Ca2+ sensitivity of the contractile apparatus can be regulated by a number of agents such as RhoA (3Gong M.C. Iizuka K. Nixon G. Browne J.P. Hall A. Eccleston J.F. Sugai M. Kobayashi S. Somlyo A.V. Somlyo A.P. Proc. Natl. Acad Sci. U. S. A. 1996; 93: 1340-1345Crossref PubMed Scopus (266) Google Scholar, 4Somlyo A.P. J. Physiol. 1999; 516: 630Crossref PubMed Scopus (12) Google Scholar, 5Uehata M. Ishizaki T. Satoh H. Ono T. Kawahara T. Morishita T. Tamakawa H. Yamagami K. Inui J. Maekawa M. Narumiya S. Nature. 1997; 389: 990-994Crossref PubMed Scopus (2555) Google Scholar), cyclic nucleotides (6Wu X. Somlyo A.V. Somlyo A.P. Biochem. Biophys. Res. Commun. 1996; 220: 658-663Crossref PubMed Scopus (139) Google Scholar, 7Nishimura J. van Breemen C. Biochem. Biophys. Res. Commun. 1989; 163: 929-935Crossref PubMed Scopus (192) Google Scholar), arachidonic acid (8Gong M.C. Kinter M.T. Somlyo A.V. Somlyo A.P. J. Physiol. 1995; 486: 113-122Crossref PubMed Scopus (64) Google Scholar), and diacylglycerol (8Gong M.C. Kinter M.T. Somlyo A.V. Somlyo A.P. J. Physiol. 1995; 486: 113-122Crossref PubMed Scopus (64) Google Scholar). Dephosphorylation of the regulatory myosin light chains by SMPP-1M can be inhibited by a G-protein-coupled pathway that can increase the level of MLC20 phosphorylation, with no change in cytosolic calcium (Ca2+ sensitization) (1Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1733) Google Scholar, 9Somlyo A.P. Kitazawa T. Himpens B. Matthijs G. Horiuti K. Koayashi S. Goldman Y.E. Somlyo A.V. Adv. Protein Phosphatases. 1989; 5: 181-195Google Scholar, 10Somlyo A.P. Wu X. Walker L.A. Somlyo A.V. Rev. Physiol. Biochem. Pharmacol. 1999; 134: 201-234PubMed Google Scholar), and we suggested that the reverse mechanism, activation of SMPP-1M, could lead to Ca2+ desensitization. Stimulation of permeabilized smooth muscle with 8-Br-cGMP relaxes submaximal Ca2+-induced contractions and can also reverse G-protein-coupled inhibition of SMPP-1M (6Wu X. Somlyo A.V. Somlyo A.P. Biochem. Biophys. Res. Commun. 1996; 220: 658-663Crossref PubMed Scopus (139) Google Scholar), suggestive of a mechanism mediated by activation of SMPP-1M. Telokin, also known as kinase-related protein, is a low molecular mass (17 kDa) protein whose sequence is identical to the C terminus of MLCK (11Ito M. Dabrowska R. Guerriero Jr., V. Hartshorne D.J. J. Biol. Chem. 1989; 264: 13971-13974Abstract Full Text PDF PubMed Google Scholar) and is independently expressed at high concentrations in some smooth muscles (12Gallagher P.J. Herring B.P. J. Biol. Chem. 1991; 266: 23945-23952Abstract Full Text PDF PubMed Google Scholar) through a promoter located within an intron of the MLCK gene (13Herring B.P. Smith A.F. Am. J. Physiol. 1996; 270: C1656-C1665Crossref PubMed Google Scholar). Telokin binds to the S1/S2 region of unphosphorylated smooth muscle myosin (14Shirinsky V.P. Vorotnikov A.V. Birukov K.G. Nanaev A.K. Collinge M. Lukas T.J. Sellers J.R. Watterson D.M. J. Biol. Chem. 1993; 268: 16578-16583Abstract Full Text PDF PubMed Google Scholar, 15Masato T. Numata T. Katoh T. Morita F. Yazawa M. J. Biochem. 1997; 121: 225-230PubMed Google Scholar) and prevents the folding of the 6 S myosin into 10 S conformation, thus stabilizing filamentous myosin in solution (14Shirinsky V.P. Vorotnikov A.V. Birukov K.G. Nanaev A.K. Collinge M. Lukas T.J. Sellers J.R. Watterson D.M. J. Biol. Chem. 1993; 268: 16578-16583Abstract Full Text PDF PubMed Google Scholar). Stabilization of unphosphorylated myosin filaments has been suggested to be a physiological function of telokin in smooth muscle (14Shirinsky V.P. Vorotnikov A.V. Birukov K.G. Nanaev A.K. Collinge M. Lukas T.J. Sellers J.R. Watterson D.M. J. Biol. Chem. 1993; 268: 16578-16583Abstract Full Text PDF PubMed Google Scholar), and its acidic C terminus was believed necessary for high affinity binding to myosin and filament stabilization. Furthermore, it has been shown (16Rusconi F. Potier M.C. Le Caer J.P. Schmitter J.M. Rossier J. Biochemistry. 1997; 36: 11021-11026Crossref PubMed Scopus (11) Google Scholar) that there are at least 6 C-terminal variants of telokin, differing from each other in the number of C-terminal glutamates (from 0–5), and it was suggested that the heterogeneity of the C terminus may play an important role in the regulation of macromolecular protein complexes at the structural level (16Rusconi F. Potier M.C. Le Caer J.P. Schmitter J.M. Rossier J. Biochemistry. 1997; 36: 11021-11026Crossref PubMed Scopus (11) Google Scholar). The concentration of endogenous telokin is high (70–80 μm) in some phasic smooth muscles, and its loss is accompanied by a linearly related loss of 8-Br-cGMP-induced relaxation and an enhanced responsiveness to exogenously added native and recombinant telokin that can relax permeabilized smooth muscle and reduce MLC20 phosphorylation (17Wu X. Haystead T.A. Nakamoto R.K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 11362-11369Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The relaxant effect of telokin is potentiated by 8-Br-cGMP and cyclic GMP-dependent kinase (PKG), and we suggested that phosphorylation of telokin by PKG (or cAMP-dependent protein kinase) modulates desensitization of smooth muscle to calcium, as telokin is the major cytosolic protein phosphorylated during cGMP-induced relaxation and dephosphorylation of MLC20(17Wu X. Haystead T.A. Nakamoto R.K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 11362-11369Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Most recently, we have mapped the in vivo site of phosphorylation of mammalian telokin to Ser-13 in intact, forskolin-stimulated and permeabilized, 8-Br-cGMP-stimulated rabbit ileum (18MacDonald J.A. Walker L.A. Nakamoto R.K. Gorenne I. Somlyo A.V. Somlyo A.P. Haystead T.A.J. FEBS Lett. 2000; 479: 83-88Crossref PubMed Scopus (35) Google Scholar). Therefore, to determine whether Ser-13 is also the functionally relevant site of telokin phosphorylation, we have produced mutant telokins with replacement of Ser-13, Ser-12, and Ser-19 with either an aspartate or alanine to determine their respective contributions to calcium-independent effects on force and MLC20 phosphorylation. Additionally we have expressed a telokin mutant lacking the acidic C terminus to examine the other proposed functions (14Shirinsky V.P. Vorotnikov A.V. Birukov K.G. Nanaev A.K. Collinge M. Lukas T.J. Sellers J.R. Watterson D.M. J. Biol. Chem. 1993; 268: 16578-16583Abstract Full Text PDF PubMed Google Scholar, 19Sobieszek A. Andruchov O.Y. Nieznanski K. Biochem. J. 1997; 328: 425-430Crossref PubMed Scopus (13) Google Scholar, 20Silver D.L. Vorotnikov A.V. Watterson D.M. Shirinsky V.P. Sellers J.R. J. Biol. Chem. 1997; 272: 25353-25359Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) of telokin. Longitudinal ileal smooth muscle was removed from rabbits anesthetized with halothane and killed by exsanguination according to approved animal protocols. Small strips (250 μm × 2 mm) were dissected and permeabilized with Triton X-100 (0.1%) in a Ca2+-free solution containing 1 mm EGTA (G1) for 30 min. For storage, muscle strips were washed with relaxing solution (containing 10 mm EGTA and 40 mmreduced glutathione), placed in the same relaxing solution containing 50% glycerol, and stored at −20 °C. of the for of permeabilized smooth muscle of muscles, and the of of endogenous telokin have been (17Wu X. Haystead T.A. Nakamoto R.K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 11362-11369Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, T. M. Somlyo A.P. Proc. Natl. Acad Sci. U. S. A. 1991; PubMed Scopus Google Scholar). For force strips were removed from the washed in and with to the of was and the other was to a force The was in a on a to solution and were to were at The telokin of strips was determined by and For in of exogenously added telokin, permeabilized, strips were in a with submaximal calcium, washed in solution containing mm and for 10 in the same solution with strips were by the of either wild-type or mutant μm) telokin in the or of 8-Br-cGMP and 10 strips were and in and by were to and placed on for were and for telokin. For strips of ileal smooth muscle were to as fixed in acid by with and and in strips as telokin was as a from Herring of (12Gallagher P.J. Herring B.P. J. Biol. Chem. 1991; 266: 23945-23952Abstract Full Text PDF PubMed Google Scholar). The was by chain F. The Google Scholar) the site and a that to the The chain was into P.J. Protein 1999; PubMed Scopus Google Scholar) from (from the placed the telokin sequence of the and an with the sequence from native telokin by the and The sequence was by mass determined that the molecular of the protein was as chain F. The Google Scholar) was to and of Ser-13, Ser-12, and Ser-19 or a in of each the was to that no were by the molecular were as J. T. A Scholar). Telokin was expressed by with the recombinant were at in with S. J. 93: PubMed Google Scholar) to an at of Protein was for 2 by the of 1 mm were by through a at into a containing mm mm glycerol, and 1 mm at and were removed by a × for 1 The was a with the same the protein was with 10 mm in mm the mm 1 and 10 of protein of was and the was for at at °C. the by 30 mm the telokin was by a × with by a or the The were by The was to 30 mm mm mm for the of telokin to muscle The protein was a Protein concentrations were determined from the at the of (14Shirinsky V.P. Vorotnikov A.V. Birukov K.G. Nanaev A.K. Collinge M. Lukas T.J. Sellers J.R. Watterson D.M. J. Biol. Chem. 1993; 268: 16578-16583Abstract Full Text PDF PubMed Google Scholar). (18MacDonald J.A. Walker L.A. Nakamoto R.K. Gorenne I. Somlyo A.V. Somlyo A.P. Haystead T.A.J. FEBS Lett. 2000; 479: 83-88Crossref PubMed Scopus (35) Google Scholar), mutant telokin was phosphorylated for 2 with of PKG in a containing mm 1 mm and mm The protein was with and the were by reverse The was and reverse high and its acid sequence was determined by and each light chain phosphorylation was determined as T. Somlyo A.P. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). the same smooth muscle strips for were in at and removed from the were to acid in and stored for a of and to washed with and were in a containing glycerol, mm and were by and to as T. Somlyo A.P. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). The were with and were by The phosphorylation was expressed as the unphosphorylated light chain and and phosphorylated light A of was are the and is the number of was at the of to in rabbit telokin. Protein kinase and 8-Br-cGMP were from other were from shown (17Wu X. Haystead T.A. Nakamoto R.K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 11362-11369Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), recombinant telokin rabbit ileal smooth muscle in a the of 8-Br-cGMP, 10 μm telokin relaxation of a submaximal calcium contraction and μm telokin by with 8-Br-cGMP for potentiated the effect of telokin and the relaxation by 10 μm telokin to and μm to that of the contractile to submaximal calcium. 10 μm S13D mutant telokin the charge on phosphorylated ileum to and μm S13D telokin by 8-Br-cGMP not significantly change the relaxation by S13D telokin at any concentration 10 μm Ser-13A telokin phosphorylation of ileum by and was not affected by with 8-Br-cGMP. determine whether phosphorylation of the to Ser-13, by in phosphorylation of the the relaxant of telokin, we both the and S12D the of 8-Br-cGMP, the mutant permeabilized ileum by and its effect was potentiated to by 8-Br-cGMP the S12D in the of 8-Br-cGMP, smooth muscle by and was potentiated to are not significantly different from of that is not a relevant phosphorylation site in the of other phosphorylation. A telokin mutant with a at the mitogen-activated protein kinase in a relaxation of strips at 10 μm and the S19D mutant in that phosphorylation of Ser-19 may a physiological of effect. Therefore, we a mutant with an at and an at 10 ileum by not significantly different from 8-Br-cGMP and PKG the relaxation by this mutant μm) to either a site of phosphorylation of the telokin or the of that, can with telokin that is not phosphorylated at determine whether of the relaxant effect of telokin by 8-Br-cGMP was related to phosphorylation of this mutant at a site other than Ser-13 or we the in of the the of exogenously added telokin, phosphorylation of the of endogenous telokin that in strips was both in the and of 8-Br-cGMP. the of an increase in into endogenous telokin in the of 8-Br-cGMP that this telokin is not a for PKG or is added and telokin were phosphorylated in the of 8-Br-cGMP that the mutant could be phosphorylated at the site of phosphorylation of the telokin, the recombinant mutant was phosphorylated in and as for site (18MacDonald J.A. Walker L.A. Nakamoto R.K. Gorenne I. Somlyo A.V. Somlyo A.P. Haystead T.A.J. FEBS Lett. 2000; 479: 83-88Crossref PubMed Scopus (35) Google Scholar, Jr., J. Biol. Chem. Full Text PDF PubMed Google Scholar). phosphorylation, the site phosphorylated on the mutant was this site is not phosphorylated in in 8-Br-cGMP or forskolin-stimulated (18MacDonald J.A. Walker L.A. Nakamoto R.K. Gorenne I. Somlyo A.V. Somlyo A.P. Haystead T.A.J. FEBS Lett. 2000; 479: 83-88Crossref PubMed Scopus (35) Google Scholar) or in recombinant rabbit telokin the Ser-13 is to either or S13D that it is not the of Ser-13 that to a kinase and the was phosphorylated in the we the and both of mutants were potentiated by 8-Br-cGMP in the of 8-Br-cGMP, a submaximal calcium contraction by and by with 8-Br-cGMP the relaxation by mutants to and Furthermore, neither of the mutants was phosphorylated in smooth muscle strips were with in the and of 8-Br-cGMP and the mutant protein we were to both mutants with cAMP-dependent protein kinase or PKG shown in and S13D telokin μm) relaxation of ileum smooth Therefore, we the phosphorylation of the regulatory light chains of myosin and relaxation to be with a decrease in MLC20 phosphorylation. a decrease in phosphorylation from in the of submaximal calcium to and S13D telokin a decrease in MLC20 phosphorylation to 10 min. phosphorylation in the stored ileum muscles in the of calcium was high level of phosphorylation in the of calcium may be to a decrease in endogenous phosphatase in smooth muscles C. J. Proc. Biol. PubMed Google Scholar) and/or the of a kinase as as can in on J. Biol. Res. Google Scholar, S. J. Biochem. PubMed Scopus Google Scholar), and it has been suggested D.L. Vorotnikov A.V. Watterson D.M. Shirinsky V.P. Sellers J.R. J. Biol. Chem. 1997; 272: 25353-25359Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) that the C-terminal to is the site of telokin and whether the C-terminal of telokin had relaxant as the protein through either a effect on the phosphatase or through binding to we a acid peptide to the C terminus (residues 142–155) of rabbit telokin The of the acidic C-terminal peptide μm) of telokin not relax submaximal contractions of rabbit ileum not nor it with relaxation by 10 μm telokin not a truncated telokin the acidic C terminus ileum at constant calcium in a with no from wild 10 μm truncated telokin a submaximal calcium contraction by and was potentiated to in the of μm 8-Br-cGMP, and in the of 8-Br-cGMP and was potentiated to MLC20 phosphorylation is regulated by both and we the that telokin was MLCK A. J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), it not the of (17Wu X. Haystead T.A. Nakamoto R.K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 11362-11369Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). rabbit ileum strips were in solution μm telokin for 10 min. μm) was added to SMPP-1M. the muscle was with submaximal calcium. of stored rabbit ileum with 10 not the nor change the of contraction in the of to telokin inhibited it be to the and/or magnitude of in the of a phosphatase has been suggested that major role of telokin is of myosin filaments in vivo (14Shirinsky V.P. Vorotnikov A.V. Birukov K.G. Nanaev A.K. Collinge M. Lukas T.J. Sellers J.R. Watterson D.M. J. Biol. Chem. 1993; 268: 16578-16583Abstract Full Text PDF PubMed Google we the filament in and smooth ileum smooth muscle was permeabilized with Triton X-100 as for telokin showed that more than of the telokin was removed within in Triton of of ileum strips a number and of myosin filaments by filaments and of with in the and were with the effects of the major that of the in and/or kinase site of phosphorylation of telokin to an its to relax smooth muscles, whereas of Ser-13 to not its relaxant not the relaxant of either or of the to either an or an not change the of and the acidic C-terminal peptide of telokin not mimic the relaxant effect of telokin, whereas the truncated mutant is as effective as telokin in relaxation of smooth We also that of endogenous telokin not lead to the loss or of myosin that Ser-13 is a relevant in vivo phosphorylation site with cyclic relaxation and that the acidic C terminus of telokin is neither necessary nor for its relaxant effect. A of Ser-13 to to mimic a mitogen-activated protein kinase to a an in phosphorylation the physiological role of phosphorylation of is The are with (17Wu X. Haystead T.A. Nakamoto R.K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 11362-11369Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) that cyclic relaxation is mediated at least in by phosphorylation of telokin and of myosin light chain phosphatase (17Wu X. Haystead T.A. Nakamoto R.K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 11362-11369Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The at that is a in vivo site of phosphorylation by mitogen-activated protein kinase (17Wu X. Haystead T.A. Nakamoto R.K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 11362-11369Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) as not an increase in phosphorylation rabbit ileal smooth muscle was with either or permeabilized ileal muscle was with 8-Br-cGMP (18MacDonald J.A. Walker L.A. Nakamoto R.K. Gorenne I. Somlyo A.V. Somlyo A.P. Haystead T.A.J. FEBS Lett. 2000; 479: 83-88Crossref PubMed Scopus (35) Google Scholar). of Ser-19 to either an or an are with phosphorylation of this site a site the S19D mutant can be phosphorylated on Ser-13 or Furthermore, it has been shown A.V. V.P. 2000; Google Scholar) that of either smooth muscle or with in an increase in phosphorylation of telokin at an as site that smooth muscle not with in the regulation of and phasic smooth phosphorylation of Ser-13, it to be shown whether Ser-19 phosphorylation is relevant in the of physiological concentrations of in muscles or telokin may be a for other signaling We the effects of the of telokin within the of the native telokin a by an acid and C-terminal acid the N nor the C terminus is in either the at M. Hartshorne D.J. I. J. Biol. PubMed Scopus Google Scholar) or a more at as the of of The that the acidic C terminus is neither nor necessary for the relaxant effect of telokin not the of in such the truncated is also acidic with a acidic M. Hartshorne D.J. I. J. Biol. PubMed Scopus Google Scholar). of the N terminus that the phosphorylation not the relaxant effect of telokin (17Wu X. Haystead T.A. Nakamoto R.K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 11362-11369Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), it its by 8-Br-cGMP. charge modification of acidic of the may be in the structural for the relaxant of telokin. Furthermore, on the telokin mutants in the as suggested by a increase in the the and S19D telokin mutants no by A. A. V. and A. and of Ser-19 to an alanine the to it is that a the it is that of the Ser-19 and and the native by 8-Br-cGMP and PKG an with the or Therefore, we are to a physiological function to the effects of the of a phosphatase of ileal strips with 10 μm wild-type telokin not change either the or of the (17Wu X. Haystead T.A. Nakamoto R.K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 11362-11369Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) that telokin through or activation of the phosphatase than inhibition of myosin light chain kinase A. J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). we have thus been to in a effect of telokin on phosphatase (18MacDonald J.A. Walker L.A. Nakamoto R.K. Gorenne I. Somlyo A.V. Somlyo A.P. Haystead T.A.J. FEBS Lett. 2000; 479: 83-88Crossref PubMed Scopus (35) Google Scholar). may the to the structural myosin phosphatase and and telokin. is also that is in the phosphorylation of telokin and relaxation activation of the of smooth A of in to telokin are phosphorylated during cyclic relaxation not to and and A. J. A. X. R. K. T. A. J. A. V. and A. The that or more of are in with telokin to the myosin phosphatase is we of no to that either or with telokin, can SMPP-1M. telokin is in phosphorylated by cyclic G. M. PubMed Scopus Google Scholar, J. Biol. Google Scholar, U. F. J. Biol. Chem. Full Text PDF PubMed Google Scholar) are to be relevant to telokin The that the physiological role of telokin is to unphosphorylated myosin filaments through binding its acidic to myosin (14Shirinsky V.P. Vorotnikov A.V. Birukov K.G. Nanaev A.K. Collinge M. Lukas T.J. Sellers J.R. Watterson D.M. J. Biol. Chem. 1993; 268: 16578-16583Abstract Full Text PDF PubMed Google Scholar, 20Silver D.L. Vorotnikov A.V. Watterson D.M. Shirinsky V.P. Sellers J.R. J. Biol. Chem. 1997; 272: 25353-25359Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) is not with the myosin filament in with Triton X-100 and of endogenous telokin (17Wu X. Haystead T.A. Nakamoto R.K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 11362-11369Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) and with the of effect of of the acidic C terminus on the relaxant effect of telokin We the that in to the structural and SMPP-1M, and in there was a of telokin that is in of the low of telokin to myosin than μm that the of telokin in permeabilized is for filament we that the of phosphorylation of the Ser-13 of telokin in cyclic dephosphorylation of MLC20 and relaxation of smooth

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